FACTOR VIII COLLECTION BY PHERESIS

671 Donors

Communications

Preliminary

FACTOR VIII COLLECTION BY PHERESIS RICHARD J. SASSETTI MILA I. PIERCE

BRUCE C. MCLEOD EDMOND R. COLE

Section of Hematology, Department of Medicine, Rush-Presbyterian St. Luke’s Medical Center, Chicago, Illinois, U.S.A.

A

new blood donation procedure for obtaining selectively the proteins in cryoprecipitate consists of sequential automated plasma exchanges, in which the donor’s fresh plasma is replaced with the autologous cryoprecipitate supernatant from the previous exchange donation. Fresh plasma is processed into

Summary

and supernatant, both of which are frozen and stored. Six donors have undergone a total of twenty-six exchange donations of 1.5 to 2 litres. No adverse effects have been encountered. The yield of factor VIII per unit of plasma processed decreases during a donation but remains substantial in the last unit of plasma obtained from a 2 litre exchange. The average total yield of factor VIII from a donation was 730 U. The increased yield of factor VIII per donor may reduce the donor exposure, and hence the hepatitis risk, associated with factor VIII replacement

cryoprecipitate

therapy. INTRODUCTION

AN important change in blood collection procedures in the past decade has been the use of individual blood donors for production of single cellular blood components via "pheresis" donations. We extended the pheresis concept so that only the plasma proteins present in cryoprecipitate (CP) are separated from a blood-donor’s circulation. We have focused on the effects of the procedure on the donor and on the yield of factor VIII (AHF). METHODS

Donation Procedure The

procedure consists of an automated plasma exchange blood-separator instrument in which fresh plasma is removed from the donor and replaced with autologous CP supernatant. The fresh plasma is processed into CP and supernatant plasma (SP). CP has thereby been donated, and the SP is frozen and stored until the next donation, when the process is repeated. Before the first exchange, donors undergo three 600 ml plasma donations so that sufficient SP is stored for the first exchange. using

new

a

We have used

a

’Hsemonetics Model 30’ blood processor for

plasma exchanges. The anticoagulant has been 1 part ACD (formula A) to 8 parts blood. We have done weekly exchanges of 1500 - 2000 ml plasma, which require 1 t - 2h.

Cryoprecipitate Production Plasma collected in 200 ml increments in plastic bags having an integral satellite bag was frozen within 2 h in a dry-ice acetone bath, thawed overnight at 4°C, and centrifuged at 4°C to separate CP.l All SP was expressed into the satellite bag. The CP was dissolved in 20 ml saline and a portion was taken for AHF assay. After separation, both CP and SP were frozen. The total duration of the periods of liquid storage did not exceed 24 h for either CP

SP; storage in the non-frozen state may increase risk of bacterial growth, but because all of the bags of CP produced in the study were entered for assay none of them 7ere transfused. or

Six donors, between the ages of 24 and 39,have given two to five donations each, making a total of twenty-six exchange donations.

Coagulation Assays Coagulation assays were performed by standard techniques used in the hospital’s clinical laboratory. Baseline values were obtained for prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), fibrinogen and factor VIII coagulant activity (factor VIII:C, AHF), and the tests were repeated before and after each donation. AHF was measured separately in the CP from the first and last 200 ml bags from a donation, and in the pooled CP from intermediate bags. von Willebrand factor was measured before and after a donation in four donors. Other Donor Studies A complete blood count (CBC) and platelet count were done before and after each donation. Total serum protein, serum protein electrophoresis, urinalysis, and biochemical measurements included in a multichannel analyser (SMA-12) were determined weekly or at each donation. Before each donation, donors were questioned for evidence of unusual bleeding, bruising, or other side-effects associated with the previous donation. Measurement of vital signs and inspection for petechix and purpura were carried out at each donation.

RESULTS

Response and Physical Findings In general, the donation procedure has been well tolerated. Donor complaints were infrequent and were the same as those made by cytopheresis donors. Hxmostasis at venepuncture sites was easily achieved after donation. No donor reported unusual bleeding or bruising after a donation, and no evidence of bleeding was found in repeated physical examinations. Donor

Coagulation Studies PTT, PT, and TT lengthened slightly during donation but post-donation values were normal or nearly normal in

Donor

every case. All three variables returned toward the baseline in the interval between donations. Thus standard screening tests of coagulation suggested that a bleeding tendency would not occur in the donors, even in the immediate post-

donation interval. AHF and fibrinogen decreased by 30 to 50% during donation, and post-donation values often reached statistically subnormal levels. However, the levels remained above those which have been associated with a bleeding tendency in patients with congenital deficiencies (25-3007o for AHF2 and 50 - I00 mg/dl for fibrinogen3) and returned to the normal range by the time of the next donation. Changes in AHF are shown in fig. 1. Both AHF and fibrinogen levels rose rapidly during the 48 h interval after donation (fig. 2). AHF was measured on the day after a donation at least once in four of the six donors, and had returned to normal in every case. von Willebrand factor decreased during a donation but normal levels were found before subsequent donations. Other Donor Studies

Changes in CBC and platelet count after donation were trivial. Serial SMA-12 determinations remained normal except for slight increases in transaminases (SGOT and SGPT) in two donors after unaccustomed muscular exer-

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Several factors were found to influence the yield of AHF from an exchange. The yield correlated with the predonation AHF level and with the efficiency of isolation of AHF in CP. It increased with the volume of plasma exchanged, but not in a linear fashion, because the yield of AHF per volume of plasma gradually declined during the procedure; in the last 200 ml withdrawn the yield was usually 40-50% of that in the first 200 ml. The yield of AHF from the first 200 ml averaged 107 U for these donations. Thus the yield from an exchange donation was approximately seven times that which would have been obtained from a whole blood donation by the same donors. DISCUSSION

Our immediate questions about the plasma exchange donation procedure concern donor safety and product

Fig. I-Change in donor factor VUI:C with repeated plasma exchange donation of cryoprecipitate. * baseline level; closed circle predonation level, open circle postdonation level. Broken lines enclose the statistical normal range. Data from individual donations are shown in temporal sequence; the actual interval between donations was variable but was usually about one week. =

=

tion and trauma (skiing and horseback riding). Transient enzyme elevation is expected after such activities,4 and in both cases the enzymes returned to normal despite further donations. Urinalysis, total serum protein, and serum protein electrophoretic pattern remained normal in all donors.

Fig. 3-Total yield of factor VIII:C in 21 plasma exchange donations (2 of 1500 ml, 6 of 1600 ml, 13 of 2000 ml). Horizontal bar indicates the average yield of 730 U.

Factor VIII Yield Yields of AHF from 21 exchanges of 1.5 to 2 litres in which total yield was evaluable ranged from 426-1895 U (fig. 3). The average AHF yield was 730 U.

Fig. 2-Time course of recovery of donor factor VHI:C (closed circles) and fibrinogen (open circles) levels after cryoprecipitate donation. The donation

began

at

"-2"

on

the x-axis and ended

at

"0".

(AHF) yield, and so far the new method of donation has seemed promising on both accounts. It has produced neither immediate side-effects nor a bleeding tendency or other delayed adverse effects in donors. It yields considerably more AHF than is expected from routine whole blood (70-100 U) or plasma (140-200 U) donations.5 Our average yield of 730 U could probably be bettered by selection of large donors or donors with spontaneously high or stimulated6,7 AHF levels, and by more efficient methods for CP isolation, such as the thaw-siphon technique.8 Further studies will be needed to confirm the safety of the procedure and to determine the maximum allowable frequency of donation; however, the rapidity with which AHF and fibrinogen return to baseline levels suggests that more than one donation per week will be biologically feasible. An important problem with the use of AHF preparations derived from whole blood and plasma donations is the transmission of hepatitis,9 since thousands of donors may be needed to satisfy an individual patient’s needs, and AHF products are often pooled before administration. Hepatitis is a common cause of death in young hxmophiliacsio and chronic liver disease is a growing source of morbidity in those patients who now survive their bleeding tendency into adulthood.l1-13 An AHF preparation that is less likely to

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transmit hepatitis is a pressing need for future generations of haemophilic patients. 14 Pheresis donations have brought new or improved blood products, such as granulocytes,15 HLA-matched platelets,l6 and young red cells (neocytes),17 to many patients whose special transfusion needs could not be satisfactorily met by routine blood donations. The approach to AHF collection described here may be a partial solution to the hepatitis problem in haemophilia by increasing the yield of AHF per donor and hence reducing the number of donors needed to supply a given amount of AHF. The donor exposure associated with AHF replacement could be drastically reduced if the products of sequential donations by the same donor were segregated in storage and given to a single patient. For example, when the average yield is 700 U per donation, a mild to moderate hxmophiliac with an AHF requirement of 30 000 U per year could be supported by the CP from one person donating only once a week. Material obtained from plasma exchange donations would not have to be transfused as CP. CP from one or a restricted number of selected donors could be processed to produce AHF of any desired degree of purity. Selective depletion of one or a few proteins from an individual’s plasma has been accomplished previously with immunoadsorbent columns placed directly in the plasma circuit of a blood separator instrument. These elegant devices have been used to remove anti-DNA antibodies in systemic lupus erythematosus,18 and anti-ABH antibodies from recipients of ABH-incompatible bone-marrow transplants.l9 A staphylococcal protein A adsorbent can remove all species of IgG. 20 We have taken a different approach and have tried to separate a desired material from stored plasma obtained by sequential plasma exchanges on the same individual. This requires less complex instrumentation and can make use of slower separatory processes. We have recently reported our experience with a procedure in which cryoglobulin-depleted autologous plasma is returned to a patient in a sequential plasma exchange programme.21This selective depletion process has proved valuable in the treatment of cryoglobulinsemia. In this report, we describe the first instance of selective depletion of plasma proteins in the context of a blood donation. This method takes advantage of the rapid regeneration of cryoprecipitable proteins relative to more abundant plasma constituents. It could bring the benefits of a single-donor blood-product to haemophiliacs, and to patients with fibrinogen deficiency and von Willebrand’s disease. Requests for reprints should be addressed to B. C. McL., Rush-Presbyterian-St. Luke’s Medical Center, 1753 West Congress Parkway, Chicago, Ilhnois 60612, U.S.A.

REFERENCES

1. Pool

JG, Shannon AE. Production of high-potency concentrates of antihæmophilic globulin in a closed-bag system. N Engl J Med 1965; 273:

1443-47. C. Hemophilia and related conditions. In: Williams WJ, Beutler E, Erslev AJ, Rundles RW, eds. Hematology, New York: McGraw-Hill, 1977: 1404-1422. 3. Gralnick HR. Congenital disorders of fibrinogen. In: Williams WJ, Beutler E., Erslev AJ, Rundles RW, eds. Hematology, New York: McGraw-Hill, 1977: 1423-1431.

2. Houge

RADIOIMMUNOASSAY OF SERUM CREATINE KINASE-BB AS A TUMOUR MARKER IN BREAST CANCER

R.

EILEEN D. RUBERY HILARY M. JONES

J. THOMPSON

Department of Clinical Biochemistry, School of Clinical Medicine, Addenbrooke’s

Hospital, Cambridge CB2 2QR

Brain type creatine kinase-BB (CPK-BB) measured by radioimmunoassay in the serum of 113 women with breast cancer and 354 female controls. 80% of women with metastatic breast cancer had levels above 3 ng/ml (control range 0·5-3·7 ng/ml); the highest level was 23 ng/ml. 60% of women with local disease but no evidence of distant metastases showed levels above 3 ng/ml, the highest being 9·0 ng/ml. Of women who had presented with stage I, II, or III disease and postoperatively had no evidence of persistent disease 30% had levels above 3 ng/ml. Serial measurements in 31 patients indicated that the serum CPK-BB correlated with clinical response to treatment.

Summary

was

INTRODUCTION

EvERY year in England and Wales about 17 000 new cases of breast cancer in women are registered and some 12 000

4. Fowler

WM, Gardner GW, Kazerunian HH, Lauvstad WA. The effect of

exer-

enzymes. Arch Phys Med 1968: 49: 554-65. 5. Langdell RD. Safety and efficacy of blood products. In: Morrison FS, ed. Hemophilia, a technical workshop, Washington DC: American Association of Blood Banks, 1978: 43-48. 6. Strand CL, Beene JR, Geiger T, Eckel MO, Kunkel K, Bull G. Production of high-potency cryoprecipitate from exercised blood donors and the treatment of hemophilia A with this material. Am J Clin Path 1974; 62: 496-501. 7. Nilsson IM, Walter H, Mikaelsson M, Vilhardt H. Factor VIII concentrate prepared from DDAVP stimulated blood donor plasma. Scand J Hœmatol 1979; 22: 42-46. 8. Mason EC. Thaw-siphon technique for production of cryoprecipitate concentrate of factor VIII. Lancet 1978; ii: 15-17. 9. Lewis JH, Maxwell NG, Brandon JM. Jaundice and hepatitis B antigen/antibody in hemophilia. Transfusion 1974; 14: 203-11. 10. Lewis JH, Spero JA, Hasiba U. Deaths in hemophiliacs. In: Aronson DC, Frantantoni J, eds. Unsolved therapeutic problems in hemophilia (DHEW Publication No. NIH 77-1089), Washington DC: Government Printing Office, 1976: 29-33. 11. Hasiba U, Spero JA, Lewis JH. Chronic liver dysfunction in multi-transfused hemophiliacs. Transfusion 1977; 17: 490-94. 12. Hilgartner MW, Giardina P. Liver dysfunction in patients with hemophilia A, B and von Willebrand’s disease. Transfusion 1977; 17: 495-99. 13. Lesesne HR, Morgan JE, Blatt PM, Webster WP, Roberts HR. Liver biopsy in hemophilia A. Ann Intern Med 1977; 86: 703-07. 14. Spero JA, Lewis JH, Van Thiel AH, Hasiba U, Rabin BS. Asymptomatic structural liver disease in hemophilia. N Engl J Med 1978; 298: 1373-78. 15. Higby DJ, Burnett D. Granulocyte transfusion: current status. Blood 1980; 55: 2-8. 16. Yankee RA, Grumet FC, Rogentine GN. Platelet transfusion therapy. The selection of compatible platelet donors for refractory patients by lymphocyte HLA typing. N Engl J Med 1969; 281: 1208-12. 17. Propper RD, Button LN, Nathan DG. New approaches to the transfusion management of thalassemia. Blood 1980; 55: 55-60. 18. Terman DS, Buffaloe G, Mattioli C, et al. Extracorporeal immunoadsorption: Initial experience in human systemic lupus erythematosus. Lancet 1979; ii: 824-27. 19. Besinger WI. Immune adsorption of anti-A and anti-B antibodies. Paper presented at a national symposium on progress in plasmapheresis, March 1980. 20. Bansal SC, Bansal BR, Thomas HL, et al. Ex vivo removal of serum IgG in a patient with colon carcinoma. Cancer 1978; 42: 1-18. 21. McLeod BC, Sassetti RJ. Plasmapheresis with return of cryoglobulin-depleted autologous plasma (cryoglobulinpheresis) in cryoglobulinemia. Blood 1980; 55: 866-70. cise on serum