Since 1980, Clinical and Laboratory Standards Institute (CLSI) has recommended a correction for citrate blue-top Vacutainer-type tubes for patients with high hematocrit greater than 55%. The correction applies to routine coagulation tests and only to patients with high hematocrits and not to patients with low hematocrits.¹ The idea is to adjust the amount of citrate anticoagulant so that the anticoagulant:plasma ratio stays fairly constant (1:9). In blood samples with elevated hematocrit values, the plasma is less, and therefore the citrate concentration is more in the plasma in the collection tube (fairly constant anticoagulant:plasma ratio of 1:9 is not maintained); and the citrate is present in great excess after binding the free-ionized calcium of blood. When the plasma is added to the clotting test reagents subsequently, the residual excess citrate in the plasma binds a significant amount of calcium that is added to the clotting test reaction. This causes an artificial increase in clotting time or prolongation of coagulation test results. ^1–5 Thus, either the amount of citrate in the tube should be reduced or more blood should be added to the tube in order for a valid result to be obtained.

Over the last few decades, several articles and studies on this gray zone have come to form the basis for the continued emphasis on the practice of correction of citrate levels for polycythemic patients. However, all the studies deal only with artificially manipulated tubes, and none of them deal with actual polycythemic patients.

Later, Marlar et al.² studied and directly compared the effect of adjusted and non-adjusted citrate concentrations on coagulation test results in blood samples from patients with high hematocrit values. With a direct study, Marlar et al. emphasized the fact that adjusting the citrate concentrations and volume for a high hematocrit value is valid to determine the prothrombin time (PT) and activated partial thromboplastin time (APTT).

The concept of correction undergoes decades of “carryover” as authors of books and articles repeat the statement without verification. The CLSI relies on statements and inferences from earlier books and articles, none of which deal with actual polycythemic patients.^1,2 It is an unproven leap of faith to assume that if artificially manipulated samples have prolonged APTTs and PTs, then actual patient samples will also have in a similar manner.²

Researchers manipulate samples with normal hematocrit and extrapolate test results to actual patients with high hematocrit. ^4–6 They equate underfilled citrate blue top Vacutainer tubes with polycythemia and demonstrate that underfilled tubes (or polycythemia) produce prolonged coagulation test results.⁶

Moreover, there is minimal evidence to support the concept that patients with high hematocrit have prolonged APTTs and PTs if not corrected. There are hardly any studies on actual samples with high hematocrit values to validate the need for citrate adjustment in this setting.

Hence, this is a reasonable assumption for our research into this issue, simply because laboratory policy should not be based on assumptions but built on concrete evidence.

AIMS AND OBJECTIVES

The aims are to

• Compare the effect of adjusted and non-adjusted citrate concentrations on coagulation test results (PT and APTT) in samples from patients with high hematocrit values.

• To evaluate the usefulness of the practice of adjusting citrate volume in patients with hematocrit measurements more than 55%.

MATERIALS AND METHODS

Study Population

Specimens were obtained from selected patients at a rural tertiary hospital, Chettinad Hospitals and Research Institute, Kelambakkam, during the month of April and May 2011. Patients with high hematocrit value, ie, those patients with hematocrit values greater than 55%, were selected for the study. Patients included were trauma patients, patients from local area, and patients referred from outlying areas for specialized surgical and medical treatments. The causes for high hematocrit were as follows—dehydration, smoking, hypoxia due to high altitude, polycythemia vera, and cyanotic congenital heart disease. The present study has the approval of the institutional ethics committee.

Specimen Collection

Hematocrit values were obtained on a Coulter Ac.T 5diff AL using a standard instrument protocol. Patients with high hematocrit values (more than 55%) were identified, and after getting their informed consent, two samples, ie, citrate adjusted and non-citrate adjusted blood specimens, were obtained in random order from the patient:

Non-citrate adjusted samples, ie, one standard 2.7 ml fill, 3.2% sodium citrate tube (Becton Dickinson Vacutainer System) with 0.3 ml of trisodium citrate (final citrate:blood ratio: 1:9) and

Citrate adjusted sample, ie, one standard 2.7 ml fill, 3.2% sodium citrate tube (Becton Dickinson Vacutainer System) with citrate adjusted based on hematocrit value (according to CLSI guidelines) were obtained at the same time.

For adjusted citrate tubes, a sterile tuberculin syringe was inserted through the stopper and the appropriate volume of anticoagulant was removed. The amount of citrate to be present in the blood drawing tube is

C = (1.85 * 10⁻³) (100 − Hct)(Vblood), where

C = volume of citrate remaining in tube;

Hct = hematocrit of patient;

V = volume of blood to be added.

The vacuum on the adjusted tube was retained and a normal fill volume was obtained.

Both the samples were centrifuged immediately (2500 g for 15 minutes) and plasma was removed from the cell mass and maintained at room temperature (20°C–22°C) for determination of PT and APTT. The PT and APTT were ascertained using coagulation reagents Thromborel S and Actin FSL T, respectively, and testing was done on Sysmex CA 50 Automated Coagulation Analyzer. The APTT and PT assays for adjusted and non-adjusted samples were performed in duplicates, and the results were averaged and noted.

Each patient sample pair was evaluated by determining the change in the coagulation value between the citrate adjusted sample and the non-citrate adjusted sample. Statistical significance of this difference was determined using the Wilcoxon matched pairs signed-rank test. The percentage change was calculated for each sample pair using the following equation:

%Change = (PT adjusted-PT non-adjusted)/ PT adjusted + 100%:

A clinically relevant difference was defined as change between the citrate adjusted coagulation test values of greater than 10%.

In the present study, patients were further subdivided into two subgroups based on Hct values: (1) patients with moderately elevated hematocrit (Hct: 55% to 59.99%) and (2) patients with extremely elevated hematocrit (Hct ≥ 60%). The statistical association between the hematocrit levels and clinically relevant difference in the coagulation values were ascertained using Fisher's exact test. For the current study, a P value less than 0.05 was considered statistically significant. The statistical software SPSS version 16.00 was used for the statistical analysis.

RESULTS

Paired citrate adjusted and non-adjusted citrate blood specimens were compared for 18 cases. The specimens obtained for the PT assay ranged from 10.20 seconds to 19.90 seconds with a mean PT of 13.72 seconds while the specimens obtained for the APTT assay ranged from 18.50 seconds to 35.10 seconds with a mean APTT of 25.53 seconds in the citrate adjusted tubes. On the contrary PT assay ranged from 11.60 seconds to 42.30 seconds with a mean PT of 17.82 seconds while the specimens obtained for the APTT assay ranged from 19.55 seconds to 48.30 seconds with a mean APTT of 31.17 seconds in the non-adjusted citrate tubes (Table 1).

The mean difference in the PT between the citrate adjusted and non-adjusted citrate samples was 4.11 seconds with a 95% confidence interval (0.95865 to 7.25256). The mean difference in the APTT between the citrate adjusted and non-adjusted citrate samples was 5.64 seconds with a 95% confidence interval (2.57274 to 8.70504) (Table 2). The difference in PT and APTT values ascertained with citrate adjusted and non-citrate adjusted tubes was statistically significant with P values of <.001 and .007, respectively (Table 2).

The average reduction in PT between the adjusted and non-adjusted citrate tubes in patients with elevated hematocrit was around 27.01%, the reduction being really high in the order of 40.59% in the extremely elevated hematocrit patients as compared to the low of 13.31% in the moderately elevated hematocrit patients. Although the average reduction in APTT was just hovering over the clinically relevant difference at 10.92% in the moderately elevated hematocrit patient subgroup, the difference was strikingly high at the order of 35.89% in the extremely elevated hematocrit patient subgroup. Overall, the average reduction in APTT between the citrate adjusted and non-citrate adjusted tubes in patients with elevated hematocrit was 23.41% (Table 3).

More than three fourths of patients (77.77%) with extremely elevated hematocrit had a clinically relevant difference in APTT between the adjusted and non-adjusted citrate tubes, whereas only 22.23% of patients with moderately elevated hematocrit had a clinically relevant difference. More than half the patients (66.67%) with extremely elevated hematocrit had a clinically relevant difference in PT between the adjusted and non-adjusted citrate tubes. Although there was a difference in the clinically relevant PT and APTT change distribution among moderately and extremely elevated hematocrit patients, it proved to be statistically not significant (Table 4).

DISCUSSION

There are various articles in the clinical laboratory literature recommending adjustment of citrate anticoagulant concentration in the blood collection tube to compensate for the decreased amount of plasma present in the blood sample of patients with high hematocrit values (>55%). However, the recommendations are based on indirect evidence such as short draws to simulate the increased concentrations found in samples with high hematocrit ^5–7 or artificially constructed hematocrit values^5,7,8 and not on direct evidence to demonstrate the necessity for citrate concentration adjustment in blood samples with high hematocrit. It is an unproven leap of faith to assume that if artificially manipulated samples have prolonged APTT and PT, then actual patients will also.

Due to lack of adequate direct research for establishing the standard of adjusting the citrate concentrations in blood coagulation tubes for patients with high hematocrit values, the best indirect evidence was used, which has been the basis for this practice for decades.

The present study makes a direct comparison of routine coagulation test values obtained with adjusted and non-adjusted citrate blood samples in patients with high hematocrit values.

Across all the 18 subjects, the PT dropped 4.11 seconds on an average between the non-adjusted citrate tubes and citrate adjusted tubes. This mean difference in the PT at Pearson correlation of .745 was statistically significant. The mean difference in the APTT between the citrate adjusted and non-adjusted citrate samples was 5.64 seconds with a 95% confidence interval (2.57274 to 8.70504). This difference in APTT was also proved to be statistically significant with a correlation of .611.

This difference in the PT and APTT between the citrate adjusted and non-adjusted citrate tubes in patients with elevated hematocrit shows the effect of increasing levels of citrate in the tubes apart from having a dilutional effect on the test, which also interferes with the coagulation reactions. Hence, the CLSI recommendation of adjusting the final citrate concentration in the light blue capped tubes for patients with elevated hematocrit (>55%) is correct, and the adjustment method described in the appendix of the CLSI document must be followed using a normogram or a mathematical formula.¹

In the present study, an exact half of the samples (50%; 9/18 for PT and APTT) had a clinically relevant difference between their adjusted and non-adjusted citrate tubes. However, the samples with clinically relevant difference in PT (66.67%; six out of nine) and APTT (77.78%; seven out of nine) in the extremely elevated hematocrit subgroup by far outnumbered the samples in the moderately elevated hematocrit group (33.33% for PT, 22.22% for APTT). The odds of the patient with extremely elevated hematocrit having a clinically relevant APTT change between citrate adjusted and non-adjusted citrate tubes is 12.25-fold more compared to clinically relevant APTT change in moderately elevated hematocrit patients. The odds of the patient with extremely elevated hematocrit having a clinically relevant PT change between adjusted and non-adjusted citrate tubes is four times high compared to clinically relevant PT change in moderately elevated hematocrit patients. This difference in distribution is not significant, though. However, the small sample size being a limitation is to be considered. Probably, the vast change in PT and APTT results in extremely high hematocrit is due to a synergy of dilutional effect and interfering action of the excess citrate in the coagulation test tubes and the moderate difference in the test results in moderately high hematocrit patients is due to lone dilutional effect of the excess citrate.

CLSI recommends adjustment of citrate in light blue capped tubes for all patients with hematocrit more than 55%. But, we found that the probability of having a clinically relevant difference in PT and APTT was far greater among patients with extremely high hematocrit (≥60%) when compared to patients with moderately high hematocrit (55% to 59.99%).

Hence, we would like to throw light in a different angle into this not so vastly researched field. The recommendation to adjust the citrate in light blue capped tubes for estimation of routine coagulation test is mandatory for patients with hematocrit greater than equal to 60% whereas it is not so essential to adjust the citrate for patients with hematocrit between 55% and 59.99%.

CONCLUSION

The CLSI recommendation of adjusting the final citrate concentration in the light blue capped tubes for patients with elevated hematocrit (>55%) to estimate the routine coagulation tests PT and APTT is valid; the adjustment method described in the appendix of the CLSI document must be followed using a normogram or a mathematical formula. However, we suggest that adjusting the citrate concentration in light blue capped tubes for patients with extremely high hematocrit (≥60%) is mandatory, whereas for patients with moderately high hematocrit (55% to 59.99%), it is optional to give accurate PT and APTT. However, our sample size is small to draw a definitive conclusion and further direct research with a larger sample size is warranted to validate the latter suggestion.

RECOMMENDATIONS

Based on the results of the present study, we suggest to reconsider the practice of adjusting citrate concentrations in all patients with elevated hematocrit (>55%). Instead we would recommend

• Mandatory adjustment of the citrate concentrations in light blue capped tubes before blood collection for patients with extremely high hematocrit (>60%).

• Optional adjustment of the citrate concentrations in light blue capped tubes before blood collection for patients with moderately high hematocrit (55% to 59.99%).

Acknowledgements: We thank ICMR (Indian Council of Medical Research)-STS (Short term student project) for funding this study.

REFERENCES

1. Adcock D, Hoefner D, Kottkke-Marchant K, et al. Collection, transport and processing of blood specimens for coagulation testing and performance of coagulation assays: approved guideline. NCCLS document. Wayne, PA: NCCLS; 2003. p. H21–A4.
2. Marlar R, Potts R, Marlar A. Effect on routine and special coagulation testing values of citrate anticoagulant adjustment in patients with high hematocrit values. Am J Clin Pathol. 2006;126:400–5.
3. Adcock D, Kressin D, Marlar R. Minimum specimen volume requirements for routine coagulation testing: dependence on citrate concentration. Am J Clin Pathol. 1998;109:595–9.
4. Pai S, Michalaros K. Effect of sample volume on coagulation testing. Lab Med. 1990;6:371–3.
5. Koepke J, Rodgers J, Ollivier M. Pre-instrumental variables in coagulation testing. Am J Clin Pathol. 1975;64:591–6.
6. Reneke J, Etzell J, Leslie S. Prolonged prothrombin time and activated partial thromboplastin time due to under-filled specimen tubes with 109 mmol/L (3.2%) citrate anticoagulant. Am J Clin Pathol. 1998;109:754–7.
7. Adcock D, Kressin D, Marlar R. Effect of 3.2% vs 3.8% sodium citrate concentration on routine coagulation testing. Am J Clin Pathol. 1997;107: 105–10.
8. Peterson P, Gottfried E. The effects of inaccurate blood samples volume on prothrombin time (PT) and activated partial thromboplastin time (APTT). Thromb Haemost. 1982;47:101–3.

Recombinant activated factor VII (rFVIIa, NovoSeven, Novo Nordisk, Denmark) is an effective hemostatic agent in hemophilic patients with inhibitors, congenital FVII deficiency, and Glanzmann thrombastenia ⁴. Recently, the off-label use of rFVIIa has ensured benefits in many nonhemophilic and excessive bleeding diseases such as intracranial hemorrhage, severe thrombocytopenia, major surgery, trauma, bleeding in association with hepatic failure, gynecologic and obstetric hemorrhages, and coumadine-induced hemorrhage that were unresponsive to conventional therapy to control massive bleeding ⁵. The rFVIIa shows the hemostatic effect through two mechanisms. The first pathway is a tissue factor (TF)-dependent mechanism and a TF-FVIIa complex is formed following a vessel injury and this complex causes the protrombinase complex (FXa/FVa) assembly on an activated platelet surface leading to thrombin generation and a stable fibrin clot. The second pathway is a TF-independent mechanism. The rFVIIa activates FIX and leading to an additional burst of thrombin by following FX ⁶.

Here we report the effect of rFVIIa treatment in a 13-year-old girl patient with T-cell NHL who developed a massive intra-abdominal and intra-thorasic hemorrhage unresponsive to conventional therapies such as fresh frozen plasma (FFP) and thrombocyte transfusions followed by high-dose MTX treatment. A discussion on the off-label use of rFVIIa treatment in the patients with excessive bleeding unresponsive to conventional therapies is reported on.

CASE

A 13-year-old girl patient with stage-IV T-cell NHL was admitted to our Pediatric Hematology-Oncology Ward for receiving a consolidation-I treatment of LMT-89 chemotherapy protocol. The patient had previously received COP, COPADM1, and COPADM2 treatments. A physical examination revealed bilateral cervical lenfadenomegaly in a diameter of 4×5 cm, palpable splenomegaly 3 cm below the left costae margin and palpable hepatomegaly 3 cm below the right costae margin. Laboratory findings were as follows: the hemoglobin was 12.6 g/dl, leukocytes 5100/µl, thrombocytes: 122 000/µl, absolute neutrophil count (ANC) 3500/µl, 4% blasts were seen on the peripheral blood smear. Bone marrow aspiration was not performed. Creatinine clearance was 97 ml/dk 1.73 m². Abdominal ultrasonography (USG) showed normal findings except for hepatosplenomegaly. The chest X-ray showed mediastinal enlargement and hilary lenfadenomegaly.

Other biochemical findings were between normal limits. The LMT-89 consolidation-I treatment protocol was initiated, but the patient received MTX in a dose of 5 g/m² instead of a dose of 2 g/m² because she was thought to be resistant to chemotherapy. In addition, the patient received cytosin arabinoside (100 mg/m²) and etoposid (100 mg/m²) at the first and the second days of the treatment. Folinic acid rescue (15 mg/m² every 6 h for eight doses) was initiated at 18 h after the completion of MTX treatment and the patient was hydrated (3000 ml/m²/d and 2 mEq/kg/d NaHCO₃). The laboratory findings on the second day of chemotherapy indicated tumor lysis syndrome. Chemotherapy was stopped and intravenous fluid replacement was increased to 3500 ml/m²/d. Itching and hyperemia on her face, hands, neck, and chest of the patient developed on the fifth day after completing the MTX treatment. Intravenous diphenhydramine was initiated, the response was not obtained, and MTX toxicity was diagnosed.

These lesions were converted to the bullous form after 2 days. Unfortunately, the serum MTX level was not calculated at this time because of laboratory technical problems. Leucovorin rescue (15 mg/m²/d) was complemented to 20 doses. Grade IV mucositis developed and a local oral supportive treatment initiated. Febrile neutropeni was developed on the 10th day of hospitalization and ceftazidime (40 mg/kg/g), netilmycine (4 mg/kg/g), teicoplanin (2.5 mg/kg/d), and granulocyte colony stimulating factor (G-CSF: 5 µg/kg/d) was started because serum BUN and creatinine levels were 46 mg/dl and 3 mg/dl, respectively. The patient received supportive treatment such as packed red blood cell platelet transfusion and FFP (15–20 ml/kg three to four times a day). Retrosternal pain and dysphagia were developed on the 11th day of the neutropenic period and fungal esophagitis was diagnosed, flucanasole (6 mg/kg/d) was started. On the 15th day, flucanasole was changed to caspofungin (2 mg/kg/d) because of resistant hypokalemia and persistent dysphagia. Carbapenem (40 mg/kg/d) instead of ceftazidim and dopamin (5 µg /kg/min.) was started on the 14th day of hospitalization because resistant fever and hypotension had developed.

Oxygen saturation was decreased to 80% and respiratory sounds were decreased on the lower right lung area on the 15th day. Pleural fluid was determined on the chest X-ray, 450 ml fluid was drained by thoracentesis. The biochemical characteristic of the pleural fluid was compatible with transudate. At the 19th day, oxygen saturations were again decreased, tachypne and abdominal distention developed. Serum BUN was 119 mg/dl, creatinine 2.9 mg/dl, chest X-ray showed bilateral lung edema and pleural fluid. At this time, abdominal distention developed and abdominal and thoracal USG showed diffuse intra-abdominal fluid and pleural fluid in a diameter of 20 mm on the right pleura and in a diameter of 7.5 mm on the left pleura. We drained 400 ml of hemorrhagic fluid by paracentesis and a catheter was inserted.

Hemoglobin decreased to 4.8 g/dl following paracentesis, platelets were 26 000/µl and D-dimer was 19.89 while the patient received FFP, packed red blood cell and thrombocyte transfusions. The rFVIIa (50 µg/kg every 2 h for three doses) was started because hemorrhagic fluids were obtained by paracentesis and the vital signs of the patient were becoming worse. Hemorrhagic fluid drainage was stopped after the rFVIIa treatment. One day later, 300 ml hemorrhagic fluid was again drained, rFVIIa was repeated at the previous dosage. At last, hemorrhagic fluid was not observed while 300 ml fluid was drained by paracentesis. After three days, the paracentesis catheter was taken out because no fluid had drained. Hemoglobin and thrombocytes increased, mucosal and skin findings improved and the patient was discharged on the 30th dayof hospitalization. The patient received a total dose of rFVIIa of 300 µg/kg.

The patient received chemotherapy according to LMT 89 NHL treatment protocol and now the patient is being cared for at the out-patient part of our Pediatric Hematology-Oncology ward.

DISCUSSION

Hemato-oncologic diseases are quite common complicated by severe hemorrhagic episodes, which may be due to a primary disease or chemotherapy-induced toxicity. Thrombocytopenia is usually a common cause of hemorrhage in hemato-oncological patients.

Until 4 to 5 years ago, rFVIIa was generally prescribed by the hematologist in the treatment of hemophilic patients with inhibitors, FVII deficiency, and Glanzman thrombastenia whereas now it is being prescribed by the surgeon, hepatologists, anesthetists, hepatologists, and so on. These authors used rFVIIa in patients without congenital bleeding disorders such as thrombocytopenia, liver failure, cardiopulmonary bypass/extracorporeal membrane oxygenation, prolonged INR, central nervous system bleeding, and prolonged aPTT ⁶. The off-label use of rFVIIa may increase the risk of a thrombotic complication (myocardial infarction, stroke, pulmonary embolism, and deep venous thrombosis) in patients without congenital bleeding disorders ^7–9 .

Recently, rFVIIa has been used in the treatment of severe bleeding refractory to the standard transfusional treatment in patients with hemato-oncological diseases ^{10, 11}. White et al was the first to use rFVIIa successfully in the treatment of pulmonary hemorrhage secondary to Aspergillus in a patient with acquired FVII deficiency undergoing treatment for acute myeloid leukemia (AML) ¹². The rFVIIa, in a dose of 85 µg/kg (range 18 to 1040 µg/kg), was used as a median number of 1.6 doses (range 1–8) in the treatment of hemorrhage in thrombocytopenic patients with hematologic malignancies. Bleeding stopped in 46% of the patients, markedly decreased by 33%, and decreased 17%. Authors suggest the beneficial effect of rFVIIa in this clinical setting ^{10, 13}. Ischemic stroke was developed in a patient because of the use of rFVIIa ¹³. Franchini et al collected a total of 22 articles that included 42 patients with hemorrhages due to the chemotherapy-induced mucosal damage ¹⁰.

The bleeding site most frequently involved was the gastrointestinal tract (46.9%). The mean dose of rFVII administered was 82.5 µg/kg, a single dose of rFVII was used in 42.9% of the patients, some patients required the additional doses to achieve hemostasis (median of 1.6 doses per episode). The rFVIIa for off-label indications has been used in severe hemorrhages occurring in bone marrow transplantation (BMT). Transplant-related thrombocytopenia, liver toxicity, gastrointestinal bleeding, intracranial hemorrhage, hemorrhagic cystitis, diffuse alveolar hemorrhage, epistaxis, consumption coagulapathy, mucosal bleeding, and uterine and cutaneous bleeding usually occurred in patients following by BMT. These patients received rFVIIa at initial doses ranging between 18 and 1040 µg/kg at doses of 1–56 ^{10, 13}.

Chuansumrit et al treated and discussed the effectiveness of rFVIIa in three children with acute bleeding resulting from liver failure and disseminated intravascular coagulation (DIC) ¹⁴. Two of the cases were diagnosed as Dengue fever and prolonged shock, and the other underwent left lobe hepatectomy for a hepatoblastoma. This case was complicated by myoglobinüria, hepatic and renal failure, and DIC. The patients were treated with blood component replacement in addition to rFVIIa administration in a dose of 40–80 µg/kg b.w. followed by 16.5–33 µg/kg b.w. per hour continuous infusion. The authors suggested that rFVIIa was effective in controlling acute bleeding in children with liver failure and DIC.

We used a rFVIIa treatment in a dose of 50 µg/kg every 2 h three times in a patient with T-Cell NHL who developed intra-abdominal and intra-pleural hemorrhages followed by high-dose MTX therapy. The patient received rFVIIa at a dose of 50 µg/kg for six doses in 2 days (total 300 µg/kg) in addition to blood component replacement. Hemorrhagic shock and acute renal failure (ARF) developed in the patient following by high-dose MTX therapy. We think that the cause of ARF was hypovolemia following by massive hemorrhage and diffuse mucositis due to high-dose MTX therapy. The ARF improved after fluid replacement treatment (3000 ml/m²/d) and stopped the massive hemorrhage with rFVIIa treatment. The bleeding was stopped after 3 days of the rFVIIa treatment and the patient was discharged after 30 days of hospitalization.

CONCLUSION

In conclusion, rFVIIa was shown in the effectiveness for controlling massive intra-abdominal bleeding due to high-dose MTX therapy in a patient with T-cell NHL while the patient received FFP (15–20 ml/kg every 6–8 h) and thrombocyte transfusions. No thromboembolic complication developed in the patient that received rFVIIa at a total dose of 300 µg/kg. The effect of rFVIIa is not surprising in the case because the use of rFVIIa as a bypassing agent in various coagulation diseases has been described and illustrated but as far as this author knows, this is the first report of the effectiveness of rFVIIa in massive intra-abdominal bleeding in a patient with T-cell NHL caused by high dose MTX.

Thus, further more comprehensive studies are needed to establish the appropriate doses and to better assess the safety and effectiveness of the off-label use of rFVIIa treatment in excessive bleeding unresponsive to conventional therapy.

Disclosure: The authors declare no conflict of interest.

REFERENCES

Hemophilia A is classified as a mild, moderate or severe bleeding disorder based on circulating coagulation factor FVIII levels of 0.05 IU/mL (>5%), 0.01 to 0.05 IU/mL (1–5%), and 0.01 IU/mL (<1%), respectively.^{1, 2} Recurrent bleeding episodes into the joints, typically beginning from an early age in life (children aged 1–2 years), are the hallmark of severe hemophilia,^{3, 4} and a proportion among moderate hemophilia population are affected.⁵ Joint bleeds (hemarthroses) often occur spontaneously or with minimal or unknown trauma. Repeated bleeding events into a joint lead to progressive joint damage (hemophilic arthropathy), debilitating dysfunction, chronic pain, and reduced health related quality of life (HRQoL).^{3, 6} Compared to patients with moderate/mild hemophilia and to non-hemophiliacs, individuals with severe hemophilia have statistically lower levels of HRQoL.⁶

Currently, start of early prophylaxis is recommended as standard of care for young children with severe hemophilia A to reduce risk of bleeding and related morbidity in countries where safe factor VIII concentrates are available, as supported by numerous retrospective cohort studies.^7–9 The rationale for prophylaxis is that patients with mild and moderate hemophilia A (FVIII >0.05 IU/mL) rarely develop chronic arthropathy.^{10, 11} One large prospective, multicenter, randomized trials conducted in the USA (the Joint Outcome Study) ¹³ supports the long-term efficacy of prophylaxis compared with on-demand treatment in the prevention of hemarthroses and improved outcome of joint status in young boys (prophylaxis initiated age ≤2.5 years) with hemophilia A.

Currently, no universal prophylactic regimen has been adopted; however, several primary prophylactic strategies including Swedish, Dutch, Canadian, and French models have been compared.^{10, 11, 13–15} The initial prophylaxis regimen was pioneered in Sweden and involves infusions of 20–40 IU of FVIII per kilogram body weight on alternative days (minimum three times per week) for severe hemophilia A subjects.¹⁰ Due to the high costs and the demanding impact on the patient's veins of alternate-day injections, there is a controversial debate whether less frequent prophylaxis regimens may be sufficiently beneficial for specific patient groups.^{11, 16}

This report of a prospective study aims to provide additional evidence regarding the extent to which different prophylaxis regimens prevent joint bleeds in children with severe/moderate hemophilia A and how they influence joint status and quality of life (QoL).

PATIENTS AND METHODS

Study Design

This multicenter trial was designed as a nonrandomized, open label, parallel-group study. Four Russian Hemophilia Centers participated in this clinical study. The study conformed to international standards of Good Clinical Practice and was approved by the Institutional Review Board of each participating center.

Eligibility and Exclusion Criteria

Eligibility criteria were an age between 1 and 12 years, severe (<1% factor VIII activity [FVIII:C]) or moderate hemophilia A [1–5% FVIII:C], and previous treatment with >20 exposure days [ED] with any FVIII product). Exclusion criteria were a history or detectable levels of factor VIII inhibitor (inhibitor titer>0.6 Bethesda Units [BU]), possibility of requiring surgery during the study duration, other coagulation disorder than hemophilia A, treatment with blood product other than with an anti-hemophilic factor VIII virally inactivated product, known or suspected hypersensitivity to any FVII, product, or abnormal renal or liver function.

Treatment

Eligible subjects were assigned to receive one of three prophylactic treatment regimens with recombinant full-length factor VIII formulated with sucrose (rFVIII-FS; Kogenate® FS; Octocog alfa, Manufacturer: Bayer Healthcare AG),as follows: regimen 1: 70 IU/kg once per week; regimen 2: two doses per week (one dose of 30 IU/kg and a second of 40 IU/kg after 3 days), and regimen 3: 3 times a week 25 IU/kg rFVIII-FS. The assignment was based on the patient's previous treatment situation prior to study inclusion.

Dose escalation was permitted for regimens 1 and 2: for regimen 1 subjects after the first joint bleed during the study, the dosage was changed and increased to 35 IU/kg twice a week and further escalated to 25 IU/kg three times a week after another joint bleed; for regimen 2 subjects after the first joint bleed during the study, dosage was allowed to be escalated to 25 IU/kg three times a week. On-demand treatment was administered to all patients who developed joint or other bleeds during the study. The number of follow-up treatments was decided by the investigator based on severity and location of bleeding. Following baseline assessment, patients were assessed at week 2, at months 3, 6 and 9, or at any time following a serious adverse event (SAE). Treatment was administered either at home or at a participating clinic.

Outcome Measures

The primary efficacy outcome was the proportion of subjects with less than two joint bleeds during the 9-month observation period. Secondary efficacy variables were the number of bleeds; change in clinical joint status; the number of patients in each group at the end of the study; total monthly rFVIII-FS consumption; and change in QoL. All patients who received study drug were evaluated for safety.

The number and type of bleedings and infusions were documented on infusion report forms. Clinical examination of joints with assessment of hemorrhages, pain, gait, strength, and joint function were evaluated using the Stockholm Hemophilia Joint Score according to the recommendation of the European PedNet for Hemophilia Management.¹⁷ A total score of “0” corresponds to the best possible joint condition, and “25” (for one single joint) up to the maximum of “150” for the worst possible condition (all six joints). Health-related QoL was determined by the disease-specific Haemo-Qol questionnaire.¹⁸ Two age-specific versions were used for self-administration in children: one for children aged 4–7 years and another for children between 8 and 16 years. Raw values of the Hemo-QoL were standardized on a scale from 0 to 100, where 0 represents the best condition and 100 the worst condition. Results for children and parents were analyzed separately. Blood was collected every 3 months for factor VIII inhibitor testing, FVIII activity at least 48 hours after last injection, and serologic tests for hepatitis B and C, human immunodeficiency, and parvovirus. Titers of FVIII inhibitors were determined by local laboratories according to usual practice with definition of positivity by the local laboratory. In case of a positive test result, samples were re-tested in a Central Laboratory (Prof. Budde, Hamburg, Germany<xref ref-type="fn" rid="NOTE0001">1</xref>) with the use of the Nijmegen modified Bethesda assay.¹⁹

Statistical Analysis

Statistical evaluation was performed using the software package SAS release 9.1 (SAS Institute Inc., Cary, NC, USA). All variables were analyzed by descriptive statistical methods. All analyses were performed according to the regimen assignment at baseline. Efficacy and safety analysis were conducted for all patients who started treatment.

RESULTS

Patient Demographics and Baseline Disease Characteristics

Thirty-two children were enrolled in the study between June 2007 and December 2008. Since each patient was followed for 9 months, the study period ran until Sep 2009. Eleven subjects were allocated to regimen 1, 13 subjects to regimen 2, and 8 subjects to regimen 3. All 32 patients completed the 9-month treatment period and were included in both the safety and the efficacy analysis.

The baseline demographics for the three regimen groups are shown in Table 1 . All participants were Caucasian, with the exception of one Asian patient in regimen 3. The patients’ mean age was 5.1±3.5 years. As anticipated, younger subjects were preferentially allocated to regimen 1 (mean age: 3.3±3.2 years) with once weekly dosing, and older children to regimen 2 (mean age: 6.1±3.5 years) or regimen 3 (mean age: 6.0±3.1 years). This imbalanced but expected distribution also explains differences in other patient characteristics at baseline: older participants in regimen 3 had more target joints, lower median factor VIII level at diagnosis as well as at baseline before first injection, and already 3× weekly injections during 6 months prior to study inclusion. One previously untreated patient was included and treated until the end of the 9 month observation period, even though less than 20 Exposure Days was an exclusion criterion.

None of the subjects had a reported history of inhibitor. Most children enrolled in this study (78.1%) were previously treated with plasma-derived FVIII only.In the nine months prior to study entry, the study subjects had a mean of 10.2±11.6 bleeds and 18.7% subjects had no joint bleeds.

Efficacy Analysis

Primary Efficacy Outcome

Results for the primary efficacy analysis are shown in Table 2 and Figure 1 . The overall proportion of patients with <2 joint bleeds during the observation period was 78.1%, corresponding to 72.7% subjects in regimen 1, 84.6% subjects in regimen 2, and 75.0% subjects in regimen 3.

Secondary Efficacy Outcomes

Secondary efficacy variables are also shown in Table 3 . The median number of joint bleeds was 0.0 in all regimens. In total, 71.9% (23/32) subjects had no joint bleeds. There were 72.7% (8/11), 69.2% (9/13) and 75.0% (6/8) of patients without any joint bleeds in regimens 1, 2, and 3, respectively. Approximately 34.4% of the total population did not experience any bleeds during the study ( Figure 2 ), with the lowest percentage in regimen 1 (27.3%) and the highest in regimen 3 (50%) subjects ( Table 1 ). More than 10 bleeds were reported in regimen 1 only (data not shown). The median number of all bleeds ranged from 1.5 (regimen 3) to 3 (regimen 1). There was one patient (group 3) with a target joint who had more than eight joint bleeds during the study period. Excluding this patient, the maximum number of joint bleeds in group 3 would have been two joint bleeds. Overall, these data compared favorably to the subjects’ bleeding history in the 6- to 9-month period prior to study entry ( Table 1 ).

Changes in Joint Score

The clinical status was evaluated at baseline and after 3, 6, and 9 months using the Stockholm Hemophilia Joint Score System ¹⁷ ( Table 4 ). Mean total score at baseline was 5.5±5.2 points. Joint scores were lowest in the regimen 1 group (3.9±4.8 points) and the highest in regimen 3 group (7.1±4.6 points) at baseline. Mild improvements or stable condition were observed in all three regimens. As shown in Table 4 , the improvements mainly occurred within the first 3 months of treatment. Thereafter, only minor changes were observed and the overall condition maintained up to the end of the study period at month 9.

Quality of Life Scores

Due to the small subject numbers in the three regimen subgroups, Haemo-QoL ¹⁸ values for the total sample only are shown in Figure 2 . Although only data from 10 children and 10 guardians were available for every visit, data clearly showed improvements in QoL for these individuals. Improvements were observed within the first 3 months of prophylactic treatment and maintained over the complete 9-month treatment period ( Figure 2 ).

Study rFVIII-FS Dosing and Consumption

The median weekly rFVIII-FS dose for prophylaxis slightly exceeded the planned dose in each regimen set, mainly due to the rounding up to full vial sizes and a temporary lack of 250 IU and 500 IU vials at participating centers. In regimen 1 (planned 70 IU/kg dosing once per week), regimen 2 (planned 30+40 IU/kg dosing twice per week), and regimen 3 (planned 3×25 IU/kg dosing three times per week), the overall median weekly dose for prophylaxis during the study was 78.2, 77.7 and 91.1 IU/kg, respectively. The higher mean FVIII usage in regimen 3 could be explained by the temporary unavailability of small vial sizes at the centers and the higher likelihood of rounding effects in 3 times weekly dosing. The median treatment duration was 275, 276, and 274 days in regimens 1, 2, and 3, respectively (range: 253 to 288 days).

The mean monthly rFVIII-FS consumption due to acute bleeds was small and ranged between 2±5 IU/kg (regimen 3) and 21±28 IU/kg (regimen 1).

Treatment regimens at the End of the Study

Number of patients in each treatment regimen at the end of the study can be seen in Figure 3 . Escalation due to one joint bleed was applicable for 7 of 32 patients: 3 subjects in regimen 1 and 4 subjects in regimen 2. Two of the three patients with joint bleeds in regimen 1 were switched to regimen 2 after their first joint bleed, and one was switched to regimen 3 after their second joint bleed (data not shown). One patient in regimen 1 switched despite no joint bleed, based on doctor's estimate of patients condition. In regimen 2, only two of the four subjects with joint bleeds were switched to regimen 3. One patient from regimen 3 was switched to regimen 2 on their guardian's request. All patients who were actually switched had target joints.

Adverse Events

Eleven (34.4%) of the 32 subjects experienced a total of 25 AEs; 23 of these reported AEs were rated as not related to the study drug by the treating physician. All AEs were mild or moderate in intensity and mostly resolved during the study treatment period. Two drug-related AEs (ADR) occurred in two subjects in regimen 1: one ADR was a case of moderate allergic dermatitis. The other was a case of transient FVIII inhibitor in a previously untreated subject after 13 ED to study medication. This 15-month-old male patient with severe hemophilia A was allocated to the rFVIII-FS prophylaxis regimen 1 with 70 IU/kg body weight (bw) administered once per week. The FVIII-level of this participant at diagnosis and at baseline was indicated as 1.0%. Laboratory tests six days after last injection at month 3 showed an inhibitor titer of 3.5 BU (Bethesda assay). and an FVIII level of 0.9%. This participant had experienced his last bleed between screening and baseline visit and did not experience any bleeds during the study. After 40 ED at last visit, the inhibitor titer was measured as negative (0.5 BU). The patient completed the 9-month treatment period as planned. No other patient tested positive for inhibitor during the study. There were no deaths or treatment discontinuations during this study. Three patients (9.4%) experienced a total of five SAEs, four of them not drug related. One SAE was the aforementioned case of a transient low titer FVIII inhibitor in a previously untreated patient

DISCUSSION

This study was conducted to assess the efficacy and safety of three prophylactic treatment regimens with rFVIII-FS in children with severe or moderate hemophilia A. The number of patients with less than two joint bleeds during the 9-month treatment period clearly demonstrated the efficacy of all three prophylaxis regimens. Overall, 71.9% of study children did not experience any joint bleeds, and 6.3% experienced only 1. The data support the current evidence of prophylaxis as a method to prevent joint bleeds.^{10, 13–15} The incidences of adverse events were as expected in the age group and for hemophilia. There were two drug related adverse reactions reported: 1) moderate allergic dermatitis and 2) one single case of a low titer transient inhibitor in a previously untreated patient. The latter was a protocol deviation and confirms the risk of inhibitor development especially in the previously untreated patient population.²³ There are current discussions in the scientific community whether once weekly FVIII prophylaxes with low doses might be beneficial with regard to inhibitor development.²⁴(Kurnik et al, 2009), but further evidence on larger sample size is required. Since this Russian study was designed for bleed prevention, the once weekly dosage was high compared to the recommended dose of 25 IU/kg.

A major concern of an optimal prophylaxis regimen in very young children with poor venous access is the need for frequent intravenous injections due to the short biological half-life of coagulation factors.²⁰ Although no definitive treatment allocations were made, younger children in this study population were preferentially allocated to regimen 1, with once weekly dosing in order to avoid venipuncture until increased bleeding frequency required more frequent factor injections. The median number of joint bleeds during the treatment period was 0 in each group. During this period before study entry, only 6 subjects (18.8%) had not experienced any joint bleeds, and the remaining 26 children had experienced a total of 178 joint bleeds (a mean of approximately 7 joint bleeds per patient) ( Table 2 ). Such a reduction in joint bleeds to median values of 0 is concurrent with data reported from other retrospective cohort studies.^{11, 21} The prospective, randomized Joint Outcomes Study ¹³ showed a comparably low value of (Median=0.63) joint bleeds per year in the prophylaxis arm, respectively. Regimen 3 had the highest percentage of subjects without any bleeds or any joint bleeds at all during the study period ( Table 3 ). Hence, the total number of bleeds in all study regimens during prophylactic treatment with rFVIII-FS ( Table 3 ) was markedly reduced compared to the total number of bleeds in the 6 months prior to study entry ( Table 2 ). This finding on bleeding rate reduction corresponds to other studies which compared prophylactic treatment with episodic treatment regimens.^{11, 13}

The efficacy of the three regimens used for prophylaxis was also reflected in the improvements in clinical status as determined with the observed Stockholm Hemophilia Joint Scores.¹⁷ Increased age of subject had a more pronounced impact on joint functioning, with pre-study scores being worst in the slightly older children in regimens 2 and 3, with 87.5% of subjects in regimen 3 having at least one target joint before study entry at a mean age of 6 years (corresponding percentages of patients with target joints were 36.4% and 53.8% for regimens 1 and 2, respectively); this finding is also reflected in the higher Stockholm Joint Scores at baseline. The majority of subjects either did not show any deteriorations or showed improvements by up to 16 points during the study period. As shown in the baseline characteristics, some had pathological joint scores already before baseline. The beneficial effect of rFVIII-FS treatment on clinical status was already observed within the first 3 months ( Table 4 ). An average score improvement from baseline was reached in regimen 3 (median=−1) ( Table 4 ).

In the case of hemophilia, where cure is currently not attainable and lifelong therapy is needed, QoL is an essential outcome parameter.⁶ The effective bleeding protection observed in this study under prophylaxis with rFVIII-FS also influenced not only the childrens’ but also their guardians’ QoL as known from other hemophilia studies measuring QoL.²² Although evaluable data, extracted from the hemophilia-specific HRQoL questionnaires,¹⁸ were available from only 10 parents and 10 children in total, all parents and the majority of children reported improvements in QoL under rFVIII-FS study treatment within the first 3 months, and the effect was maintained until study end.

Despite the numerical differences in several subject characteristics between the study regimens but especially between regimen 1 versus regimens 2 and 3, a direct comparison is problematic due to the small sample sizes of the regimens and the different levels of the children's age-dependent physical activity and subject characteristics at baseline. Furthermore, dose escalations due to joint bleeds were not followed in all cases; some children were not switched despite joint bleeds, and others were switched despite absence of joint bleeds. However, when taking these differences in age, severity of disease, etc., into account, a trend in favor of three times weekly doses instead of one weekly high dose can be inferred. This trend is reported also in other research literature and clinical studies on prophylaxis in severe hemophilia A.^{11, 16}

CONCLUSIONS

All three prophylaxis regimens were well tolerated. The single case of transient FVIII inhibitor development in a previously untreated patient with a high dose of 70IU/kg per injection confirmed the risk in this patient population and potentially also the risk of such a high-dose regimen.

All of the three prophylaxis regimens were effective, but the results suggested regimen 3 to be more beneficial in preventing joint bleeds and preserving the joint status in previously treated children with severe or moderate hemophilia A.

Keywords

recombinant factor VIII, FVIII, prophylaxis in children, hemophilia A, arthropathy, hemarthroses, joint bleeds

Acknowledgements: This study was fully sponsored by Bayer Pharma AG. Klara Belzar PhD provided medical writing assistance for this manuscript.

The authors are very grateful for the support by following four Russian sites:

Prof. Dr. Vdovin V.V., Dr. Shiller E.E., Dr. Svirin P.V. and sites staff from Izmailovskaya Children's Clinical Hospital, Center of Hemophilia treatment, Moscow, Russia Prof. Dr. Andreeva T.A., Dr. Lavrichenko I.A. and site staff from City Polyclinic #37, City Hemophilia Centre, St. Petersburg, RussiaDr. Chernova T.A. and site staff from Scientific Research Institute of Hematology and Blood Transfusion, Kirov; Russia Dr. Perina F.G. and site staff from Regional Children's Clinical Hospital #1, Children's Center of Oncology and Hematology, Ekaterinburg, Russia

Disclosure: All investigators received investigator payments from BAYER HEALTHCARE AG in this study. The investigators declare no further conflict of interest. Dr. Monika Maas-Enriquez and Dr. Stephan Rauchensteiner are employees of BAYER HEALTHCARE AG.

Notes

1. Prof. U. Budde, Coagulation Laboratory, Aescu Labor Hamburg, Haferweg 36, 22769 Hamburg, Germany.

REFERENCES

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3. Rodriguez-Merchan EC. Effects of hemophilia on articulations of children and adults. Clin Orthop Relat Res. 1996;328:7–13.
4. Pollmann H, Richter H, Ringkamp H, Jurgens H. When are children diagnosed as having severe haemophilia and when do they start to bleed? A 10-year single-centre PUP study. Eur J Pediatr. 1999;158(Suppl 3): S166–S170.
5. Ingrid den Uijl DBDGKF. Outcome in moderate haemophilia [Abstract# O-MO-076, ISTH Kyoto 2011]. J Thromb Haemost. 2011.
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7. Ota S, McLimont M, Carcao MD, et al. Definitions for haemophilia prophylaxis and its outcomes: the Canadian consensus study. Haemophilia. 2007;13(1):12–20.
8. Berntorp E, Astermark J, Bjorkman S, et al. Consensus perspectives on prophylactic therapy for haemophilia: summary statement. Haemophilia. 2003;9(Suppl 1):1–4.
9. Richards M, Williams M, Chalmers E, et al. A United Kingdom Haemophilia Centre Doctors’ Organization guideline approved by the British Committee for Standards in Haematology: guideline on the use of prophylactic factor VIII concentrate in children and adults with severe haemophilia A. Br J Haematol. 2010;149(4):498–507.
10. Nilsson IM, Berntorp E, Lofqvist T, Pettersson H. Twenty-five years’ experience of prophylactic treatment in severe haemophilia A and B. J Intern Med. 1992;232(1):25–32.
11. Fischer K, Astermark J, van der Bom JG, et al. Prophylactic treatment for severe haemophilia: comparison of an intermediate-dose to a high-dose regimen. Haemophilia. 2002;8(6):753–760.
13. Manco-Johnson MJ, Abshire TC, Shapiro AD, et al. Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia. N Engl J Med. 2007;357(6):535–544.
14. Carcao M, Chambost H, Ljung R. Devising a best practice approach to prophylaxis in boys with severe haemophilia: evaluation of current treatment strategies. Haemophilia. 2010;16(Suppl 2):4–9.
15. Meunier S, Trossaert M, Berger C, et al. French guidelines. Long-term prophylaxis for severe haemophilia A and B children to prevent haemophiliac arthropathy. Arch Pediatr. 2009;16(12):1571–1578.
16. Ljung R. Prophylactic therapy in haemophilia. Blood Rev. 2009;23(6): 267–274.
17. Hill FG, Ljung R. Third and fourth Workshops of the European Paediatric Network for Haemophilia Management. Haemophilia. 2003;9(2):223–228.
18. Bullinger M, von Mackensen S, Fischer K, et al. Pilot testing of the ‘Haemo-QoL’ quality of life questionnaire for haemophiliac children in six European countries. Haemophilia. 2002;8(Suppl 2):47–54.
19. Kasper CK, Aledort L, Aronson D, et al. Proceedings: a more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh. 1975;34(2):612.
20. Fischer K, van der Bom JG, Mauser-Bunschoten EP, et al. The effects of postponing prophylactic treatment on long-term outcome in patients with severe hemophilia. Blood. 2002;99(7):2337–2341.
21. Berntorp E. Prophylactic therapy for haemophilia: early experience. Haemophilia. 2003;9(Suppl 1):5–9.
22. Gringeri A, von Mackensen S, Auerswald G, et al. Health status and health-related quality of life of children with haemophilia from six West European countries. Haemophilia. 2004;10(Suppl 1):26–33.
23. Gouw SC, van den Berg HM, le Cessie S, van der Bom JG. Treatment characteristics and the risk of inhibitor development: a multicenter cohort study among previously untreated patients with severe hemophilia A. J Thromb Haemost. 2007;5(7):1383–1390.
24. Kurnik K, Bidlingmaier C, Engl W, Chehadeh H, Reipert B, Auerswald G. New early prophylaxis regimen that avoids immunological danger signals can reduce FVIII inhibitor development. Haemophilia 2009.

Over the last decades, clinical hemostasis and thrombosis has evolved as an interdisciplinary practice involving an ever-widening array of diagnostic methods and therapeutics. In addition to the well-characterized inherited bleeding disorders such as hemophilia and von Willebrand disease and the management of patients with venous thromboembolic diseases, the practice of clinical hemostasis and thrombosis increasingly requires interactions with many disciplines including cardiology, neurology, gastroenterology, gynecology, obstetrics, hemato-oncology, transfusion medicine, pneumology, orthopedics, oral surgery, and genetics. Moreover, with the advent of thrombophilia and a better awareness of bleeding disorders, screening for a bleeding tendency and testing for thrombophilia are increasingly requested for a growing number of medical and surgical patients. Many tertiary hospitals have set up thrombosis and hemostasis centers or units, usually closely linked to a hemophilia center, with the purpose of providing a comprehensive and multidisciplinary approach to the diagnosis and management of patients with a wide variety of hemostatic and thrombotic abnormalities.

A European curriculum describing the skills and competences needed by the specialists in hemostasis and thrombosis has recently been proposed ¹. Several training programs in hemostasis have been developed in the United States and Europe in order to enhance the clinical expertise and the professional development of individuals dedicated to treating patients with coagulation disorders ². Moreover, a European Association for Hemophilia and Allied Disorders has recently been created to promote collaboration in clinical practice, research, pharmacovigilance, dissemination of knowledge, education, and training ³.

The present study was undertaken to evaluate the impact of this evolution on the activity and organization of specialized clinics. Such information appears critical to better define the areas of expertise required for the thrombosis and hemostasis specialists, optimize interactions with other disciplines and anticipate clinical and biological needs and developments.

POPULATION AND METHODS

The reasons for referral and final diagnosis of all out-patients referred over a 24-month period (from January 2006 until December 2007) to the Haemostasis and Thrombosis Unit and Haemophilia Center, Division of Adult Haematology of the Cliniques Universitaires Saint-Luc, Brussels, Belgium were reviewed by two clinicians of the thrombosis and hemostasis unit (CL and CH). The Haemostasis and Thrombosis unit is a dual-consultant clinic, part of a tertiary care faculty hospital located in Brussels, Belgium. Patients are referred either from primary care practices or by internal or external specialists. Patients regularly followed in the anticoagulation clinic were not included.

The reasons, for referral and the blood tests subsequently performed, were extracted from the electronic medical file using the internal medical software (Medical Explorer) for each visit. The results of the laboratory work-ups were correlated with the clinical data and critically reviewed. The proportion of work-ups showing abnormal results was evaluated.

The reasons for referral were classified into eight main categories: bleeding disorders, venous thromboembolic (VTE) disease, complicated pregnancies, nonthrombotic venous disease, arterial thrombosis, general hematology, management of antithrombotic agents, and miscellaneous.

Patients were categorized as belonging to the “bleeding disorders” group if they had been referred for assessment of a bleeding tendency, preoperative evaluation, follow-up of a known inherited bleeding disorder (hemophilia A and B, von Willebrand disease, other coagulation factor deficiency) or family screening of a bleeding disorder.

The venous thromboembolic disease group involved patients referred for diagnosis and/or follow-up of superficial or deep venous thrombosis (DVT) or pulmonary embolism (PE), thrombophilia testing because of a past history of venous thrombosis (DVT, PE, retinal, or splanchnic venous thrombosis), familial thrombophilia screening, counseling about thrombophilia and advice on antithrombotic prophylaxis.

The group of complicated pregnancies included all patients referred for thrombophilia screening because of a past history of poor pregnancy outcome (recurrent miscarriages, unexplained intrauterine fetal death, intrauterine growth retardation, abruptio placenta, preeclampsia or HELLP syndrome) as well as monitoring of antithrombotic therapy (mainly low-molecular weight heparin) during pregnancy.

The arterial thrombosis group involved all patients referred by neurologists, cardiologists, and vascular surgeons to further investigate arterial thrombotic events (stroke, myocardial infarction, peripheral arterial disease) that were either recurrent, affected young individuals, or were unexplained by classical cardiovascular risk factors.

Patients with nonthrombotic venous disease were referred for primary or secondary venous insufficiency (post-thrombotic syndrome).

The last group involved patients referred for management of antithrombotic agents including adaptation of anticoagulant therapy and referral to the anticoagulation clinic, bridging of antithrombotic therapy before invasive procedures and advice or counseling regarding the optimal use of multiple antithrombotic agents.

Routine bleeding work-up included the following tests: fibrinogen level (150–450 mg/dl), prothrombin time (9–14 sec), thrombin time (15–24 sec), APTT (20–33 sec), platelet optical aggregometry (using ADP, collagen, and ristocetin as agonists), closure time evaluated with PFA-100, assays of coagulation FVIII (50%–150%), FXI (70%–150%), FIX (50%–150%), FVII (70%–140%), FV (70%–140%), FX (70%–140%), and FII (if indicated) levels, von Willebrand factor antigen and activity (50%–150%). In a subset of patients, specific assays were performed: platelet nucleotides, evaluation of platelet glycoproteins by flow cytometry, platelet morphology by light microscopy, whole blood thromboelastography (Rotem Pentapharm) ⁴, Euglobulins Lysis Time (ELT; 180–360 min), PAI-1 assay (in case of ELT shortening; 4–43 ng/ml), FXIII assay. Abnormal results were defined as a single or multiple clotting factor deficiency, platelet functional defect, increased fibrinolysis, or isolated disturbancies of whole blood thromboelastography. For all patients with abnormal findings, in particular, platelet functional defects and hyperfibrinolysis, a second confirmatory test was requested.

Thrombophilia testing included activated protein C resistance (normalized activated protein C ratio <0.9) with a screen for the factor V Leiden mutation if positive, prothrombin G20210A gene mutation analysis, antithrombin functional assay (70%–130%), protein C functional assay (70%–130%), free protein S antigenic assay (70%–170%) with a measurement of total protein S level (70%–170%) in case of free protein S deficiency, screen for antiphospholipid antibodies (lupus anticoagulant and anticardiolipin antibodies), and in some patients, homocystein (5–15 µmol/L) and factor VIII level (50%–150%) were also measured. For patients with a past history of splanchnic thrombosis, screening for JAK2V617F mutation was also performed.

RESULTS

In total, 4502 clinics (appointments) during a 2-year study period were reviewed. They accounted for 23% and 0.45% of all visits in the division of adult hematology and the hospital, respectively.

As shown in Table 1, a majority (52.7%) of patients were referred for thromboembolic diseases involving venous thrombosis, thrombophilia testing for obstetric complications and monitoring of anticoagulation during pregnancy, arterial thrombosis, and family screening for thrombophilia. A smaller group (23.7%) was referred for evaluation of bleeding symptoms and follow-up of hereditary bleeding disorders (hemophilia A and B patients, hemophilia carriers, and von Willebrand disease). Twenty percent of the patients were referred for primary or secondary venous insufficiency, general hematology, or for inappropriate indications.

Abnormal results were found in 17.9% of the patients with bleeding symptoms using routine tests (Table 2). Von Willebrand disease and hemophilia A or B were found in 34.8% and 17.8% of the patients, respectively. Platelet functional defect was diagnosed in 30.3% (n=34) of the abnormal hemostasis work-ups. Disturbed platelet aggregation studies were much more prevalent with positive findings in 107 patients (58%) referred for evaluation of a bleeding disorder. Explanations for this discordance are the absence of confirmatory tests in many patients (n=58) and intake of medications interfering with platelet function. Deficiency of factor II, V, VII, X, XI, or hypo- or dysfibrinogenemia accounted for 16.9% of abnormal findings.

In patients with a clear positive bleeding history and a negative routine assessment (n=250), RoTEM analysis, measurement of the ELT and the PAI-1 level were in addition performed. Hyperfibrinolysis identified either by a shortening of the ELT, an abnormal RoTEM, or a decrease of the PAI-1 level was found in 181 patients with a bleeding tendency. Shortening of the ELT was confirmed on repeated testing in 77 patients only. Sixty-nine patients had an isolated abnormal RoTEM tracing that could not be explained by a primary hemostatic or coagulation defect.

A thrombophilic abnormality was identified in 27.5% of the patients referred for thrombophilia testing (Table 2). A thrombophilic trait was found in 35% of patients with a DVT and/or PE history, in 24% with obstetrical complications, in 29% with splanchnic thrombosis, and in only 2% with retinal vein thrombosis. Among patients referred for arterial thrombosis, one-fourth of patients had an abnormal finding (Table 2). As expected, the factor V Leiden mutation was the most frequent thrombophilic abnormality, except in women with obstetrical complications in whom the most prevalent abnormality was protein S deficiency, which in most cases, was acquired and explained by hormonal influence (estro-progestative pill, pregnancy).

DISCUSSION

This study provides original data about current clinical practice of thrombosis and hemostasis in a tertiary hospital. It highlights the importance, the wide spectrum, and complexity of this speciality.

Although the conclusions cannot be applied to all centers, the clinical practice of thrombosis and hemostasis represents a significant fraction of the outpatient consultations (clinics) not only of the hematology division (nearly one-fourth) but also of the entire hospital (0.45%).

As clearly shown by our study, most patients were referred because of thrombotic or hemorrhagic manifestations associated with a large variety of nonhematological disorders. Their appropriate management requests a broad knowledge of clinical medicine with an in-depth understanding of the physiology of normal hemostasis and its disturbances in most pathological situations. By contrast with inherited bleeding disorders, care of patients with venous or arterial thrombotic diseases requires interactions with many specialists and an expertise in the diagnosis and management of organ-specific thrombotic manifestations. The recently published European curriculum for thrombosis and hemostasis integrates this holistic approach of clinical practice of hemostasis and thrombosis and emphasizes the need for optimal interactions with a wide array of specialties.

Our study confirms that thrombotic diseases represent a predominant reason for referral compared to hemorrhagic disorders. Moreover, we found that classical indications for referrals such as hemophilia, Von Willebrand disease and venous thromboembolic disease represent a small proportion of the global activity in comparison with other more recent indications for referral including complicated pregnancies, monitoring and adaptation of antithrombotic agents, as well as evaluation of unexplained or unusual arterial thromboses.

Our study shows the importance of increasing the awareness of the indications and precautions (adequate timing) of thrombophilia testing, especially in women with complicated pregnancies. Thrombophilic work-up was indeed requested by experienced obstetricians for many women with a past history with complicated pregnancy in the absence of guidelines recommending extensive thrombophilia screening with the exception of antiphospholipid antibodies ⁵. Moreover, the work-up was frequently requested during pregnancy or in women taking hormones, accounting for the high prevalence of acquired protein S deficiency in our study. Our study highlights the need for validated and practical recommendations in order to avoid misuse and over diagnosis of thrombophilia screening.

With respect to bleeding disorders, most patients were referred for evaluation of a bleeding tendency. Management and/or diagnosis of hemophilia and von Willebrand disease as well as other rare bleeding disorders represented a small fraction of the total activity. The high prevalence of platelet functional disorders and hyperfibrinolysis should be interpreted with caution given the absence of control population, the possible influence of confounding factors, the poor reproducibility, and the analytical limitations of the used tests. Our study also emphasizes the importance of confirmatory tests that could not be obtained in a large group of patients during the study period. The large proportion of patients with an isolated abnormal thromboelastogram highlights potential limitations of currently used assays.

Our study has some important limitations. It was conducted in a single center. Our results are influenced by the local organization and long-standing collaborations with specific disciplines and divisions within our hospital. Our conclusions can, therefore, not be generalized to other centers and countries. The number of patients referred for thrombotic diseases was most likely underestimated. Our study provides useful information regarding the proportion of abnormal findings. It was, however, not intended to review and audit the appropriateness of bleeding and thrombophilia screenings and their agreement with current guidelines and recommendations.

In conclusion, despite some limitations, this study provides original and valuable information on the size and wide spectrum of clinical practice of hemostasis and thrombosis. It highlights the need to implement the European curriculum through appropriate educational programs. Such evolution appears instrumental for the future of hemophilia care as well as the diagnosis and management of the increasing number of patients with thrombotic or hemorrhagic disorders.

Disclosure: The authors declare no conflict of interest.

Keywords

hemophilia, thrombosis, clinical practice

REFERENCES

1. Astermark J, Negrier C, Hermans C, et al. European curriculum for thrombosis and haemostasis. Haemophilia. 2009;15(1):337–344.
2. Berntorp E, Gomperts E, Hoots K, Wong WY. The next generation of hemophilia treatment specialists. Semin Thromb Hemost. 2006;32(suppl 2): 39–42.
3. Ludlam CA, Mannucci PM. Proposal to establish a European association for hemophilia and allied disorders. J Thromb Haemost. 2006;4(10): 2270–2271.
4. Sorensen B, Johansen P, Christiansen K, Woelke M, Ingerslev J. Whole blood coagulation thrombelastographic profiles employing minimal tissue factor activation. J Thromb Haemost. 2003;1(3):551–558.
5. Bates SM, Greer IA, Pabinger I, Sofaer S, Hirsh J. Venous thromboembolism, thrombophilia, antithrombotic therapy, and pregnancy: American College of Chest Physicians evidence-based clinical practice guidelines (8th ed.). Chest. 2008;133(suppl 6):844S–886S.

The development of inhibitors is one of the most challenging adverse consequences of factor replacement therapy in hemophilia. Inhibitor development in hemophilia B is relatively rare, affecting around 2%–4% of individuals¹ in contrast to hemophilia A, which is associated with the development of inhibitors in 20%–30% of patients.² Around half of all inhibitors are detected before the age of 10 years, with studies suggesting a peak in incidence at around 2 years of age.² The majority of inhibitor patients (~80%) are high responders² and are associated with major gene deletions.

The management of hemophilia B patients is complicated by the occurrence of anaphylactic reactions to factor IX (FIX) infusions, which may accompany or precede inhibitor development—especially when patients are exposed to intensive high-dose treatment.³ Anaphylactic reactions are most prevalent in patients with severe hemophilia B (<1% FIX).³

Treatment of patients with hemophilia B and inhibitors is determined, at least in part, by the inhibitor titer and the nature of the immune response. Low-titer, low-responding (nonanamnestic) inhibitors (<5 Bethesda Units [BU]) can often be overcome with higher than usual doses of FIX concentrates to ensure hemostasis. This option may be limited due to the risk of severe allergic reactions upon re-exposure to FIX concentrates. Immune tolerance induction (ITI), which is recommended for all patients with severe hemophilia A and inhibitors, is markedly less effective in hemophilia B⁴ and the inherent risk of both anaphylaxis and potentially irreversible nephrotic syndrome⁵ has limited the use of ITI in hemophilia B patients.⁶

The management of high-titer and high-responding FVIII or FIX inhibitors (≥5 BU) requires alternative strategies including the use of bypassing agents.⁷ Factor Eight Inhibitor Bypassing Activity (FEIBA, Baxter AG, Vienna, Austria) is an established bypassing agent that has been used extensively and successfully in the management of patients with hemophilia A and inhibitors for more than 30 years.⁸ However, the clinical efficacy and safety of FEIBA in patients with hemophilia B and FIX neutralizing antibodies might be expected to differ from that observed in hemophilia A as the product contains significant amounts of FIX protein, potentially putting susceptible patients at risk of developing FIX-mediated immune responses.

Unfortunately, due to the rarity of hemophilia B patients with inhibitors (only 94 such individuals have so far been reported to the international FIX inhibitor registry⁴), the conduct of prospective efficacy and safety studies with bypassing agents in this patient group is not feasible. There are, however, increasing numbers of published reports suggesting that FEIBA is being used in the treatment of hemophilia B patients with inhibitors despite the availability of alternative therapies.

In order to better understand when and how FEIBA is being applied in clinical practice and to provide insight into the treatment's efficacy and safety when used in hemophilia B patients with inhibitors, we have reviewed published case reports and data presented at international congresses since FEIBA was first introduced for therapy. In this report we present an overview of the cases identified and discuss the efficacy and safety implications of what has been reported.

CASE REPORTS OVERVIEW: 1979–2010

A total of 37 reports on the use of FEIBA in 46 hemophilia B patients with inhibitors have been identified (Table 1)^{5, 6, 9–42}: 16 of the reports have been published, 19 papers were presented at international congresses, and 2 cases were reported to Baxter Healthcare Corporation directly. The nature of the reports varies considerably, ranging from single case studies in which FEIBA had been used at some point in an individual's treatment path, with little or no detail provided, to well-reported retrospective surveys assessing the efficacy and safety of FEIBA in patients with hemophilia B and inhibitors. Of the 37 reports identified, 23 (62%) relate primarily to the on-demand use of FEIBA to treat acute bleeds,^{5, 6, 9–29} eight (22%) describe the use of FEIBA for surgical coverage,^30–37 four (11%) present data on FEIBA prophylaxis,^{32, 38–40} and two (5%) describe the use of FEIBA to induce immune tolerance (Table 1).^{41, 42} The majority of patients had severe hemophilia B and high-responding inhibitors; the age range was 2 – 70 years.

EFFICACY OF ON-DEMAND USE OF FEIBA

The first report on the use of FEIBA to treat a bleeding episode in an individual with hemophilia B and inhibitors was published in 1979 in Japan (Table 2).¹⁴ A 6-year-old boy who required hospital admission after a knee joint bleed was found to have an inhibitor of 14 BU/mL. He was treated with FEIBA infusions of 1000 units (U) twice daily for 3 days with good clinical outcome. No changes in FIX activity or inhibitor titer were noted. Two years later, Iizuka and Nagao¹⁶ reported positive outcomes in five out of six bleeding episodes (four episodes of hemarthrosis, one major soft tissue hemorrhage, and one life-threatening GI tract bleed) experienced by two young cousins, aged 6 and 7 years. Doses of FEIBA ranged from 42 to 50 U/kg—the major bleeds required a total of three and five infusions to achieve hemostasis. Efficacy was achieved irrespective of the inhibitor titer, which was 360 BU/mL in one patient.

During the 1980s and 1990s, the successful on-demand treatment of an additional nine hemophilia B patients with inhibitors was reported,^{10, 13, 15, 19, 20, 23, 26, 27} including one patient with a life-threatening intracranial bleed²⁶ and one who had failed ITI with high doses of FIX.¹³

In one report,²⁷ single infusions using the same dose of FEIBA (50 U/kg) were self-administered at home successfully by a 23-year-old high responder with severe hemophilia B to treat spontaneous soft tissue and joint bleeds. At the time of publication, the patient had been using FEIBA for over 4 years, successfully treating two bleeds per month on average and required nine hospital admissions amounting to a total of 32 days for the management of large bleeds during that time.

Négrier and coworkers have more recently reported the results of several multicenter, prospective surveys assessing the efficacy and safety of FEIBA in patients with FVIII and FIX inhibitors.^{11, 22} In the most recent study involving 63 inhibitor patients with either hemophilia A (n=60) or B (n=3), physicians rated the treatment outcome with FEIBA as good or excellent in 82% of acute bleeding episodes and in 91% of all surgical treatments.¹¹ The median cumulative dose of FEIBA administered per bleeding episode was 167–223 U/kg depending on the site of the bleed. The mean total dose of FEIBA used in major and minor surgical procedures was 1174 U/kg and 748 U/kg, respectively. Although the results for one surgical procedure and four acute bleeds in the three hemophilia B patients treated with FEIBA were not described separately, the authors concluded that: “FEIBA appears to have been equally effective in patients with hemophilia A and B.”

SEQUENTIAL COMBINED BYPASSING THERAPY

Two papers have been presented describing the efficacy and safety of on-demand sequential combined bypassing therapy (SCBT) with FEIBA and rFVIIa in the treatment of severe bleeding in hemophilia B patients with inhibitors who were previously refractory to monotherapy.^{28, 29} In the first paper, a 38-year-old male with severe hemophilia B and a long-standing high-responding inhibitor failed to respond to treatment with rFVIIa (90 µg/kg every 2 hours) after presenting with a spinal mass and developing progressive spinal cord compression requiring evacuation of the epidural tissue. The patient developed persistent epidural bleeding and deteriorated despite increasing doses of rFVIIa to 120 µg/kg every 2 hours. Subsequently, a sequential therapy was initiated using FEIBA 80 U/kg every 12 hours and rFVIIa 120 µg/kg six times a day. The bleeding diminished significantly within 24 hours of initiating sequential therapy, FEIBA was discontinued after 4 days, and the patient was discharged on postoperative day 9 on a home regimen of once-daily FEIBA 70 U/kg for 4 days and once daily rFVIIa 90 µg/kg/day for 4 days. Bleeding did not recur and there were no thromboembolic complications.

Gringeri et al²⁹ reported on two patients with hemophilia B who had received SCBT (alternating one FEIBA dose to three rFVIIa doses) for unresponsive major bleeds. FEIBA dosing ranged from 20 to 80 U/kg every 8 to 12 hours, rFVIIa dosing ranged from 80 to 270 µg/kg every 3 to 12 hours. Bleeding control was achieved within 12–24 hours of SCBT initiation in both patients.

EFFICACY OF FEIBA DURING SURGERY

Several detailed accounts of the use of FEIBA treatment during surgery in patients with FIX inhibitors have been published (Table 2)^{34–36, 40} and presented at congresses,^{31, 33} the first as early as 1983.³³ Stine and coworkers³⁵ described the successful use of FEIBA in a 14-year-old boy with a low-titer inhibitor (0.43 BU/mL) who required port removal and replacement. FEIBA was administered preoperatively as a single dose of 48 U/kg (with FIX 119 IU/kg) and two doses of 48 U/kg each every 8 hours postoperatively, followed by 48 U/kg every 12 hours for four doses, then 48 U/kg QD for 4 days plus FIX 119 IU/kg QD for 3 days, then FIX 72 IU/kg QD for 3 days. Hemostatic control was rated as excellent, no unexpected bleeding occurred, and there were no thrombotic complications.

Tjønnfjord (2004)³⁶ reported on the Norwegian experience of using FEIBA in 15 minor and 6 major surgical and invasive diagnostic or therapeutic procedures in 10 inhibitor patients, one of whom had acquired hemophilia B and an inhibitor titer of 37 BU/mL at the time of the report. This 58-year-old individual underwent a trephine biopsy with FEIBA coverage at a dose of 55 U/kg/day with an excellent hemostatic outcome and no complications. A preoperative loading dose of 100 U/kg was administered. In total, the patient received 4000 units of FEIBA.

In Turkey, Zulfikar et al (2008)³⁷ analyzed the outcomes of 27 surgical procedures performed in patients with hemophilia A or B and inhibitors using FEIBA as the primary hemostatic agent. One patient in this series—a 48-year-old individual—had hemophilia B and an inhibitor titer of 60 BU/mL at the time of undergoing a transurethral resection. A FEIBA dose of 112.6 U/kg/day was used for this patient with a good hemostatic outcome.

The SURgical Interventions with F¯EIBA (SURF) study, which involves 23 international centers, recently reported data from two surgeries in hemophilia B patients with inhibitors.^{31, 32} One patient underwent a right hip replacement using a FEIBA dose of 216 U/kg/day given as two infusions per day and requiring a total dose of 7140 units.³¹ Hemostatic efficacy was reported to be excellent and there were no adverse events documented. The second patient had a knee arthroplasty during which 186 U/kg of FEIBA were infused, a total of 1042 U/kg was also administered postoperatively in decreasing doses.³² Efficacy was rated good and no product-related adverse events were reported. This second patient had undergone three previous surgical interventions—two of which with good results using FEIBA. The FEIBA doses utilized and the surgical outcomes reported for both patients are similar to those in hemophilia A patients undergoing major orthopedic surgeries.

EFFICACY OF FEIBA PROPHYLAXIS

Four cases were identified describing long-term prophylactic treatment with FEIBA in hemophilia B patients with inhibitors (Table 2).^{32, 38–40} Most recently, Klamroth et al (2010)³⁸ reported the successful use of FEIBA in a dose of 20 U/kg every other day in a 24-year-old male with an inhibitor titer ranging from 5 to 35 BU/mL. This patient had failed ITI with low-dose FIX (24 IU/kg/day) and was experiencing one to two acute bleeds per month. One breakthrough bleed was reported during 3 months of FEIBA prophylactic treatment, which was treated successfully with rFVIIa 200 µg/kg.

Valentino published the results of a retrospective analysis of six patients with hemophilia A or B and high-titer inhibitors treated prophylactically with FEIBA.⁴⁰ The only hemophilia B patient was an 8-year-old boy of mixed race with a long-standing inhibitor (historic peak of 29 BU/mL) and a history of frequent and extended hospitalizations for uncontrolled bleeding in one knee and both elbows. FEIBA prophylaxis was initiated at a dose of 100 U/kg/day for 15 months, after which he underwent arthroscopic synovectomy. Following surgery and during the subsequent 6 months of aggressive physical therapy, the boy received FEIBA prophylaxis at a dose of 75 U/kg every third day. Administration of FEIBA over more than 4 years has reduced his bleeding episodes from 113 to 16 per year—an 85% reduction from before the use of FEIBA. The inhibitor titer was 16 BU/mL at the last evaluation. While receiving prophylactic treatment with FEIBA, no hospitalizations or visits to the emergency department were reported, the mobility improved, as did his school attendance and academic performance.

The benefits of short-term FEIBA prophylaxis have been reported by Komrska et al³⁹ and Kupesiz.³² A 19-year-old male with hemophilia B developed a high-titer FIX inhibitor (13.6 BU/mL) at 4 years of age.³⁹ He was treated with both FEIBA and rFVIIa at different times with varying degrees of success. He developed a large hematoma and hypertrophy of the synovia in his left knee joint for which surgery was considered. To improve the patient's condition but avoid surgery, FEIBA prophylaxis at a dose of 85 U/kg three times a week was administered for 4.5 weeks. During this period, the patient underwent intensive rehabilitation and aqua therapy. No bleeding episodes were reported. The joint edema regressed, the mobility significantly improved, and the patient began walking without assistance.

A 4-month course of prophylactic treatment with FEIBA 56 U/kg twice weekly was initiated in a 12-year-old boy; the patient experienced only one joint bleed in the 4-month period.³²

USE OF FEIBA TO INDUCE IMMUNE TOLERANCE

Continuous use of FEIBA has been reported to induce immune tolerance (ITI) in hemophilia B patients with inhibitors.^{41, 42} Meunier et al⁴¹ described ITI with FEIBA in a boy with severe hemophilia B who developed neutralizing antibodies to FIX at the age of 2 years. The patient was a high responder (historic peak of 43 BU/mL) who developed an allergic reaction to plasma-derived FIX. Bypassing agents were used to control acute bleeding episodes subsequent to inhibitor development. After being admitted for an intra-abdominal hematoma, rFVIIa treatment was initiated for bleeding control that was switched to FEIBA in order to start secondary prophylaxis. FEIBA doses, which were initially 80 U/kg every 12 hours, were progressively tapered off mimicking a prophylactic regimen (80 U/kg every 3 days). After a transient anamnestic response (peak 1 BU/mL), the inhibitor remained undetectable. The patient was subsequently switched to plasma-derived FIX, the inhibitor remained undetectable, and the FIX recovery remained normal.

Watanabe et al⁴² reported a similar success in an 11-year-old boy with severe hemophilia B and a high-titer inhibitor (historic peak of 65 BU/mL). Subsequent to inhibitor formation at the age of 2 years, bleeding episodes were treated with either FEIBA or rFVIIa on demand, however, rFVIIa eventually became ineffective and ITI was initiated with high-doses of FIX plus cyclophosphamide: the inhibitor titer fluctuated between 20 and 60 BU/mL. FEIBA was then initiated at a dose of 30–50 U/kg administered two to three times per week for 2 years. The inhibitor titer gradually decreased after a transient rise to 39 BU/mL. The continued administration of FEIBA resulted in the elimination of the inhibitor. No anaphylaxis or nephrotic syndromes were observed in this patient.

SAFETY AND TOLERABILITY OF FEIBA

Overall, FEIBA appears to have been well tolerated in most of the 46 patients who received treatment using a variety of different doses and treatment modalities. A total of 10 adverse events were reported in association with the use of FEIBA: six cases of anaphylaxis,^{5, 6, 12, 18, 25} one case each of thrombosis,²⁴ disseminated intravascular coagulation (DIC),²⁶ and myocardial infarction (MI),³⁰ and one case of pruritis/rash³⁷ (Figure 1, Table 3).

Anaphylactic Reactions

Dioun et al¹² reported the outcome of two boys, aged 9 and 2 years, with severe hemophilia B and high-titer inhibitors who had been treated successfully with either FEIBA on-demand or FIX concentrate prophylactically plus FEIBA on-demand before developing severe anaphylaxis to FEIBA and FIX, respectively. In the first boy, other than a mild rash during an infusion at 2 years of age, FEIBA infusions had been well tolerated until 7 years of age, when he developed generalized urticaria, angioedema of the lips and eyelids, cough, abdominal discomfort, and tachycardia within 10 minutes of receiving an intravenous (IV) infusion of FEIBA. His symptoms resolved with IV diphenhydramine and dexamethasone. In the second boy, FIX infusions (100 U/kg) every other day had been well tolerated until 10 months of age, when he developed a generalized maculopapular rash during a FIX infusion. One month later, he had generalized urticaria, angioedema, projectile vomiting, and wheezing within 5 minutes of an IV administration of FEIBA. This anaphylactic reaction was treated with epinephrine, methylprednisolone, and diphenhydramine and the patient was admitted to the intensive care unit for 1 day. Both boys subsequently underwent successful desensitization to FIX using a modified version of the Jamieson protocol⁴³ and they were subsequently kept on FIX to maintain the desensitization. FEIBA was used on-demand for treatment of hemarthroses without further adverse reactions.

Successful desensitization was also reported by Siripassorn and Chantaphakul²⁵ in an 18-year-old male with hemophilia B (body weight not reported) and a high-titer inhibitor who had an anaphylactic reaction (hypotension and urticaria) 20 minutes after FEIBA was administered to treat bleeding from a fracture site. FEIBA desensitization was achieved over 5 days using an initial dose of FEIBA of 0.0025 units, stepped up every 15 minutes to reach 0.3 units in 3 hours. FEIBA was subsequently administered at a rate of 250 U/hour and finally increased to 750 U/hour. The total FEIBA dose used was 2000 units; no further anaphylaxis was observed.

A fourth young patient with severe hemophilia B who developed a FIX inhibitor at the age of 5 years (historic peak of 6.2 BU/mL) was treated successfully with FEIBA on several occasions before he experienced a severe allergic reaction resulting in hypovolemic shock.⁶ On-demand treatment with rFVIIa was initiated. The boy subsequently underwent successful ITI with a combination of rituximab, myophenolate mofetil, dexamethasone, IV immunoglobulins, and high-dose FIX.

Ewenstein et al⁵ reported the case of a 2-year-old boy who at the age of 11 months had developed a large intrathoracic and mediastinal hemorrhage after placement of a central venous catheter. After 2 weeks of intensive FIX treatment, an inhibitor with a peak titer of 67 BU/mL was detected and the patient concomitantly developed an urticarial rash after FIX administration. Soon afterwards, the patient developed a life-threatening anaphylactic reaction to FEIBA (dose not reported). Desensitization was subsequently attempted, however, the boy developed reversible nephrotic syndrome some months later after further FIX infusions.

Finally, Laguna and Klukowska¹⁸ presented the case of an 8-year-old boy who had developed a FIX inhibitor of 125 BU/mL at age 5 and had used FEIBA on-demand for 2 years. The boy had been admitted to the hospital with suspected gangrenous appendicitis and bleeding into the abdomen. Towards the end of a FEIBA infusion, severe anaphylaxis occurred (shivering, fever, respiratory complications, and tachycardia). The patient was operated on successfully after rFVIIa administration.

Thrombotic Complications

Detailed and often extended laboratory investigations were performed in several of the identified cases^{14, 15} with no evidence of intravascular coagulation or clinically significant alterations in platelet counts or fibrinogen levels. Gringeri et al²⁹ reported a rise in D-dimer levels in three out of five tested patients, but without consumption of platelet and/or fibrinogen. In their series of 11 hemophilia A and B patients with high-titer inhibitors who underwent SCBT with FEIBA and FVIIa, no clinical adverse events were observed in any patient.

Taki et al²⁶ reported the successful treatment of a life-threatening intracranial hemorrhage in a patient with hemophilia B and a high-responding inhibitor with high-dose FEIBA. However, the patient was reported to have developed DIC during treatment. The level of antigen of antithrombin III (ATIII) remained within normal limits during treatment, although the level of activity of ATIII was decreased.

Penner and Hassouna²⁴ described the case of a 63-year-old man with hemophilia B and inhibitor. The patient received 10000 units of FEIBA within 14 days to treat a knee bleed. Following his discharge, he had seizures and his condition worsened with the appearance of superior vena cava syndrome. The resulting occlusion of his vena cava by thrombosis was treated successfully with urokinase.

A single case of MI in a patient with pre-existing thrombotic risk factors was reported by Mizon et al in 1992.³⁰ The patient was a 41-year-old male with severe hemophilia B and an inhibitor titer of 6 BU/mL. He was mentally retarded, a smoker, obese, HCV positive, and hyperlipidemic. FEIBA (7000 units; 82 U/kg) was initially administered 1 hour prior to multiple dental extractions, which was well tolerated. However, 1 hour after a second infusion of the same dose, the patient complained of severe chest pain, and an acute inferior MI was diagnosed. At the time, coagulation tests revealed only a slight increase in fibrin/fibrinogen degradation products and the presence of soluble complexes. During the infusion, the inhibitor titer was reduced to undetectable levels with a rise in plasma FIX activity that may have increased the thrombotic risk. The patient eventually recovered after suffering a second MI on the third day and, as the inhibitor remained undetectable, he resumed treatment with FIX concentrates.

The published reports outlined above correspond with Baxter's pharmacovigilance data in that the safety profile of FEIBA when used for the treatment of inhibitor patients is comparable in hemophilia A and hemophilia B patients.³² This is true with the exception of an increased risk of allergic and anaphylactic reactions in hemophilia B patients with inhibitors.

DISCUSSION

Depending on the country of licensure, the indications for FEIBA may include treatment of spontaneous bleeding and use during surgery in hemophilia A or B patients with FVIII or FIX inhibitors and in nonhemophiliacs with acquired inhibitors to FVIII or FIX.⁴⁴ FEIBA is also licensed in various countries for prophylaxis in hemophilia A or B patients with high-responding inhibitors.⁴⁴

FEIBA has been used for over 30 years to manage hemophilia A patients with inhibitors, either for on-demand or for prophylactic treatment. FEIBA has been shown to be more effective than prothrombin complex concentrate (PCC) in a comparative study in hemophilia A patients with inhibitors⁴⁵ and response rates have been reported to be as high as 65%–95%.^46–48

Hemophilia A treatment guidelines offer recommendations for the use of bypassing agents in the management of acute bleeds and for surgical prophylaxis.⁸ The guidelines highlight the clinical observation that patients who fail to respond to rFVIIa may still respond to FEIBA and vice versa, emphasizing that if there is no response to one bypassing agent, the alternative may be used.⁸ Although no specific recommendations are given for the use of bypassing agents for prophylaxis in hemophilia A patients with inhibitors, the guidelines suggest that FEIBA may be a useful prophylactic agent due to its relatively long half-life of thrombin generation.⁸

Recommendations for the management of patients with hemophilia B and inhibitors are somewhat less clear.^{8, 49} While early ITI is recommended for all patients with severe congenital hemophilia and a confirmed FVIII or FIX inhibitor, caution is urged in patients with hemophilia B, given the low success rate and risk of anaphylaxis and nephrotic syndrome.^{4, 5, 8, 49} For the management of bleeding episodes, the guidelines acknowledge a lack of licensed options and a paucity of published data, but, in light of the observed risk of FIX-related allergic responses, rFVIIa is recommended for bleeding in patients with high-responding FIX inhibitors or those who have had allergic reactions to FIX-containing therapeutics.⁵⁰

Over the last 6 years, increasing numbers of clinicians have been reporting the use of FEIBA in the context of modern treatment strategies in their hemophilia B patients with inhibitors, despite the potential risk of adverse events. This includes surgical or long-term prophylaxis, and in a few cases, the induction of immune tolerance and sequential combined treatment of bypassing agents. According to the reports described in this review, FEIBA doses of 25–100 U/kg, similar to the doses recommended in hemophilia A patients with inhibitors, effectively and safely reversed a range of soft tissue and joint bleeds. This included several severe and life-threatening bleeding episodes. These doses are comparable to those used in hemophilia A patients with inhibitors. When used for surgical prophylaxis, FEIBA doses ranging from 50 to 216 U/kg/infusion provided excellent hemostatic control including major surgeries. Dosages above 100 U/kg/infusion are outside the labeled dose range. Nevertheless, side effects were not reported in these cases. In most of the cases reported in this review, FEIBA was the initial treatment of choice; in some cases FEIBA was used successfully after insufficient responses to rFVIIa.^{41, 42} In three cases, where allergic reactions to FEIBA treatment had developed, the subsequent initiation of rFVIIa was reported to be successful.^{6, 18, 39} Two cases were managed with sequential treatment after monotherapy failed.^{28, 29}

The safe and efficacious use of FEIBA as a prophylactic agent in the four cases of hemophilia B with an inhibitor we are aware of is encouraging, given the potential benefits to long-term joint status of a prophylactic approach. However, the optimal doses and dosing intervals have not yet been defined. Possibly, the control of breakthrough bleeds into joints may be more problematic if a patient is receiving FEIBA prophylaxis compared to the on-demand situation.

Acquired hemophilia is frequently associated with severe and life-threatening bleeding that requires aggressive treatment.⁸ Within his case series, Tjønnfjord³⁶ described a patient with acquired hemophilia B and high-titer inhibitors who responded well to FEIBA use during surgery.

Reports of the novel and effective use of FEIBA in SCBT and ITI, although intriguing, nevertheless strongly suggest that the risks and benefits are assessed carefully before any consideration is given to attempting either experimental strategy.

FEIBA was well tolerated in the majority of cases reported in this case series. Anaphylaxis, which is a well-known phenomenon with the use of high-dose FIX treatment (including FEIBA and FIX concentrates), occurred in six patients, and was effectively managed in three of them by desensitization. Three patients continued with FEIBA, whereas one was switched to rFVIIa. Desensitization or ITI appear to be options to eliminate allergic responses to FIX protein, specifically, but not only, in patients refractory to rFVIIa. One case of MI presented with multiple predisposing cardiovascular risk factors, which may have complicated treatment. There were no reports of nephrotic syndrome after FEIBA in this series of 46 patients, although some of them received relatively high doses of FEIBA over extended periods.

Thrombotic events represent a rarely reported but important side effect associated with bypassing agents including FEIBA.^{51, 52} While it is commonly thought that congenital hemophilia may protect against thrombotic phenomena, this may be true only for venous thrombosis, since several sporadic cases of MI have been reported.⁵³ Comorbidities common in the general male population (ie, smoking and hyperlipidemia) and the high prevalence of infections (ie, HCV) in aging hemophilia patients represent confounding and predisposing risk factors for the development of such thromboembolic events.^{54, 55}

CONCLUSION

In this series of 46 patients with mainly severe hemophilia B and high-titer inhibitors, FEIBA was effective and well tolerated when used on-demand for perioperative coverage and as long-term prophylaxis. Although accepting the limitations of using a case series approach to evaluate the efficacy and safety of FEIBA, the relatively consistent positive findings and the detail with which many of these cases have been reported may assist in managing these clinically challenging individuals in the future.

Keywords

FEIBA, hemophilia B, inhibitors, bypassing agents

Acknowledgement: We thank Dr. A. Kupesiz and Dr. C. Negrier for kindly sharing with us unpublished data to complete the current database.

Disclosure: A. Schoppmann, K. Jaeger, R. Berg and B. Abbuehl are all employees of Baxter Innovations GmbH, Vienna, Austria and this literature review has been sponsored by Baxter.

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29. Gringeri A, Fischer K, Karafoulidou A, et al. Sequential combined bypassing therapy (SCBT) is safe and effective in the treatment of unresponsive bleeding in adults and children with haemophilia and inhibitors. Poster presentation at the 51st American Society of Hematology Annual Meeting; December 5–8, 2009; New Orleans, LA. Abstract 1294.
30. Mizon P, Goudemand J, Jude B, et al. Myocardial infarction after FEIBA therapy in a hemophilia-B patient with a factor IX inhibitor. Ann Hematol. 1992;64(6):309–311.
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33. Perez Bianco R, Schachter S, Elgue G, et al. Successful effect of FEIBA plus factor IX in a patient with inhibitor to factor IX. Presentation at the XVth World Federation of Hemophilia Congress; June 27-July 1, 1983; Stockholm, Sweden. Abstract 180.
34. Satomura A, Takahashi S, Fujita T, et al. Anasarca improved by extracorporeal ultrafiltration through an internal shunt in a case of severe haemophilia B with inhibitor and steroid-resistant nephrotic syndrome. Haemophilia. 2006;12(1):103–105.
35. Stine KC, Shrum D, Becton DL. Use of FEIBA for invasive or surgical procedures in patients with severe hemophilia A or B with inhibitors. J Pedriatr Hematol Oncol. 2007;29(4):216–221.
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Osteoporosis is widely recognized as a major health issue, and tremendous resources are spent screening, diagnosing, and treating this disease in postmenopausal women. According to the National Institutes of Health (NIH) Consensus Development Panel on Osteoporosis, 10 million people in the USA have osteoporosis and almost twice as many have decreased bone mass (1). Osteoporosis is defined as a decline in bone density and bone quality resulting in an increased risk for fragility fractures ¹. It can be separated into primary osteoporosis resulting from gradual bone loss with aging and secondary osteoporosis due to a variety of medical disorders including chronic corticosteroid use, hypogonadism, alcoholism, and hyperthyroidism. The final common pathway for both primary and secondary causes of osteoporosis is increased bone resorption in relation to new bone formation.

Diagnosis and screening for osteoporosis in men and women rely on the estimation of bone mineral density (BMD) most commonly achieved through the use of dual-energy X-ray absorptiometry (DXA) ^{1, 2}. The World Health Organization (WHO) defines osteoporosis and osteopenia on the basis of the T-score (the number of standard deviations above or below the average BMD value for young healthy white women) and the Z-score (the number of standard deviations above or below the average BMD for age and sex matched controls). WHO defines osteoporosis as a T-score <2.5 and osteopenia as a T-score −1 to −2.5 ³. These definitions were developed for estimating fracture risk in the postmenopausal female population and have since been extrapolated to the population at large.

While much of the medical literature has focused on the diagnosis and treatment of osteoporosis in postmenopausal women, men are also at risk. In fact, a 60-year-old white man has approximately a 25% lifetime risk of osteoporotic fracture ². Secondary causes of osteoporosis including hypogonadism, glucocorticoid use, and alcoholism contribute to approximately 50% of the cases of osteoporosis in men ¹. There are compelling reasons to treat or prevent osteoporosis in men as the 1-year mortality after hip fracture is twice as high in men compared to women ⁴.

Adults with hemophilia have unique risks for developing significant bone loss and osteoporosis. These include decreased physical activity, especially weight bearing exercise during childhood that leads to decreased peak bone mass, and infection with Hepatitis C virus (HCV) and/or human immunodeficiency virus (HIV) ^5–7. In this article we review the prevalence, risk factors, and pathogenesis of bone loss in adults with hemophilia. We also present recommendations for diagnosing and treating osteopenia and osteoporosis in this at-risk population.

PREVALENCE

One of the first studies demonstrating an association between hemophilia and osteoporosis came from a cross-sectional study of 19 adult males with severe hemophilia A from England ⁸. BMD as measured by DXA was significantly lower in the lumbar spine in hemophilia patients (1.109±0.042 g/cm²) compared to age matched controls (1.234±0.027 g/cm²) ⁸. BMD was similarly reduced at the femoral neck. No differences were found between the two groups with regards to several serum and urine markers of bone resorption and remodeling including serum calcium, parathyroid hormone, and 1,25 dihydroxyvitamin D3 and urinary hydroxyproline, pyridinoline, and deoxypyridinoline ⁸.

Children with hemophilia have also been reported to have an increased prevalence of low BMD. In a cross-sectional survey of 19 Australian children with severe hemophilia, Barnes et al ⁹ reported that bone mineral apparent density (BMAD) was significantly reduced compared to control subjects. The BMAD is an approximation of the volumetric density of bone calculated using bone mineral content and the projected volume of bone. The authors considered BMAD to be a more accurate measurement of bone density in children. Decreased BMAD was correlated with severity of joint disease, but not with HCV status, height, or weight ⁹. The use of BMAD to estimate bone density has not been routinely accepted, leading to criticism of this study ¹⁰.

Tlacuilo-Parra et al similarly performed a case-control study to evaluate the frequency of low BMD in 62 children with hemophilia in Mexico ⁵. Among these patients, 38% had low BMD defined as a Z-score below −2.0 compared to 16% in healthy sex, race, and aged matched controls (odds ratio 2.86, 95% confidence interval 1.17–7.41; P=.014). The above findings have important implications for adult bone health as low BMD in childhood negatively impacts peak bone mass and thereby increases the risk of osteoporosis later in life ¹.

In the adult literature there have been three recent studies examining the association between osteopenia/osteoporosis and hemophilia in developed countries ^{6, 7, 11}. In 2007, Wallny et al ¹¹ published the results of a cohort study of 62 German adult patients with severe hemophilia A and found osteopenia in 44% and osteoporosis in 26% of the patients. Katsarou et al ⁷ evaluated the prevalence of osteoporosis in a large cohort of 90 hemophilia patients from Greece. At the femoral neck, osteopenia was observed in 31% and osteoporosis in 56%. Osteopenia of the lumbar spine was observed in 52% and osteoporosis in 13%. Our group has previously described the prevalence and risk factors associated with decreased BMD ⁶. In our cohort, 43% of patients had osteopenia and 27% had osteoporosis. A recently published meta-analysis pooling 212 patients with severe hemophilia found that BMD of the lumbar spine was significantly lower in both adults and children ¹². However, as T- and Z-scores were not reported in all studies, the authors were unable to determine the prevalence of osteoporosis as it is commonly defined.

The clinical significance of reduced peak BMD in childhood and the subsequent development of osteopenia/osteoporosis by DXA criteria in young adults with hemophilia is still controversial. The diagnosis of osteoporosis in a woman indicates that a patient meets not only the above criteria by DXA, but more importantly implies an increased risk for fragility fractures. Fragility fractures are defined as those occurring from a fall from a standing height or less and without associated major trauma such as a motor vehicle accident. The association of osteoporosis with fragility fractures is less strong in men. Several of the studies described above note the number of patients sustaining a fracture. For example, Gallacher et al ⁸ reported that 7 of the described 19 hemophilia patients suffered a peripheral fracture as compared to zero in the control group. However, no data were provided on whether or not these fractures were trauma related. In our cohort, six patients (20%) had a history of prior long bone or compression fracture ⁶. Of these patients, three had osteopenia, two had osteoporosis, and one had normal BMD. Two of the fractures were trauma related, including in the patient with a normal BMD. Two patients, ages 27 and 66, suffered from fragility fractures of the femoral neck. Both of these patients were osteopenic on DXA. In a case control study of 50 adult patients with hemophilia A or B from India, Nair et al ¹³ reported a higher rate of fractures in patients with hemophilia compared to controls (12% vs. 0%, respectively) with five fractures of the femur and one of the radius and ulna. All patients with fractures had osteoporosis in their lumbar spine, and both of the patients with femoral neck fractures demonstrated osteoporosis in their hip, supporting a diagnosis of fragility fractures ¹³. However, no history was provided regarding the specific circumstances of the fractures and thus the possibility that some of the fractures were trauma related cannot be excluded. In another case series from India, 20 out of 500 patients with severe hemophilia sustained fractures in a 5-year period ¹⁴. The average age at the time of fracture was 28 years (range of 14 and 46 years) and 16 of the patients sustained fracture after a fall from a standing position, consistent with fragility fractures. The DXA scan data were available for six of these patients, all of whom had osteoporosis. These last two studies from India did not comment on access to factor replacement therapy, and thus these results may not be directly relevant to patient populations receiving on-demand factor replacement treatment.

BMD alone is not the sole predictor of fragility fractures. Patient age and frailty also play a major role in predicting fracture risk as a given T-score in a 50-year-old person is associated with a much lower risk of fracture compared to the same T-score in an 80-year-old person ¹⁵. In his recent review article, Kovacs discussed the importance of distinguishing low peak bone mass achieved in childhood from a loss of normal peak bone mass, as occurs in older adult women with osteoporosis ¹⁰. Individuals with low peak bone mass from childhood still have normal bone microarchitecture and thus the risk of fracture below age 50 should remain low ¹⁰. The increase risk of fracture that occurs with aging is multifactorial and includes bone-independent factors such as decreased muscle strength and coordination that increase risk of falls ¹⁰. Thus, it is unclear if decreased peak bone mass and/or low BMD by DXA in young adults with hemophilia results in an increase in fragility fractures or requires therapy. Increased understanding of the clinical consequences of decreased peak bone mass is necessary for determining appropriate guidelines on screening and treatment for patients with hemophilia.

RISK FACTORS

Several of the above studies have attempted to identify risk factors associated with the development of osteoporosis (see Table 1). Details describing our cohort of adult patients with moderate or severe hemophilia A or B followed at the Arizona Hemophilia and Thrombosis Center have been previously described ⁶. In the initial univariate analysis of our 30 patients, variables associated with low BMD included low serum 25-hydroxyvitamin D levels (P=.03), lower activity scores (P=.02), decreased joint range of motion (P=.046), HIV (P=.03) and HCV (P=.02) infection, history of a factor inhibitor (P=.01), lower BMI (P=.047), and older age (P=.03) ⁶. In our patients, bone loss was not associated with alkaline phosphatase levels, TSH, serum calcium, or testosterone levels. We have recently performed a multivariate analysis on our patient data using a stepwise ordinal logistic regression. We used a self-rated activity score (Table 2) as a surrogate measure of joint arthropathy. This simple 5-point scale was strongly correlated with joint range of motion and BMD in our patient population. In our multivariate analysis, lower activity score (P=.006), low BMI (P=.02), and HCV infection (P=.035) remained independent risk factors for abnormal BMD (osteopenia or osteoporosis) (Table 3). Of these, a low activity score and a low BMI showed the strongest association with bone loss.

In the largest published study, Katsarou et al ⁷ identified osteopenia/osteoporosis in over 80% of the 90 patients studied. On multivariate analysis, only HIV infection (OR 3.765, 95% CI 1.026–13.820; P=.05) and degree of hemophilic arthropathy (OR 2.42 per 10-unit increase in WFH clinical score, 95% CI 1.436–4.102; P=.001) were significant with the degree of arthropathy being the strongest predictor for osteoporosis. Because over 95% of the patients in this study were infected with Hepatitis C, the impact of HCV on osteoporosis development could not be directly tested ⁷.

The association of decreased BMD with severity of joint arthropathy and/or its complications including decreased range of motion and decreased activity has been consistent in published studies. (Table 1) ^{5–7, 9, 11}. Recurrent joint bleeds can lead to prolonged convalescence, decreased range of motion, chronic pain, and fear of future bleeding, resulting in decreased activity levels both as children and adults. Because of the importance of weight bearing exercise in reaching peak BMD as well as slowing the rate of bone loss in adults, lack of exercise may largely contribute to the low BMD found in patients with hemophilia. Data to support this come from a study by Khawaji et al ¹⁶ comparing 26 Swedish adult patients with severe hemophilia receiving regular prophylactic treatment to 16 patients with mild hemophilia. In this study no difference was found between the two groups with regards to BMD. Additionally, in all patients, the Z-score by DXA was greater than −1, indicating that BMD was comparable to the reference population. There was also no difference in physical activity or joint disease between the two groups ¹⁶. It is now established that the regular use of scheduled factor replacement therapy or prophylaxis treatment in children can prevent joint damage ¹⁷. Additional studies suggest that children who receive prophylactic factor therapy can safely participate in athletics ¹⁸. Together these studies suggest that joint disease is the most important risk factor for decreased BMD in patients with hemophilia and provide further justification for the early initiation of primary prophylaxis to prevent joint arthropathy and subsequent bone loss.

The role of chronic hepatitis C infection in the development of osteoporosis in patients with hemophilia is unclear. Several studies, including our own, have found an increased rate of osteoporosis in patients with hemophilia and hepatitis C infection. Metabolic bone disease, primarily in the form of osteoporosis and rarely as osteomalacia, is common in patients with chronic liver disease and referred to as hepatic osteodystrophy ^{19, 20}. Several risk factors for osteoporosis are more common in patients with liver disease including alcohol abuse, malnutrition, hypogonadism, vitamin D deficiency, and corticosteroid use. However, chronic infection with hepatitis B or C, with or without associated cirrhosis, also appears to be an independent risk factor ^{21, 22}. Viral hepatitis is thought to increase RANKL (receptor activator of NF-κB ligand) mediated osteoclast activity through the action of inflammatory mediators including interlukin-1 and tumor necrosis factor-alpha (TNF-α), which are frequently elevated in patients with hepatitis ²⁰. Decreased hepatic clearance of intact parathyroid hormone and decreased levels of insulin-like growth factor 1 (IGF-1) have also been implicated in hepatic osteodystrophy ^{21, 22}. In one study of 32 male patients with viral cirrhosis and no history of alcohol intake, abnormal BMD was correlated with severity of liver disease, low serum IGF-1, and urine deoxypyridinoline levels; but not with other serum markers of bone metabolism including bone Gla-protein, alkaline phosphatase, 25-hydroxyvitamin D, intact PTH, and free testosterone index ²². Suboptimal vitamin K levels may further increase the risk of abnormal bone metabolism in patients with liver disease as discussed below ²⁰.

HIV infection may also play a role in the development of osteoporosis in patients with hemophilia. While antiretroviral therapy has been associated with decreased BMD, HIV infection itself is thought to play a role in abnormal bone metabolism ²³. As in HCV, this may be mediated by immune activation and increased production of various proinflammatory cytokines including TNF-α, IL-1, and IL-6 that promote RANKL release and increased osteoclast mediated bone resorption ²⁴. In addition, HIV is often associated with known risk factors for osteoporosis including decreased physical activity, malnutrition, decreased BMI, smoking, alcohol use, use of other medications (eg, corticosteroids, anticonvulsants), and hypogonadism. A comprehensive review of the many factors that can impact BMD in HIV-infected patients is provided by Glesby ²⁴. Similar to the situation in hemophilia, the clinical significance and treatment of low BMD in younger patients with HIV remain an area of debate and controversy.

Little is known about bone markers of resorption and formation in patients with hemophilia. Katsarou et al ⁷ found that patients with hemophilia had increased levels of markers associated with bone resorption (osteoclastic activity) including N-terminal cross-linked telopeptide of collagen I (NTX), C-terminal cross-linking telopeptide of collagen I (CTX), and tartrate-resistant acid phosphatase isoform-5b (TRACP-5b), but no parallel increase in markers of bone formation, suggesting that pathologic bone metabolism may play a role in the increased incidence of osteopenia and osteoporosis. Interestingly, levels of osteoclast regulators including RANKL, osteoprotegerin (OPG), and TNF-α were not significantly different compared to controls. In addition, markers of osteoclast activity did not differ significantly between HIV-positive and HIV-negative patients with hemophilia, though the presence of HIV infection had independent prognostic value with regards to osteoporosis development ⁷. To explain these results, the authors hypothesized that systemic serum levels of RANKL/OPG may not accurately reflect levels in the bone microenvironment and that the lower BMI found in patients with HIV may explain the increased risk of osteoporosis in HIV-infected patients.

In addition to the above relationships, there may be direct associations between coagulation and osteoporosis. Long-term treatment with heparin has been associated with osteoporosis ²⁵. This correlation was initially recognized in pregnant patients requiring long-term heparin therapy ²⁶. A proposed mechanism involves interactions between heparin and the OPG/RANKL pathway ²⁷. Vitamin K is also recognized as playing a role in multiple aspects of bone metabolism. Shearer and Newman have recently published a thorough review of the metabolism and cell biology of vitamin K, including its role in bone metabolism ²⁸. In both human and animal studies, vitamin K supplementation has been shown to positively impact BMD (28). One molecular form of vitamin K, menaquinone-4 (MK-4) has activity on osteoclast and osteoblast function, suggesting multiple possible roles in bone metabolism ²⁸. For example, in vitro studies of cultured osteoblasts have demonstrated that MK-4 inhibits the production of the bone-resorption inducing agent prostaglandin E2 ²⁹. Additionally, MK-4 has been shown to increase 1,25-dihydroxyvitamin D₃-induced mineralization in vitro ³⁰.

Recently, a direct association between factor VIII-von Willebrand factor complexes and RANKL has also been observed ³¹. Factor VIII-von Willebrand factor complexes can bind directly to OPG and RANKL thereby inhibiting RANKL-induced osteoclastogenesis ³¹. Thus, patients deficient in factor VIII may perturb OPG and RANKL function resulting in increased osteoclast activity and bone loss. Hemophilic arthritis is often associated with an inflammatory cellular infiltrate. This infiltrate may release cytokines that activate RANKL and increase bone resorption, suggesting another etiology for the strong association between joint arthropathy and bone loss in patients with hemophilia ³².

SCREENING

When screening for osteopenia or osteoporosis, it is important to remember that bone density is a surrogate for predicting fracture risk, and the purpose of screening is to identify those patients at highest risk for fracture. Several guidelines have been published regarding BMD screening recommendations, although many, such as those by the United States Preventive Services Task Force, are limited to women ³³. In men, there are limited data correlating BMD with fracture risk ³⁴. Some published guidelines, however, do include recommendations that apply to patients with hemophilia and suspected low BMD. The American Association of Clinical Endocrinologists recommends further evaluation and possible intervention in any adult who sustains a low-trauma fracture ³⁵. The National Osteoporosis Foundation recommendations for BMD screening with DXA include men over 70, men aged 50–69 with concerning risk factor profiles, adults who have a fracture after age 50, and adults with a condition or taking medication associated with low bone mass or bone loss ³⁶. The foundation includes hemophilia in their list of conditions that cause or contribute to osteoporosis and fracture ³⁶.

While the above guidelines are helpful, there are currently no guidelines for osteoporosis screening in adult patients with hemophilia. Case finding should be done to identify and consider treatment for those patients with hemophilia and high risk for osteoporosis and future fractures. From our own data and the available literature, we would recommend DXA screening be performed in all patients with hemophilia and a documented fragility fracture. In addition, screening should be strongly considered in those patients with significant joint arthropathy. Patients over 50 years of age with additional risk factors including HIV or HCV infection and/or low BMI should also be considered for screening. Screening for osteoporosis should be avoided in patients felt to be at low risk for fracture based on age and comorbidities or in patients unreceptive to possible therapies or interventions.

TREATMENT

Given the paramount role of hemophilic arthropathy in the development of osteoporosis, the initiation of early factor replacement treatment (primary prophylaxis therapy) in children will likely have a significant preventative effect on the development of osteoporosis as adults ^{16, 17}. Recombinant and virally inactivated factor concentrates and the enhanced screening of blood products have virtually eliminated the risk of HIV and HCV transmission to patients that may also impact the development of osteopenia/osteoporosis. However, there are many adult patients with hemophilia who currently meet DXA criteria for osteoporosis. The optimal treatment of these patients, especially those under age 50, remains controversial. Studies to date looking at the association of hemophilia and osteoporosis have focused on defining prevalence and risk factors. There have been no studies testing specific treatment interventions in this population.

In our cohort, several patients have been followed after therapy with either bisphosphonates or calcium and vitamin D supplementation. Our anecdotal results suggest that bisphosphonate therapy can be effective at improving T-scores. Calcium and vitamin D supplementation alone was ineffective in our patients, but poor compliance may account for these results. Figure 1 shows preliminary data from two patients treated with weekly alendronate over the course of 6 years. Patient A began treatment at age 30 due to severe osteoporosis on DXA, severe hemarthroses (wheelchair bound), and HCV and HIV infection. His baseline T-scores at the lumbar spine increased by over 28% after 6 years of oral bisphosphonate therapy (T-score increased from −4.8 to −3.3). Patient B began treatment at age 44 due to severe hemarthroses and HCV. His baseline T-score at L1-L4 was −1.6 consistent with osteopenia, though T-scores at the femoral neck and hip were −2.8 and −2.7, respectively consistent with osteoporosis. He also showed improvement in BMD over the course of treatment with a 6-year follow-up DXA demonstrating resolution of osteopenia at the L-spine by DXA criteria (repeat T-score 0.8) and improvement at both the femoral neck (repeat T-score −2.3) and hip (repeat T-score −2.2) as well.

Adequate amounts of vitamin D and calcium intake, weight bearing physical activity, and treating diseased joints to maintain mobility are reasonable and safe recommendations that should improve BMD and the quality of life in all patients with hemophilia, regardless of age and baseline BMD ¹⁰. In those patients who have known low bone mass, active loss of bone mass, or skeletal fragility, Kovacs advises calcium and vitamin D supplementation in addition to the above interventions ¹⁰. Additional comorbidities that might contribute to reduced BMD such as hyperthyroidism, hyperparathyroidism, and hypogonadism should be ruled out when clinically suspected and treated if confirmed on laboratory testing.

The role of antiresorptive medications including bisphosphonates remains untested. Such medications will not increase bone mass, and their perceived benefits should be weighed against potential harm ^{6, 10}. Known severe complications associated with bisphosphonate use include osteonecrosis of the jaw, renal insufficiency, and gastrointestinal side effects including esophagitis ³⁷. While bisphosphonates are commonly used in older women, their long-term side effects are still being defined. Bisphosphonates are retained in the bone for decades, and the long-term implications of this, especially in younger patients, are unknown. Their use is not recommended for low peak bone mass alone in patients between 20 and 50 but reserved for those with a history of fracture or significant risk factors for fracture and confirmed bone loss by DXA ¹⁰. Given similar concerns regarding long-term complications, newer agents including teriparatide and strontium ranelate should also be reserved for patients with severe osteoporosis.

Because of the emerging role of RANKL in the pathogenesis of hemophilia-associated osteoporosis, the use of RANKL inhibitors such as denosumab is an intriguing potential treatment option for adults with osteoporosis. RANKL inhibitors act by reversibly inhibiting osteoclast-mediated bone resorption. Though head-to-head studies are lacking, recent data suggest that such agents are as effective as bisphosphonates in reducing fragility fractures and may have a more favorable side effect profile ³⁸. As with bisphosphonates, data regarding long-term toxicities of denosumab are lacking. However, such agents do not remain in the bone long term, and their shorter half lives make them more attractive for use in younger patients.

CONCLUSIONS

It is now well documented that low BMD is more common in both children and adults with hemophilia. In adults, severity of hemophilic joint arthropathy appears to be the strongest predictor of osteopenia and osteoporosis. The relative contribution of other risk factors, including HIV and HCV, is less well defined, but likely plays a contributory role. The clinical significance of decreased BMD by DXA to fracture risk, especially in younger patients, remains poorly defined. In addition, there are no published guidelines regarding optimal screening and treatment of low BMD in patients with hemophilia. We recommend screening be considered only in those patients with documented fragility fractures, in those with severe arthropathy, and in those patients >50 years of age with additional risk factors such as HIV or HCV infection or low BMI. Adequate vitamin D and calcium intake, weight bearing physical activity and interventions to maintain joint mobility are recommended for all hemophilia patients. The role of antiresorptive therapies, including bisphosphonates, remains poorly defined and should be reserved for patients with severe osteoporosis, with additional risks for fracture, or with documented fragility fractures.

Keywords

hemophilia, joint arthropathy, osteoporosis, osteopenia, bone mineral density, fragility fracture

Acknowledgement: We would like to thank our patients and to recognize the dedication and hard work of our co-workers at the Arizona Hemophilia and Thrombosis Center.

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18. Ross C, Golgenberg N, Hund D. Athletic participation in severe hemophilia: bleeding and joint outcomes in children on prophylaxis. Pediatrics. 2009;124:1267–1272.
19. Bonkovsky HL, Hawkins M, Steinberg K, et al. Prevalence and prediction of osteopenia in chronic liver disease. Hepatology. 1990;12:273–280.
20. Collier J. Bone disorders in chronic liver disease. Hepatology. 2007;46: 1271–1278.
21. Schiefke I, Fach A, Wiedmann M, et al. Reduced bone mineral density and altered bone turnover markers in patients with non-cirrhotic chronic hepatitis B or C infection. World J Gastroenterol. 2005;11:1843–1847.
22. Gallego-Rojo FJ, Gonzalez-Calvin JL, Munoz-Torres M, et al. Bone mineral density, serum insulin-like growth factor I, and bone turnover markers in viral cirrhosis. Hepatology. 1998;28:695–699.
23. Duvivier C, Kolta S, Assoumou L, et al. Greater decrease in bone mineral density with protease inhibitor regimens compared with nonnucleoside reverse transcriptase inhibitor regimens in HIV-1 infected naý¨ve patients. AIDS. 2009;23:817–824.
24. Glesby MJ. Bone disorders in human immunodeficiency virus infection. CID. 2003;37(suppl 2):S91–S95.
25. Griffith GC, Nichols G, Asher JD, et al. Heparin Osteoporosis. J Med Assoc. 1965;193:91–94.
26. Dahlman TC. Osteoporotic fractures and the recurrence of thromboembolism during pregnancy and the puerperium in 184 women undergoing thromboprophylaxis with heparin. Am J Obstet Gynecol. 1993;168:1265– 1270.
27. Vik A, Brodin E, Sveinbjornsson B, Hansen JB. Heparin induces mobilization of osteoprotegerin into the circulation. Thromb Haemost. 2007;98:148–154.
28. Shearer MJ, Newman P. Metabolism and cell biology of vitamin K. Thromb Haemost. 2008;100:530–547.
29. Koshihara Y, Hoshi K, Shiraki M. Vitamin K2 (menatetrenone) inhibits prostaglandin synthesis in cultures human osteoblast-like periosteal cells by inhibiting prostaglandin H synthase activity. Biochem Pharmacol. 1993;46:1355–1362.
30. Koshihara Y, Hoshi K, Ishibashi H, et al. Vitamin K2 promotes 1alpha25(OH)2 vitamin D3-induced mineralization in human periosteal osteoblasts. Calcified Tissue International. 1996;59:466–473.
31. Baud’huin M, Duplomb L, Teletchea S, et al. Factor VIII-von Willebrand factor complex inhibits osteoclastogenesis and controls cell survival. J Biol Chem. 2009;284:31704–1713.
32. Lafeber FP, Miossec P, Valentino LA. Physiopathology of haemophilic arthropathy. Haemophilia. 2008;14(suppl 4):3–9.
33. Screening for Osteoporosis in Postmenopausal Women, Topic Page. September 2002. U.S. Preventive Services Task Force. Agency for Healthcare Research and Quality, Rockville, MD.
34. Raisz LG. Clinical practice. Screening for osteoporosis. N Engl J Med. 2005;353:164–171.
35. Hodgson SF, Watts NB, Bilezikian JP, et al. American Association of Clinical Endocrinologists medical guidelines for clinical practice for the prevention and treatment of postmenopausal osteoporosis: 2001 edition, with selected updates for 2003. Endocr Pract. 2003;9:544–564.
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37. Yarom N, Yahalom R, Shoshani Y, et al. Osteonecrosis of the jaw induced by orally administered bisphosphonates: incidence, clinical features, predisposing factors and treatment outcomes. Osteoporos Int. 2007;18: 1363–1370.
38. Cummings SR, San Martin J, McClung MR, et al. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med. 2009;361:756–765.

Treatment of hemophilia A entails replacing missing or low levels of clotting factor VIII (FVIII). The evolution of FVIII replacement therapy began more than 60 years ago with infusion of fresh or frozen plasma to replace FVIII,¹ but only low FVIII concentrations could be achieved with this method because of the risk of hypervolemia.¹ Higher FVIII concentrations were subsequently achieved in the 1960s through use of cryoprecipitation and glycine precipitation techniques.¹ However, the source of FVIII remained as fresh human plasma, placing recipients of FVIII replacement therapy at risk for blood-borne diseases.² In the late 1970s and early 1980s, transmission of hepatitis B and C virus and HIV reached epidemic levels among patients with hemophilia treated with plasma-derived FVIII (pdFVIII) products, which, at that time, did not undergo a viral inactivation step during manufacture.³

To decrease the risk of pathogen transmission associated with blood-derived products, recombinant DNA technology, in which mammalian cells lines are genetically altered to express FVIII,⁴ began to be used to manufacture FVIII. First-generation recombinant FVIII (rFVIII) products, which required use of plasma-derived proteins only in the culture medium and final formulation, were introduced in the 1990s. Subsequent refinements allowed further reduction of the use of human plasma; with second-generation rFVIII products, plasma protein is used only in the culture medium. In the early 2000s, the first third-generation rFVIII product was marketed; with these products, no human or animal blood-based proteins are used in the cell culture or formulation stages, but a murine monoclonal-antibody-based purification step is still used in one third-generation product (Advate).^4–8

Pathogen transmission has been minimized through careful screening of blood donations,⁹ virus inactivation methods, and use of rFVIII products, but safety issues remain. Although the risk appears to be low, transmission of the abnormal prion proteins associated with development of variant Creutzfeldt-Jakob disease¹⁰ currently outweighs concerns over transmission of hepatitis and HIV, and transmission of disease-causing pathogens for which screening techniques do not yet exist remains a possibility with any product manufactured using human plasma.⁴ Use of plasma- and albumin-free culture medium in the manufacture of third-generation rFVIII products has further decreased the risk of transmitting known blood-borne viral pathogens.⁷

At present, the principal safety concern in treating hemophilia A is the potential development of FVIII antibodies or inhibitors.^{5, 11, 12} These inhibitors neutralize the effects of FVIII, rendering replacement therapy ineffective in maintaining hemostasis.^{5, 13} Based on results from a systematic review, prevalence of FVIII inhibitor development is about 5% to 7% in the overall hemophilic patient population but about 12% to 13% in patients with severe hemophilia.¹⁴ Why inhibitors develop in some patients but not others is not completely clear.¹⁵ The risk of inhibitor development is highest soon after a patient's first exposure to FVIII replacement therapy and tends to decrease after 20 exposure days (EDs) or when patients reach ages 6–10 years.¹⁴ Among previously untreated patients (PUPs) receiving rFVIII products, the incidence of inhibitor development in pivotal trials ranged from 15 to 32%,^16–21 compared with an incidence of approximately 1%–3% for previously treated patients (PTPs).^19–27 Inhibitors developing soon after initial exposure are likely a primary immune system response to the foreign antigen, while inhibitors that develop after years of FVIII exposure probably reflect a breakdown of immune system tolerance.²⁸

Once inhibitors develop, the efficacy of rFVIII products in preventing and controlling bleeds is compromised, especially in patients with high-titer inhibitors.¹³ Although bleeding episodes in patients with inhibitors can be managed with bypassing agents, these products are generally not as effective as FVIII in the long term.¹³ Compared with those without inhibitors, hemophilic patients with inhibitors are more vulnerable to development of substantial clinical morbidity including increased risk for infection and orthopedic disability.¹³ For patients with persistent inhibitors, immune tolerance therapy is currently the only means of eradicating inhibitors and restoring the efficacy of FVIII replacement products.¹³

Studies in PUPs or minimally treated patients (MTPs) treated with pdFVIII products have reported inhibitor incidence rates ranging from 3% to 33%.^29–34 Some studies have indicated that inhibitor development in PUPs, particularly children and patients with severe hemophilia A, may be more likely with rFVIII products than with pdFVIII products, although between-trial comparisons are difficult given the diversity of study design, inhibitor testing frequency, and patient populations in the various studies.^{14, 35, 36} Other work indicates that the risk of inhibitor development in PUPs is no greater with rFVIII products than with pdFVIII products.^36–38 Risk of de novo inhibitor development in PTPs also appears to be unaffected by the type of FVIII product used.¹¹ Whether the risk of inhibitor development is greater with rFVIII products than with pdFVIII products cannot yet be definitively determined because of the current lack of uniform and systematic collection of inhibitor data in observational studies and the need for randomized, controlled trials evaluating inhibitor development with rFVIII and pdFVIII products.

Given that the risk of pathogen transmission has been mitigated through use of recombinant methods of producing exogenous FVIII, this review focuses on inhibitor development, which currently is the principal safety issue associated with rFVIII use. Because the evolution of the safety of FVIII products is tied to changes in manufacturing techniques, understanding how such changes might affect inhibitor development and other safety issues is important.

MANUFACTURING PROCESSES FOR RECOMBINANT FACTOR VIII (FVIII) PRODUCTS

Several rFVIII products are currently marketed including the second-generation products Kogenate FS (KOGENATE Bayer in Europe; also sold as Helixate FS/Helixate NexGen by CSL Behring; sucrose-formulated rFVIII [rFVIII-FS]) and ReFacto (B-domain-deleted rFVIII [BDD-rFVIII]) and the third-generation products Advate (recombinant antihemophilic factor, plasma/albumin-free method [rAHF-PFM]) and ReFacto AF/Xyntha (albumin-free BDD-rFVIII [BDD-rFVIII AF]). The first-generation product Recombinate (rFVIII) remains on the market, but first-generation Kogenate (Bayer's rFVIII) is no longer manufactured.³⁵ Each product is unique in regard to the combination of cell line, culture medium, viral inactivation methods, and product stabilizer used in its manufacture; product half-lives and recovery percentages are somewhat similar ( Table 1 ).^{7, 39–42}

Recombinant FVIII (rFVIII)-sucrose formulated (FS)

Second-generation rFVIII-FS is the successor to Bayer's first-generation rFVIII product. The rFVIII-FS is produced by inserting the full-length DNA sequence of human FVIII into baby hamster kidney (BHK) cells engineered to express FVIII. To begin rFVIII-FS production, frozen BHK cells from the master cell bank are thawed, cultured, and expanded into fermentation vats using a defined growth medium. The cell culture medium contains human plasma protein solution and recombinant insulin but no proteins derived from animal sources.⁴¹ The culture media are harvested and undergo filtration, centrifugation, or both. Viable cells are returned to the fermentation vats along with new growth medium.⁴³ Continuous perfusion of the BHK cells affords efficient rFVIII production because of optimized high cell growth concentrations and relatively short protein residence time.⁴⁴

A stepwise process is employed for purification. Using anion exchange, monoclonal antibody immunoaffinity chromatography, and size exclusion chromatography, impurities such as BHK proteins, BHK DNA, and murine immunoglobulin are removed. The purification process also includes a solvent/detergent step for viral inactivation.⁴¹ Once purified, the product is formulated with stabilizers (sucrose, glycine, and histidine but not human serum albumin [HSA])^{41, 45} and lyophilized in dose vials. The manufacturing process takes approximately 2 weeks, with cell expansion, scale up, fermentation, and filling/freeze-drying occurring under sterile conditions to prevent microbial contamination.⁴³

The manufacture of rFVIII-FS differs from that of Bayer's rFVIII product in that HSA is not used in the purification and formulation steps, and a solvent/detergent treatment replaced heat processing as the means of viral inactivation. The purification process also was simplified by reducing the number of chromatographic steps, which decreased process time, limited the opportunity for proteolytic degradation, and increased overall yields per milliliter of culture medium.⁴³

B-domain-deleted (BDD)-recombinant FVIII (rFVIII)

The BDD-rFVIII is a second-generation product still on the market but being phased out in favor of BDD-rFVIII AF. Deletion of the B-domain⁴⁰ facilitates secretion of FVIII by Chinese hamster ovary (CHO) cells; B-domain deletion also allows for a more stable product because of decreased susceptibility to proteolysis.³⁵ The cell culture medium used for BDD-rFVIII contains HSA and recombinant insulin but no proteins derived from animal sources. The FVIII protein is purified using chromatography processes and a solvent/detergent step is used for viral inactivation. Sucrose is used as a product stabilizer and the final formulation contains no preservatives or added human or animal components.⁴⁰

Recombinant antihemophilic factor (rAHF)-plasma/albumin-free method (PFM)

Third-generation rAHF-PFM, the successor to first-generation rFVIII, is a full-length FVIII product manufactured using a CHO cell line adapted to grow in a protein-free cell culture medium containing no human- or animal-derived additives. Production consistency and stability are afforded through use of a continuous (chemostat) perfusion bioreactor culture to harvest FVIII with minimal environmental changes to the cell line. The methods used to purify rAHF-PFM are based on those that were used for its first-generation predecessor (rFVIII). The cell culture medium is filtered to remove CHO cells and the remaining FVIII-containing medium undergoes a sterile filtration step. The purification process involves immunoaffinity chromatography to remove non-FVIII proteins (with monoclonal antibodies expressed in plasma- and albumin-free conditions), and ion exchange (to eliminate other impurities using electrical charge differences). A solvent/detergent treatment is used for viral inactivation, followed by anion exchange chromatography for further purification. The final formulation contains no sucrose or human/animal-derived additives; mannitol, trehalose, and buffered salts are used as stabilizers instead of albumin.^{4, 10, 42}

B-domain-deleted (BDD)-recombinant antihemophilic factor (rFVIII) albumin-free (AF)

BDD-rFVIII AF, a third-generation rFVIII product,³⁹ is a successor to the second-generation product BDD-rFVIII.⁶ BDD-rFVIII AF is manufactured using CHO cells grown in a cell culture medium that contains recombinant insulin but no materials derived from animal or human sources.³⁹ Purification of BDD-rFVIII AF differs from that of BDD-rFVIII in that the murine monoclonal antibody used in BDD-rFVIII affinity chromatography is replaced by a synthetic peptide affinity ligand, and an additional 35 nm viral filtration step is added during purification.⁶ A solvent/detergent process is used for viral inactivation and sucrose is included as a stabilizer.³⁹

INHIBITOR DEVELOPMENT

The manufacturing processes used for second- and third-generation FVIII products have minimized the risk of pathogen transmission, making inhibitor formation the principal safety concern. The incidence of inhibitor development is typically reported in clinical trials and postmarketing surveillance studies, but conclusions regarding the risk of inhibitor formation associated with individual rFVIII products are difficult to make given variations in patient population, study design, and frequency of inhibitor testing.^{5, 28} Data from head-to-head trials, in which FVIII products are compared in the same study using a randomized patient population, are needed to fully understand whether the risk of inhibitor development differs among different products.⁵ At present, data from pivotal trials (see Table 2 for a summary of inhibitor incidence in pivotal trials of second- and third-generation rFVIII products) may provide the most accurate picture of the true incidence of inhibitor formation given that postmarketing data may not be collected according to strict criteria or uniform guidelines.⁵ Also, trials in PTPs who are tolerant of FVIII products and have not developed inhibitors, as well as trials in PUPs, are necessary to determine the risk of inhibitor development associated with new rFVIII products.^{10, 45, 46}

Recombinant antihemophilic factor (rFVIII)-sucrose formulated (FS)

Previously untreated patients (PUPs)/minimally treated patients (MTPs)

In an open-label, multicenter pivotal trial enrolling 61 patients,¹⁸ 37 PUPs and 24 MTPs (≤4 prior EDs) with severe hemophilia A were treated with rFVIII-FS for a median of 114 EDs (range, 4 to 478). Inhibitors developed in 9 of 60 patients (15%); 5 of these patients developed low-titer inhibitors (peak, ≤23 Bethesda units [BU]), and 4 developed high-titer inhibitors (peak, ≥110BU). By comparison, in the pivotal trial in PUPs using Bayer's first-generation rFVIII product, inhibitors developed in 21 of 102 evaluable patients (21%) and in 19 of 65 patients (29%) with severe hemophilia tested for them.¹⁷

Inhibitors developed in 15 of 43 evaluated patients (35%) in a Japanese surveillance study of 47 PUPs who received Bayer's rFVIII product.⁴⁶ High-titer inhibitors developed in only 5 of 43 patients (12%), an incidence similar to that observed in the PUP pivotal trial of Bayer's rFVIII product (12 of 102 patients [12%]).¹⁷

Previously treated patients (PTPs)

The first rFVIII-FS clinical trial was an open-label pivotal study conducted in Europe and North America enrolling 71 PTPs with severe hemophilia A.²⁶ Mean±SD Eds were 216±179 and 125±68 for patients in Europe and North America, respectively. No evidence of de novo inhibitor development was observed over 24 months of follow-up. Inhibitors developed in one patient (an incidence of 1%) who had low-titer inhibitors at baseline (0.39 BU); the observed peak titer (1.6BU) in this patient was low.⁴⁵ The lack of de novo inhibitor formation in this rFVIII-FS PTP trial matches what was observed in the pivotal PTP trial using Bayer's rFVIII product.²⁵

In a pivotal multicenter Japanese study of 20 PTPs (mean age, 26.8 years) with moderate or severe hemophilia A,⁴⁷ no patient developed inhibitors after ≥24 weeks of treatment.

In a study of 65 boys (mean age, 1.6 years) randomly assigned to prophylaxis or enhanced episodic (on demand) therapy with Bayer's rFVIII product or rFVIII-FS,⁴⁸ high-titer inhibitors developed in 2 of 32 patients (6%) receiving prophylaxis and none of the 33 patients treated with enhanced episodic therapy.

Among 123 PTPs in a Japanese surveillance study,⁴⁹ inhibitors developed in 7 of 103 patients (7%) who tested negative for inhibitors before rFVIII administration, but clinically meaningful titers developed in only 3 of these patients (3%).

Patients undergoing surgery

rFVIII-FS was evaluated in a study of 15 PTPs and 7 PUPS or MTPs undergoing surgery.⁵⁰ All patients received bolus administration of rFVIII-FS. No inhibitors were reported after the surgical procedure; transient inhibitor development was noted in 1 MTP 10 months before surgery and in 1 PUP 12 days before surgery.⁵⁰

B-domain-deleted (BDD)-recombinant antihemophilic factor (rFVIII)

Previously untreated patients (PUPs)/minimally treated patients (MTPs)

In an open-label, multinational pivotal trial of 101 PUPs (mean age, 10 months) with severe hemophilia A,¹⁹ inhibitors developed in 32 patients (32%) after a median of 12 EDs (mean, 32 EDs); high titers (≥5 BU) were reported in 50% of cases.

Previously treated patients (PTPs)

In a multinational PTP study,²⁰ inhibitors developed in 1 of 113 patients (1%). This patient had a low-titer inhibitor (0.6 BU) at baseline but developed a high-titer inhibitor (~13 BU) after 14 months of treatment; he was withdrawn from the study 19 months after the inhibitor was detected.

Recombinant antihemophilic factor (rAHF)-plasma/albumin-free method (PFM)

Previously untreated patients (PUPs)/minimally treated patients (MTPs)

No finalized inhibitor data from pivotal trials of rAHF-PFM in PUPs or MTPs have been published to date, although an ongoing PUP study indicated that inhibitors developed in 5 of 25 patients (20%) after a median of 11 EDs, including high-titer inhibitors in 4 patients.⁴²

Previously treated patients (PTPs)

The pivotal trial for rAHF-PFM was conducted in PTPs. A total of 111 patients with severe hemophilia A were enrolled, of whom 108 were treated. No high-titer inhibitors developed in any patient after a median of 117 EDs. One patient (1%) developed a transient low-titer inhibitor (2.0 BU) after 26 EDs.²⁷ In pooling data from five rAHF-PFM clinical trials and registries of 208 PTPs, of whom 198 had ≥10 EDs or 6 months of observation, transient inhibitors developed in one patient, for an overall incidence of <1% (95% confidence interval, 0.03 to 2.9%).¹⁰

In a postmarketing surveillance study of rAHF-PFM in hemophilic patients with varying degrees of disease severity, low-titer inhibitors developed in 3 of 402 patients (0.8%) with >50 EDs and moderately severe disease (FVIII levels ≤2%) and in 3 of 332 patients (1%) with >50 EDs and severe disease (FVIII levels <1%).⁵¹ The de novo inhibitor incidence in this patient population was 0.3% (1 of 348 patients).

Patients undergoing surgery

In a study assessing use of rAHF-PFM, either as bolus administration or continuous infusion, in patients undergoing surgical, dental, or other invasive procedures, no patient developed an FVIII inhibitor after surgery.⁵²

B-domain-deleted (BDD)-recombinant antihemophilic factor (rFVIII) albumin-free (AF)

Previously untreated patients (PUPs)/minimally treated patients (MTPs)

No inhibitor data from pivotal trials of BDD-rFVIII AF in PUPs/MTPs have been reported as yet.

Previously treated patients (PTPs)

Two pivotal trials for BDD-rFVIII AF were conducted in PTPs. The first trial enrolled 94 patients, who remained in the study for a median of 34.4 weeks (range, 21.3 to 43 weeks).⁶ In the per-protocol population (restricted to patients with ≥50 EDs), inhibitors developed in 2 of 89 patients (2%); both patients developed transient, low-titer inhibitors. The second trial enrolled 110 patients, who participated in the study for a median of 22.4 weeks (range, 4.1 to 78 weeks).⁶ In this study, inhibitor development was reported in three patients (3%), but only one of these patients (<1%) developed de novo inhibitors.⁶

Patients undergoing surgery

Use of BDD-rFVIII AF was assessed in 30 patients undergoing surgery, 22 of whom received bolus infusions and 8 of whom received continuous infusions; a single case of low-titer inhibitor development (incidence, 3%) was reported in a patient given a bolus infusion.⁵³

CONCLUSIONS

With the introduction of second- and third-generation rFVIII products, the safety of FVIII replacement therapy for hemophilia A has increased considerably. The risk of pathogen transmission is minimal, given the steps taken to screen blood donations, inactivate viruses, purify products that use plasma-derived proteins, and improve delivery systems. Inhibitor development remains a safety concern with all rFVIII products, particularly in PUPS. However, the risk of developing inhibitors decreases substantially as patients accumulate a higher number of EDs to rFVIII products.

Keywords

factor VIII, hemophilia, inhibitor, recombinant, pathogen safety

Disclosure: The author declares no conflict of interest.

Acknowledgements: I would like to thank Karen Zimmermann and Chris Kirk, PhD, from Complete Healthcare Communications, Inc., for medical writing assistance. Medical writing assistance was fully funded by Bayer HealthCare Pharmaceuticals.

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Hemostatic challenges occur throughout the period of bone marrow transplantation (BMT). Endothelial cell injury is a dominant contributing factor to the hemostatic impairments via chemotherapy ¹, growth factors ¹, intravenous catheters, and graft versus host reaction ². Thrombocytopenia is also a major contributor to the bleeding manifestations. A number of changes in procoagulant, anticoagulant and, to a lesser extent, fibrinolytic factors have been described. These include reduction in levels of factor VII, factor X, protein C, antithrombin III, and plasminogen ^3–7. Conversely, levels of fibrinogen, von Willebrand factor (VWF), plasminogen activator inhibitor (PAI-1), and tissue plasminogen activator (t-PA) are increased ^{3–5, 8, 9}. Collectively, major changes in the coagulation system predispose BMT patients to hemostatic complications. In a retrospective analysis of 364 allogeneic and 83 autologous transplants, 83% presented with at least one hemostatic complication from the start of conditioning therapy and 4–10 years follow-up ¹⁰. Most bleeding episodes occurred within the first 4 weeks after transplantation and were relatively mild. However, 27% of the patients hemorrhaged severely, doubling the overall mortality of the BMT recipients. Bleeding was strongly associated with prolonged thrombocytopenia and graft versus host disease (GVHD) ¹⁰. Thromboembolic events, hepatic veno-occlusive disease (VOD), and thrombotic microangiopathy (TMA) occurred most frequently in allogeneic transplant recipients, for whom the incidence was 15%, 4.7%, and 14.6%, respectively, leading to an increased overall mortality ¹⁰.

THROMBOTIC COMPLICATIONS

Hepatic Veno-Occlusive Disease (VOD)

The VOD is one of the most common and important regimen-related toxicities experienced after allogeneic and autologous BMT ^11–14. VOD is a clinical syndrome characterized by painful hepatomegaly, jaundice, ascites, fluid retention, and weight gain ^11–14. The onset is usually before day +35 after stem cell reinfusion ^11–14. VOD develops in 1%–22% of patients after BMT and ranges in severity from mild, reversible disease to a severe syndrome associated with multiorgan failure and death ¹². The incidence and severity of VOD can be influenced by differences in pretransplantation patient characteristics, transplantation conditioning regimens, and source of graft with allogeneic transplantation conferring a threefold increase in risk compared to autologous transplantation ^{15, 16}.

Pathogenesis

The VOD is believed to be caused by primary injury to sinusoidal endothelial cells and hepatocytes with subsequent damage to the central veins in zone 3 of the hepatic acinus ^{17, 18}. Early changes include deposition of fibrin within veins and sinusoids. Subendothelial edema, collagen deposition, sclerosis, and fibrosis of the venous zone are followed by fibroblast cells proliferation and collagen accumulation in the extracellular matrix ^18–20. As the process of veins microthrombosis, ischemia, and fibrosis advances, widespread zonal disruption leads to portal hypertension, hepato-renal syndrome, multiorgan failure, and death ¹⁷. Injury to sinusoidal endothelial cells and hepatocytes by high-dose alkylating agents appears to be the primary event in the pathogenesis of VOD ^21–23. Detoxification of these chemotherapy agents in the liver is achieved primarily via cytochrome P450 and glutathione-S-transferase enzymes, which are present at high concentrations within zone 3 of the liver acinus ²⁴. Depletion of glutathione stores has been shown to predispose hepatocytes to necrosis, whereas the addition of glutathione mono-8-diester can selectively protect hepatocytes from high-dose alkylator injury ^{25, 26}. Several markers of endothelial injury and adhesion molecules are upregulated in patients with VOD. These include plasma thrombomodulin (TM), P- and E-selectins, tissue factor pathway inhibitor (TFPI), soluble tissue factor, and PAI-1 ^{9, 27}. In vitro studies show that endothelial PAI-1 production is triggered by cytokine transforming growth factor beta (TGF-β) released from activated platelets ²⁸. Pretransplantation elevated plasma TGF-β levels in patients have been associated with the development of hepatic VOD ²⁹. Additionally, proinflammatory cytokines may also contribute to the initial endothelial injury in VOD. Increased concentrations of tumor necrosis factor (TNF-α), interleukin (IL)-6, IL-8, and IL-1β have been associated with jaundice, renal dysfunction, and pulmonary disease in BMT ^{4, 30}.

The role of thrombophilic factors in the pathogenesis of the thrombotic complications in BMT was not investigated intensely. In one small retrospective study the prothrombin gene 20210 G-A mutation was found to be a predisposing factor for VOD ³¹. An additional prospective study in children revealed a strong association between factor V Leiden mutation and VOD ³². More studies are needed to confirm the association between thrombophilia and VOD.

Therapy

The most established practice in VOD prevention has been the use of pharmacokinetics to monitor chemotherapeutic drug levels to minimize hepatic injury. The prophylactic administration of ursodeoxycholic acid, a hydrophilic water-soluble bile acid, has been studied in randomized placebo controlled prospective trials, some showing a statistically significant benefit in patients predicted to be at a high risk of VOD ^33–35. However, a large phase III study did not demonstrate significant benefit ³⁶. Anticoagulation, with low dose heparin or low molecular weight heparin (LMWH), alone or in combination with other agents has been also studied as VOD prophylaxis. Only one randomized study demonstrated a beneficial effect of low-dose continuous heparin prophylaxis ³⁷. The LMWH seem to be relatively safe and may have some effect in the prevention of VOD ^38–41, however, well-designed, multicenter, randomized studies are needed to confirm these results.

Despite recent advances to date, there is no FDA-approved therapy for hepatic VOD. The therapeutic approaches for established VOD are based on supportive care and on the use of drugs with anticoagulant or fibrinolytic activity; that is, LMWH or recombinant tissue plasminogen activator (rt-PA) ^{37, 38, 42–44}. Their use is suggested by the fact that VOD is associated with the obliteration of hepatic venules or sinusoids by deposition of fibrin ^{12, 18, 20}. A major drawback in the use of rt-PA and heparin is the high risk of life-threatening bleeding due to thrombocytopenia and lack of increment with platelet transfusions due to portal hypertension and splenic sequestration as well as antiplatelets autoantibodies ^{43, 45}.

The use of defibrotide, a single-stranded polydeoxyribonucleotide that has specific binding sites on vascular endothelium, has shown promise in the treatment of VOD ^46–48. Defibrotide upregulates the endothelial release of prostacyclin (PG I₂), prostaglandin E₂, thrombomodulin (TM), and t-PA both in vivo and in vitro ^49–51. Moreover, it has been shown to decrease thrombin generation, tissue factor expression, PAI-1 release, and endothelin activity ^{49, 52}. Preclinical studies have also demonstrated profibrinolytic effects and inhibition of fibrin deposition with selective activity on small vessels ⁵³. No significant effect on systemic coagulation has been shown in either preclinical studies or clinical trials of defibrotide ⁵⁴. In addition, it was shown that defibrotide may have antiangiogenic potential in endothelial cells and in an animal model ⁵⁵. Initial clinical reports of defibrotide use as a treatment for severe VOD have recorded complete resolution in 36% to 42% of patients with most surviving past day 100+ ^{46, 47, 56}.

In a recent randomized phase II dose-finding trial to determine the efficacy of defibrotide in patients with severe VOD following BMT, adult and pediatric patients received either lower dose (arm A: 25 mg/kg/day; n=75) or higher dose (arm B: 40 mg/kg/day; n=74) i.v. defibrotide administered in divided doses every 6 hours for more than or equal to 14 days or until complete response, VOD progression, or any unacceptable toxicity occurred. In the absence of any differences in activity, toxicity, or changes in PAI-1 level, defibrotide 25 mg/kg/day was selected for phase III trials ⁵⁷. In a prospective case series study of pediatric BMT using preemptive antithrombin III replacement and combined antithrombin III/defibrotide therapy, excellent remission and survival rates were yielded ⁵⁸. In another report of 58 adult during BMT, no patient developed VOD following the use of defibrotide prophylaxis without concurrent heparin ⁵⁹. Cappelli et al reported on 57 children affected by beta thalassemia at very high risk for developing VOD (liver fibrosis, iron overload, hepatitis C virus infections, and busulphan-based conditioning). All patients received oral defibrotide (40 mg/kg per day, final dose) as VOD prophylaxis from median day –9 to median day +29. Defibrotide was well tolerated. Only one patient developed VOD. This patient had discontinued defibrotide 6 days prior to VOD onset, due to a high risk of hemorrhage⁶⁰. Thus far, no phase III randomized studies were published on the use of defibrotide as a prophylaxis or treatment of VOD; nevertheless, these studies are currently ongoing. Table 1 summarizes the current available treatments in VOD.

Transplantation-related thrombotic microangiopathy (TMA)

Transplantation-related TMA is a devastating complication of allogeneic BMT. Diagnosis of TMA is principally based on the presence of thrombocytopenia and microangiopathic hemolytic anemia in the absence of an alternative clinically apparent etiology ^{61, 62}. Similar to idiopathic thrombotic thrombocytopenia purpura (TTP), transplantation-related TMA is also associated with development of renal dysfunction and neurologic complications ⁶². However, since both thrombocytopenia and fragmentation of red blood cells are extremely common after BMT and renal dysfunction and neurologic complications can occur secondary to a diverge range of etiologies, a definitive diagnosis of transplantation-related TMA is often somewhat uncertain ^{63, 64}. Transplantation-related TMA can be caused by extensive prior therapy, graft versus host disease (GVHD), reactivation of cytomegalovirus, and drugs (Cyclosporine-A, tacrolimus) ^65–67. The incidence of TMA after allogeneic and autologous transplantations is 0.5%–15% and 0.1%–0.25%, respectively ^{10, 65, 68}. Cyclosporine A is widely used as an immunosuppressive agent and is the most commonly reported cause of drug induced TMA ⁶⁹. The microangiopathic injury caused by this drug is not limited to bone marrow transplantation. In renal transplant recipients treated with Cyclosporine-A, TMA occurs in 3%–5% of the patients, but the prognosis in these cases is generally good ⁷⁰. Transplantation-related TMA is associated with shorter overall survival but due to the multifactorial causes, the exact prognosis is difficult to assess ⁷¹.

Pathogenesis

Posttransplantation TMA was found not to be associated with severe vWF-cleaving protease deficiency. The elevated level of vWF antigen—found in this condition—is probably consistent with diffuse endothelial injury likely to be caused by multiple interacting factors as illustrated in Figure 1, ^{65, 72}. Cyclosporine A is cytotoxic to cultured endothelial cells at concentrations similar to the peak plasma levels achieved in vivo ⁷³. During episodes of Cyclosporine-A induced microangiopathy, plasma levels of vWF and endothelin are elevated ⁷⁴, as well as levels of prostacyclin and thromboxane A2 released by endothelial cells ⁷³.

In patients with BMT-related TMA and treatment with tacrolimus, a rise in serum levels of endothelin and several cytokines (IL-10, IL-12, tumor necrosis factor-a, interferon-gamma) were found. These data are consistent with the mechanism of endothelial cell injury accompanied by cytokines that affect the expression of various adhesion molecules ⁷⁵. In addition, Cyclosporine-A was demonstrated to enhance platelet aggregation and platelet thromboxane A2 release. Platelet hyperaggregability in renal allograft patients on long-term Cyclosporine-A therapy tended to revert toward normal following the replacement of the drug with azathioprine. These observations suggest that Cyclosporine-A mediated platelet activation may contribute to the pathogenesis of the thromboembolic phenomena associated with the use of this drug ⁷⁶.

Therapy

Optimal management of transplantation-related TMA is currently not defined. The suspected offending drug should be discontinued. Because of its clinical similarity to idiopathic TTP, it has been traditionally managed by plasma exchange. However, this modality has limited efficacy, with relatively low response rates (<20%−50%) in comparison to idiopathic TTP (80%) ^{61, 64, 77}. Other modalities of treatment (ie, defibrotide, immunoglobulin G) have been tried with variable success ^{78, 79}.

HEMORRHAGIC COMPLICATIONS

Diffuse Alveolar Hemorrhage (DAH)

Respiratory insufficiency due to lung injury is a major cause of death in BMT patients. Both infectious and noninfectious findings are frequently found at autopsy of BMT recipients. In a recent retrospective cohort study the medical and autopsy records of 71 autologous or allogeneic BMT recipients with pulmonary complications were studied ⁸⁰. Infectious cause was found in 27 (37%). Among the noninfectious causes were diffuse alveolar damage, amyloidosis, leukemia/lymphoma, bronchitis obliterans, bronchiolitis obliterans, pulmonary alveolar proteinosis, acute organizing pneumonia, and aspiration pneumonia. Hemostatic problems were identified in 15 (21%) patients, including alveolar hemorrhage (10, 14%) and pulmonary embolism (5, 7%) ⁸⁰.

Diffuse alveolar hemorrhage is a major complication of BMT recipients with a reported mortality rate of approximately 80% ^81–83. The reported incidence of DAH varies from 1%–21% in autologous and from 2%–17% in allogeneic BMT recipients ^{81, 84–87}. Thus, the present data suggest that in DAH the type of transplantation does not play a major role. The DAH can present with symptoms mimicking infection or be associated with infection. In a recent study, symptoms of hypoxemia, pulmonary infiltrates, and progressive bloody alveolar lavage were documented in 116 patients ⁸⁸. Among these, 71 (61%) had documented associated infectious cause. The probability of a 60-day survival from onset of hemorrhage was 16% for the DAH and 32% for the infection associated alveolar hemorrhage group ⁸⁸. Allogeneic BMT using reduced intensity conditioning (RIC) has lower morbidity and mortality compared to transplantation using myeloablative conditioning (MAC). In a prospective cohort study alveolar hemorrhage occurred in 18/206 RIC (8%) and 85/1112 MAC (7%, p=0.56), indicating that reducing the intensity of the preparative regimen does not protect against transplantation-related alveolar hemorrhage ⁸⁹. White blood cell recovery and renal insufficiency—but not prolonged prothrombin time, partial thromboplastin time, or low platelets counts—were associated with the development of DAH ^{90, 91}. Although most patients with DAH have thrombocytopenia, the DAH is not corrected with platelet transfusion ⁹¹.

Pathogenesis

Vascular abnormalities, in the form of endothelial swelling and thrombi, are found in autopsies of BMT recipients with acute hemorrhagic pulmonary edema;⁹² although the resulting clinical presentation is hemorrhagic, thrombi found at autopsy may imply to a thrombotic etiology. The incidence of DAH is high in BMT recipients with acute GVHD ⁹³. In addition to the toxicity from therapy for GVHD, antigen-specific injury to endothelium may be a contributing factor to the development of DAH ⁹⁴. Mice receiving bone marrow cells with T lymphocytes have been shown to develop alveolitis, characterized by alveolar hemorrhage, increased alveolar leukocytes, platelet microthrombi, and damage to endothelial and epithelial cells during the acute phase of the graft versus host reaction ². The use of dimethyl sulfoxide for cryopreservation of blood stem cells has been implicated in causing damage to the alveolar endothelial lining thereby leading to the development of DAH ^{86, 95}. Hematopoietic growth factors, such as granulocyte colony-stimulating factor, may also play a role in worsening of the alveolar damage and capillary leakage by increasing neutrophils infiltration into the lungs ^{96, 97}. Cytokines release may mediate the development of DAH ⁹⁸. It is speculated that damage to alveolar capillary endothelial membranes begins during preparative chemotherapy or total body irradiation and results in a release of inflammatory mediators ⁹⁸. The response may be amplified after the release of endotoxin into the circulation from the gut after injury from mucositis ⁹⁸.

Therapy

There are no prospective, randomized trials addressing the treatment of DAH in BMT recipients. As the pathogenesis of DAH is considered to be an inflammatory response to various insults and based on anecdotal experience and retrospective studies, BMT recipients with DAH are treated with systemic corticosteroids ^{81, 86, 98, 99}. Fresh frozen plasma transfusion and plasmapheresis have been tried in one study with inconclusive results ¹⁰⁰. The reports on off-label use of recombinant factor VIIa (rFVIIa) in patients with DAH in BMT recipients are anecdotal and a conclusion cannot be drawn due to the limited data ^{101, 102}. In a phase II multicenter trial in BMT patients with major bleeding, a total dose of 280–1120 µg kg⁻¹ rFVIIa was used ¹⁰³. The regimen included three arms of 40, 80, or 160 µg/day, receiving seven successive doses every 6 hours for 36 hours, compared to a placebo. In this study 7 patients out of 100 suffered from DAH. Six thromboembolic events were observed in the treated group. This study could not reveal a significant benefit of rFVIIa in the setting of BMT-related severe bleeding.103 The use of antifibrinolytic agents such as tranexamic acid and aprotinin is not well documented. Solomonov et al reported on six patients with significant hemoptysis, two who bled during bronchoscopy biopsy, and four with spontaneous bleeding (lung cancer, diffuse alveolar hemorrhage, idiopathic pulmonary bleeding, and metastatic thyroid carcinoma). For the two who bled during bronchoscopy, they used a bolus of tranexamic acid 500 mg/5 mL through the bronchoscope working channel, while the latter four received aerosolized transexamic acid 500 mg/5 ml three to four times a day. In all cases, the bleeding stopped with the first dose of TA, and the treatment was well tolerated without adverse events ¹⁰⁴. Thus, therapy in DAH warrants further investigation in controlled clinical trials.

HEMORRHAGIC CYSTITIS

Hemorrhagic cystitis occurs as microscopic hematuria (grade I) or as gross hematuria with clots and urinary tract obstruction (grades II-IV) in 5%–40% of BMT recipients and causes significant morbidity and mortality ¹⁰⁵.

Pathogenesis

Histological analyses reveal bladder mucosal microscopic alterations such as edema, necrosis, and hemorrhage ¹⁰⁶. Acute GVHD, the use of alkylating agents in the conditioning regimen, and viral reactivation have been implicated in the pathogenesis of hemorrhagic cystitis ^{107, 108}. Reactivation of the BK virus, and less frequently, adeno and herpes simplex viruses have been associated with late onset (>2 weeks posttransplant) of hemorrhagic cystitis ^108–110. In a recent study, it was demonstrated that hemorrhagic cystitis was less common in patients with related donors than in those with unrelated donors, and was less common in patients receiving reduced intensity conditioning regimen rather than full conditioning ¹¹¹.

Therapy

Various palliative treatments, including hydration, hyperbaric oxygen, antiviral antibiotic, estrogen, selective embolization of the internal iliac arteries, intravesicle instillation of materials as alum, silver nitrate, and formalin, showed variable success in alleviating a patient's symptoms ^{110, 112–115}. In the previously mentioned multicenter randomized trial in BMT patients with major bleeding ¹⁰³, 26 patients with intractable hemorrhagic cystitis were included. In the subgroup of patients with hemorrhagic cystitis, the study was also unable to demonstrate clinical benefit for rFVIIa in terms of bleeding control ¹⁰³. In another report, four patients with severe hemorrhagic cystitis after BMT were treated with rFVIIa. In this study, a total dose of 150–1200 µg kg⁻¹ was found to be effective with no documented thrombotic event ¹¹⁶. Treatment options are summarized in Table 2.

CONCLUSIONS

In view of the possibility that DAH is also a thrombotic event, the three typical hemostatic complications in BMT (namely, VOD, TMA, and DAH) are microcirculation thrombosis. Hemorrhagic cystitis is probably a primary epithelial injury. Endothelial cells line the vascular bed. Each vascular bed has unique structural and functional properties and understanding of these properties holds important clues to site-specific diagnostics and therapeutics. An example for the hemostatic endothelial heterogeneity is high vWF in mice endothelium of lung and lowest in liver ¹¹⁷, while the anticoagulant TFPI is highest in lung and undetectable in liver ¹¹⁸. Widening the understanding of the specific changes at the endothelium in each thrombotic condition will enable us better treatment strategies. Currently, hemostatic complications cause high morbidity and mortality in BMT patients. The use of more intensive conditioning regimens and mismatched donors increase the incidence of bleeding and thrombosis. Since the ability to treat the patient in the presence of severe thrombocytopenia—frequently refractory to platelets transfusions—is limited, the need to search for hemostatic agents that does not interfere significantly with the coagulation system, such as defibrotide, is essential.

Keywords

bone marrow transplantation, diffuse alveolar hemorrhage, hemorrhagic cystitis, veno-occlusive disease, thrombotic microangiopathy

Disclosure: Nadir Yona is the principal investigator for this report.

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54. Palmer KJ, Goa KL. Defibrotide. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in vascular disorders. Drugs. 1993;45:259–294.
55. Koehl GE, Geissler EK, Iacobelli M, et al. Defibrotide: an endothelium protecting and stabilizing drug, has an anti-angiogenic potential in vitro and in vivo. Cancer Biol Ther. 2007:6.
56. Richardson PG, Murakami C, Jin Z, et al. Multi-institutional use of defibrotide in 88 patients after stem cell transplantation with severe veno-occlusive disease and multisystem organ failure: response without significant toxicity in a high-risk population and factors predictive of outcome. Blood. 2002;100:4337–4343.
57. Richardson PG, Soiffer RJ, Antin JH, et al. Defibrotide for the treatment of severe hepatic veno-occlusive disease and multiorgan failure after stem cell transplantation: a multicenter, randomized, dose-finding trial. Biol Blood Marrow Transplant. 2010;16:1005–1017.
58. Haussmann U, Fischer J, Eber S, Scherer F, Seger R, Gungor T. Hepatic veno-occlusive disease in pediatric stem cell transplantation: impact of pre-emptive antithrombin III replacement and combined antithrombin III/defibrotide therapy. Haematologica. 2006;91:795–800.
59. Dignan F, Gujral D, Ethell M, et al. Prophylactic defibrotide in allogeneic stem cell transplantation: minimal morbidity and zero mortality from veno-occlusive disease. Bone Marrow Transplant. 2007;40:79–82.
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61. Batts ED, Lazarus HM. Diagnosis and treatment of transplantationassociated thrombotic microangiopathy: real progress or are we still waiting? Bone Marrow Transplant. 2007;40:709–719.
62. Daly AS, Xenocostas A, Lipton JH. Transplantation-associated thrombotic microangiopathy: twenty-two years later. Bone Marrow Transplant. 2002;30:709–715.
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64. Martinez MT, Bucher C, Stussi G, et al. Transplant-associated microangiopathy (TAM) in recipients of allogeneic hematopoietic stem cell transplants. Bone Marrow Transplant. 2005;36:993–1000.
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69. Atkinson K, Biggs JC, Hayes J, et al. Cyclosporin A associated nephrotoxicity in the first 100 days after allogeneic bone marrow transplantation: three distinct syndromes. Br J Haematol. 1983;54:59–67.
70. Zent R, Katz A, Quaggin S, et al. Thrombotic microangiopathy in renal transplant recipients treated with cyclosporin A. Clin Nephrol. 1997; 47:181–186.
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72. Teshima T, Miyoshi T, Ono M. Cyclosporine-related encephalopathy following allogeneic bone marrow transplantation. Int J Hematol. 1996; 63:161–164.
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74. Fogo A, Hakim RC, Sugiura M, Inagami T, Kon V. Severe endothelial injury in a renal transplant patient receiving cyclosporine. Transplantation. 1990;49:1190–1192.
75. Burke GW, Ciancio G, Cirocco R, et al. Microangiopathy in kidney and simultaneous pancreas/kidney recipients treated with tacrolimus: evidence of endothelin and cytokine involvement. Transplantation. 1999;68:1336–1342.
76. Grace AA, Barradas MA, Mikhailidis DP, et al. Cyclosporine A enhances platelet aggregation. Kidney Int. 1987;32:889–895.
77. Nakamae H, Yamane T, Hasegawa T, et al. Risk factor analysis for thrombotic microangiopathy after reduced-intensity or myeloablative allogeneic hematopoietic stem cell transplantation. Am J Hematol. 2006; 81:525–531.
78. Corti P, Uderzo C, Tagliabue A, et al. Defibrotide as a promising treatment for thrombotic thrombocytopenic purpura in patients undergoing bone marrow transplantation. Bone Marrow Transplant. 2002; 29:542–543.
79. Alessandrino EP, Martinelli G, Canevari A, et al. Prompt response to high-dose intravenous immunoglobulins given as first-line therapy in post-transplant thrombotic thrombocytopenic purpura. Bone Marrow Transplant. 2000;25:1217–1218.
80. Sharma S, Nadrous HF, Peters SG, et al. Pulmonary complications in adult blood and marrow transplant recipients: autopsy findings. Chest. 2005;128:1385–1392.
81. Metcalf JP, Rennard SI, Reed EC, et al. Corticosteroids as adjunctive therapy for diffuse alveolar hemorrhage associated with bone marrow transplantation. University of Nebraska Medical Center Bone Marrow Transplant Group. Am J Med. 1994;96:327–334.
82. Sisson JH, Thompson AB, Anderson JR, et al. Airway inflammation predicts diffuse alveolar hemorrhage during bone marrow transplantation in patients with Hodgkin disease. Am Rev Respir Dis. 1992;146:439– 443.
83. Witte RJ, Gurney JW, Robbins RA, et al. Diffuse pulmonary alveolar hemorrhage after bone marrow transplantation: radiographic findings in 39 patients. AJR Am J Roentgenol. 1991;157:461–464.
84. Ho VT, Weller E, Lee SJ, Alyea EP, Antin JH, Soiffer RJ. Prognostic factors for early severe pulmonary complications after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant. 2001;7:223–229.
85. Baker WJ, Vukelja SJ, Burrell LM, Lee N, Perry JJ. High-dose cyclophosphamide, etoposide and carboplatin with autologous bone marrow support for metastatic breast cancer: long-term results. Bone Marrow Transplant. 1998;21:775–778.
86. Chao NJ, Duncan SR, Long GD, Horning SJ, Blume KG. Corticosteroid therapy for diffuse alveolar hemorrhage in autologous bone marrow transplant recipients. Ann Intern Med. 1991;114:145–146.
87. Frankovich J, Donaldson SS, Lee Y, Wong RM, Amylon M, Verneris MR. High-dose therapy and autologous hematopoietic cell transplantation in children with primary refractory and relapsed Hodgkin’s disease: atopy predicts idiopathic diffuse lung injury syndromes. Biol Blood Marrow Transplant. 2001;7:49–57.
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91. Robbins RA, Linder J, Stahl MG, et al. Diffuse alveolar hemorrhage in autologous bone marrow transplant recipients. Am J Med. 1989;87:511– 518.
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93. Wojno KJ, Vogelsang GB, Beschorner WE, Santos GW. Pulmonary hemorrhage as a cause of death in allogeneic bone marrow recipients with severe acute graft-versus-host disease. Transplantation. 1994;57:88– 92.
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98. Haselton DJ, Klekamp JG, Christman BW, Barr FE. Use of high-dose corticosteroids and high-frequency oscillatory ventilation for treatment of a child with diffuse alveolar hemorrhage after bone marrow transplantation: case report and review of the literature. Crit Care Med. 2000;28:245–248.
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Normal pregnancy leads to several alterations in hemostatic parameters. The plasma concentrations of a number of coagulation factors increase, coagulation inhibitors decrease, and fibrinolytic activity is reduced.¹ Moreover, a gradual increase in maternal plasma lipid concentrations may also increase cardiovascular risk. In a previous study we followed 22 women with familial hypercholesterolemia (FH) through pregnancy and observed a 30% increase in low density lipoprotein (LDL)-cholesterol (range 6.7 to 8.6 mmol/L) and a 116% increase in fasting triglycerides (range 1.5 to 3.0 mmol/L) from gestational week 17–20 compared to gestational week 36.² The increase in plasma LDL-cholesterol was higher in FH women than in pregnant controls (1.9 mmol/L vs 0.8 mmol/L) (2). There is a close interaction between plasma lipids and several mediators in the coagulation cascade.³ Further, elevated LDL-cholesterol can lead to the formation of foam cells in the endothelium. Any atherosclerotic plaque may trigger coagulation and pregnancy-associated thromboembolism in veins or arteries is a cause of significant maternal morbidity.⁴ Since women with familial hypercholesterolemia (FH) are more prone to atherosclerosis than healthy women, they may be particularly vulnerable to alterations in hemostatic parameters during the pregnancy and some case reports have indeed suggested this.^5–7 From experience during many years in our Lipid Clinic, we have learned that many women with FH are concerned about the cardiovascular risk in pregnancy. Lack of data have made evidence-based answers difficult to provide. Pregnancy usually will result in a two- to threefold increase in LDL-cholesterol in FH women, since they need to stop taking lipid lowering medications. There are many difficult considerations in the treatment of pregnant women with FH and a possible accelerating atherosclerosis in the mother must be weighed against the very important concern for fetal safety. The UK National Institute for Health and Clinical Excellence (NICE) advice is that women with FH who are considering pregnancy should be provided expertise in both cardiology and obstetrics.⁸ Clearly, the special challenges associated with pregnancy in women with FH makes it relevant to review the literature in this area, especially in a clinical perspective. The specific issues related to the very rare homozygote FH will not be discussed in the present paper.

SEARCH METHOD

A search in PubMed for “acute myocardial infarction AND pregnancy” resulted in 433 references. The papers were sorted after categories provided by PubMed like “Clinical Trial, Editorial, Letter, Meta-Analysis, Practice Guideline, Randomized Controlled Trial, Case Reports, Clinical Conference, Comment and Comparative Study.” The majority of the articles fell in the category reviews, case reports, or journal articles see Table 1. Of a total of 18 clinical trials identified none were found relevant for FH, but 34 of 109 review articles, 5 of 20 letters, and 1 of 4 editorials were found to be relevant for the present review. Many of the articles did, however, focus on quite specific medical conditions of little relevance to FH such as congenital heart disorders, different valvular diseases, atrial fibrillation, surgical procedures, different coagulation disorders, causes and management of coronary dissection, different anticoagulation therapy, cocaine abuse, and specific diagnostic tools and procedures. These articles were not included in the present review. Regarding the 195 individual case reports identified in the present search, Roth and Elkayam thoroughly reviewed 95 cases of pregnancies complicated by myocardial infarction (MI).⁹ Therefore, we did not analyze the individual case reports identified in the present search.

THE RISK FOR CARDIOVASCULAR DISEASE IN PERSONS WITH FH

Heterozygous FH is an autosomal dominant disease usually caused by a mutation in the LDL receptor gene. Approximately 1:500 people are affected.^{10, 11} It is estimated that more than 10 million people have FH in a global perspective.^11–13 The disease results in significantly elevated plasma cholesterol value, not only from birth, but even in utero.^{12, 13} Plasma cholesterol is elevated due to a high level of the unfavorable LDL-cholesterol.¹⁰ Untreated FH leads to an increased risk of cardiovascular morbidity and mortality from vascular causes with the increase in relative risk highest in young adults where the risk of vascular disease a priori is very low. Untreated FH results in close to 100 times increased risk of dying from coronary heart disease in the age group 20–39 years,¹⁴ but at increasing age the relative risk drops down to about four times higher in the age group 40–59 years, and for those older than 60 FH does not give any notably increased risk at all.¹⁴ In older studies, from the period before HMG-CoA reductase inhibitors (statins) were available, 75% of men with untreated FH had experienced MI before 60 years of age.¹⁵ The risk was lower in women, 15% had a MI before the age of 60.¹⁵ Other studies have reported that coronary heart disease occurs on average about 10 years earlier in men than in women. ^16–18 At the beginning of the 1990s, the first statins were approved for use. This meant a revolution in the treatment of FH. For the first time it was possible to normalize plasma cholesterol in the vast majority of patients with FH as statins, in principle, repair the cellular defect of a too low number of LDL receptors. Observational studies have indicated that FH patients treated with statins now have approximately the same risk of vascular disease as the general population.^{15, 19, 20} There are, however, no controlled prospective trials proving the efficacy of statins on hard endpoints in a FH population.

Regarding the very rare and serious homozygous hypercholesterolemia, MI has been described as occurring as early as 3 years of age in untreated patients, but there are also reports of untreated patients who have not had MI before 30 years of age. Correct treatment of these patients normally involves LDL-apheresis and there are several case reports of successful treatment during pregnancy.

THE RISK FOR CARDIOVASCULAR DISEASE IN PREGNANT WOMEN

Acute myocardial infarction in pregnancy is uncommon. Two US population-based studies,^{21, 22} one expert consensus²³, and several reviews^24–28 have reported that it occur in about 3–10 cases per 100·000 deliveries. The physiological and hemodynamic change in the direction of a hypercoagulable state induced during pregnancy is possibly mediated by hormonal changes. The risk for MI has been reported to be three- to fourfold higher in pregnant vs nonpregnant women in a population-based study²¹ meaning that pregnancy itself does increase the risk for cardiovascular disease. When MI occurs during pregnancy, the fatalities have been reported to be from 5.1%²¹ to 11%⁹ and up to 37%.²⁸ This large variation in fatality rate may be related to the type of population studied and how old the data are. Treatment has improved during the years, the criteria for diagnosing MI have changed and the sensitivity of the diagnostic test has improved. Fetal and neonatal mortality after a maternal MI has been reported to range from 13%–34%^{9, 22} and this underlines that MI is a very serious medical condition in pregnancy.

THE RISK FOR CARDIOVASCULAR DISEASE IN PREGNANT WOMEN WITH FH

In a prospective study, we have previously reported some changes in hemostatic balance and endothelial activation in 22 pregnant FH women compared to 149 healthy controls from gestational week 17–20 to week 24 and week 36.²⁹ The concentration of circulating prothrombin fragments 1+2, a marker of thrombin generation, was significantly higher in the FH group compared to the controls, whereas the amount of free tissue factor pathway inhibitor (TFPI)-type 1 antigen and activity increased from week 17–20 to week 36 only in healthy controls. Further, vascular cell adhesion molecule (VCAM)-1 rose significantly during pregnancy by 120% in the FH group only, it remained unaltered in the reference group. We did also observe a possible attenuated decrease in the umbilical artery pulsatility index in FH women reflecting an increased uteroplacental vascular resistance³⁰ suggesting a possible unfavorable hemostatic and endothelial homeostasis during pregnancy in FH women.

Women with FH usually initiate lipid lowering drug treatment later in life than men³¹ possibly due to the a priori lower risk for cardiovascular disease in women. As it is advised to stop statin treatment from the time a pregnancy is being planned and until the breastfeeding is finished, this may result in many years with severely elevated LDL-cholesterol. Current recommendation is to stop taking statins 3 months before conception.⁸ Assuming that it on average will take 3 months to become pregnant and that after 9 months of pregnancy the women will breastfeed for 6 months, each delivery will result in 21 months with severely elevated plasma cholesterol. Some couples need much more than 3 months to become pregnant and some wish to breastfeed for more than 6 months. Pregnancy may, therefore, result in a substantial number of years with severely elevated plasma cholesterol. Men at the same age will have the opportunity to receive effective treatment. Modern treatment of FH thus facilitates preventive drug treatment for men over that for women. This should be taken into consideration for the interpretation of older studies from the time before there was effective treatment for patients with FH. Such studies, showing lower risk in FH women than in FH men may not represent the situation of today where fertile men will have more years on effective statin therapy than fertile women.

DISCUSSION IN A CLINICAL PERSPECTIVE

FH women in fertile age have some frequently asked questions related to pregnancy that are not always easy to answer. Some of these will be discussed in this section.

Should I use oral contraceptives despite having FH and high blood cholesterol?

The package leaflets for oral contraceptives include a warning against the use if a person has a disease that can increase the risk of developing a thrombosis in the arteries. A very high fat level in the blood (cholesterol or triglycerides) is mentioned specifically in addition to high blood pressure. An intuitive, but not evidence-based, answer is that the use of a statin will normalize blood cholesterol so that the FH woman will no longer have high cholesterol and the warning against contraceptives is then no longer appropriate. Nevertheless, one should be aware that the decision about whether the use of the p-pill is advisable or not should always be based on a global risk estimate. This involves knowledge about important predisposing factors in the individual patient such as smoking, type 2 diabetes, blood pressure and, most importantly, the age. In a study of case reports, Roth and Elkayam identified 103 pregnancies complicated by acute MI.⁹ They observed a relatively high incidence of well-known risk factors for MI among the patients. Forty-five percent were smokers, 24% had hyperlipidemia, 22% had family histories of premature MI, 15% had hypertension, and 11% had diabetes. When choosing a type of oral contraceptive for women with FH, those with no estrogen may be preferable because it is the estrogen component that is responsible for the increase in cardiovascular risk accompanying p-pill use. The so called mini-pill does, however, not contain estrogen.

I used statins for the first weeks of pregnancy, could the embryo have taken damage—should I have an abortion?

Statin treatment is an automatic routine in the daily life and, not surprisingly, some women have used statins during the first weeks before being aware of their pregnancy. Data on the safety of statins during pregnancy in humans is limited. In rats, increased incidence of skeletal abnormalities have been detected with lovastatin³² and apoptosis in neonatal rat cardiac myocytes has been described with fluvastatin.³³ Simvastatin, lovastatin, and atorvastatin all achieve embryoplacental concentrations similar to those of maternal plasma. Lipophilic statins have been shown to have adverse reproductive effects in the axial skeleton, viscera, or central nervous system in animal studies. However, information obtained from a worldwide, postmarketing surveillance based on 137 reports to the manufacturer of simvastatin and lovastatin during pregnancy did not show any adverse pregnancy outcome. It should be noted that the study was rather small and not conclusive, although the data support the view that these two statins do not increase the risk for any adverse pregnancy outcome dramatically.³⁴ Among 14 infants exposed to pravastatin no malformations were reported, supporting the view that this statin results in a very high risk for adverse pregnancy outcome.

In a prospective, observational cohort study of 64 pregnant women taking statins compared with 64 controls, birth weight was lower in the statin group.³⁵ Women in the statin group were exposed to various statins; atorvastatin (n=46), simvastatin (n=9), pravastatin (n=6), or rosuvastatin (n=3) during the first trimester. The rate of major malformations did not differ in the two groups (1/46 live birth in the statin group vs 1/52 live birth in the comparison group (p=0.93). Further, there was no difference in live births (71.9 vs 81.2%), spontaneous abortions (21.9 vs 17.2%), therapeutic abortions (4.7 vs 0: 0%), and stillbirths (1.5 vs 1.6%). Gestational age at birth (38.4±2.8 weeks vs 39.3±1.3 weeks: Mean±SD, p=0.04) and birth weight (3.14±0.68 kg vs 3.45±0.42 kg, p=0.01) was, however, lower in the statin group.

Another study assessed the manufacturer pharmacovigilance database for reports of exposure to simvastatin or lovastatin during pregnancy.³⁶ There were 477 reports (386 prospective and 91 retrospective) that were identified with 225 prospective outcomes reported: 154 live born infants, 49 elective abortions, 18 spontaneous abortions, and 4 fetal deaths. The rate of congenital anomalies (congenital anomalies/live births plus fetal deaths) was 3.8%, which was not different from the background population rate (3.2%). In 13 retrospective reports, a range of congenital anomalies were reported but no specific pattern of anomalies was seen. Rates for other outcomes were similar to background rates. The authors concluded that there was no evidence of a notable increase in congenital anomalies in women exposed to simvastatin or lovastatin versus the general population.

First-trimester statin exposure was reported in 178 cases to the US Food and Drug Administration (FDA) from 1987 through 2001. These reports were reviewed for patterns suggesting possible drug-related effects on embryogenesis.³⁷ Fifty-two of these cases were considered evaluable and there were 20 reports of malformation including 5 severe defects of the central nervous system and 5 limb deficiencies; one patient had both of these malformations. In all cases of adverse outcomes at birth, the associated statin was lipophilic.

The prevalence use of prescription drugs among pregnant women was studied in 33343 deliveries.³⁸ Nearly 1% of the women were exposed to drugs contraindicated in pregnancy and 0.6% received these drugs during the first trimester. Several statin medications were among the most common contraindicated drug exposures.

Drugs should be used only if the benefits outweigh the risks. Therefore, since statins inhibit the synthesis of mevalonic acid, important both for the synthesis of steroids in fetal development and in DNA replication, the use is not recommended in pregnancy.

I had my cholesterol measured after I became pregnant and it has never been that high. Should I start treatment with statins again?

Based on the precautionary principle, statin therapy should not be started. The only lipid-lowering drugs that can be used in pregnancy are bile-acid binding resins. Animal studies with resins do not indicate any harmful effects with respect to pregnancy or embryonic development. There is, however, some difference in the summary of product characteristics (SPC) text between the different resins regarding pregnancy,although the resins have similar mechanisms of action. The SPC of cholestipol states that “safe use during pregnancy has not been established due to insufficient experience” and conclude that cholestipol should not be used during pregnancy. In the SPC of another resin, colestyramin, it is stated that “clinical experience in pregnant women is limited and that animal experimental data are incomplete regarding pregnancy.” It states that the drug is not excreted in human milk and that caution should be taken when prescribing colestyramine to pregnant women. In the SPC text of the new low-dose resin, colesevelam, it is stated that “animal studies do not indicate harmful effects with respect to pregnancy, embryonic, birth, or postnatal development but that caution should be exercised when prescribing to pregnant women and when prescribing to lactating women, as safety is not established.” Thus, according to the SPC text, colestyramin is the resin to choose in pregnant or lactating women. Constipation is a very common side effect with the old resins and colesevelam is an alternative. Unfortunately, a reduction in LDL-cholesterol of more than 15% is seldom seen. However, plant sterols and dietary fiber can in general contribute with an additional 10% reduction.

In general, women with FH should be advised not to measure their cholesterol value during pregnancy according to the NICE guidelines.⁸ The LDL-cholesterol is expected to increase with about 30% in the third trimester and the triglyceride value is expected to double.² After delivery it will return to normal within 3 weeks. Since there is no therapeutic option with an effect in the magnitude of statins for pregnant women, a cholesterol measurement will not help in a therapeutic perspective but will certainly have the potential of frightening the FH patient.

I want to become pregnant, but I am afraid to stop taking my cholesterol lowering medication. Can I take any other medication to reduce my risk?

Diet and lifestyle are important for the cholesterol levels in a pregnant women with FH. Taking into account the increased risk of FH, it is very important not only for the fetus but for the cardiovascular risk of the mother as well to not smoke. Furthermore, a healthy diet and adequate exercise is of great importance. One possible way to reduce the risk for arterial thrombosis is to take aspirin. However, aspirin can contribute to maternal and fetal bleeding and it does cross the placenta. It has not been associated with congenital anomalies, but an increased risk of vascular disruptions—like possibly premature closure of the ductus arteriosus—has been reported by inhibiting prostaglandin synthesis³⁹ and aspirin has induced such anomalies in chick embryos⁴⁰ as well as nonstereoid anti-inflammatory drugs like indometacin that have induced such changes in rat.⁴¹ Still, large trials demonstrate low-dose aspirin is relatively safe in pregnancy.⁴²

Women with FH frequently ask if pregnancy and delivery leads to an increased risk of heart attack for them. A usual answer to give is that it does not, but again the evidence behind this answer is unfortunately somewhat unclear. No studies have yet been able to answer the question properly. It is, however, worrying that a few case reports have described the unstable angina or cardiac infarction in pregnant women with FH.^{6, 7, 43} It is well known that pregnancy causes the activation of both the coagulation system and the endothelium as well as an increase plasma triglyceride, total cholesterol, LDL cholesterol, and HDL cholesterol in normal^{44, 45} as well as in FH women.^{2, 29} The pathogenesis underlying MI in pregnancy may have several other causes than atherosclerosis. Hence, atherosclerosis was found in 41 cases (40%), and definite or probable coronary thrombus without evidence of atherosclerotic disease was present in 8% in pregnant women with MI.⁹ The coronary arteries were described as normal in 13% of the cases.

CONCLUSION

Available data on pregnant women with FH support the view that MI is very uncommon during pregnancy. The increase in the relative risk for FH women must be considered together with the low risk for cardiovascular disease in absolute terms. There is no contraindication to pregnancy or breastfeeding for the majority of woman with FH. Women with additional risk factors like elevated lipoprotein (a), hypertension, smoking, or diabetes will probably benefit from early re-starting of the statin therapy after delivery and will then need to stop for the breastfeeding. The FH will probably involve a slightly increased risk in itself, and this should be taken into account in relation to all other known risk factors. Women with FH should be offered an opportunity to consult a lipid clinic for discussing medical care in pregnancy.

Keywords

familial hypercholesterolemia, myocardial infarction, cholesterol, statins, pregnancy

Disclosure: The authors declare no conflict of interest

REFERENCES

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The majority of critically ill patients have coagulation abnormalities.¹ These abnormalities range from subtle activation of coagulation that can only be detected by sensitive markers for coagulation factor activation to somewhat more stronger coagulation activation that may be detectable by a small decrease in platelet count and subclinical prolongation of global clotting times to fulminant disseminated intravascular coagulation (DIC), characterized by simultaneous widespread microvascular thrombosis and profuse bleeding from various sites.² Patients with severe forms of DIC may present with manifest thromboembolic disease or clinically less apparent microvascular fibrin deposition that predominantly presents as multiple organ dysfunction.^{3, 4} Alternatively, severe bleeding may be the leading symptom,⁵ but quite often a patient with DIC has simultaneous thrombosis and bleeding. Bleeding is caused by consumption and subsequent exhaustion of coagulation proteins and platelets due to the ongoing activation of the coagulation system.⁶

CLINICAL SETTING OF DIC

It is important to emphasize that DIC is not a disease in itself but is always secondary to an underlying disorder.⁷ The underlying disorders most commonly known to be associated with DIC are listed in Table 1.

Bacterial infection, in particular septicemia, is often associated with DIC.⁸ However, systemic infections with other microorganisms such as viruses and parasites may lead to DIC as well.⁹ Factors involved in the development of DIC in patients with infections may be bacterial endotoxins (eg, from Gram-negative bacteria) or exotoxins (eg, staphylococcal alpha toxin).⁸ These components may cause a generalized inflammatory response, characterized by the systemic occurrence of cytokines. Cytokines are mainly produced by activated mononuclear cells and endothelial cells and are responsible for the derangement of the coagulation system in DIC.¹⁰

Severe trauma is another clinical condition frequently associated with DIC.¹¹ A combination of mechanisms—including release of tissue material (fat, phospholipids) into the circulation, hemolysis, and endothelial damage—may contribute to the systemic activation of coagulation. In addition, there is solid evidence that cytokines play a pivotal role in the occurrence of DIC in trauma patients as well. In fact, systemic cytokine patterns have been shown to be virtually identical in trauma patients and septic patients.¹²

Both solid tumors and hematological malignancies may be complicated by DIC.^{13, 14} There is increasing evidence that tissue factor expressed by cancer cells (for example, glioblastoma or adenocarcinoma) is important in the pathogenesis of DIC in cancer.¹⁵ Solid tumor cells can also express other procoagulant molecules such as cancer procoagulant, a cysteine protease with factor X activating properties. A specific form of DIC is frequently encountered in acute promyelocytic leukemia, which is characterized by a severe hyperfibrinolytic state in addition to an activated coagulation system.¹⁶ Although clinically bleeding predominates in this situation, disseminated thrombosis is found in a considerable number of patients at autopsy.

Acute DIC occurs in obstetrical calamities such as placental abruption and amniotic fluid emboli.¹⁷ Amniotic fluid has been shown to be able to activate coagulation in vitro, and the degree of placental separation correlates with the extent of DIC, suggesting that leakage of thromboplastin-like material from the placental system is responsible for the occurrence of DIC. Although the coagulation system may be activated in patients with pre-eclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, it is subclinical and overt DIC only occurs in a small percentage of patients, usually occurring in patients with an abruptio placentae or some other complication.

Vascular disorders, such as large aortic aneurysms or giant hemangiomas (Kasabach-Merritt Syndrome), may result in local activation of coagulation.¹⁸ Activated coagulation factors can ultimately “overflow” to the systemic circulation and cause DIC, but more common is the systemic depletion of coagulation factors and platelets as a result of local consumption.

Microangiopathic hemolytic anemia represents a group of disorders comprising thrombocytopenic thrombotic purpura (TTP), hemolytic uremic syndrome, chemotherapy-induced microangiopathic hemolytic anemia, malignant hypertension, and the HELLP syndrome.¹⁹ Although some characteristics of microangiopathic hemolytic anemia and the resulting thrombotic occlusion of small and midsize vessels leading to organ failure may mimic the clinical picture of DIC, these disorders in fact represent a distinct group of diseases.²⁰

RELEVANCE OF COAGULATION ABNORMALITIES IN DIC

There is ample evidence that activation of coagulation in concert with inflammatory activation can result in microvascular thrombosis and thereby contributes to multiple organ failure in patients with severe sepsis.²¹ Firstly, extensive data has been reported on postmortem findings of patients with coagulation abnormalities and DIC in patients with severe infectious disease.^{22, 23} These autopsy findings include diffuse bleeding at various sites, hemorraghic necrosis of tissue, microthrombi in small blood vessels, and thrombi in midsize and larger arteries and veins. The demonstration of ischemia and necrosis was invariably due to fibrin deposition in small and midsize vessels of various organs.²⁴ Importantly, the presence of these intravascular thrombi appears to be clearly and specifically related to the clinical dysfunction of the organ. Secondly, experimental animal studies of DIC show fibrin deposition in various organs. Experimental bacteremia or endotoxemia causes intra- and extravascular fibrin deposition in kidneys, lungs, liver, brain, and various other organs. Amelioration of the hemostatic defect by various interventions in these experimental models appears to improve organ failure and, in some but not all cases, mortality.^25–28 Interestingly, some studies indicate that amelioration of the systemic coagulation activation will have a profound beneficial effect on resolution of local fibrin deposition and improvement of organ failure.^{5, 29} Lastly, clinical studies support the notion of coagulation as an important denominator of clinical outcome. Disseminated intravascular coagulation has shown to be an independent predictor of organ failure and mortality.^{3, 30} In a consecutive series of patients with severe sepsis, the mortality of patients with DIC was 43%, as compared with 27% in those without DIC. In this study, the severity of the coagulopathy was also directly related to mortality in septic patients.³¹

Apart from microvascular thrombosis and organ dysfunction, coagulation abnormalities may also have other harmful consequences. The relevance of thrombocytopenia in patients with sepsis is in the first place related to an increased risk of bleeding. Indeed, in particular critically ill patients with a platelet count of <50 × 10⁹/l have a four- to fivefold higher risk for bleeding as compared to patients with a higher platelet count.^{32, 33} The risk of intracerebral bleeding in patients at the intensive care unit is relatively low (0.3%-0.5%), but in 88% of patients with this complication the platelet count is less than 100 × 10⁹/l.³⁴ Regardless of the cause, thrombocytopenia is an independent predictor of ICU mortality in multivariate analyses with a relative risk of 1.9 to 4.2 in various studies.^{32, 33, 35} In particular, a sustained thrombocytopenia during more than 4 days after ICU admission or a drop in platelet count of >50% during ICU stay is related to a four- to sixfold increase in mortality.^{32, 36} In particular, the combined presence of thrombocytopenia and the use of anticoagulant agents increases the risk of bleeding.³⁷ The platelet count was shown to be a stronger predictor for ICU mortality than composite scoring systems such as the Acute Physiology and Chronic Evaluation (APACHE) II score or the Multiple Organ Dysfunction Score (MODS). Also low levels of coagulation factors in patients with sepsis, as reflected by prolonged global coagulation times, may be a risk factor for bleeding and mortality. A PT or aPTT ratio >1.5 in critically ill patients was found to predict excessive bleeding and increased mortality.^{38, 39}

PATHOGENETIC PATHWAYS IN DIC

Several simultaneously occurring mechanisms play a role in the pathogenesis of DIC (Figure 1). Fibrin deposition is a result of tissue factor-mediated thrombin generation that is insufficiently balanced by dysfunctional physiologic anticoagulant mechanisms such as the antithrombin system and the protein C system. In addition to enhanced fibrin formation, fibrin removal is impaired due to depression of the fibrinolytic system. This impairment of endogenous thrombolysis is mainly caused by high circulating levels of the fibrinolytic inhibitor PAI-1. As mentioned before, in exceptional forms of DIC fibrinolytic activity may be increased and contribute to bleeding. Somewhat more in detail, the following processes are involved.

Thrombin generation. There is ample evidence for a pivotal role of the tissue factor/factor VIIa system in the initiation of thrombin generation. Firstly, experiments of human endotoxemia or in humans infused with the endotoxin-induced mediator TNF-α did not show any change in markers for activation of the contact system.^{40, 41} Furthermore, abrogation of the tissue factor/factor VII(a) pathway by monoclonal antibodies specifically directed against tissue factor or factor VIIa activity resulted in a complete inhibition of thrombin generation in endotoxin-challenged chimpanzees and prevented the occurrence of DIC and mortality in baboons that were infused with E. coli.^{27, 42, 43} Indeed in most patients with DIC, tissue factor antigen is detectable in plasma.⁴⁴ Tissue factor may be expressed on mononuclear cells in vitro and tissue factor expression on circulating monocytes of patients with severe infection has indeed been demonstrated.⁴⁵ In addition, tissue factor may be expressed on endothelial cells,⁴⁶ although the importance of endothelial cell tissue factor expression in vivo and its role in the pathogenesis of DIC is disputed. Another source of tissue factor may be its localization on polymorphonuclear cells and other cell types,⁴⁷ although it is unlikely that these cells actually synthesize tissue factor in substantial quantities.⁴⁸ Based on the observation of transfer of tissue factor from leucocytes to activated platelets on a collagen surface in an ex vivo perfusion system, it is hypothesized that this “blood borne” tissue factor is transferred between cells through microparticles derived from activated mononuclear cells.⁴⁹

• Dysfunctional physiological anticoagulant pathways. An impaired function of various natural regulating pathways of coagulation activation may amplify the further thrombin generation and contribute to fibrin formation.⁵⁰ Plasma levels of the most important inhibitor of thrombin, antithrombin III, are usually markedly reduced in septic patients. This reduction is caused by a combination of consumption due to ongoing thrombin generation, degradation by elastase, which is released from activated neutrophils, and impaired synthesis. Low antithrombin III levels in DIC are associated with increased mortality.^{30, 51} The fact that low levels of antithrombin precede the clinical manifestation of sepsis in prospective studies suggests that antithrombin is indeed involved in the pathogenesis of this disease and associated organ dysfunction.⁵¹

In addition to the decrease in antithrombin III, a significant depression of the protein C system may occur. This impaired function of the protein C pathway is mainly due to downregulation of thrombomodulin expression on endothelial cells by proinflammatory cytokines like TNF-α and IL-1β.^52–54 The downregulation of thrombomodulin has been confirmed in studies in patients with meningococcal sepsis.⁵⁵ This, in combination with low levels of zymogen protein C (due to similar mechanisms as described for antithrombin), results in diminished protein C activation, which will enhance the procoagulant state. Animal experiments of severe inflammation-induced coagulation activation convincingly show that compromising the protein C system results in increased morbidity and mortality, whereas restoring an adequate function of activated protein C improves survival and organ failure.⁵⁶ Interestingly, experiments in mice with a one-allele targeted deletion of the protein C gene (resulting in heterozygous protein C deficiency) have more severe DIC and organ dysfunction and a higher mortality than wild type littermates.⁵⁷ Besides being implicated in the physiological regulation of thrombin formation, activated protein C probably also has important inflammation-modulating effects, which may be of relevance in the pathogenesis of DIC.^{52, 58}

The third significant inhibitor of coagulation is tissue factor pathway inhibitor (TFPI). The role of TFPI in the pathogenesis of DIC is not completely clear. Experiments showing that administration of recombinant TFPI (and thereby achieving higher than physiological plasma concentrations of TFPI) blocks inflammation-induced thrombin generation in humans and the observation that pharmacological doses of TFPI are capable of preventing mortality during systemic infection and inflammation suggest that high concentrations of TFPI are capable of modulating tissue factor mediated coagulation.^{25, 59} However, the endogenous concentration of TFPI is presumably insufficiently capable of regulating coagulation activation and downstream consequences during systemic inflammation.⁶⁰

• Impaired fibrinolysis. Experimental models indicate that at the time of maximal activation of coagulation, the fibrinolytic system is largely shut off. Experimental bacteremia and endotoxemia results in a rapidly occurring increase in fibrinolytic activity, most probably due to the release of plasminogen activators from endothelial cells.⁴² However, this profibrinolytic response is almost immediately followed by a suppression of fibrinolytic activity, due to a sustained increase in plasma levels of plasminogen activator inhibitor, type 1 (PAI-1).^{61, 62} Interestingly, it has been shown that a mutation in the PAI-1 gene, the 4G/5G polymorphism, not only influenced the plasma levels of PAI-1, but was also linked to clinical outcome of meningococcal septicemia.⁶³

• Two-way interaction between coagulation and inflammation. Coagulation activation yields proteases that not only interact with coagulation protein zymogens, but also with specific cell receptors to induce signaling pathways that mediate inflammatory responses. Coagulation proteins, such as factor Xa, thrombin, and fibrin, can activate endothelial cells, eliciting the synthesis of proinflammatory cytokines and growth factors.⁶⁴ The most important mechanisms by which coagulation proteases influence inflammation is by binding to so-called protease activated receptors or PARs, of which four types (PAR 1-4) have been identified, all belonging to the family of transmembrane domain, G-protein coupled receptors.^{64, 65} PARs 1, 3, and 4 are thrombin receptors whereas PAR 2 cannot bind thrombin but can be activated by the tissue factor-factor VIIa complex, factor Xa, and trypsin. PAR 1 can also serve as receptor of the tissue factor-factor VIIa complex and factor Xa. PARs are localized in the vasculature on endothelial cells, mononuclear cells, platelets, fibroblasts, and smooth muscle cells.⁶⁴ Recently, the important role of dendritic cells as an interface for these mechanisms of coagulation and inflammation have been identified.⁶⁵ In vivo evidence for a role of coagulation-protease stimulation of inflammation comes from recent experiments showing that the administration of recombinant factor VIIa to healthy human subjects causes a small but significant three- to fourfold rise in plasma levels of IL-6 and IL-8.^{6658, 67} Indeed, activated protein C has been found to inhibit endotoxin-induced production of TNF-α, IL-1β, IL-6, and IL-8 by cultured monocytes/macrophages.^{68, 69} Infusion of activated protein C abrogates inflammatory activity and improves organ function and survival in an experimental E. coli sepsis model in baboons.⁵⁶ It is likely that the effects of activated protein C on inflammation are mediated by the endothelial protein C receptor (EPCR).⁶⁷ In addition, all three physiological anticoagulant pathways are capable of influencing inflammatory activity. This is most prominently shown for the protein C pathway.

DIAGNOSTIC APPROACH TO DIC

It is important to realize that apart from DIC there are several other reasons for coagulation abnormalities in critically ill patients (Table 2). Although thrombocytopenia is common in patients with severe sepsis, this may also be caused by other (sometimes simultaneously occurring) diseases such as immune thrombocytopenia, medication-induced bone marrow depression, heparin-induced thrombocytopenia, or thrombotic microangiopathies.^{70, 71} It is very important to properly diagnose these causes of thrombocytopenia, since they may require distinctive treatment strategies.⁷² Laboratory tests can be helpful in differentiating the coagulopathy in sepsis from various other hemostatic disorders such as vitamin K deficiency or liver failure. However, since such conditions may also occur simultaneously with, for example, DIC, this differentiation is not always simple.⁷³

Tests for intravascular fibrin formation and fibrin degradation products

According to the current understanding of DIC-associated coagulation abnormalities, the determination of soluble fibrin in plasma appears to be crucial.^74–77 In general, the sensitivity of these assays for severe coagulation activation or DIC is relatively higher than the specificity. Indeed, initial clinical studies indicate that if the concentration of soluble fibrin has increased above a defined threshold, a diagnosis of DIC can be made.^{74, 76, 78, 79} Most of the clinical studies show a sensitivity of 90%-100% for the diagnosis of DIC but a rather low specificity.⁸⁰

Fibrin degradation products (FDPs) may be detected by specific ELISAs or by latex agglutination assays, allowing rapid and bedside determination in emergency cases.⁸¹ None of the available assays for fibrin degradation products discriminates between degradation products of cross-linked fibrin and fibrinogen degradation, which may cause spuriously high results.^{82, 83} The specificity of high levels of fibrin degradation products is therefore limited and many other conditions such as trauma, recent surgery, inflammation, or venous thromboembolism are associated with elevated FDPs. Assays for D-dimer, resulting from the plasmin-degraded cross-linked γ-chain of fibrin better differentiate degradation of cross-linked fibrin from fibrinogen or fibrinogen degradation products.⁸⁴ D-dimer levels are high in patients with DIC, but also poorly distinguish patients with DIC from patients with venous thromboembolism, recent surgery, or inflammatory conditions.^{81, 85}

Markers for thrombin generation and coagulation activation

Activation peptides that are released upon the conversion of a coagulation factor zymogen to an active protease are sensitive markers for coagulation activation. Examples of such markers are prothrombin activation fragment F1+2 (F1+2), and the activation peptides of factors IX and X.^86–88 Indeed, these markers are markedly elevated in most patients with sepsis. Elevated plasma concentrations of thrombin–antithrombin complexes may well reflect the increased generation of thrombin and thrombin-mediated fibrinogen to fibrin conversion can be monitored by increased levels of fibrinogen activation peptide fibrinopeptide-A (FPA).^{89, 90} The most important disadvantage of these tests may be that their use is limited to specialized coagulation laboratories and that they are not available for routine use in most clinical centers. Thus, although these tests are very relevant for research on the pathogenesis of coagulation disturbances in sepsis and the effect of specific interventions in the coagulation cascade of patients with sepsis, their practical use in clinical medicine is limited so far.

Platelet count and coagulation factors in patients with DIC

The platelet count in DIC is correlated with markers of thrombin generation, since thrombin-induced platelet aggregation is for a large part responsible for platelet consumption.⁷¹ Since the normal platelet count may vary considerably, a single determination is often not very helpful but a continuous drop in platelet count, determined in septic patients at intervals of about 4 hours, may indicate the generation of thrombin causing intravascular platelet aggregation. As mentioned before, however, a low or decreasing platelet count is not very specific for DIC.

Consumption of coagulation factors leads to low levels of coagulation factors in patients with DIC. In addition, impaired synthesis, for example due to impaired liver function or a vitamin K deficiency, and loss of coagulation proteins, due to massive bleeding, may play a role as well.⁹¹ The low level of coagulation factors is reflected by prolonged coagulation screening tests such as the prothrombin time or the activated partial thromboplastin time (aPTT). Plasma levels of factor VIII are paradoxically increased in most patients with DIC, probably due to massive release of von Willebrand factor from the endothelium in combination with acute phase behavior of factor VIII.⁹² Measurement of fibrinogen has been widely advocated as a useful tool for the diagnosis of DIC but in fact is not very helpful to diagnose DIC in most cases.⁶ Fibrinogen acts as an acute-phase reactant and despite ongoing consumption plasma levels can remain well within the normal range for a long period of time. In a consecutive series of patients, the sensitivity of a low fibrinogen level for the diagnosis of DIC was only 28% and hypofibrinogenemia was detected in very severe cases of DIC only. Sequential measurements of fibrinogen might be more useful and provide diagnostic clues.

Plasma levels of physiological coagulation inhibitors such as antithrombin or protein C are useful indicators of ongoing coagulation activation.^{30, 51} Antithrombin is the principal inhibitor of thrombin and may be readily exhausted during continuous thrombin generation. Plasma levels of antithrombin have been shown to be potent predictors for survival in patients with sepsis and DIC.⁵¹ Levels of protein C may also indicate the severity of the DIC. In patients with meningococcal septicemia, very low plasma levels of protein C are observed and this may play a pivotal role in the occurrence of purpura fulminans in these patients.^{93, 94} In fact, the plasma level of protein C also may be regarded as a strong predictor for the outcome in DIC patients.⁹⁵

Diagnostic management in clinical practice

For the diagnosis of overt DIC, a simple scoring system has been developed by the subcommittee on DIC of the International Society on Thrombosis and Hemostasis (ISTH).⁹⁶ The score can be calculated based on routinely available laboratory tests (ie, platelet count, prothrombin time, a fibrin-related marker [usually D-dimer], and fibrinogen). Tentatively, a score of five or more is compatible with DIC, whereas a score of less than five may be indicative but is not affirmative for nonovert DIC. For nonovert DIC, more refined scoring systems have been developed, which are currently being evaluated.⁹⁷ A recent study showed that the INR can be used (instead of PT prolongation), further facilitating international exchange and standardization.⁹⁸ By using receiver-operating characteristics curves, an optimal cutoff for a quantitative D-dimer assay was determined, thereby optimizing sensitivity and the negative predictive value of the system.⁸⁰ Prospective studies show that the sensitivity of the DIC score is 93% and the specificity is 98%.^{99, 31} Linking prognostic determinants from critical care measurement scores such as Acute Physiology and Chronic Health Evaluation (APACHE-II) to DIC scores is an important means to assess prognosis in critically ill patients. Similar scoring systems have been developed and extensively evaluated in Japan.¹⁰³ The major difference between the international and Japanese scoring systems seems a slightly higher sensitivity of the Japanese algorithm, although this may be due to different patient populations (Japanese series typically include relatively large numbers of patients with hematological malignancies).¹⁰⁴

SUPPORTIVE TREATMENT OF DIC

Key to the treatment of DIC is the specific and vigorous treatment of the underlying disorder. However, additional supportive treatment, specifically aimed at the coagulation abnormalities, may be required.¹⁰⁵

Low levels of platelets and coagulation factors may increase the risk of bleeding. However, plasma or platelet substitution therapy should not be instituted on the basis of laboratory results alone, but is only indicated in patients with active bleeding and in those requiring an invasive procedure or otherwise at risk for bleeding complications. The threshold for transfusing platelets depends on the clinical situation of the patient. Based on expert opinion, platelet concentrate is, in general, transfused to patients who bleed and who have a platelet count of <50 × 10⁹/l. In nonbleeding patients, a much lower threshold for platelet transfusion is used (usually <10-20 × 10⁹/l), which is based on randomized controlled trials in patients with thrombocytopenia following chemotherapy. It may be necessary to use large volumes of plasma to correct the coagulation defect. Coagulation factor concentrates such as prothrombin complex concentrate will overcome this obstacle, but these compounds lack essential factors such as factor V.

Based on the notion that DIC is characterized by extensive activation of coagulation, anticoagulant treatment may be a rationale approach. Experimental studies have shown that heparin can at least partly inhibit the activation of coagulation in DIC. A large trial in patients with severe sepsis supports a slight (nonsignificant) benefit of low dose heparin on 28-day mortality and underscored the importance of not stopping heparin in patients with DIC and abnormal coagulation parameters.¹⁰⁶ Theoretically, the most logical anticoagulant agent to use in DIC is directed against tissue factor activity. Phase II trials of recombinant TFPI in patients with sepsis showed promising results but a phase III trial did not show an overall survival benefit in patients that were treated with TFPI.

The use of agents that are capable of restoring the dysfunctional anticoagulant pathways in patients with DIC has been studied relatively intensively. Antithrombin concentrate has been available since the 1980s and most trials with this compound showed some beneficial effect in terms of improvement of laboratory parameters, however, none of the trials demonstrated a significant reduction of mortality.³¹ Later studies confirmed the ability of activated protein C to normalize coagulation activation during severe sepsis. Of note, activated protein C appears to be relatively more effective in higher disease severity groups and a prospective trial in septic patients with relatively low disease severity did not show any benefit of activated protein C.¹⁰⁹

In general, the use of prohemostatic agents in patients with DIC is not recommended, since this may theoretically worsen the coagulopathy. There are some reports of the successful use of prohemostatic agents, in particular recombinant factor VIIa in patients with DIC and life-threatening bleeding, but the efficacy and safety of this treatment in DIC is unknown. Interestingly, the administration of factor VIIa seemed not to result in an aggravation of the DIC in these patients.

CONCLUSION

Disseminated intravascular coagulation is a syndrome characterized by systemic intravascular activation of coagulation, leading to widespread (micro)vascular deposition of fibrin, thereby contributing to multiple organ dysfunction. The ongoing activation of coagulation may result in exhaustion of platelets and coagulation factors, which may cause bleeding. Invariably, DIC is seen as a complication of a variety of disorders, most commonly severe infection or inflammation, cancer, or trauma. A diagnosis of DIC can be made by a combination of routinely available laboratory tests, for which diagnostic algorithms have become available. Recent knowledge on important pathogenetic mechanisms that may lead to DIC has resulted in novel supportive therapeutic approaches to patients with DIC. Strategies aimed at the inhibition of coagulation activation may theoretically be justified and are being evaluated in clinical studies. These strategies comprise anticoagulant agents or agents that may restore physiological anticoagulant pathways.

Keywords

disseminated intravascular coagulation, thrombosis, bleeding, platelets, coagulation factors, antithrombin, protein C, tissue factor, fibrinolysis

Disclosure: The author declares no conflict of interest.

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