August 200545813621372Original Article
Blackwell Science, LtdOxford, UKTRFTransfusion0041-11322005 American Association of Blood Banks
PCT-FFP FOR COAGULATION FACTOR DEFICIENCIESDE ALARCON ET AL.
BLOOD COMPONENTS Fresh frozen plasma prepared with amotosalen HCl (S-59) photochemical pathogen inactivation: transfusion of patients with congenital coagulation factor deficiencies Pedro de Alarcon, Richard Benjamin, Marion Dugdale, Craig Kessler, Rinah Shopnick, Peter Smith, Thomas Abshire, Julie Hambleton, Prasad Matthew, Idith Ortiz, Alice Cohen, Barbara A. Konkle, Michael Streiff, Martin Lee, David Wages, and Laurence Corash
BACKGROUND: Photochemical treatment (PCT) with amotosalen HCl (S-59) was developed to inactivate pathogens and white blood cells in plasma (PCT-FFP) used for transfusion support. STUDY DESIGN AND METHODS: An open-label, multicenter trial was conducted in patients with congenital coagulation factor deficiencies (factors [F]I, FII, FV, FVII, FX, FXI, and FXIII and protein C) to measure the kinetics of specific coagulation factors, hemostatic efficacy, and safety of PCT-FFP. Posttransfusion prothrombin time (PT), partial thromboplastin time (PTT), and clinical hemostasis were evaluated before and after PCT-FFP transfusions. RESULTS: Thirty-four patients received 107 transfusions of PCT-FFP for kinetic studies or therapeutic indications (mean dose, 12.8 ± 8.5 mL/kg). Incremental factor recoveries ranged from 0.9 to 2.4 IU per dL per IU per kg (FII, FV, FVII, FX, FXI, and protein C). Mean pretransfusion PT (20.7 ± 22.2 sec) corrected after PCT-FFP (13.8 ± 2.4 sec, p < 0.001). Mean pretransfusion PTT (51.2 ± 29.3 sec) corrected after PCT-FFP (32.0 ± 5.1 sec, p < 0.001). Thirteen patients required 77 transfusions for therapeutic indications. PCT-FFP provided effective hemostasis and was well tolerated. CONCLUSIONS: Replacement coagulation factors in PCT-FFP exhibited kinetics and therapeutic efficacy consistent with conventional FFP.
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espite continued improvements in screening and testing of donated blood, there is residual risk of transfusion-transmitted infections.1 Although the per donation risks for selected pathogens for which tests are in place are very low, patients may require multiple blood components, thus increasing
ABBREVIATIONS: AE(s) = adverse event(s); MB = methylene blue; PCT = photochemical treatment; PCT-FFP = amotosalen HCl and plasma treated with amotosalen HCl and long-wavelength ultraviolet light; PT = prothrombin time; PTT = partial thromboplastin time. From the Department of Pediatric Hematology, University of Virginia, Charlottesville, Virginia; the Department of Pathology, Brigham & Women’s Hospital, Boston, Massachusetts; the University Oncology & Hematology Center, University of Tennessee, Memphis, Tennessee; the Georgetown University Medical Center, Washington, DC; the Hemophilia & Thrombosis Center of Nevada, Las Vegas, Nevada; the Department of Pediatrics, Rhode Island Hospital, Providence, Rhode Island; the Department of Pediatrics, Emory University, Atlanta, Georgia; the Department of Medicine, University of California, San Francisco, California; the Department of Pediatrics, University of New Mexico, Albuquerque, New Mexico; the Center for Investigational Clinics, University of Puerto Rico, Rio Piedras, Puerto; the Newark Beth Israel Medical Center, Newark, New Jersey; Hematology/ Oncology, University of Pennsylvania, Philadelphia, Pennsylvania; the Department of Medicine, Johns Hopkins University, Baltimore, Maryland; the Department of Biostatistics, University of California, Los Angeles, California; and Cerus Corporation, Concord, California. Address reprint requests to: Laurence Corash, MD, Cerus Corporation, 2411 Stanwell Drive, Concord, CA 94520; e-mail:
[email protected]. Received for publication December 8, 2004; revision received January 20, 2005, and accepted January 31, 2005. doi: 10.1111/j.1537-2995.2005.00216.x TRANSFUSION 2005;45:1362-1372.
PCT-FFP FOR COAGULATION FACTOR DEFICIENCIES
the risk of infection on a per-transfusion basis. Even the most sensitive tests may not detect all infectious donors.2 More importantly, new infectious agents, such as West Nile virus, may enter the blood donor population before sensitive tests can be implemented.3 To improve transfusion safety, technologies have been developed to treat plasma for inactivation of pathogens that may remain undetected before transfusion.4,5 A photochemical treatment (PCT) with the psoralen compound, amotosalen HCl (formerly known as S-59), and long-wavelength ultraviolet light (UVA)6,7 has been developed and shown to inactivate a broad spectrum of viruses, bacteria, protozoa, and residual white blood cells (WBCs) in platelets (PLTs) and plasma8 with preservation of physiologic function.9,10 Fresh frozen plasma (FFP) is indicated for the treatment of selected congenital coagulopathies, acquired complex coagulopathies, and therapeutic plasma exchange.11 Patients with these disorders may require large volumes of FFP during a single treatment episode, and many of these patients will require repeated FFP transfusions resulting in exposures to many different blood donors. In the development of processes to inactivate infectious agents that may contaminate donor plasma, it is critical to evaluate the therapeutic efficacy and safety of treated plasma. Preclinical studies demonstrated high safety margins for amotosalen HCl and plasma treated with amotosalen HCl and UVA light (PCTFFP).12-15 Subsequently, studies in healthy subjects were performed with PCT-FFP to measure the clearance of residual amotosalen HCl and the kinetics of FVII replacement after anticoagulation with warfarin.9 In this study, the posttransfusion kinetics and hemostatic efficacy of specific coagulation factors in PCT-FFP were evaluated in patients with a spectrum of congenital coagulation factor deficiencies treated with plasma transfusion.
MATERIALS AND METHODS Study design A multicenter, single-arm, open-label, Phase III clinical trial was conducted at 13 sites in the United States and Puerto Rico to evaluate the efficacy and safety of PCT-FFP. Analysis of the study was conducted on an intent-to-treat basis. The study had two objectives: 1) to characterize the posttransfusion kinetics of specific replacement coagulation factors in PCT-FFP during steady-state conditions and 2) to assess the hemostatic efficacy of PCT-FFP administered for prophylaxis of invasive procedures or to treat active bleeding. The study was designed to enroll a spectrum of patients with congenital coagulopathies treated with FFP. Patients were recruited from the Hemophilia Research Society registry with congenital deficiencies of either FI (fibrinogen), FII, FV, FVII, FX, FXI, or FXIII who required therapeutic or prophylactic transfusion of FFP. Patients with protein C and protein S deficiencies also
were eligible for enrollment. Owing to the rarity of patients with these factor deficiencies, an open-label study design was selected. There were no concurrent control groups in this study. The results of PCT-FFP transfusions were compared to reported data for conventional FFP. All patients were treated in accordance with each institution’s standard of care, with informed consent in accordance with local investigational review board procedures. Patients of either sex, age 2 years or older (including pregnant patients), diagnosed with at least one congenital coagulation factor deficiency were eligible for enrollment. The coagulation deficiency was confirmed by a screening functional coagulation test before study transfusions. Patients were excluded for the following conditions: receipt of an investigational blood product within 45 days before enrollment, receipt of a blood component proximate to PCT-FFP transfusion, or evidence of a specific coagulation factor inhibitor. No other concomitant therapies were excluded.
Transfusions for elective kinetic studies Each patient received at least one elective PCT-FFP transfusion for measurement of posttransfusion recovery and the kinetics of clearance of the specific deficient coagulation factor. Transfusions for these kinetic studies were based on established procedures for measurement of coagulation factor kinetics16,17 and were administered when patients were not bleeding or subjected to hemostatic challenges. PCT-FFP was transfused at a suggested dose of 15 to 20 mL per kg at a rate of 10 to 15 mL per min. Physicians were permitted to adjust the dose and rate of FFP administration per individual patient requirements. A blood sample was obtained within 8 hours before transfusion to provide pretransfusion values for the prothrombin time (PT), partial thromboplastin time (PTT), and the specific relevant coagulation factor. Posttransfusion blood samples were collected during the first hour after transfusion at 10 to 15, 30, and 60 minutes after transfusion. Depending on the specific coagulation factor deficiency, four more blood samples were drawn over a period equal to 2 to 2.5 t1/2 of the relevant coagulation factor, based on reported values. Determination of posttransfusion coagulation factor kinetics was based on use of all posttransfusion blood samples. Posttransfusion samples drawn 60 minutes after transfusion were used to assess response of the PT, PTT, and to calculate immediate posttransfusion recovery of the relevant coagulation factor.
Transfusions for clinical indications During the study, enrolled patients received PCT-FFP for clinical indications for either prophylaxis against hemostatic challenges or to treat active bleeding. PCT-FFP was administered at the same suggested dose and rate as for Volume 45, August 2005
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kinetic studies, with physician based adjustments for specific patient conditions. A blood sample was obtained within 8 hours before transfusion to provide pretransfusion values for the PT, PTT, and the specific relevant coagulation factor. The PT, the PTT, and the specific deficient coagulation factor were measured within 1 hour after transfusion to determine the effect of replacement PCTFFP on global tests of coagulation and to calculate recovery of the relevant coagulation factor. For patients who received transfusion of PCT-FFP for therapeutic or prophylactic purposes, clinical hemostatic evaluations were performed before and after transfusion.
Additional study procedures Measurement of posttransfusion amotosalen HCl (S-59) levels. For all patients, samples for measurement of amotosalen HCl plasma levels were obtained with the first coagulation sample at completion of transfusion and 24 hours after transfusion. Detection of antibodies directed against potential amotosalen HCl–related neoantigens. For all enrolled patients, a blood sample was drawn before the first transfusion and at the end of the study to test for antibodies to putative amotosalen HCl neoantigens. The assay was based on a sensitive enzyme-linked immunosorbent assay (ELISA) previously used in a Phase III clinical trial of PCT PLTs.10 Safety assessments. For all patients, adverse events (AEs) were recorded for 1 week after each transfusion, and serious AEs were recorded through 2 weeks after transfusion. Chemistry and hematology panels were obtained 24 hours after transfusion. Acute transfusion reactions were defined as AEs within 6 hours of plasma transfusion. AEs were reported and analyzed with established criteria.18
Preparation of PCT-FFP The investigational product, PCT-FFP (Cerus Corporation, Concord, CA), consisted of FFP prepared with PCT with the synthetic psoralen S-59 (amotosalen HCl; 3-[(2-aminoethoxy)methyl]-2,5,9-trimethyl-7H -furo[3,2g][1]benzopyran-7-one hydrochloride). Single units of plasma used for this study were prepared from wholeblood collections or by approved plasmapheresis methods. Donors met all of the AABB’s qualifications for volunteer blood donors. Approximately 15 mL of amotosalen HCl was mixed with approximately 250 mL of plasma, and a preillumination sample was collected. The remaining plasma was then illuminated with UVA light (320-400 nm) with a 3.0 J per cm2 treatment and transferred to an amotosalen HCl compound adsorption device. The treated plasma was incubated at room temperature for 1 hour with agitation to reduce the residual levels of amotosalen HCl, and transferred to a final con1364
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tainer for freezing and storage within 8 hours of collection. PCT-FFP was prepared at six regional blood centers to support this trial, studies of patients with acquired coagulopathy, and a trial of therapeutic plasma exchange. PCTFFP units were stored frozen at -18∞C for up to 1 year, shipped from processing centers to patient-care sites as required for transfusion, thawed, and administered according to standard blood banking procedures at each clinical site. Approximately 9000 PCT-FFP units were prepared during the course of these studies. Before freezing PCT-FFP units, samples for coagulation testing were removed, frozen at -35∞C or colder, and stored at -18∞C or colder for up to 1 year from the date of collection. Coagulation factor activities were measured in 275 units randomly selected from the PCT-FFP inventory with standard laboratory methods (Cerus Corporation). Reference range values and mean values for untreated plasma were determined by measuring coagulation factor activities with aliquots obtained from plasma units before PCT processing (Table 1). The reference range for conventional plasma was expressed as the 95 percent central interval (2.5-97.5 percentile) of the distribution for the values of untreated plasma. Antithrombotic protein activities were measured in 14 units of PCT-FFP (Table 2). Samples of untreated plasma were used to obtain mean values for plasma before PCT. Reference range values for antithrombotic proteins were obtained from literature citations (Table 2).
TABLE 1. Coagulation factor activity of PCT-FFP Factor Fibrinogen FII FV FVII FVIII FIX FX FXI FXIII
Untreated FFP 272 ± 30 94 ± 15 131 ± 27 134 ± 27 165 ± 27 115 ± 11 95 ± 9 92 ± 17 108 ± 11
PCT-FFP 218 ± 47 91 ± 13 109 ± 18 92 ± 24 117 ± 34 93 ± 19 89 ± 16 94 ± 18 107 ± 11
Reference range* 183-403 79-137 82-160 66-180 73-253 68-150 73-136 71-156 65-165
* Coagulation factor activity was measured with standard functional assays for retained samples of plasma prior to treatment and from PCT-FFP taken from 274 to 277 randomly selected units after preparation and before freezing and storage. Approximately 9000 units of PCT-FFP were prepared during the course of the study. The mean ± SD was calculated and compared to a reference range for conventional FFP. Activity of fibrinogen was expressed as mg per dL and as IU per dL for all other factors. FXIII activity was based on analysis of 14 samples of untreated plasma and PCT-FFP units. Reference ranges for conventional FFP were determined with samples obtained from plasma units (n = 274-277) obtained after collection and prior to PCT processing and freezing. The reference range was defined at the 95 percent central interval for coagulation factor activity in plasma derived from citrate phosphate dextrose-A whole blood with approved collection systems (Transfusion Therapies, Baxter Healthcare Corporation, Deerfield, IL). All PCT-FFP units were frozen within 8 hours of collection.
PCT-FFP FOR COAGULATION FACTOR DEFICIENCIES
TABLE 2. Antithrombotic protein factor activity of PCT-FFP* Factor a2-Antiplasmin Protein C Protein S Antithrombin
Untreated FFP 95 ± 5 105 ± 19 113 ± 9 93 ± 6
PCT-FFP 85 ± 6 105 ± 18 116 ± 9 85 ± 5
Reference range 72-132 58-164 56-168 80-125
* Antithrombotic protein activities were measured with standard assays for retained samples of plasma before treatment and from PCT-FFP taken from 14 plasma units after preparation and before freezing. Activity was expressed as IU per dL. The mean ± SD was calculated and compared to reported reference ranges for conventional FFP.47
Analysis of response to PCT-FFP transfusion Assay methods for coagulation factors. Posttransfusion blood samples were collected in conventional citrate anticoagulant and processed with standard methods for preparation of plasma in each center’s clinical laboratory. All measurements were performed with conventional coagulation factor assays in study center clinical laboratories with appropriate certification for the conduct of coagulation factor assays. Fibrinogen levels were measured with the Clauss method for determination of clottable fibrinogen (mg/dL). FII, FV, FVII, F IX, and FXI were assayed by a one-stage clotting endpoint assay with factor-deficient substrate plasma. FXIII was assayed with a chromogenic assay (Dade Behring, Deerfield, IL). For FXIII samples with activity below 0.1 IU per dL, an ELISA-based method was used to determine FXIII protein levels. Protein C levels were determined with an aPTT-based assay. PT and PTT assays were conducted with the conventional methods in use at each study center laboratory. Posttransfusion coagulation factor activity in response to PCT-FFP transfusion. The primary efficacy endpoint was the incremental response of coagulation FI, FII, FV, FVII, FX, FXI, FXIII, and protein C after transfusion of PCT-FFP. Incremental coagulation factor recoveries were calculated based on the peak coagulation factor response within the first hour after transfusion based on a weight-adjusted dose of PCT-FFP with the formula16 Incremental factor recovery = Peak incremental response (IU/dL ) . Total factor dose (IU) / body weight (kg )
(1)
Coagulation factor recoveries also were calculated as the proportion of the infused factor dose recovered in the first hour after transfusion based on the formula Factor recovery = Peak incremental response (IU/dL ) ¥ PV (dL ) . (2) Total factor dose (IU)
Plasma volume (PV) was estimated based on patient weight, where PV = blood volume - red blood cell (RBC) mass. Blood volume was estimated at 66.5 mL per kg and RBC mass was estimated at 25 mL per kg. Kinetics of transfused coagulation factors. Additional efficacy endpoints for patients who received PCTFFP for factor kinetic assessments included the t1/2.16 Specific coagulation factor activities were calculated based on measurements at up to seven time points after transfusion, which encompassed 2 to 2.5 t1/2 of the relevant coagulation factor. Coagulation factor t1/2 were calculated with a series of piecewise log-linear regressions with robust estimation techniques.16,17,19 The factor t1/2 was determined from the slope of the regression function for the phase of interest. Results from the kinetic studies were analyzed to fit the regression function and to determine t1/2. Posttransfusion PT and PTT response to PCT-FFP transfusion. The PT and PTT are global tests of coagulation function used to manage FFP transfusion therapy. Measurements of the change in the PT and PTT in response to PCT-FFP transfusion were secondary efficacy variables for all patients. Clinical hemostatic assessments. Pre- and posttransfusion hemostatic evaluations were conducted for patients who received PCT-FFP for prophylaxis or therapy of an active bleeding event. Potential anatomic sites of bleeding were examined and scored for minor and major bleeding with a modified version of the World Health Organization (WHO) bleeding scale.10,20 Bleeding at these sites was assessed for the 24-hour period before transfusion through 24 hours after transfusion. In addition, bleeding identified by the investigators as clinically significant was recorded as an adverse hemorrhagic event with established reporting criteria.18
Statistical analysis All calculations of coagulation factor activities were performed in accordance with ISTH recommendations and compared to relevant medical literature for coagulation factor activity in conventional FFP. All other laboratory results were summarized with summary statistics that included the mean, standard deviation, and range. AEs were coded according to the COSTART dictionary and by body system and preferred term. The frequency of each AE was calculated and reported as a percentage of patients treated. Owing to the rarity of these coagulation factor deficiencies, no formal sample size calculations were generated. Statistical analyses were conducted with a two-sided paired t test with a 5 percent level of significance. Missing data were not estimated or imputed in statistical analyses. Volume 45, August 2005
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RESULTS Transfusion experience Thirty-four patients were enrolled. Of these, 24 patients initially received elective PCT-FFP transfusions for coagulation factor kinetic studies. Three of these patients received subsequent PCT-FFP transfusions for therapeutic indications. Ten patients initially received PCT-FFP transfusions for prophylaxis or therapy for active bleeding. In this cohort, three patients subsequently received elective PCT-FFP transfusions for elective factor kinetics. All patients in the study who received transfusions were included in the safety analysis, and all patients completed the trial for safety monitoring. The 34 enrolled patients received a total of 107 transfusions with PCT-FFP. Thirty-three patients received 77 transfusions with sufficient data for calculation of incremental factor recoveries (Table 3). Sufficient data were available from 22 transfusions in 22 patients to permit measurement of factor kinetics (Table 6). Among patients who received PCT-FFP for prophylaxis or therapy to treat an active bleeding condition, data were available from 77 transfusions to examine the PT and PTT response (Table 5) and clinical hemostasis (Table 7).
Patient demographics, baseline characteristics, and transfusion exposure The mean age for enrolled patients was 33.4 years (range, 3-77 years) and 22 of 34 were female. Baseline vital signs
TABLE 3. Incremental dose and proportional recovery of coagulation factors after transfusion of PCT-FFP* Factor Fibrinogen FII FV FVII FX FXI FXIII Protein C
N (T) 2 (3) 3 (3) 7 (24) 3 (3) 1 (1) 11 (37) 3 (3) 3 (3)
Recovery (IU/dL/U/kg) 0.79 ± 0.373 1.33 ± 0.091 2.03 ± 0.358 0.90 ± 0.356 2.37 1.69 ± 0.358 2.26 ± 0.346 0.90 ± 0.232
Recovery (%) 32.7 ± 15.5 56.2 ± 4.1 87.1 ± 15.5 53.2 ± 3.2 103.8 72.2 ± 16.4 93.1 ± 16.3 41.2 ± 13.6
Reference values (%) 50 50-100 50-100 100 50-100 100 50-100 No data
* Thirty-three patients received 77 PCT-FFP transfusions for determination of posttransfusion coagulation factor recovery. The number of patients studied with each type of factor deficiency (N) and the number of transfusions (T) used to calculate dose adjusted incremental factor recovery (IU/dL/IU/ kg body weight) and unadjusted recovery are indicated. Unadjusted recovery was determined with an estimated blood volume for each patient based on body weight, total dose of replacement factor infused, and incremental coagulation factor activity at the maximal level within the first 60 minutes after transfusions (after transfusion - before transfusion) and expressed as the proportion of the administered dose (%). Means ± SD are presented for all factors with multiple determinations. Reference values for proportional posttransfusion recovery were obtained from reported sources for comparison.24
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(blood pressure, heart rate, respiratory rate, and temperature) were within normal limits. Twenty-seven patients (79.4%) were routinely treated with medications, including analgesics, psychoanaleptics, nasal preparations, antihistamines, or vitamins. Patients with documented congenital deficiencies of coagulation factors FI, FII, FV, FVII, FX, FXI, and FXIII, and/or protein C were enrolled. One patient presented with combined deficiencies of FV and FVIII, and one patient was enrolled with a FV New Brunswick variant deficiency. Five patients were enrolled without screening for factor inhibitors. Of the remaining 29 patients, none had documented factor inhibitors at enrollment. The mean volume of PCT-FFP administered with elective transfusions (n = 29) was 17.1 ± 4.0 mL per kg. For transfusions to support therapeutic indications (n = 77), the mean volume of PCT-FFP transfused was 11.2 ± 9.2 mL per kg. For all transfusions (n = 107), the mean volume of PCT-FFP transfused was 12.8 ± 8.5 mL per kg.
Posttransfusion factor recovery Incremental factor recovery (IU/dL/IU/kg body weight) and proportional factor recovery were determined for 25 elective transfusions and 52 transfusions in support of therapeutic indications (Table 3). Proportional factor recovery data were compared with reported values.21 Four patients received five or more transfusions of PCT-FFP for kinetics and therapeutic indications (Table 4). No coagulation factor inhibitors were observed in any patients exposed to multiple units of PCT-FFP.
Response of the PT and PTT to PCT-FFP transfusions The responses of the PT and the PTT were determined for all transfusions (Table 5). As expected, patients exhibited large variation in the level of factor deficiencies resulting in large variation of baseline PT and PTT values. The response to PCT-FFP transfusion was examined for all transfusions (n = 107), for elective transfusions for kinetics
TABLE 4. Coagulation factor proportional recovery in response to multiple transfusions of PCT-FFP* Patient: factor (number of transfusions) 531-301: FV (12) 541-301: FV (5) 533-301: FXI (7) 541-302: FXI (18)
Recovery 90 (70-107) 103 (94-114) 55 (43-62) 78 (62-108)
* Four patients had five or more transfusions of PCT-FFP during the course of the study. Patient study number, type of factor deficiency, and the number of transfusions administered are indicated. Mean recovery for each patient and the range of observed values are expressed as the proportion of the infused factor dose (%) recovered at the peak value within 60 minutes after transfusion.
PCT-FFP FOR COAGULATION FACTOR DEFICIENCIES
TABLE 5. Response of the PT and PTT to transfusions of PCT-FFP in patients with congenital factor deficiencies* Patient group All transfusions PT PTT Elective transfusions PT PTT Therapeutic transfusions PT PTT FII, FV, FVII, and FX replacement PT FII, FV, FX, and FXI replacement PTT
Pre-PCT-FFP transfusion
Post-PCT-FFP transfusion
p value†
Reference range‡
20.7 ± 22.18 (n = 80) [10.8-200] 51.2 ± 29.32 (n = 81) [22.3-173.0]
13.8 ± 2.39 (n = 67) [11.0-23.8] 32.0 ± 5.10 (n = 67) [22.8-42.0]
