Acta Clinica Belgica International Journal of Clinical and Laboratory Medicine

ISSN: 1784-3286 (Print) 2295-3337 (Online) Journal homepage: http://www.tandfonline.com/loi/yacb20

Laboratory And Clinical Evaluation Of Concentrates For Treatment Of Haemophilia M. Verstraete & J. Vermylen To cite this article: M. Verstraete & J. Vermylen (1975) Laboratory And Clinical Evaluation Of Concentrates For Treatment Of Haemophilia, Acta Clinica Belgica, 30:5, 437-448, DOI: 10.1080/17843286.1975.11717033 To link to this article: https://doi.org/10.1080/17843286.1975.11717033

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437

LABORATORY AND CLINICAL EVALUATION OF CONCENTRATES FOR TREATMENT OF HAEMOPHILIA

M . VERSTRAETE and J . VERMYLEN*

LABORATORY AND CLINICAL EVALUATION OF CONCENTRATES FOR TREATMENT OF HAEMOPHILIA A AND B

The treatment of haemophilia has improved considerably since the late Judith Pool described a procedure which allows large scale production of a factor- VIII concentrate, without skilled labour or wastage of human plasma (36). In essence, the concentrate is the material that does not go into solution when frozen plasma is thawed at refrigerator temperature; this cryoprecipitate is a by-product of plasma fractionation. A major drawback of the cryoprecipitated factor-VIII preparation is that it is not a standardized product as each blood donation is subject to the variation in factorVIII activity among random donors (50 to 200 %); moreover, processing variables are considerable and in part undefined often resulting in a low yield (35 to 50%)(49). Another disadvantage is that storage of this non-lyophilized factor- VIII concentrate requires constant freezing at - 30°C. In clinical practice, treatment of haemophilia with cryoprecipitated factor- VIII has given satisfactory results; during the last decade this material has become the most widely used factor-VIII concentrate. Considering the present trend to avoid hospitalization of haemophilic patients by making immediate outpatient infusions readily availa* Department Medische Navorsing. Laboratorium Bloedstolling. Universiteit Leuven, Belgium.

ble and to stimulate home transfusion of haemophiliacs by general practicioners or even lay members of the family, there is a need for lyophilized concentrates of factor- VIII and -IX facilitating stockpiling of reserve supplies, and transport. Moreover frequent laboratory surveillance is impossible in non-hospitalized patients stressing the need for well standardized concentrates. The purposes of this study were to evaluate the in vitro properties and potency and in vivo recovery and half-disappearance time of the lyophilized and standardized factor- VIII (Kryobulin) and -IX (Bebulin) preparations, developed by Immuno A.G., Vienna. MATERIALS AND METHODS

The different batches of Kryobulin and Bebulin were donated by lmmuno A.G. (Vienna, Austria) and lyophilized cryoprecipitated factor-VIII was purchased from the Belgian Red Cross, Leuven; all materials were stored at 4°C until use. These- different concentrates were reconstituted according to the manufacturers' instructions: 20, 50 or 100 ml of pyrogen-free distilled water for the two factor- VIII products of Immuno, 10 ml for the factor- IX preparation Bebulin, and 50 ml for the lyophilized factor- VIII of the Red Cross, Leuven. Factor-VIII was assessed using a simple and sensitive two-stage method which allows a reproducible, quantitative assay to be performed on 20 ,ul capillary blood or plasma (Vermylen et al., 1968). This assay is based on Acta C/inica Belgica. 30. 5 (19 75)

438 the principle that a linear relationship between the prot hrombin converting principle "prothrombinase" and factor-Vlll exists in the 0-4 per cent factor- VIII range. Activated bovine and human serum mixed with cephalin provide all the coagulation factors req uired for prothrombinase formation , except factor- VIII. All blood samples were assayed immediately after collection. One unit of factor-VIII is defined as the activity present in 1 ml of average normal pooled human citrated plasma, assessed less than one hour after blood sampling. The " AHF reference normal plasma (dried)" of Hyland Co. was used. Factor-IX was assessed in a one-stage procedure. The substrate was deep- frozen citrated factor-IX deficient plasma from a severe haemophilia B patient. To this substrate, eq ual volumes of phospholipid (Thrombofax I:200 in citrated Verona! buffer) and kaolin (0.5 % in Verona! buffer) were added. The mi xture was incubated for 5 min at 37°C and thereafter stored in an ice-bath during testing. Serial di lutions of the sample to be tested were made in BaS0 4-adsorbed oxalated bovine plasma. An aliquot of the diluted sample to be tested was added to the pre-warmed substrate, incubated for I min at 3rc and the coagulation time determined after add ition of CaCI 2 M/20. One should realize that some authors found 50 % higher factor-IX acti vity in concentrates when using a 2-stage assay than when using a onestage assay . All blood samples for factor- IX determinations were obtained by separate venipunctures. A plasma pool of at least 5 normal donors was used as reference plasma. A unit of factorIX is arbitrarily defined as that amount of factor-IX activity contained in 1 ml of pooled normal human citrated plasma. Activated factor-X was assessed jn the Be-

PLASMACONCENTRATES IN HAEMOPHILIA

bulin preparation using as substrate haemoph ilia A plasma and phospholipid (Thrombofax di luted I :5 in Verona! buffer). After incubation of this mixture for 6 min at 3rC, the coagulation time was meas ured after addition of CaCI2. For blood grouping the technical methods and procedures of the American Association of Blood Banks (2) were used. The presence of Australia antigen was investigated by a radioimmunoassay. Patients with severe or moderately severe haemophiliaA or B were selected at random for the in vivo investigations. All were well documented cases of congenital haemophilia and none had demonstrable inhibitors to factor- VIII or factor-IX . The concentrates were usually ad ministered over 15-20 min into a superficial arm vein through a filt er needle. The metabolic half-disappearance times for the factor-Vlll or -IX concentrates were determined in haemophilic patients with a moderate haemarthrosis without temperature rise or major bleeding. The blood levels of each subject were plotted on a logarithmic scale against time on a linear scale. The factor-VIII levels during the rapid but sometimes irregular first phase of the biphas ic die-away curve (first 90 min) represent most likely the summation of a rapid component due to an eq uilibration and diffusion between the intravascular and extravascular distribution spaces and a more gradual component due to biological degradation . The second prolonged part of the biphasic decrease is thought to represent the true biological half-life. For the purpose of this study, the half-disappearance time was calculated on the prolonged part of the disappearance curve when the circulating level of factor-VIII or -IX was between 50 and 5% (best fitt ed line).

Formulas used: Blood volume (ml): 71 xbodyweight in kg Plasma volume (ml): 41 xbodyweight in kg Observed increase of factor-VIII in U/100 ml blood : post-infusion (10 min) - pre-infusion levels Acta Clinica Belgica, 30, 5 (1975)

PLASMACONCENTRATES IN HAEMOPHILIA

439

Expected rise of factor-VIII in U/100 ml blood : total dose administered (U)x 100 blood volume (ml) . (U) observed increase in Uxblood volume (ml) Factor- VIII gam : --------:-:~-----~____:_ 100 factor-VIIIgainxiOO . (%): _ _ _ _ _____::__ _ __ In v1vo recovery total dose administered (U) Observed % increase of factor- VIII per unit factor- VIII administered per kg, or dose response

RESULTS

I. Properties of the factor- VIII and -IX concentrates in vitro The solubility of the two lyophilized factorVIII preparations of Immuno was reasonable; a minimum of 15 min of gentle rotating and shaking was required to obtain satisfactory solubilization after reconstitution with the solvent. The lyophilized cryoprecipitated factorVIII prepared by the Belgian Red Cross was most often more readily soluble. Bebulin required at least 30 min before a satisfactory solubilization was obtained even if the diluent was pre-warmed to 3rc. It therefore appeared essential to use a filter for administration. If the concentrate is not completely dissolved, active material will be removed by the filter. The presence of soluble A and B antigen was negligible. Titres of a and f3 isoagglutinins were in the expected low range of 1:32 or below, which is considered a clinically insignificant level; these concentrates can therefore be used without typing or crossmatching. Irregular blood group antibodies were absent in all but one preparation when tested in a saline test at 20° and 37°C, in high protein medium at 37°C, or with the antiglobulin test according to the prescriptions of the American Association of Blood Banks (2). Only one bottle of Kryobulin contained a high titre of anti-D antibodies. This is of particular importance considering the relatively long half-life of gammaglobulin during prolonged periods of intensive treatment.

=

observed increase of factor- VIII in % dose ad ministered per kg

Using a radioimmunoassay there was no evidence in any of the vials tested for the presence of Australia antigen . Per unit factor- VIII present in Kryobulin (label activity) there is about 9.3 mg protein (mean of 69 vials) and 2. 7 mg fibrinogen (mean of 10 vials). As in plas ma there is 14.2 U factor- VIII per g protein and in Kryobulin 100 U per g protein, the latter material is 7 times purified. The factor- VIII content of the Kryobulin bottles pertaining to different production lots was assessed and compared with the activity as stated on the label : 75 vials of Kryobulin were assessed, the total label activity was 47,000 U and the assessed ac~ivity 41,169-U . This means th at according to our experience the factor- VIII content of the vials was about 12 % lower than specified by the manufacturer. Lechner (27) assessed the activity of 50 vials of Kryobulin labelled 500 Units, he found 476 U ±64 % which corresponds to a 5% difference. The problems inherent in the assay of factor- VIII concentrates are well known and have recently been discussed (3). The factor-IX content in Bebulin vials was found to be higher than indicated on the label (8,970 U assessed verws 5,200 u· label value) in 6 vials. This preparation also contains per 100 label U factor-IX about 24 U prothrombin and 83 U factor- X (means of 4 vials) but almost no factor- VII. The protein content was 360 mg per vial of W ml (500 label units) and the preparation was devoid of fibrinogen . The specific activity was 1.3 U per mg protein which repreActa Clinica Belgica. 30, 5 (19 75)

440

PLASMA CONCENTRATES IN HAEMOPHILIA

sents a 91-fold concentration in terms of plasma proteins. After 7 hours incubation of the suspended material at Jrc no thrombin activity co uld be assessed. There was only a limited amount of activated factor-X in this preparation . As Bebulin is free of thrombin acti vity, no heparin need be added before use. 2. In vivo activity of/actor- VIII concentrates The in vivo recovery was assessed I 0 to 15 minutes after completion of the intrave nous infusion las ting 15-20 min. The recovery was calculated by relating the factor-VIII gain in the intravascular blood volume to the amount of factor- VIII ad ministered . Table I gives the results obtained after 53 infusions with factorVIII concentrates; the figures are gi ven for the labe l activity as stated by the manufacturer and for the assessed factor-VIII activity . The response of haemophilia A patients to 53 infusions with factor- VIII concentrates is given in table II. The plas ma volume increase on giving these concentrates is assumed to be negligible. It appears that a dose of 20 U fac tor-VIII administered per kg body weight raises the factor- VIII by about 40 %. The mean rise in factor-VIII immediately after the end of

Table I In vivo recovery values obtained with lmmuno factor- VIII concentrates and e~yoprecipitates from the Red Cross, Leuven Dose In vivo infused recovery per kg

Number of infusions Kryobulin Non-lyophilized cryoprecipiate Belgian Red Cross, Leuven Lyophilized cryoprecipitate Belgian Red Cross, Leuven

42

label assessed

19.04 u 143.796 16.53 u 165.096

8

assessed

35.9

u

116.796

3

label assessed

33.4 31.8

u u

76.896 77.6 96

Acta Clinica Be/gica. 30. 5 (1975)

the perfusion in general exceeds the expected rise. Figure I shows the relationship between the amount of factor- VIII activit y ad ministered, as determined in vi tro, and the amou nt recovered in vivo 10 minutes after injecti on of the factor- VIII concentrate of Immuno. It appears th at a dose of 20 U Kryobulin adm inistered per kg body weight raises the factor-VIII by about 40 %. The mean in vivo factor-VIII increase per unit ad ministered factor-VIII of Kryobulin was 2.06 (label ac ti vity) or 2.51 (assessed activity). Multiple blood collections are required to compute with some validity the distribution and degradation of factor-VIII in quiescent haemophilia. To this end, 7 haemophilia patients without actu al bleed ing or with minor haemorrhage (mainl y haemart hrosis) were admitted to the hospital for one week for the sake of this stud y. Factor-VIII levels were determined at regular intervals during the first 48 hours after infusion of Kryobulin; a few days later the same experiment was repeated after adm inistration of cryoprecipitated factorVIII (Belgian Red Cross) (Table IV). These studies required about 30 separate factor- VIII assays on each patient. In a few patients, not included in this report , an exceptionally rapid clearance of factor- VIII revealed the presence of an iso-immune antibody against factor- VIII . As factor- VIII is a large molecule the diffusion phase is slow but degradation is rapid . It appears that the mean metabolic half-disappearance time of factor- VIII is about 8.5 hours after transfusion of the Belgian Red Cross preparation and about 9 hours after administration of K.ryobulin. Most authors find a half-life between 12 and 14 hours when factor- VIII is assessed as a procoagulant.

3. In vivo activity ofthefactor-IX concentrate Bebulin In total, 18 infusions of Bebulin were given. When based on label activity, the mean dose infused was 30.71 U per kg; the mean factorIX increase was only 15.73 % (2.8-30 %) whereas the expected increase was 74.8 %. The mean

441

PLASMACONCENTRATES IN HAEMOPHILIA

Table II

Response ofpatients with haemophilia A to 53 infusions with concentrated/actor- VIII. Comparison between the observed rise and expected rise o.f.factor- VIII Expected rise

Number of infusions

Observed rise (mean)

42

38.3 (7.5-73)

Infusions with non-l yophilized cryoprecipitate RC

8

50.2 (28-67)

45.3 (0.4- 143.8)

Infusions with lyophilized cryopreci pitate RC

3

34.0 (15-62.2)

47.02 (41.2-56.3)

Infusions with Kryobulin

Based on label activity

Based on assessed ac tivity

27.8 (6. 7-89.4)

24.3 (2 .6-75 .6)

Table III

Table IV

Factor- VIII gain per unit factor- VIII administered per kg bodyweight

Half-disappearance time offactor- VIII a.fier rapid infusion of two commercial .factor- VIII concentrates in 7 patients with haemophi/ia A.

Number of infusions Kryobulin Immuno Non-lyophilized cryoprecipitate Belgian Red Cross, L.euven Lyophilized cryoprecipitate Belgian Red Cross , L.euven

Based on label activit y

Based on assessed activity

42

2.37 2.03 (0.38-5. 13) (0.38-3.64)

8

1.66 (0.67-3. 73)

3

0.95 (0.45-1.6)

Haemophilia A I. J.P. S.

2. 3. 4. 5. 6. 7.

S.V. B. R. P. R. P. J. s. L. S. M.V.

Kryobulin Lyophilized Cryoprecipitate lmmuno preparation of factor-VIII Belgian Red Cross 8

8 II

3.2 II II 7

7.5 6.5 18.5 5.4 10.5 7.5 7

4. Haemostatic efficacy in vivo recovery was 16.9% (3.2-31.2 %) with an actual mean gain per unit factor-IX admin istered of 0.41 % (0.11-0. 76 % ). Figure 2 shows the relationship between the amount of factorIX activity injected as determined in vitro, and the amount recovered in vivo 10-15 minutes after injection of the concentrate. The half-disappearance time of Bebulin was determined in 2 haemophilia B patients. When plasma was administered the half-disappearance time was 16 and 12.5 hours; after infusion of Bebulin the values found in the same patients were 16 and 14 hours respectively.

In these experiments, the factor-VIII and-IX concentrates were mainly administered to patients with recent haemarthrosis, often on an outpatient basis. In most cases single doses of therapeutic material proved sufficient to alleviate symptoms and permit early movements and mobilisation of the affected joint; early haematomas resolved promptly after treatment. No patients with more serious lesions such as acute abdominal or cerebral haemorrhage were treated with these factor- VIII concentrates. Two haemophilia B patients were subjected to surgery (elongation of the Achilles tendon and correction of an inguinal hernia); Acta C/inica Be/giro. 30, J. 0 .975)

PLASMACONCENTRATES IN HAEMOPHILIA

442 50

40 ~

1 'b == 0.0. 67 980

I

12 ~

a

= 17.943

>-

0

0

ID

()

30

"' ~ "'.... ~

"'z ::; 0 -

0

0

ID

()

..... "'0

....

"' w

~

20

~

z

~

0



"'

0 ~ u ~

10

u.

0

"' !:: z

:::> 10

20

NET FACTOR VII I INCREASE

30

40

50

10 MIN. AFTER END OF IN FUS ION

60

70

80

KRYOBULIN CONCENT RA TE.

85

PLASMACONCENTRATES IN HAEMOPHILIA

satisfactory haemostasis was ensured with Bebulin.

5. Side effects No major side effects were noted in the course of the 42 infusions with Kryobulin . Neither temperature rise nor haemolytic transfusion reactions were noted, probably because the titre of incomplete antibodies and haemolysins · are very low in these factor- VIII concentrates. We have seen in 3 instances a brief anaphylactoid reaction with sinus tachycardia, dysphoea, dizziness and marked anxiety lasting 5 to 10 minutes. In two instances, the Kryobulin administration had been too rapid; other patients receiving the same batches had no reaction. It is of interest that a similar complication is known to occur with rapid infusions of human globulin (4). So far 3 haemophiliacs in this study group have developed overt hepatitis; ample opportunity existed for our patients to become infected because of the large number of transfusions with concentrates but also with other plasma fractions (non-lyophilized and lyophilized cryoprecipitate) or whole blood during the last months before hepatitis became manifest. In a few cases we have seen transient episodes of abnormal liver tests; because of the proper time relations this could be secondary to the treatment with blood or blood products.

DISCUSSION

The correspondence between label and assessed activity is fair for factor- VIII concentrates of Immuno (12 % variance with the label activity) and also for the limited number of vials of Bebulin (factor-IX concentrate) tested. It also appears that Lechner found a lower factor- VIII activity than stated on the label (observed value 476 Units per bottle of "Kryobulin500") (27). The latter investigator uses a onestage method for factor- VIII assay. A definite advantage of commercial factor- VIII concentrates over home-made cryoprecipitated factorVIII is that the potency of the first are stated;

443 the potency of the latter may show considerable variation depending on the activity of the individual donors (12 , 47). We have found a mean in vivo factor-VIII increase per unit of factor- VIII of the Kryobulin concentrate of 2.06 (label activity) or 2.51 (assessed activity). These values are higher than those found by Lechner (27), who found an increase of 1.56 (0.52-3.26) per unit of Kryobulin factor- VIII. In the latter centre the second blood sample was collected 30 min instead of 15 min after the end of the infusion. The values obtained by us are closer to the theoretical increase of 2.3% calculated by Rizza (37) for administration of concentrated factor-VIII preparations. The percentage of the administered factorVIII that could be recovered immediately after administration varied considerably from patient to patient and there were considerable variations between the calculated levels and the levels actually observed. The same phenomenon was also observed in this laboratory with non-lyophilized cryoprecipitate (mean of 187 infusions: 85.2 %, SO 53.5) and antihaemophilic factor (human) Method Four Hyland (28 infusions, mean recovery 100.3%)(30). In 22 studies with the factor- VIII concentrate manufactured by Courtland Laboratories, Smith et al. (44) found a recovery of between 51 % and 166 % with a mean of 97 % whereas in 20 instances in which the factor- VIII concentrate Hemofil (Hyland) was used, the recovery ranged between 28 % and 117% with a mean of only 62 %. It was suggested that the reported differences in in vivo recovery and survival of concentrated factor- VIII could be related to its purification, as the highest purified also showed diminishing in vivo stability. This rather pessimistic view was however not confirmed by the experiments of Kasper et al. (2 I) . who found that factor- VIII of greater or lesser purification appears equally capable of survi'ving in vivo although immediate recovery (1 0 min) varies significantly among brands of concentrate. These immediate recoveries also exceed 100% (Hemofil, Hyland concentrate: 120±20; Humafac, Parke Davis concentrate: 122 ± 18; Acta C/inica Belgica, 30, 5 (19 75)

444 Red Cross concentrate: 130±28 ; Abbott AHF concentrate: 167±33). Recoveries greater than 100% can be explained on the basis of errors in the assay system and in the estimated blood volume from body weight; plasma volume may vary considerably depending on such factors as the state of hydration and the lean body mass . Furthermore, the diffusion compartment may be more variable than postulated. In our experiments, infusit•ns of concentrated factorVIII were given at several days interval; it was stated that overall recovery with purified concentrates can be higher on the second or third day ~han on the first, possibly related to the factor- VIII space or pool, rather than to the metabolic degradation (20). The mean in vivo recovery of Bebulin was 16.9 % (3.2-31.2 %), a low value was also observed with other factor-IX concentrates: 17.333.6 % with Proplex, Hyland (32) ; 39 % (1669 %) with a Swedish Kabi concentrate (33) and 40 % with Hemoplex, Cutter (19). Also the Oxford group (6, 13) has reported an in vivo recovery of between 18 and 62 %. Considering that the in vivo recovery of factor-JX is usually less than half the calculated value, it is assumed that because of its small molecular weight this factor is distributed throughout the intravascular and extravascular fluid spaces (which is 2 to 7 times larger than the plasma volume) .whereas factor- VIII is retained within the circulation (1, 16, 19, 29, 37). It is also possible that factor-IX is rapidly activated and consumed. Similar low in vivo yields of factorIX have also been observed after plasma transfusion and are therefore not a function of the purification process. The range of response of each patient to separate infusions was found to be as wide as the range of all responses of all patients for other factor-IX concentrates (32). The discrepancy between the factor-IX units measured in vitro (label activity) and the in vivo factor-IX activity recovered in the patient is confusing to medical personnel accustomed to the closer correlation between in vitro and in vivo recovery of factor- VIII concentrates. The half-disappearance time of factor- VIII Acta C/inica Belgica. 30, 5 (19 75)

PLASMACONCENTRATES IN HAEMOPHILIA

was determined with Kryobulin and of the Belgian Red Cross concentrate in the same patients and found to be similar (Kryobulin 8.5 hours , Belgian Red Cross concentrate 9 hours). Most often the half-disappearance time of factor- VIII is reported to be between 7 and 15 hours (35). This variability may be due to a greater lability of a portion of the factor- VIII as factor- VIII also deteriorates biphasically in vitro (II, 23, 34, 48). We only performed survival studies in non-operated haemophiliacs without fever; it is known that an accelerated disappearance of factor- VIII is found postoperatively, and in asociation with tissue necrosis, fever and severe bleeding. The reported half-disappearance time for factor- IX is 17-29 hours; our observed value for Bebulin (13 hours) and plasma (15 hours) was similar when tested after a suitable interval in the same haemophilia 8 patients. Nilsson (33) found , for the Swedish factor- IX concentrate of Kabi, a longer half-disappearance time of 20 hours. As can be seen in figure I the observed rise after administration of Kryobulin was about the initial dose multiplied by 2. This rule of thumb is valid for haemophilia A patients without circulating inhibitor to factor- VIII. The same dose was usually repeated at 24 hourly intervals depending on the severity and site of bleeding. The mean initial dose of Bebulin used in our experiments was 30 U per kg body weight resulting in a mean factor-IX increase of 15 %. In comparison to the larger factor- VIII molecule, the loading dose of factor-IX must be higher because of the greater diffusion compartment (7). In most instances the subsequent maintenance doses can be reduced to 15 U per kg body weight at 24 hour intervals. Bebulin contains a limited amount of prothrombin (for 100 U factor-IX we found 25 U prothrombin). Concentrates of factor-IX are prepared from plasma and great care must be taken that no thrombin forms in the concentrate with resulting activation of factor-X which may induce a state of hypercoagulability both in haemophilic and non-haemophilic pa-

PLASMACONCENTRATES IN HAEMOPHILIA

tients. Those preparations not containing less prothrombin may contain activated factor-IX, activated factor-X or both as shown by in vitro and in vivo tests (9, 25). Most preparations without heparin are thrombogenic in rabbits at doses which are used in patients and there is a suggestion that heparin should be added to all factor-IX concentrates (3 !). The wide-spread therapeutic use of fractions prepared from large portions of plasma has increased the risk of exposure to serum hepatitis. A sharp increase in the incidence of jaundice after infusion of factor- VIII and also after factor-IX concentrates from various sources has been reported (lO, 13, 18, 21, 25, 28, 50). The measured titres of Australia antigen in Kryobulin or Bebulin were not meas urable. However, as Schroeder et al. (42) have pointed out, there is no evidence to date to indicate that the titres of Australia antigen (HAA) and its antibody as measured in factorVIII or -IX concentrates are a predictive measure of their clinical infectivity. In all probability the titre of HAA in pooled products is too low to be detected by the usual methods including counter-immuno-electrophoresis (28). It was stated recently that with present methods only 25 to 50 per cent of hepatitis-contaminated bloods can be diagnosed; moreover there are some false-positive reactions due to other conditions (14). There is also the possibility that the presence of antibody or excess of normal gamma-globulin might inactivate HAA contributed by one donor to a pool of plasma (8). Until a reliable serological test for viral hepatitis is available, the donor with anicteric hepatitis will go undetected and remain on the donor lists. Therefore anti-haemophilic factorVIII and -IX concentrates will remain a potential source for the transmission of hepatitis virus until previous attacks of this form of hepatitis can be reliably diagnosed or an effective means of sterilization without altering the factor-VIII or -IX content is produced. Even if the Australia antigen could be reliably detected, immunity to serum hepatitis (SH) but not to infectious hepatitis (IH) would be obtained. We have observed the occurrence of hepa-

445 titis in 3 out of 33 haemophiliacs followed in this study; in a few cases there were transient episodes of abnormal li ver tests . The likelihood of subclinical or overt hepatitis is proportional to the number of donors represented in the factor-VIII concentrate, to the adeq uacy of screening HAA-positive donors and to the susceptibility of patients to develop hepatitis. It is known that patients receiving only small amounts of HAA do not develop clinical hepatitis (5, 43) but the development of antibodies against HAA in a high proportion of haemophiliacs suggests that infection does occur. This may be a fortun ate si tuation as the resulting resistance to the hepatitis antigen renders further exposure less pathogenic. Until effective measures for eliminating. the hepatitis virus are discovered, the risk of subclinical or clinical hepatitis seems inevitable with frequent ad ministration of factor- VIII or -IX concentrates. The negligible titre of soluble A and B antigen and A and B antibodies in Kryobulin and Bebulin is very fortun ate. Indeed, iatrogenic Coomb's positive haemolytic anaemi·a has been described in haemophilic patients with a blood group other than type 0 intensively treated with factor-VIII concentrates containing a high titre of anti-A and anti-B antibodies (24, 40). The presence in one bottle of lmmuno factor- VIII of a high titre of an ti-D antibody is embarassing; it could have possibly induced a serious auto-immune haemolytic anaemia if several bottles of the same lot had been given to a Rh positive patient. Severe haemolytic anaemia during treatment with Cohn fraction I containing anti-D has been described (41). Paradoxical bleeding tendency in haemophilic patients in face of an adequate level of factor-VIII has puzzled many clinicians .using large amounts of home made or commercial factor- VIII concentrates. It was found that the fibrinogen level can rise to three times its initial value due to their high fibrinogen content: 250±30 mg/donor-unit (about 100 U factorVIIO of cryoprecipitate according to Soloway et al. (48) and 1690-1900 mg/100 ml concentrates Acta C/inica Belgica. 30, 5 (1975)

446 of Hyland and Courtland according to Hathaway et al. (17). Also Kryobulin still contains 2,700 mg fibrinogen per 100 U. Possible explanations of this bleeding tendency are that fibrin split products, fibrinogen monomer or complexes or other platelet coating proteins may decrease the platelet function or interfere with polymerization of fibrinogen . During intensive therapy, an increase in fibrinogen degradation products was observed in the circulating blood in most of the patients during bleeding episodes and its level correlated with the abnormal tests of pl atelet fun ction (17). KEYWORDS Factor-VIII - Factor-IX - Hae mophilia A - Haemophilia B - Bleeding disorders - Treatment. SUMMARY A fai r correspondence was found between the label and assessed factor-VIII and -IX of the Immuno concentrates Kryobulin (factor-VIII) and Bebulin (factor- IX). The mean in vivo recovery after 42 infusions of Kryobulin in 26 haemophilia A patients was 143 % ; after 8 infusions of a non-l yophilized cryoprecipitate in 8 haemophilia A patient~ 116 %. The observed in vivo facto.r-VIII gain per unit of concentrated factor- VIII (Kryobulin) administered per kg body weight was 2.03. The metabolic half-disappearance time of factor- VIII when adm inistered as Kryobulin or a lyophilized preparation prepared by the Belgian Red Cross was 8.5 and 9 hours respectively . In total 18 infusions of Bebulin have been given to 7 haemophilia B patients. Based on label acti vity, the mean initial dose infused was 30.71 U per kg. The mean factor-IX rise immediately after the end of perfusion was only 15.7 % whereas the expected increase was 74.8%. The mean in vivo gain administered per unit of factor-IX (Bebulin) was 0.41 (0.11-0.76). The half-disappearance time of factor-IX was circa 13 hours after Bebulin and 15 hours after plas ma administration. Satisfactory haemostasis was obtained with Kryobulin (42 infusions) and Bebulin (18 infusions). Out of 3 haemophiliacs treated with one of the two Immuno concentrates 3 developed overt hepatitis. As other blood products were also administered, causal relationship wi_w the Immuno products remains uncertain . No other major side effects were noted. • The advantages of the lyophilized Immuno concentrate Kryobulin over fresh or deep frozen cryoprecipitate are its pre-assayed potency , small volume, virtual absence of isoagglutinin and a reasonable solubility. The same advantages hold when Bebulin is compared to plasma. Acta Clinica Belgica, 30, 5 (1 975)

PLA SMA CONCENTRATES IN HAEMOPHILIA RESUME

La correspondance entre l'activite declaree et dosee du facteur VIII et IX dans les preparations Immuno Kryobulin (concentre de facteur VIII) et Bebulin (concentre de facteur IX) est satisfaisante. Le taux moyen retrouve in vi vo ap res 42 perfusions de Kryobulin chez 26 malades atteints d' hemophile A est de 143 %, apres 8 perfusions d' un concentre non -lyophilise d'un autre concentre de facteur VIII est, chez 8 hemophiles, de 116 %. L'elevation du taux du facteur VIII par unite de facteur VIII (Kryobulin) injecte par kg de poids corpore! est de 2,03 . La vitesse de disparition du facteur VIII dans La circulation est tres voisine apres administration de Kryobulin (8,5 heures) ou d'un concentre lyophilise de Ia Croix Rouge Beige (9 heures). La dose initiale moyenne de 18 perfusions de Bebulin administrees i1 7 hemophiles B etait de 30,7 1 Un par kg de poids corpore!. L'elevation moyenne du facteur IX il Ia fin de Ia perfusion etait seulement de 15 ,7 % au lieu de !'elevation calculee de 74,8 %. Par unite de facteur IX (Bebulin) ad min istree par kg de poids corpore!, le gain in vivo etait de 0,41 (0,11 -0,76). La vitesse de disparition du facteur IX dans Ia circulation etait environ de 13 heures pour le concentre Bebulin et de 15 heures apres perfusion de plasma. Les resultats cliniques s'averent satisfai sants du point de vue hemostase dans !'ensemble de notre experience apres 42 perfusions de Kryobulin et 18 perfusions de Bebulin . Sur 31 hemophiles traites par une des deux pn!parations d'Immuno, 3 hemophiles ont fait une hepatite avec signes cliniques m a nife~s; vu que ces malades avaient egalement ete traites avec d'autres derives sanguins, il n'est pas possible d'etablir avec certitude une relation de cause a effet avec les concentres lmmuno. Les avantages du concentre lyophilise Immuno Kryobulin compare au cryo precipite frais ou congele sont son activite declaree, son petit volume, un titre extremement faible des anticorps anti-erythrocytaires anti-A et anti-B et une mise en solution raisonnable.

SAMENV ATTING Er was een bevredigende overeenstemming tussen de opgegeven en gedoseerde concentratie van factor-VIII en -IX in de Immunobereidingen Kryobulin (factor-VIII) en Bebulin (factor-IX). Na 42 infusen van Kryobul in by 26 hemofilie A patienten was de gemiddelde , in vivo recovery" 143 % en na 8 infusen van een niet-gedroogd cryoprecipitaat bij 8 hemofilie A patienten 116%. Per eenheid factorIII Kryobulin die per kg werd toegediend was de in vivo winst 2.03 %. De halveringstijd van factor-VIII , toegediend als Kryobulin of als concentraat van het Belgische Rode Kruis was respectievelijk 8.5 en 9 uur. In totaal werden 18 infusen Bebulin aa n 7 hemofilie B patienten toegediend. De gemiddelde startdosis was 30.7 E per kg. De toename in factor-IX concentratie na toedien-

PLASMACONCENTRATES IN HA EMOPHILIA

lng van de startdosis was slechts 15.7% en niet 74.8 % zoals verwacht. Per eenheid factor-IX Bebulin die per kg werd toeged iend , was de in vivo winscOAl (0.11 -0.76). Ongeveer dezelfde halveringswaarde van factor-IX werd gevonden na behandeling met Bebulin (13 uur) als met plasma (15 uur). Een bevredigende hemostase werd bekomen zowel met Kryobulin (42 infusen) als met Bebulin (I 8 infusen). Op de 31 hemofiliepatienten die met een van beide concentraten werden behandeld, zijn er 3 die klinische symptomen van hepatitis hebben ontwikkeld. Aangezien deze patienten ook met andere bloedbereidingen werden behandeld is het causaal verband met de lmmunobereidingen niet bewe~en; geen andere majeure bijwerkingen werden genoteerd. De voordelen van het gedroogd lmmuno concentraat Kryobulin over fris of ingevroren cryoprecipitaatbereidingen is, dat de factor- Vlll activiteit gedoseerd is, het klein volume, de virtuele afwezigheid van isoagglutininen tegen rode bloedcellen en een bevredigende oplosbaarheid van de bereiding. Dezelfde voordelen gelden eveneens voor Bebulin t.o.v. plas ma. REFERENCES I. ADELSON, E., RHEINGOLD, J.J. , PARKER, 0 .,

STEINER, M., and KIRBY ; J.C. (1963)- The survival of factor VIII (antihemophilic globulin) and factor IX (plasma thromboplastin component) in normal humans. J. clin. Invest. , 42, 1040. 2. American Association of Blood Banks. (1970), Technical methods and proceedings of the AABB. 5th edition . Twentieth Century Press Inc. Chicago, p. 1-12. 3. BANGHAM, D.R., BIGGS, R., BROZOVIC, M. , DENSON, K.W.E., and SKEGG, J.L. (1971)- A biological standard for measurement of blood coagulation factor VIII activity. Bull. Wid. Hlth. Org., 45 337. 4. BARANDUN, S., KISTLER, P., JEUNET, H., and !SLIKER, H. (1962) - Intravenous administration of human y-globulin. Vox Sang., 7. 157. 5. BARKER, L.F., SHULMAN, N.R., MURRAY, R., HIRSCHMAN, R.J., RATNER, F., DIEFENBACH , W.C.L., and GELLER, H.M. (1970) - Transmission of serum hepatitis. J. amer. med. Ass., 211. 1509. 6. BIDWELL, E., BOOTH, J.M., DIKE, G.W .R., and DENSON, K.W.E. (1967)- The preparation for therapeutic use of a concentrate of factor IX containing also factors II, VII and X. Brit. J. Haemat .. 13, 568. 7. BIGGS, R., and MATTHEWS, J.M . (1966) - The plasma concentration ,of factor VIII in the treatment of haemophilia. In: Biggs R., and Macfarlane R.G. (Eds.): Treatment of haemophilia and other coagulation disorders, Blackwell, Oxford, 107. 8. BIGGS, R. (I 972) - Can hemophilic patients be adequately maintained with cryoprecipitates? Or is it desirable or even necessary to manufacture and ad min ister highly concentrated AHF products ? Vox Sang., 22, 554.

447 9. BLATT, P.M., LUNDBLAD, R.L., KINGDON, H.S., McLEAN, G., and ROBERTS, H.R. (1974)- Thrombogenic material in prothrombin complex concentrates. Ann. intern. Med., 81. 766. 10. BOKLAN, B.F. (1971)- Factor IX concentrate and viral hepatitis (letter). Ann. intern. Med., 74, 298. II. BOWIE, E.J. W., THOMPSON, J.H. Jr., DIDISHEIM , P., and OWEN C.A . Jr. (1967)- Disappearance rates of coagulation factors : transfusion studies in factor-deficient patients. Trans/itsion. Phi/ad .. 7. 174. 12. BRITTEN, A.F.H . (1973) - Cryoprecipitate. In : Haemophilia. F. Ola and K.W.E. Denson (Eds.). Excerpta Medica Amsterdam, p. 155-162. 13. DEL DUCA , V. , and EPPES, R.B. (1966) - Hepatitis transmitted by anti -haemophilic globulin New Engl. J. Med., 275, 965. 14. DEMIRJIAN , A. (1971) - Routine tests for hepatitis (letter). New Engl. J. Med.. 284, 1039. 15. DIKE , G.W.R. , BIDWELL, E., and RIZZA , C.R. (1972) - The preparation and clinical use of a new concentrate containing fac tor-IX. prothrombin and factor X and of a separate concentrate contai ning factor VII . Brit. J. Haemat .. 22. 469. 16. GILCHRJST, G.S.. EKERT , H., SHANBROM, E. and HAMMOND, D. (1969) - Evaluation of a new concentrate for the treatment of factor IX deficiency. New Engl. J. Med.. 280. 291. 17. HATHAWAY, W.E. , MAHASANDANA, C. , CARKE, S., and HUMBERT, J.R. (1973)- Paradoxical bleed ing in intensively transfused hemophiliacs: alteration of platelet function . Tran~/itsio n, Phi/ad.. 13, 6. 18. HELLERSTEIN, L.J ., and DEYKIN , D. (1971) - Hepatitis after Konyne ad ministration. New Engl. J . Med.. 284. 1039. 19. HOAG, M.S ., JOHNSON , F.F., ROBINSON , J.A., and AGGELER , P.M . (1969) - Treatment of hemophilia B with a new clotting factor concentrate. New Engl. J. M ed., 280, 581. 20. JOHNSON, A.J ., KARPATKIN, M.H., and NEWMAN, J. (1971) - Clinical investigation of intermediate- and high-purity antihaemophilic factor (factor Vlll) concentrates. Brit. J. Haemat. , 21. 21. 21. KASPER, C.K., and KIPNIS, S. (1972)- Hepatitis and clotting factor concentrates (letter). J. amer. med. Ass .. 221, 510. 22. KASPER, C.K., and McDONALD, J.D. (1973) - Standardization of factor VIII prepa rations (letter). New Engl. J. Med.. 288, 215 . . 23 . KETTENBORG, H.K., DE VRIES, S.l. , and VAN DER POL, E.T. (1955)- Modifications in the behaviour of coagulation factors during storage of blood and its importance for blood transfusions. Rev. beige Path .. 24, 136. 24. KING, E.G., CLARKE, .M.E., and BUCHANAN , D.l. (I 972)- Acute anemia with factor VIII therapy (letter). Ann. intern. Med .. 77. 323. Acta C/inica Belgica, 30, 5 (19 75)

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PLASMACONCENTRATES IN HAEMOPHIL/A

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raten bij hemofilie A patienten. Een klinisch biologische studie. Wedstrijd Reisbeurzen. 31. MENACHE, D, and ROBERTS, H.R. (1975)- Summary report and recommendations of the Task Force Members and Consultants. Throm b. Dialh. haemorrh .. 33, 645. 32 . MIDDLETON, S.M ., BENNETT, I.H ., and SM ITH , J .K. (I 973) - A therapeutic concentrate of coagulation

factors II , IX and X from citrated factor VIII-depleted plas ma. Vox Sang. , 24, 441. 33. NILSSON, I.M ., AHLBERG, A., and BJORLIN , G. (1971) - Clinical experience with a Swedish factor IX concentrate. Acla med. scand.. 190, 257. 34. OWEN , C.A. (1964)- Discussion. In : The Hemophilias. K.M. Brinkhous (Ed .), University of North Carol· ina Press , Chapel Hill , N.C., p. 300. 35. OWEN , C.A ., and BOWIE , E.J .W. (1975) - Infusion therapy in hemophilia A and B. In : Handbook of Hemophilia. K.M. Brinkhous and H.C. Hemker (Eds.). Excerpta M edica, Amsterdam, 449. 36. POOL, J.G., and SHANNON, A.E. (1965) - Produc-

tion of high-potency concentrates of antihemophilic globu lin in a closed-bag system. Assay in vi tro and in vivo. New Engl. J. M ed., 273, 1443. 37. RIZZA , C.R., and BIGGS, R. (1969)- In: Recent advances in blood coagulation. Edited by L. Paller, Churchill, London, 179. 38. RI ZZA, C.R., and BIGGS, R. (1969) - Treatment of congenital deficiencies of factor VIII and fac tor IX . Throm b. Diath. haemorrh .. suppl.. 35, 73 .

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39. ROBERTS, H.R.. and BLATT, PM. (1975) - Post-

transfusion hepatitis following the use of prothrombin complex concentrates. Thromb. Diath . haemorrh., 33, 6 10. 40. ROSATI, L.A ., BARNES, B., OBERMAN, H.A., and PENNER, J.A. (1970) - Hemolyt ic anemia due to

anti -A in concent rated antihemophilic factor preparations. Tran!j{usion, Phi/ad.. / 0, 139. 41. SCHRICKER, K.Th., and SCHRENK, K.H. (1972) Schwere serogene hamolytische Aniimie verursacht durch eine anti-D im Faktor-Vll1-Konzentrat. Throm b. Diath . haemorrh .. 27, 523 . 42. SCHROEDER, D.O., and MOZEN, M.M. (1970) -

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Plas ma fibrinogen levels following cryoprecipi tate infusion . Transfusion , Phi/ad. , 10, 326. 46. VERMYLEN, C., and VERSTRAETE, M. (1968) - A simple method for the assay of factor VIII , usi ng 20 microliter of capillary blood. Brit. J. Haemat., 14 , 24 1. 47. VERSTRAETE, M., LUST, A., and VERMYL EN, J. (1970) - In vi tro and in vivo recovery of cryoprecipitated factor VIII. Bib/. Ha emal. (Karger), 34, 9. 48 . WALL, R.L., SHINOWARA, G., BOURONCLE, B., DE LEEUW, N.K.M. and DOAN , C.A . (1953)- An evaluation of the preservation of human blood stored in experimental plastic containers. I. In vitro studies. J. Lab. clin. M ed .. 42, 665. 49. WAUMANS, P., VAN ITIERBEEK, R., and VERSTRA ETE, M. (1968)- The preparation of a human

plasma fraction by cryoprecipitation and its use in haemophilia. Acta clin . be/g., 23. 139. 50. WITTAKER, J.A., and BROWN , M.J . (1969), Seru m hepatitis in a haemophiliac. Brit. med. J., II , 597 .