Characteristics of a heat treated antihaemophilic cryoprecipitate

THROMBOSIS RESEARCH 45; 625-634, 1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.

CHARACTERISTICS

OF A HEAT TREATED CRYOPRECIPITATE.

ANTIHAEMOPHILIC

P. Kierulf* and H.C. Godal x Central Laboratory, Haematological Research Laboratory and Ullevsl Hospital, 0407 Oslo 4, Norway O.H.

Skjansberg,

K. Gravem,

Accepted in original form 28.11.1986 (Received 15.10.1986; by Editor F. Brosstad)

ABSTRACT In order to evaluate the influence of heat treatment (68'C for 24 or 72 hours) on the essential components of antihaemophilic cryoprecipitate, i.e. factor VIII coagulant activity (VIII:C), Willebrand factor von (VIIIR:Ag and VIIIR:RCF) and fibrinogen, ordinary lyophilized cryoprecipitate was compared to heat treated, aminoacid-enriched specimens. The median reduction in factors VIII:C, VIIIR:Ag, VIIIR:RCF and fibrinogen during lyophilization of ordinary cryoprecipitate was 26 per cent, 11 per cent, 1 per cent and 8.5 per cent, respectively. Heat treatment of such cryoprecipitate resulted in 85 to 98.5 per cent reduction in these parameters, while the reduction following lyophilization and heat treatment (24 hours) of aminoacidcontaining preparations was not significantly different from non-heated, ordinary cryoprecipitate. Following heating of aminoacid-enriched cryoprecipitate for 72 hours, only factor VIIIR:RCF was significantly reduced (32.5 per cent) compared to non-heated samples. Ordinary cryoprecipitate was almost insoluble follow ing heat treatment. Enrichment with aminoacids,however, made the heat treated cryoprecipitate fully soluble, but the content of these vials were slightly slower in dissolving than non-heated preparations. Ultracentrifugation prior to lyophilization and heating did not improve the solubility. If heat treatment proves to be efficient in inacti-

Key words:

Antihaemophilic cryoprecipitate, heat aminoacids, FVIII:C, vWF, fibrinogen. 625

treatment,

626

HEAT TREATED CRYOPREGPPITATE

Vol. 45, No. 5

vating viral agents, we conclude that heated (68°C for 24 hours), aminoacid-enriched cryoprecipitate may be a convenient for product treating haemophilia A, von Willebrand's disease and hypofibrinogenemia.

INTRODUCTION The augmented risk of infecting haemophiliacs with Human Immuno Deficiency Virus (HIV) through transfusion of contaminatVIII preparations, has led to the development of ed factor various viral inactivation methods applicable to plasma products. Heat treatment of freeze dried, highly purified factor VIII concentrates has been the method most commonly employed. Heating of less purified factor VIII preparations, such as antihaemophilic decrease in cryoprecipitate, has resulted in an unacceptable factor VIII coagulant solubility and a substantial loss of activity (factor VIII:C) (1). Thus, to be able to supply haemophiliacs with heat treated factor VIII preparations, purification of cryoprecipitate prior to heating has been regarded as Production of high-purity concentrates, however, reinevitable. quires larger amounts of plasma per unit factor VIII:C than In Norway, where production of antihaemophilic cryoprecipitate. is the factor VIII preparation locally produced cryoprecipitate of choice, re-adjustment to production of high-purity factor VIII concentrates, in order to accomplish viral heat inactiamounts of plasma. vation, would imply import of substantial both with regard to costs and Such import is unfavourable, Since the need of viral inacticontrol of donor population. vation of factor VIII preparations seems evident, we have sought to our freeze dried cryoprecipitate. suitable methods applicabe presented such a method (1). A mixture Margolis et al. recently of aminoacids was added to cryoprecipitate prior to heat treatthe decay of factor VIII:C. In the present inment, preventing the effect of heat treatment on the vestigation, we studied antihaemophilic aminoacid-enriched, components of essential von Willebrand factor and cryoprecipitate, i.e. factor VIII:C, fibrinogen.

MATERIALS

AND

METHODS

was assayed by a one(VIII:C) Factor VIII coagulant activity the effect of cryoprecistage clotting method (Z), determining time of factor VIII:C deficient pitate on the recalcification plasma (Boehringer Mannheim, GmbH, Mannheim, FRG). Cryoprecipiin imidazole buffer (pH 7.4) supplied in the tate was diluted assay kit. Pooled plasma from 20 healthy subjects was calibrated against the 1st International Reference Preparation for Factor plasma (80/511) and used as a activities in VIII-related VIII related parameters determined. reference for all factor antigen (VIIIR:Ag) was dete;;;;Td by a Factor VIII related * Cryomicrotiter ELISA technique (Boehringer Mannheim, in the kit buffer (albumin, Tween 20, precipitate was diluted

Vol. 45, No. 5

Phosphate) Factor using effect cetin

and

tested

in

two

dilutions.

VIII ristocetin cofactor activity formaldehyde fixed platelets, of cryoprecipitate on platelets (3). concentration

Fibrinogen (4).

627

HEAT TREATED CRYOPRECIPITATE

was

(VIIIR:RCF) measuring the in the presence

determined

according

Thrombin clotting time (TCT) was measured following diluted with one volume of cryoprecipitate (37”C), with Topostasine R (Hoffman-LaRoche, Basel, water, at a final concentration of 1 NIH U/ml. Total (5).

protein

concentration

was

determined

by

the

was assayed aggregating of ristoto

Jacobsson

incubation of distilled Switzerland) biuret

method

Production of antihaemophilic cryoprecipitate was carried out as previously described (6). Following separation from buffy coat plasma bags were frozen in a mixture of ethanol and red cells, to thawing at 4’C overnight. ice for 12 minutes prior and dry Cryoprecipitate was separated from the plasma supernatant by transferring the centrifugation at 4OC at 2400 g for 15 minutes, plasma to an attached bag, storing the cryoprecipitate at -2O’C. Ninety such units were used for processing 30 bottles of cryoprecipitate, each pooled from 3 single units. Five’groups (A-E), A) and B) were each consisting of 6 bottles, were established: proordinary cryoprecipitate, as a control group, A) serving cessed by dissolving 3 units of cryoprecipitate in 15 ml buffer NaCl 7.20 g/l, pH 7.0) prior to pooling in (Na-citrate 6.24 g/l, and E) were processed by C) , D) a 100 ml glass bottle. Groups dissolving 3 bags of cryoprecipitate in 9 ml of the above mentioned buffer and 6 ml of a mixture of synthetic aminoacids (Syntamin R 17, Travenol Laboratories Ltd., Thetford, Norfolk, VIII Samples for determination of Factor related England). activiies, fibrinogen, TCT and total protein were obtained from each bottle. In order to increase specific activity and solubility of the cryoprecipitate, group D-specimens were ultracentrifuged at 30000 g for 1 hour at room temperature, transferring the middle layer to 100 ml glass bottles, prior to freezing them with the other vials at -7O’C. The sparse surface layer containing lipids (confirmed by lipoprotein electrophoresis) the and bottom layer containing cellular debris and amorphous proteins (confirmed by microscopy) were discarded. Cryoprecipitate in all 30 bottles were lyophilized (GT ZOD, Leibold Hereaus, Koln, FRG), the drying time was 36 hours, the 25’C. temperature never exceeding Following lyophilization, the cryoprecipitate in groups B, C and D was heated at 68’C for 24 hours, while group E-cryoprecipitate was heated at 68’C for 72 hours. Subsequent to lyophilization and heat treatment, the content of each bottle was dissolved in distilled water at 37'C the volume equalling the pre-lyophilization volume. Samples fo; the various analyses were drawn immediately and frozen at -7O’C. Statistical test for samples.

methods. The following paired differences and

tests the

were used: Wilcoxon

The test

Wilcoxon

for

two

628

HEAT TREATED CRYOPRECIPITATE

Vol.

45, No. 5

RESULTS The process of freezing and lyophilization of small-pooled standard cryoprecipitate (group A) resulted in a 26 per cent (median) reduction of factor VIII:C (Table II). Heating at 68’C for 24 hours (group B), rendered complete dissolution of the cryoprecipitate impossible, and led to an additional 64 per cent loss of factor VIII:C (Table II). Similar heat treatment of the did not result in aminoacid-containing preparation (group C), decreased factor VIII:C content. The decay of factor VIII:C during lyophilization and heat treatment in this group, equalled that of the non-heated group A-cryoprecipitate (29.5 per cent) 1-2 min slower in dissolv(Table II). The group C-batches were ing than control batches (IO-15 minutes). Prolongation of heat treatment to 72 hours (group E) did not affect the solubility, but resulted in a brown discoloration of the cryoprecipitate and a loss of 43.5 per cent factor VIII:C (Table II). The loss of factor VIII:C in group E did not differ significantly from the Ultracentrifugation prior to lyoloss in group A (p=O.OSS). philization (group D) did not affect either factor VIII:C content or solubility. In contrast to the other preparations, group D-cryoprecipitate was transparent, enabling visual control of the product being completely dissolved. By ultracentrifugation, amorphous protein and approximately 50 per cent cellular debris, cryoprecipitate. from the the lipoproteins were removed of No significant loss of von Willebrand factor (vWF), VIIIR:RCF was observed during production of ordinary cryoand VIIIR:Ag, precipitate (group A) (Table I). During heat treatment, more than 90 per cent of vWF decayed in group B, while there was no significant loss of factor VIIIR:RCF and IO per cent loss of factor VIIIR:Ag during heating of aminoacid-containing cryoprecipitate (group C) (Tables 1,II). During heat treatment for the median loss of factor VIIIR:RCF and VIIIR:Ag was 72 hours, respectively (group E) (Table II). In the 32.5 and 26 per cent, and lyophilization ultracentrifuged preparations (group D), heating for 24 hours at 68’C resulted in a 22.5 per cent recent reduction of VIIIR:Ag duction of VIIIR:RCF and a 27 per that no significant With regard to vWF, we conclude (Table II). difference was observed between the loss of factor VIIIR:RCF and heated, and in non-heated cryoprecipitate factor VIIIR:Ag in cryoprecipitate (C,D,E), except for the aminoacid-containing reduction of factor VIIIR:RCF in group E. concentration decreased by 8.5 per cent fibrinogen The (median) during production of group A cryoprecipitate (Table II). heat treatment of group B-cryoprecipitate resulted As expected, in a pronounced fall in fibrinogen content (85 per cent), while addition of aminoacids apparently protected fibrinogen as well as factor VIII:C and vWF. The decrease in fibrinogen concentration in groups C and D was 3.5 and 15 per cent, respectively, while no decrease was observed in group E (Tables I, II). These data were not significantly different from those concerning nonheated cryoprecipitate (Table II). TCTs, which were determined in order to detect denaturation of fibrinogen, were prolonged by 27.5 per cent (median) during lyophilization of standard cryoprecipitate (group A) (Table II). Heat treatment did not cause fact, of the other groups, in additional prolongation in any

significantly

.O)

different

33.0 (27,0-37.0) 24.5 * (20.0-25.0)

35.0 (31.0-40.0) 25.5 + (21 .O-28.0)

15.6 (14.7-16.1) 15.5 (14.7-16.1)

13.5 (12.9-14.6) 11.6 * (11.3-12.2)

14.8 (13.4-15.2) 14.3 (12.0-15.1)

( 1 .:136:5)

15.1 (14.4-15.3)

16.2 (15.6-16.6) 14.8 * (14.0-15.9)

(g/l)

FIBRINOGEN

(68’C,

from post-lyophilization/heat

22.8 (18.5-25.0) 14.5 + (12.0-18.5)

24.0 (20,0-26.0) 18.5 * (16.0-20.0)

20.5 (19.0-21.5) 17.0 * (16.5-19-O)

( OY3Y5)

( o.~151*2) 34.0 (31 .o-40.0) 32.5 (27.0-33.0)

20.0 (18.0-21.0)

21.3 (18.0-22.5) 20.0 (16.0-23.0)

( U/ml >

FVIIIR:RCF

33.5 (31 .o-37.0)

35.0 (32.0-38.0) 31 .o (25.0-34.0)

(U/ml)

FVIIIR:Ag

ordinary lyophilized cryo. control group, ordinary, lyophilized and heat treated (68’C, 24 h) cryo. aminoacid-enriched, lyophilized and heat treated (68’&, 24 h) cryo. aminoacid-enriched, ultracentrifuged, lyophilized and heat treated aminoacid-enriched, lyophilized and heat treated (68”C, 72 h) cryo.

A ?? B ?? C = D = E =

values

(5.ZO10)

13.2 (7.2-14.2)

(5.Zz:o)

12.6 (6.4-15.0)

(5.ZDlO)

11.1 (7.8-13.4)

(0.::‘1:s)

(6.;%

(6.;%0)

12.9 (8.3-15.2)

(U/ml)

FVIII:C

Group Group Group Group Group

6

6

6

6

6

N

and subsequent cryoprecipitate.

pre-lyophilization within each group.

Prior to Iyophilization Following heat treatm. (72 h)

Prior to lyophilization Following heat Treatm. (24 h)

Prior to lyophilization Following heat treatm. (24 h)

Prior to lyophilization Following heat treatm. (24 h)

and

TABLE I prior given as median and range, heat treatment of antihaemophilic

* = p
Group E

Group D

Group C

Group B

Group A (control group)

parameters,

Prior to lyophilization Following lyophilization

Various

TCT (sec. >

24 h)

cryo.

treatment

15.0 (14.0-15.5) 17.0 * (16.5-18.0)

15.5 (14.5-16.5) 16.0 * (15.5-17.5)

15.8 (15.0-20.0) 17.3 (15.5-19.5)

20.5 (18.5-22.0) 19.8 (19.5-22.0)

values

51.5 (51 .o-53.0) 47.0 * (46.0-47.0)

53.0 (51 .O-56.0) 42.0 * (41 .o-43.0)

54.0 (53.0-56.0) 48.5 (46.0-54.0)

50.5 (49.0-51 .O) 16.0 * (13.0-17.0)

48.0 (47.0-49.0) 48.0 (45.0-49.0)

(g/l)

TOT.PROT.

lyophilization

22.5 (18.0-24.5) 28.3 * (23.5-32.0)

to

S d Z $: i( 2 -I 2 m

6

2

Z

I F --!

ln

0”

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HEAT TREATED CRYOPR~C~~~TAT~

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45, No. 5

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631

HEAT TREATED CRYOPRECIPITATE

C and D (Table II), were less prolonged in groups 8, Total protein remained unaltered during lyophilization of (group A) and during heat treatment of standard cryoprecipitate group C-vials (Table I). Heating for 72 hours resulted in a IO per cent decrease in total protein (group E) (Table II). Ultracentrifugation prior to heating reduced total protein by 21 per Sixtynine per cent was lost during cent (group D) (Table II). cryoprecipitate non-aminoacid-containing heat treatment of

TCTs

(group

B)

(Table

II).

DISCUSSION The philization

decay

of of

factor standard

VIII:C

observed

during

freezing

and

lyo-

cryoprecipitate, is in accordance with Following heat treatment, standard cryoprevious results (7). insoluble and lost almost all factor VIII precipitate became had a Addition of aminoacids, however, coagulant activity. as evidenced by no addistabilizing effect on factor VIII:C for 2.4 hours. tional loss following heating at 68'C A similar pattern was observed with regard to vWF and fibrinogen, no statistically significant differences could be observed between non-heated and heated, aminoacid-containing preparations. Therewe conclude that the most importent components of cryofore, precipitate, i.e. factor VIII:C, vWF and fibrinogen, are protected from heat denaturation by aminoacids, and that the biological activities of factor VIII:C and vWF seem to be preserved as well. The mechanism of this protective effect has not been fully elucidated (7). With regard to fibrinogen, TCTs were measured in to evaluate the polymerization properties order following lyophilization and heat treatment. The somewhat shorter TCTs in the starting cryoprecipitate containing aminoacids were probably due to a lower concentration of citrate (8) and to a polymerization-promoting effect of aminoacids per se, as revealed by pilot studies. A more pronounced prolongation of TCTs following lyophilization of devoid of cryoprecipitate aminoacids seemed to indicate that the polymerization properties of fibrinogen were also protected by Syntamin R 17. With regard to cryoprecipitate B, the expected prolongation of TCTs was not observed. This could be due to altered levels of fibrinogen and total protein following heat treatment of these specimens(9,lO). With regard to prolonged heat treatment (72 hours at 68'C) of aminoacid-containing cryoprecipitate, only factor VIIIR:RCF and total protein concentrations were significantly decreased compared to control cryoprecipitate. The factor VIII:C level was also decreased, but not significantly (p=O.OSS). Margolis and Eisen have pointed out that loss of factor VIII:C is accompanied progressive yellow-brown discoloration (7). This is in by a agreement with our observations. The data concerning cryoprecipitate heated for 72 hours, seem to indicate that such cryoprecipitate is of inferior quality compared to vials heat treated for 24 hours. One of the well-known problems with using freeze dried cryoprecipitate is the slow dissolution of the product prior to

632

HEAT TREATED CRYOPREC~~ITATE

Vol. 45, No. 5

treatment infusion. Heat seemed to accentuate this problem, since the heated preparations were somewhat slower in dissolving than non-heated controls. Complete dissolution, however, was achieved in all the aminoacid-enriched vials. Ultracentrifugation, which was carried-out in order to improve the solubility, did not meet with our expectations since the time needed for complete dissolution remained unchanged. Even though the specific activity (VIII:C/total protein) of the product was increased no eviand cellular debris and lipoproteins removed f we have dence for claiming that this is a better product. Transferring cryoprecipitate to ultracentrifugation tubes increases the risk of contamination, and in addition l-3 ml of cryoprecipitate was lost from each bottle during removal of the middle layer. In order to reduce the risk of transferring infective agents certain precautions ought to be considered: to haemophiliacs, 1) Screening of blood donors with regard to viral antibodies, 2) use of a limited number of donors per vial of factor VIII:C methods. concentrate and 3) employment of viral inactivation Until absolutly effective inactivation methods are available, antibody it important to stress point 1 and 2. Since seems screening may give false negative results, it is desirable to each haemophiliac to as few donors as possible. Use of expose small-pooled cryoprecipitate seems to meet with this demand (11). Both lyophilization and addition of stabilizing agents may In vitro influence the efficiency of heat treatment (12,131. studies do, however, inand preliminary in vivo (16) (14,15) dicate that use of heated, lyophilized factor VIII preparations minimize the risk of transferring infective agents. If future studies confirm that heat treatment of lyophilized, aminoacidinactivating viral cryoprecipitate is effective in enriched use of such preparations seems to be a simple and inagents, von Willeexpensive way of treating both haemophilia A (17), brand’s disease (18) and hypofibrinogenemia (19).

ACKNOWLEDGEMENTS The authors and the Fagerhol Ullev~l Immunology, technical assistance appreciated. greatly foreningen, Det

Helqe Heista, Dr. Magne to thank Dr. wish and Department of the Bloodbank staff at Hospital for their kind co-operation. The of Renate Ruyter and Anne Vaeret is also Nas jonalsupported by This was study Karsykdommer. RQd for Hjerteog Norske

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