Prepooled cryoprecipitate for treatment of hemophilia A

Lyophilized factor VIII concentrates, because of their convenience, are the most commonly used products for providing antihemophilic factor to patients with hemophilia A. Heightened concerns about associated complications indicate that cryoprecipitate usage will increase in the immediate future. We present a protocol for making cryoprecipitate more usable by "prepooling" individual units of cryoprecipitate in an "open" system. Our experience indicates this approach is safe, effective and acceptable to patients, including those on home therapy programs.     

The Effect of Cryoprecipitate Volume on Factor VIII Content

Two hundred and thirty-five cryoprecipitates were sampled and assayed for Factor VIII activity. Two hundred and twenty-five of these were given to 11 patients with hemophilia on 53 occasions. The patients' Factor VIII levels were measured just before and 15 minutes after infusion. The rise in Factor VIII activity averaged 97 per cent of the expected rise calculated from the cryoprecipitate assays and plasma volume estimates. The mean Factor VIII content per bag of cryoprecipitate was 61 units. There is a highly significant probability that the smallest volume cryoprecipitates had significantly less Factor VIII than average and that the largest volume cryoprecipitates had significantly more Factor VIII than the average.     

The influence of donor characteristics and preparation methods on the potency of human cryoprecipitate

An investigation of the influence of donor characteristics and preparative procedural variation on the potency of human cryoprecipitate was carried out on 30 whole blood and 139 plasmapheresis donors. Recovery of plasma Factor VIII in cryoprecipitate ranged from 11.2 to 89.4 per cent (average, 38 +/− 18%). The Factor VIII content of bags of cryoprecipitate ranged from 29 to 379 units (average, 111 +/− 77 units). No difference existed between whole blood donors and regular plasmapheresis donors. The only donor characteristic which was related to the potency of cryoprecipitate was the plasma concentration of Factor VIII which varied as much as sixfold on repeated visits of the same donor. The findings indicate that, within the limitations imposed by the regulations of the American Association of Blood Banks and the practicalities of an ordinary blood bank workload, no donor characteristic or variation in preparative procedures was of predictive value in obtaining cryoprecipitate of high potency. The potency of cryoprecipitate from individual donors appears to be related to factors inherent in the donor plasma itself.     

Reduction of Factor VIII Activity in Cryoprecipitate Obtained from Acidified Plasma

The rise in Factor VIII levels following paired infusions of cryoprecipitate prepared from normal and acidified plasma was studied in a group of patients with hemophilia A. In four of the six patients there was a greater than 50 per cent reduction in the in vivo recovery of Factor VIII following infusion of the acidified product. The pattern of Factor VIII disappearance over the first hour after infusion was similar in both groups. This implies that the loss in Factor VIII activity in cryoprecipitate prepared from acidified plasma cannot be restored following in vivo buffering.     

Preparation of factor VIII concentrates by cryoprecipitate sedimentation

A new method for factor VIII concentrate preparation is proposed. The method is the collection of cryoprecipitate after sedimentation at 4 C in a jacketed glass vessel. The final product is standardized (4.0 +/- 0.3 factor VIII units per ml) and can be infused in hemophilic patients regardless of their ABO blood type. Factor VIII recovery (52.2 +/- 6.4%) is comparable to other processes.     

A comparison of factor VIII activity in cryoprecipitates prepared from ACD and CPD plasma

In an attempt to develop a method to produce a cryoprecipitate with a predictable Factor VIII potency, several variables were studied and statistically analyzed. These included the hematocrit, age, blood group and Rh type of the donor; possible epinephrine release; the degree of lipemia, volume, pH and Factor VIII activity of the donor plasma; the volume of cryoprecipitates, its Factor VIII activity and the per cent yield; and the Factor VIII activity of the supernatant plasma. Cryoprecipitates were prepared by the method of Pool and Shannon from the plasmas of 40 random male donors, half the bloods being drawn in ACD and half in CPD. None of the variables had a significant influence on the per cent yield of activity, although the mean values obtained suggest that the per cent yield of Factor VIII activity in the cryoprecipitates prepared from CPD plasmas is higher than in those prepared from ACD plasma. Although the per cent yield of Factor VIII in cryoprecipitates prepared from CPD plasma is not significantly different from that of ACD plasma, the mean total units of Factor VIII activity is significantly higher in CPD cryoprecipitates. It also was confirmed that a higher per cent Factor VIII activity in the donor results in a relatively higher activity in the cryoprecipitate. The data indicate that if CPD plasma collected from donors having above a certain minimum per cent activity were used for cryoprecipitate production, one could be assured of having a minimum of 100 units of Factor VIII activity per bag.     

Dual vectors expressing murine factor VIII result in sustained correction of hemophilia A mice

Hemophilia A is a sex-linked disorder that results from a deficiency of functional factor VIII and is currently treated by protein replacement therapies. Within the past decade, gene therapy efforts have come to the forefront of novel therapeutics. In this work, a dual-vector approach was employed in which recombinant adeno-associated viral (rAAV) vectors expressing the heavy and light chains of the murine factor VIII gene were delivered either intramuscularly or intravenously to a mouse model of hemophilia A. From in vitro work, it was determined that coinfection with both vectors is required as heterodimerization of the heavy and light chains occurs intracellularly. In vivo, therapeutic levels of factor VIII expression were achieved throughout the duration of the study (22 weeks). Intravenous and intramuscular delivery resulted in a maximal average expression of 31.4 +/- 6.4 and 29 +/- 6.5% of normal murine factor VIII levels, respectively. Western blots of cryoprecipitate as well as immunostaining of injection sites with an anti-murine factor VIII light chain antibody also confirmed the expression of factor VIII. Because the murine form of the gene was used in the mouse model, less than 1 Bethesda unit of inhibitors was noted. This work demonstrates the feasibility of using rAAV vectors for the long-term treatment of hemophilia A.     

Effect of circulating immune complexes on transfusional therapy in patients with hemophilia or von Willebrand's disease

Circulating immune complexes were examined in patients with hemophilia or von Willebrand's disease in order to determine the immediate or long- term side effects after transfusion. The conglutinin binding assay which allows quantitation of C3bi-bearing immune complexes was used for 82 patients with hemophilia A. Immune complexes were detected in 37 (45%) of these cases prior to transfusion. Immune complexes also were detected in four of 11 patients with hemophilia A and factor VIII inhibitors, in five of 11 patients with hemophilia B, and in three of 10 patients with von Willebrand's disease. The levels of circulating immune complexes in 21 patients with hemophilia A and seven with von Willebrand's disease significantly increased 24 hours after concentrate or cryoprecipitate transfusions. Purified immune complexes from three patients with hemophilia A were shown to contain IgG, IgM, and complement components. No factor VIII coagulant or antigenic protein or fibrinogen was identified in the immune complexes using specific antisera. Side effects immediately after transfusion were not associated with immune complexes. The levels of factor VIII or IX after transfusion were not particularly decreased in relation to the presence of immune complexes. Finally, the presence of circulating immune complexes in the patients studied did not correlate with the number of transfusions, the units of concentrates injected, the presence of HBsAg or HbsAb, the levels of plasma aspartate transferase, or the presence of rheumatoid factor. Proteinuria was absent in all the patients studied.     

The pharmacology of a new pasteurized antihemophilic factor concentrate derived from human blood plasma

The pharmacology of a new pasteurized factor VIII (FVIII) concentrate derived from human blood plasma was studied in 23 adults with hemophilia A. In Part 1 of the study involving six nonbleeding subjects, the mean increase in FVIII activity was 1.43 +/- 0.34 U per ml 10 minutes after an intravenous dose of 50 U per kg. The intravascular survival kinetics in these six patients showed a biphasic decay curve with an initial mean half-life of 5.1 +/- 1.2 hours probably representing early redistribution, and a late half-life of 13.3 +/- 4.9 hours. In Part 2 of the study, the activity at 10 minutes was measured in another 17 patients, as well as in one patient already studied in Part 1. The mean increase in activity with the 24 observations was 1.13 +/- 0.37 U per ml with a mean FVIII dosage of 51.0 +/- 2.6 U per kg of body weight. Only one patient had an allergic reaction, which did not recur when the patient was given a second lot.     

Survival of 125iodine-labeled Factor VIII in normals and patients with classic hemophilia. Observations on the heterogeneity of human Factor VIII.

Radiolabeled human Factor VIII was used to study its survival in normals and patients with classic hemophilia, and to study the heterogeneity of Factor VIII; Purified Factor VIII was radiolabeled with 125iodine (125I-VIII) without loss of its structural integrity. The survival of 125I-VIII was studied in six normals and six hemophiliacs of whom four of the hemophiliacs had received transfusions with normal cryoprecipitate before the 125I-VIII infusion. No significant difference was observed between the disappearance of Factor VIII coagulant activity and radioactivity in these hemophiliacs. 125I-VIII in plasma showed a biphasic disappearance with an average t1/2 of 2.9 +/- 0.4 h (SEM) for the first phase and 18.6 +/- 0.7 h (SEM) for the second phase, respectively. The survival of 125I-VIII was similar comparing normals and hemophiliacs. The highest molecular weight forms of Factor VIII disappear more rapidly than the lower molecular weight ones. This was established by analysis of the fractions obtained by gel chromatography of plasma collected at several times after infusion and by analysis of the in vivo disappearance of three subfractions of Factor VIII. The fraction of 125I-VIII binding to platelets in the presence of ristocetin (containing the highest molecular weight forms of Factor VIII including the ristocetin cofactor) represented about 50% of the radioactivity present in plasma after infusion and showed a t 1/2 of 11.7 +/- 0.9 h (SEM) for the second phase. The fraction, which was recovered in cryoprecipitate of the recipient's plasma, represented about 90% of the initial radioactivity and showed a t 1/2 of 16.3 +/- 0.8 h (SEM) for the second phase. The fraction of 125I-VIII remaining in the cryosupernatant plasma (containing low molecular weight forms of Factor (VIII) showed a t 1/2 of 27.2 +/- 1.1 h (SEM). The first phase of the disappearance of 125I-VIII is caused in part by the disappearance of the highest molecular weight forms, which are possibly removed by the reticuloendothelial system.     

Comparative study of the efficacy and safety of intranasal DDAVP administered to normal blood donors

A study of the efficacy and safety of intranasal 1-deamino-8-D-arginine vasopressin (DDAVP; 300 micrograms) in normal blood donors was carried out in a double-blind, controlled, comparative study. In addition, the effect of heparin or citrate anticoagulation of blood on the recovery of factor VIII (FVIII) in plasma, cryoprecipitate, and a FVIII concentrate was assessed. Citrated plasma from placebo (CP) or DDAVP-treated donors (CD) contained 1103 +/- 73 and 1470 +/- 141 units per liter of FVIII, respectively (p less than 0.01), whereas the heparinized plasma from placebo (HP) or DDAVP-treated donors (HD) contained 1328 +/- 130 (p less than 0.01) and 2023 +/- 358 units per liter (p less than 0.01), respectively. The FVIII could be recovered in both cryoprecipitate and cold-reprecipitated cryoprecipitate (CRC) fractions. DDAVP treatment improved FVIII recovery by 41 percent in the concentrate from citrated plasma (p less than 0.01) and by 127 percent in that from heparinized plasma (p less than 0.01). The specific activity of concentrates from the CP, CD, HP, and HD groups was 0.95 +/- 0.1, 1.4 +/- 0.1 (p less than 0.01), 0.9 +/- 0.1, and 1.47 +/- 0.2 U per mg of protein (p less than 0.01), respectively. The stability of the final product was the same, regardless of the method of treatment or collection. The side effects of intranasal treatment were mild and transient and occurred with similar frequency in both placebo and DDAVP treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Unsuitability of Outdated Blood for Making Therapeutically Effective Cryoprecipitate

The factor VIII activity in cryoprecipitates prepared from plasma separated from whole blood stored for 21 days was examined. The concentration of factor VIII in plasma separated from outdated blood averaged less than 25 per cent of that found in fresh plasma, and the cryoprecipitate prepared from outdated plasma averaged only 23 factor VIII units per bag. A significant amount of free hemoglobin was present in cryoprecipitates prepared from outdated plasma. Factors in the decrease in plasma factor VIII activity in stored whole blood include both degradation and a change in the cryoprecipitability of factor VIII itself. The findings indicate that cryoprecipitate prepared from plasma separated from outdated blood is of little therapeutic effectiveness.     

Immunologic studies in asymptomatic hemophilia patients. Relationship to acquired immune deficiency syndrome (AIDS).

Asymptomatic hemophilia patients receiving Factor VIII concentrate were found to have normal natural killer (NK) cells and B cells, and an inverted T helper/suppressor ratio due to an increase in cells of T suppressor phenotype. In contrast, a hemophilia patient with acquired immune deficiency syndrome (AIDS) exhibited nonfunctional NK cells, low B cells, and an inverted T helper/suppressor ratio due to very low numbers of T helper cells. Hemophilia patients on cryoprecipitate therapy exhibited normal immune parameters. A high percentage of hemophilia patients on both treatments had antibody to hepatitis B virus. The isolated finding of elevated levels of T suppressor cells in hemophilia patients receiving Factor VIII concentrate has not been recognized as an early indicator of impending AIDS, and longitudinal studies will be required to determine its clinical significance.     

Chronic liver dysfunction in multitransfused hemophiliacs

Liver dysfunction and exposure to the hepatitis B antigen were assessed by serum transaminase (SGPT and SGOT) levels and HBsAg and anti-HBs during a three year period in a group of 118 patients with factor VIII or factor IX deficiency. The 107 HBsAg negative patients were divided into four groups according to their mode of therapy. Persistently abnormal transaminase values were present in 51 per cent of patients with a large exposure to factor VIII concentrates, in 43 per cent with a small factor VIII exposure and in 37 per cent exposed to prothrombin complexes. This was contrasted with abnormalities in 8 per cent of patients treated only with cryoprecipitate. The incidence and degree of serum transaminase abnormality appeared independent of a past history of jaundice. All patients without persistent antigenemia who had been treated with pooled plasma products showed antibodies to HBsAg. High titer anti-HBs prior to initial fraction therapy appeared protective against jaundice. The eleven patients with persistent antigenemia had significantly higher transaminase levels than the HBsAg negative group.     

Fibrinogen in cryoprecipitate and its relationship to factor VIII (AHF) levels

Very little has been published to indicate the quantity of fibrinogen in cryoprecipitates. We assayed 88 preparations from five blood banks for factor VIII(AHF) and fibrinogen to assess whether the AHF assay can predict the fibrinogen content. Cryoprecipitate was considered to be consistent with FDA standards with 80 units of factor VIII/bag (40% yield from 200 ml plasma). Fibrinogen was considered adequate if 200 mg were recovered (40% yield, 200 ml plasma, normal range 150–350 mg/dl). The mean AHF was 145 units/bag and fibrinogen. In 64/88 bags, the fibrinogen and AHF were concordant, but in 24/88 bags the results were discordant. Although it appears safe to conclude that a bag of cryoprecipitate will average 250 mg fibrinogen, adequate control may require separate assays for fibrinogen.     

Coagulation parameters of CPD fresh-frozen plasma and CPD cryoprecipitate-poor plasma after storage at 4 degrees C for 28 days

A pilot study was performed on the storage of plasma and cryosupernatant plasma at 4 degrees C for up to 28 days. Eight bags, four of CPD fresh-frozen plasma (FFP) and four of CPD cryosupernatant plasma (CSP, plasma without cryoprecipitate), were sampled during storage for assays of pH; factors V, VIII, IX, and XI; fibrinogen; prothrombin time; activated partial thromboplastin time (APTT); plasma protein electrophoresis; viscosity; and C1q binding. No changes were found in viscosity or the plasma protein electrophoretic pattern, and there was no detectable immune complex formation. The fibrinogen concentration remained constant, and the prothrombin time showed a gradual increase of 2.5 seconds for both groups of plasma. The labile coagulation factor V decreased gradually for FFP and CSP to 58 and 64 percent of its initial value, respectively (51 +/− 8% and 54 +/− 6% of the value of fresh pooled plasma). Factor VIII decreased to 36 percent of its initial value in FFP (48 +/− 14% of fresh pooled plasma). In CSP, factor VIII decreased after 28 days to 7 percent of its initial value (7 +/− 1% of fresh pooled plasma). The APTT increased for FFP from 28 to 35.8 +/− 1.1 seconds and for CSP from 36 to 49.5 +/− 4.9 seconds. The only chemical change observed for both plasmas was a rise in pH, from 7.27 to 7.56, after 28 days. The results of this pilot study indicate that FFP can be stored at 4 degrees C for 28 days with sufficient recovery of coagulation factors to maintain hemostasis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factor VIII preservation following collection into commercial plasmapheresis systems

The two-stage method of Pool and Robinson was used in determining plasma Factor VIII activity following contact with four commercially available plastic blood and pooling bag films. Employing a two-arm double plasmapheresis technique, it was shown that Factor VIII activity in whole blood initially drawn into BB-69 and PL-130 blood bags decreased only slightly from in vivo levels for activity in PL-130 blood bags (t equal 3.2317, p. less than 0.01). Factor VIII activity fell to 98 and 90 per cent preservation levels from an initial 104 per cent in vivo activity, for BB-69 and PL-130 respectively. Factor VIII activity in plasma collected in Ellay and PL-146 pooling bags, both decreased significantly to 85 and 86 per cent, respectively, after three hours storage at room temperature (24 to 25.5 C). Following five weeks storage (-30 C) in Ellay and PL-146 pooling bags, Factor VIII activity significantly (p less than 0.005) fell to 79 and 77 per cent, respectively. No significant differences were observed when Factor VIII activity levels were compared in BB-69 versus PL-130 or Ellay versus PL-146 plastic film systems.     

Evaluation of factor VIII-rich cryoprecipitate and the plasma fibronectin-rich, heparin-precipitable fraction prepared from single- donor plasma units

A plasma fibronectin-rich component was prepared by heparin-induced 4 degrees C precipitation of fresh or stored (21 days at 4 degrees C), single-donor plasma. The recovery of plasma fibronectin was 45 percent at a concentration of 0.05 mg heparin per ml (7.5 units/ml) and 75 percent at 0.1 mg per ml (15 units/ml). The biologic activity of plasma fibronectin, as assessed by the spreading of Chinese hamster ovary cells or attachment of monocytes to gelatin-coated surfaces, was similar to that of plasma fibronectin concentrates made from fresh or stored plasma. Only 20 to 30 percent of the factor VIII activity in fresh plasma was recovered in cryoprecipitate produced after the heparin-induced precipitate containing fibronectin was removed. Cryoprecipitate prepared from the supernatant plasma that remains after heparin-induced cold precipitation in the presence of CaCl2 (5 mM) contained approximately 50 percent less factor VIII. The relatively low recovery of factor VIII in cryoprecipitate prepared from fibronectin-depleted plasma makes cryoprecipitation an unsuitable method of producing fibronectin-rich and factor VIII-rich components effectively from a single unit of fresh plasma. However, heparin-induced cold precipitation provides an efficient method for preparing plasma fibronectin concentrates from small plasma pools or single units of stored or fresh plasma.     

Simultaneous collection of platelets and cryoprecipitate by plasma- exchange donation

To increase cost efficiency, the simultaneous collection of platelets during plasma-exchange donation of cryoprecipitate was investigated. Sixteen desmopressin (DDAVP)-stimulated donors underwent 90 simultaneous donations. Permanent donor plasma loss for each donation averaged 150 ml in cryoprecipitate and 151 ml in platelet concentrates. Mean factor VIII (FVIII) yield was 4699 +/- 2754 IU per donation. The mean yield in the platelet products was 4.63 X 10(11) platelets; aggregation properties and posttransfusion increments were satisfactory. White cell contamination averaged 4.05 X 10(9) but could be lowered by a secondary centrifugation. The direct cost for a single-donor platelet transfusion produced in this way is estimated at $102.19 and that for FVIII at $0.055 per IU. Simultaneous donation is technically feasible and safe for donors, and it provides functional products that are more cost-effective than apheresis platelets or cryoprecipitate donated separately.     

Immune thrombocytopenia in severe hemophilia A treated with high-dose intravenous immunoglobulin

Five patients with severe hemophilia A receiving long-term treatment with commercial factor VIII concentrates developed severe immune thrombocytopenia (ITP, platelet counts less than 20 X 10(9)/l). Concomitantly, they presented with a marked elevation of serum IgG concentrations (mean, 2364 mg/dl;range, 1712-2954 mg/dl). In four patients, the T helper to suppressor cell ratio was below 1. Treatment with high-dose intravenous immunoglobulin (IgG, 7s) at doses of 0.2g (n = 2) or 0.4g (n = 3) per kg of body weight on 5 consecutive days was effective immediately. The bleeding tendency ceased and platelet counts rose transiently. In three cases, treatment was repeatedly effective and patients underwent uneventful splenectomies. Thus, high-dose IgG therapy may serve as a life-saving agent in patients with severe hemophilia complicated by ITP.