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Advances in blood transfusion
R e v u e F r a n q a i s e de T r a n s f u s i o n et d ' I m m u n o - h ~ m a t o l o g i e T o m e X X I I . - - No 1. - - 1979
9
Advances in blood transfusion by C.F. Hi~GMAN Blood Transfusion Service University Hospital, UPPSALA.
RED CELLS it has again been emphasised that adenine is A ~ofthisgreatmeeting, value in red cell preservation, not only by improving the viability of red cells in order to prolong storage, b u t also, as shown by GUEGUEN et al. (Rennes, France) to improve the filterability of the red cells t h r o u g h 5 ~xm pore filters. It was also emphasised (DAWSON et al., Boston, USA, and LOVRIC et al., Sydney, Australia) that when storing the red cells as highly c o n c e n t r a t e d preparations (hematocrit > 0.90) it is favourable to double the initial concentration of glucose, if glucose is not supplied in other ways to the red cells. In addition to adenine, guanosine gives a further i m p r o v e m e n t (STRAUSS et al., Berlin, GRD) which confirms earlier w o r k by the SPIELMANN-SEIDLgroup (Frankfurt, FRF). Normally blood is collected in an anticoagulant which at the same t i m e serves a perservative. A new t r e n d in liquid storage, is to separate these two functions. F r o m m y own group we reported about the so-called SAG-system, Dr. L0VRIC (Sydney, Australia) reported a b o u t their ,, Circle Pack,, system and a similar system was r e p o r t e d by STRAUSS et al. (Berlin, GDR). The principle is to c o l l e c t the blood in CPD or citrate, remove the plasma, platelets and the buffy coat, suspend the cells in a suspension m e d i u m and store it for a prolonged t i m e . In our case, we store for 35 days and the suspension m e d i u m contains sodium chloride, adenine and glucose. The main p r o b l e m here is to avoid hemolysis. One of p r o b a b l y several m e c h a n i s m s which cause the hemolysis seems to
10
H O G M A N C_F.
be the following: when you reduce the protein concentration of the medium, there is an o p p o r t u n i t y for enzymes, released f r o m dead or damaged granulocytes to lyse the red cells. We have shown that the c h y m o t r y p s i n d i k e cationic protein has an affinity for the red cell m e m b r a n e , and in case there is not enough of the plasma protease inhibitors, hemolysis will occur. Therefore, it is i m p o r t a n t to remove the buffy coat when red cells are stored in a proteinpoor medium. The buffy coats can be used for a large scale p r o d u c t i o n of interferon, as has been s h o w n at the Congress by the Finnish Red Cross group (see below). Concerning the storage of red ceils in the frozen state a search for means of simplifying the p r o c e d u r e s has been going on for a long time. The addition of adenine and glucose to the glycerol freeze-preservation m e d i u m was s h o w n by MERYMAN (Bethesda, USA) to give good post-transfusion viability w h e n stored at - - 2 0 ° for at least ten months. Storage at high h e m a t o c r i t s makes the postthaw washing easier. The i m p o r t a n c e of taking the cost-benefit aspects into account was stressed at the Cryobiology S y m p o s i u m by Dr VALERI (Boston, USA). W h e n red cells are preserved in the frozen state, the very short storage period after thawing is a practical problem. The i m p r o v e d sterility of the frozen-thawed w a s h e d red cells by an aseptic fluid transfer system was described at the Congress by MYHRE et al. (Torrance, USA). The possibility of making sterile operations improves strongly the possibility to store the red cells for m o r e than 24 h o u r s at 4°C after t h a w i n g / w a s h i n g . The m a i n t e n a n c e of certain properties, for instance a s u p e r n o r m a l c o n c e n t r a t i o n of 2,3-diphosphoglycerate (DPG) is one of the aims of using frozen red cells. Certain properties can be preserved for long periods of time and the cells are then ready for use. This super n o r m a l DPG has been claimed to be of i m p o r t a n c e at certain disease states, particularly in cardiac patients (VALERI, Boston, USA). PLATELETS
The p r e p a r a t i o n of platelet concentrates can be done either f r o m o r d i n a r y blood units or by plateletpheresis. I n his review PERKINS (San Francisco, USA) emphasised that a good clinical effect can n o r m a l l y be obtained by platelets p r e p a r e d f r o m o r d i n a r y blood units. I n such cases the m o r e complicated plateletpheresis technique shall not be used because of the cost aspects. On the other hand, there are m a n y instances w h e r e the plateletpheresis technique gives
A D V A N C E S I N BLOOD T R A N S F U S I O N
11
the advantage of using well m a t c h e d donors which is i m p o r t a n t in the long-term t r e a t m e n t of patients and in patients who have already f o r m e d antibodies reacting with platelet antigens. The p r o b l e m of allo-immunization in platelet transfusion was discussed. HLA AB identical or compatible donors give a higher recovery rate after 24 hours, but interestingly enough, even with one or two incompatibilities, these latter seem to do better than the r a n d o m donors. A special case is the HLA A2 negative recipient, where you have a fairly good chance of therapeutic response, if the d o n o r is HLA A2 negative even if the antibodies have b r o a d e r s p e c i f i d t y than anti-HLA A2. More than 30 % of the transfusions from HLA identical of compatible donors are ineffective (BRAND et al., Leiden, The Netherlands). Therefore a cross-match for antibodies against platelet antigens (both HLA and non HLA) is highly desirable but it was concluded that agglutination and cytotoxicity are not sufficient. The fluorescent anti-human immunoglobulin technique, particularly in the modification described f r o m Dr. ENGELFRIET'S group (Amsterdam, The Netherlands) seems very promising. In o r d e r to avoid the p r o b l e m of non-specific staining, fixation of the platelets with 1% para f o r m a d e h y d e is p e r f o r m e d which still leaves the platelet antigens intact. Both HLA and platelet specific antibodies react. It was shown also, that about 25 % of the patients have non-HLA antibodies which p r o b a b l y are of clinical importance. This m e t h o d is also useful for the detection of auto-antibodies. The platelet microtubules disassemble after 1 h o u r at 4°C (Mc GILL, Bethesda, USA). This reaction is reversible within 18 hours. By incubation every 12 hours at 37°C for half an h o u r it was possible to reassemble the microtubules, and Dr. Mc GILL suggested that this might be a possibility to extend the storage to 4-5 days. Platelets recovered f r o m buffy coats and stored in the cold had a good m a i n t e n a n c e of osmotic shock response (PRINS et al., Amsterdam, The Netherlands). However, there were still no data showing if the viability in vivo was normal. Because of the very short time of liquid storage which can be applied in platelets, freeze-preservation is highly desired. Platelets stored with di-methyl-sulphoxide (DMSO) were shown to be clinically effective (ZARoULIS et al., Boston, USA). However, such methods have not been used to a great extent, mainly because of the w o r k l o a d needed and the relatively low yields. The glycerol technique which was d e s c r i b e d in 1973 by DAYIAN and Rowe has been remodelled slightly by DAYIAN and PERT. The trick n o w is to simplify the freezing procedure. By post-thaw dilution (so no washing is
12
H D G M A N C.F.
necessary) good in vitro function can be obtained. The incubation seems to be i m p o r t a n t in o r d e r to let the platelets recover, because they are spherical w h e n they emerge f r o m the freezing procedure. So far clinical experience is limited b u t promising. A glycerol freezing technique using a lower c o n c e n t r a t i o n of glycerol (3 %) described by HERV~ et al. (Besan~on, France) has been shown to stop clinical b l e e d i n g ; no u n t o w a r d reactions in the recipients were observed.
GRANULOCYTES
I n his survey SEIDL s u m m a r i z e d 6 controlled studies and concluded that granulocyte t r a n s f u s e d patients have a higher survival rate t h a n the control groups. However, for meaningful t r e a t m e n t considerable resources are necessary concerning staff, disposables, machines and donors. This means that t r e a t m e n t should only be instituted if it can be continued for several days. I t was r e p o r t e d that early t r e a t m e n t is favourable (RoCHANT et al., Crdteil, France). However this causes indication and resource p r o b l e m s and the increased risk of alloimmunization was considered by others to indicate a m o r e restrictive regimen (VAN DER MEER et al.). It is possible to collect a sufficient dose of granulocytes f r o m the b u l l y coat of 15-20 donors. However, m o s t people do not use buffy coats as a source of granulocytes b u t p r e f e r ' c o n t i n u o u s or discontinuous flow centrifugation, continuous flow filtration, or gravity sedimentation, w h i c h all give good yields. The new model of the IBM machine seems to give yields s o m e w h a t superior to the presently used centrifugation methods. It has been tested for a few m o n t h s in the USA, and will shortly be available in Europe. The Fenwal cell s e p a r a t o r also seems to be an interesting m a c h i n e - although there are still only experimental data available. Cross-matching for antibodies against granulocyte and m o n o c y t e antigens is possible as has been s h o w n by the ENGELFRIET group (Amsterdam, The Netherlands). Good c o r r e s p o n d e n c e between clinical o u t c o m e and serological findings were obtained with the i m m u n o f l u o r e s c e n c e technique using Fab fragments of anti-human immunoglobulins, in o r d e r to avoid binding to the white cell Fc r e c e p t o r ; like in the platelet test p a r a f o r m a l d e h y d e treated cells were used. This technique is also useful for the detection of autoantibodies.
ADVANCES
IN BLOOD TRANSFUSION
13
LEUKOCYTE D E R I V E D FACTORS
Several reports were given about leukocyte derived factors -transfer factor and interferon. Particular interest was shown the c l i n i c a l u s e of t h e l a t t e r s u b s t a n c e . L e u c o c y t e i n t e r f e r o n h a s a n t i - v i r a l a n d c y t o s t a t i c effects. H u m a n buffy coat lymphocytes can be stimulated by non-pathogenic viruses a n d a l a r g e p r o d u c t i o n c a n b e s e t u p a t a b i g b l o o d c e n t r e (KAuPPINEN & MYLLVLA, H e l s i n k i , F i n l a n d ) . T h e p r o d u c t s c a n b e u s e d in a v a r i e t y of d i s e a s e s , e.g. h e r p e s k e r a t i t i s , c h r o n i c a n d a c t i v e B h e p a t i t i s , o s t e o s a r c o m a s a n d s o m e o t h e r m a l i g n a n c i e s (CANTELL, H e l s i n k i , F i n l a n d ) . T h e i m p o r t a n c e of t h i s n e w b l o o d c o m p o n e n t is s t i l l n o t f u l l y e v a l u a t e d b u t l a r g e - s c a l e c l i n i c a l t r i a l s a r e n o w b e i n g s e t u p in m a n y c o u n t r i e s , m o s t l y u s i n g t h e p r o d u c t of t h e F i n i n i s h R e d C r o s s Blood Center.
TRANSFUSION AND TRANSPLANTATION
T h e p r o b l e m of t r a n s f u s i o n a n d t r a n s p l a n t a t i o n w a s d i s c u s s e d at a s p e c i a l s y m p o s i u m . Retrospectively, blood transfusions have a b e n e f i c i a l e f f e c t o n k i d n e y g r a f t s u r v i v a l . T h e r e a s o n f o r t h i s is still n o t c l e a r . T h e r e m i g h t h a v e b e e n a d i f f e r e n c e in t h e s e v e r i t y of t h e d i s e a s e , o r a s e l e c t i o n of g o o d m a t c h e s t o (c h i g h r e s p o n d e r s ~, o r a i m m u n o l o g i c a l i m m u n o s u p p r e s s i v e effect. T h e i m p r o v e d clin i c a l o u t c o m e may e v e n h a v e b e e n d u e to a c o m b i n a t i o n of s o m e of t h e s e f a c t o r s . S o m e p r o s p e c t i v e s t u d i e s w e r e r e p o r t e d b u t m o s t of t h e s e r i e s a r e s m a l l , so far. E x p e r i m e n t a l e v i d e n c e f r o m t h e L e i d e n g r o u p (PERSIJN et al.) i n d i c a t e s s t r o n g l y a b e n e f i c i a l e f f e c t b y t r a n s f u s i o n in m o n k e y s . H o w e v e r , a n i n t e n s i v e t r a n s f u s i o n r e g i m e n gives in h u m a n s q u i t e a h i g h f r e q u e n c y of H L A a n t i b o d i e s , s o m e 40-50 % (JEANNET et al., MANNONI et al., SUMIDA et al.) b u t n o t so m a n y , a b o u t 10 %, o f t h e s e a n t i b o d i e s a r e b r o a d s p e c t r u m ones. So a f t e r all, i t m i g h t n o t b e t o o d i f f i c u l t to f i n d d o n o r s f o r m o s t of t h e p a t i e n t s . On t h e o t h e r h a n d , t h e r e w a s e v i d e n c e f r o m J a p a n (SUMIDA et al.) t h a t f r o z e n r e d cells h a v e a p o s i t i v e e f f e c t o n g r a f t s u r v i v a l b u t give a l o w e r f r e q u e n c y o f c y t o t o x i c a n t i b o d i e s , a f i n d i n g w h i c h , h o w e v e r , is n o t in a g r e e m e n t w i t h t h a t o f o t h e r s (TERASAKI et al., Los A n g e l e s , USA). M y c o n c l u s i o n f r o m t h e d i s c u s s i o n is t h a t t h e b e s t t r a n s f u s i o n r e g i m e n still h a s to b e p r o v e n . As r e p o r t e d b y TERASAKI et al. c y t o t o x i c c o l d a n t i b o d i e s to B cell a n t i g e n s a r e posit i v e l y c o r r e l a t e d to g r a f t s u r v i v a l , w h e r e a s c y t o t o x i c w a r m antib o d i e s a r e n e g a t i v e l y c o r r e l a t e d . Dr. TERASAKI s u g g e s t e d t h a t t h e
H O G M A N C.F.
14
cytotoxic cold antibodies might be enhancing antibodies which is an interesting possibility. On the other hand, it was also shown that a p p a r e n t non-specific effects by glutaraldehyde treated red blood cell and hemoglobin depress the response in MLR. The question was raised in the discussion w h e t h e r the t r a n s f u s i o n effect in kidney t r a n s p l a n t a t i o n might be (partly ?) of a non-specific nature. The situation in bone m a r r o w t r a n s p l a n t a t i o n is c o n t r a r y to that in kidney transplantation. It is quite clear that previous transfusions in bone m a r r o w t r a n s p l a n t e d aplastic anemia patients is negatively correlated to patient survival. I n a r e p o r t f r o m the international bone m a r r o w t r a n s p l a n t registry BORTIN & RIMM (Milwaukee, USA) showed that patients who h a d received less than 15 transfusions showed a high survival rate (86 %), whereas patients with m o r e than 15 transfusions had a very p o o r survival rate (30 %).
HAEMOPOIETIC STEM CELLS
The possible use of haemopoietic stem cells collected f r o m peripheral b l o o d seems to be a new topic of some particular interest for the b l o o d banks. F r o m the FLIEDNER group in Ulm (FRG) it was s h o w n that there are some 100-300 stem cells per ml peripheral blood. A 12 litre 4 h o u r leucapheresis can give a yield of 0.8 X l0 G stem cells as d e t e r m i n e d by the colony forming technique in culture (CFUc). Two leucaphereses seem to give sufficient stem ceils to repopulate an aplastic bone m a r r o w (K/JRBLING et al., Ulm, FRG). The injection of dextran sulphate in dogs increased the yield of CFUc by 60 times. So if it can be considered ethical to use dextran sulphate in humans, the yield of peripheral stem cells would be no problem. The effect of extensive leucapheresis was studied in dogs. When the leucapheresis was continued for 24 h o u r s a very large n u m b e r of stem cells could be collected. Interestingly enough, the n u m b e r of circulating stem cells was not completely abolished w h i c h indicates that a large n u m b e r of hemopoietic stem cells can be mobilized into the circulation. F u r t h e r m o r e , the exchange between the circulation and extra-vascular sites seems to be intense. Freeze storage with 10 % DMSO as cryoprotective agent was possible with a high yield (close to 100 %) of CFUc (KifRBLING et al_). The collection and freeze storage of peripheral stem cells for autologous or allogeneic t r a n s f u s i o n seems to be an interesting possibility; then we would have not only the red cells, the different white cells and platelets in the t h e r a p e u t i c p r o g r a m m e , b u t also
A D V A N C E S IN BLOOD T R A N S F U S I O N
15
the peripheral stem cells. Whether such therapy will be practical remains to be explored in the future.
R6sumd de la r6daction Cet article rdsume les donndes les plus m a r q u a n t e s rapport6es au cours du Congr6s c o n c e r n a n t la prdparation et l'utilisation des produits cellulaires du sang. Dans le dornaine des globules rouges, l'accent a 6t6 rnis sur la valeur prdservatrice de l'ad6nine et de la guanosine ainsi que sur la tendance nouvelle h dissocier les actions anticoagulantes et prdservatrices des solutions de conservation du sang. Ces systhrnes (SAG ou Circle Pack) offrent le double avantage d'une conservation prolong6e de 35 jours et de l'utilisation de la couche leuco-plaquettaire de chaque unit6 de sang pour la p r e p a r a t i o n ~ grande 6chelle de l'interfdron. Les am41iorations techniques de la congdlation op4rationnelle des globules rouges en particulier h - - 2 0 ° C , la possibilitd d'allonger le ddlai de p6remption des globules ddcongel6s et l'utilit6 d'61ever des taux supra-normaux le 2,3 DPG intra-cellulaire ont 6t6 parrni les points les plus i m p o r t a n t s q u ' o n t abordds les sdances consacrdes /t la cryobiologie transfusionnelle. Dans le dornaine des plaquettes et des cellules blanches, la simplification et l'arn41ioration des techniques de cong41ation des plaquettes restent l'obje&if des ann6es /t venir de m a r n e que l'6tude plus approfondie des pararnhtres irnrnunologiques de l'efficacit6 des transfusions leuco-plaquettaires, lesquelles posent toujours les probl6mes des ressources limitdes en donneurs et de l'immunisation des receveurs. La pr6paration h grande 4chelle de l'interf4ron est devenue une possibilit4 mais l'4valuation clinique de son efficacit6 reste /t faire. De rn6me, la possibilit6 de recueillir par cytophdr6se les cellules souches h~mato-poidtiques constitue a u j o u r d ' h u i un rnoyen dont les applications pratiques prornetteuses restent ~ prdciser. Enfin, s'il sernble qu'en rnati6re de t r a n s p l a n t a t i o n r6nale l'effet bdn6fique des transfusions sanguines soit plus que probable, le r6gime transfusionnel ideal reste ~ trouver et contraste a v e c la nocivit6 confirrnde des transfusions sanguines chez les futurs transplant6s de rnoelle. Docteur H~JGMAN, Blood Transfusion Service University Hospital, 75014 Uppsala, SUEDE.
9
Advances in blood transfusion by C.F. Hi~GMAN Blood Transfusion Service University Hospital, UPPSALA.
RED CELLS it has again been emphasised that adenine is A ~ofthisgreatmeeting, value in red cell preservation, not only by improving the viability of red cells in order to prolong storage, b u t also, as shown by GUEGUEN et al. (Rennes, France) to improve the filterability of the red cells t h r o u g h 5 ~xm pore filters. It was also emphasised (DAWSON et al., Boston, USA, and LOVRIC et al., Sydney, Australia) that when storing the red cells as highly c o n c e n t r a t e d preparations (hematocrit > 0.90) it is favourable to double the initial concentration of glucose, if glucose is not supplied in other ways to the red cells. In addition to adenine, guanosine gives a further i m p r o v e m e n t (STRAUSS et al., Berlin, GRD) which confirms earlier w o r k by the SPIELMANN-SEIDLgroup (Frankfurt, FRF). Normally blood is collected in an anticoagulant which at the same t i m e serves a perservative. A new t r e n d in liquid storage, is to separate these two functions. F r o m m y own group we reported about the so-called SAG-system, Dr. L0VRIC (Sydney, Australia) reported a b o u t their ,, Circle Pack,, system and a similar system was r e p o r t e d by STRAUSS et al. (Berlin, GDR). The principle is to c o l l e c t the blood in CPD or citrate, remove the plasma, platelets and the buffy coat, suspend the cells in a suspension m e d i u m and store it for a prolonged t i m e . In our case, we store for 35 days and the suspension m e d i u m contains sodium chloride, adenine and glucose. The main p r o b l e m here is to avoid hemolysis. One of p r o b a b l y several m e c h a n i s m s which cause the hemolysis seems to
10
H O G M A N C_F.
be the following: when you reduce the protein concentration of the medium, there is an o p p o r t u n i t y for enzymes, released f r o m dead or damaged granulocytes to lyse the red cells. We have shown that the c h y m o t r y p s i n d i k e cationic protein has an affinity for the red cell m e m b r a n e , and in case there is not enough of the plasma protease inhibitors, hemolysis will occur. Therefore, it is i m p o r t a n t to remove the buffy coat when red cells are stored in a proteinpoor medium. The buffy coats can be used for a large scale p r o d u c t i o n of interferon, as has been s h o w n at the Congress by the Finnish Red Cross group (see below). Concerning the storage of red ceils in the frozen state a search for means of simplifying the p r o c e d u r e s has been going on for a long time. The addition of adenine and glucose to the glycerol freeze-preservation m e d i u m was s h o w n by MERYMAN (Bethesda, USA) to give good post-transfusion viability w h e n stored at - - 2 0 ° for at least ten months. Storage at high h e m a t o c r i t s makes the postthaw washing easier. The i m p o r t a n c e of taking the cost-benefit aspects into account was stressed at the Cryobiology S y m p o s i u m by Dr VALERI (Boston, USA). W h e n red cells are preserved in the frozen state, the very short storage period after thawing is a practical problem. The i m p r o v e d sterility of the frozen-thawed w a s h e d red cells by an aseptic fluid transfer system was described at the Congress by MYHRE et al. (Torrance, USA). The possibility of making sterile operations improves strongly the possibility to store the red cells for m o r e than 24 h o u r s at 4°C after t h a w i n g / w a s h i n g . The m a i n t e n a n c e of certain properties, for instance a s u p e r n o r m a l c o n c e n t r a t i o n of 2,3-diphosphoglycerate (DPG) is one of the aims of using frozen red cells. Certain properties can be preserved for long periods of time and the cells are then ready for use. This super n o r m a l DPG has been claimed to be of i m p o r t a n c e at certain disease states, particularly in cardiac patients (VALERI, Boston, USA). PLATELETS
The p r e p a r a t i o n of platelet concentrates can be done either f r o m o r d i n a r y blood units or by plateletpheresis. I n his review PERKINS (San Francisco, USA) emphasised that a good clinical effect can n o r m a l l y be obtained by platelets p r e p a r e d f r o m o r d i n a r y blood units. I n such cases the m o r e complicated plateletpheresis technique shall not be used because of the cost aspects. On the other hand, there are m a n y instances w h e r e the plateletpheresis technique gives
A D V A N C E S I N BLOOD T R A N S F U S I O N
11
the advantage of using well m a t c h e d donors which is i m p o r t a n t in the long-term t r e a t m e n t of patients and in patients who have already f o r m e d antibodies reacting with platelet antigens. The p r o b l e m of allo-immunization in platelet transfusion was discussed. HLA AB identical or compatible donors give a higher recovery rate after 24 hours, but interestingly enough, even with one or two incompatibilities, these latter seem to do better than the r a n d o m donors. A special case is the HLA A2 negative recipient, where you have a fairly good chance of therapeutic response, if the d o n o r is HLA A2 negative even if the antibodies have b r o a d e r s p e c i f i d t y than anti-HLA A2. More than 30 % of the transfusions from HLA identical of compatible donors are ineffective (BRAND et al., Leiden, The Netherlands). Therefore a cross-match for antibodies against platelet antigens (both HLA and non HLA) is highly desirable but it was concluded that agglutination and cytotoxicity are not sufficient. The fluorescent anti-human immunoglobulin technique, particularly in the modification described f r o m Dr. ENGELFRIET'S group (Amsterdam, The Netherlands) seems very promising. In o r d e r to avoid the p r o b l e m of non-specific staining, fixation of the platelets with 1% para f o r m a d e h y d e is p e r f o r m e d which still leaves the platelet antigens intact. Both HLA and platelet specific antibodies react. It was shown also, that about 25 % of the patients have non-HLA antibodies which p r o b a b l y are of clinical importance. This m e t h o d is also useful for the detection of auto-antibodies. The platelet microtubules disassemble after 1 h o u r at 4°C (Mc GILL, Bethesda, USA). This reaction is reversible within 18 hours. By incubation every 12 hours at 37°C for half an h o u r it was possible to reassemble the microtubules, and Dr. Mc GILL suggested that this might be a possibility to extend the storage to 4-5 days. Platelets recovered f r o m buffy coats and stored in the cold had a good m a i n t e n a n c e of osmotic shock response (PRINS et al., Amsterdam, The Netherlands). However, there were still no data showing if the viability in vivo was normal. Because of the very short time of liquid storage which can be applied in platelets, freeze-preservation is highly desired. Platelets stored with di-methyl-sulphoxide (DMSO) were shown to be clinically effective (ZARoULIS et al., Boston, USA). However, such methods have not been used to a great extent, mainly because of the w o r k l o a d needed and the relatively low yields. The glycerol technique which was d e s c r i b e d in 1973 by DAYIAN and Rowe has been remodelled slightly by DAYIAN and PERT. The trick n o w is to simplify the freezing procedure. By post-thaw dilution (so no washing is
12
H D G M A N C.F.
necessary) good in vitro function can be obtained. The incubation seems to be i m p o r t a n t in o r d e r to let the platelets recover, because they are spherical w h e n they emerge f r o m the freezing procedure. So far clinical experience is limited b u t promising. A glycerol freezing technique using a lower c o n c e n t r a t i o n of glycerol (3 %) described by HERV~ et al. (Besan~on, France) has been shown to stop clinical b l e e d i n g ; no u n t o w a r d reactions in the recipients were observed.
GRANULOCYTES
I n his survey SEIDL s u m m a r i z e d 6 controlled studies and concluded that granulocyte t r a n s f u s e d patients have a higher survival rate t h a n the control groups. However, for meaningful t r e a t m e n t considerable resources are necessary concerning staff, disposables, machines and donors. This means that t r e a t m e n t should only be instituted if it can be continued for several days. I t was r e p o r t e d that early t r e a t m e n t is favourable (RoCHANT et al., Crdteil, France). However this causes indication and resource p r o b l e m s and the increased risk of alloimmunization was considered by others to indicate a m o r e restrictive regimen (VAN DER MEER et al.). It is possible to collect a sufficient dose of granulocytes f r o m the b u l l y coat of 15-20 donors. However, m o s t people do not use buffy coats as a source of granulocytes b u t p r e f e r ' c o n t i n u o u s or discontinuous flow centrifugation, continuous flow filtration, or gravity sedimentation, w h i c h all give good yields. The new model of the IBM machine seems to give yields s o m e w h a t superior to the presently used centrifugation methods. It has been tested for a few m o n t h s in the USA, and will shortly be available in Europe. The Fenwal cell s e p a r a t o r also seems to be an interesting m a c h i n e - although there are still only experimental data available. Cross-matching for antibodies against granulocyte and m o n o c y t e antigens is possible as has been s h o w n by the ENGELFRIET group (Amsterdam, The Netherlands). Good c o r r e s p o n d e n c e between clinical o u t c o m e and serological findings were obtained with the i m m u n o f l u o r e s c e n c e technique using Fab fragments of anti-human immunoglobulins, in o r d e r to avoid binding to the white cell Fc r e c e p t o r ; like in the platelet test p a r a f o r m a l d e h y d e treated cells were used. This technique is also useful for the detection of autoantibodies.
ADVANCES
IN BLOOD TRANSFUSION
13
LEUKOCYTE D E R I V E D FACTORS
Several reports were given about leukocyte derived factors -transfer factor and interferon. Particular interest was shown the c l i n i c a l u s e of t h e l a t t e r s u b s t a n c e . L e u c o c y t e i n t e r f e r o n h a s a n t i - v i r a l a n d c y t o s t a t i c effects. H u m a n buffy coat lymphocytes can be stimulated by non-pathogenic viruses a n d a l a r g e p r o d u c t i o n c a n b e s e t u p a t a b i g b l o o d c e n t r e (KAuPPINEN & MYLLVLA, H e l s i n k i , F i n l a n d ) . T h e p r o d u c t s c a n b e u s e d in a v a r i e t y of d i s e a s e s , e.g. h e r p e s k e r a t i t i s , c h r o n i c a n d a c t i v e B h e p a t i t i s , o s t e o s a r c o m a s a n d s o m e o t h e r m a l i g n a n c i e s (CANTELL, H e l s i n k i , F i n l a n d ) . T h e i m p o r t a n c e of t h i s n e w b l o o d c o m p o n e n t is s t i l l n o t f u l l y e v a l u a t e d b u t l a r g e - s c a l e c l i n i c a l t r i a l s a r e n o w b e i n g s e t u p in m a n y c o u n t r i e s , m o s t l y u s i n g t h e p r o d u c t of t h e F i n i n i s h R e d C r o s s Blood Center.
TRANSFUSION AND TRANSPLANTATION
T h e p r o b l e m of t r a n s f u s i o n a n d t r a n s p l a n t a t i o n w a s d i s c u s s e d at a s p e c i a l s y m p o s i u m . Retrospectively, blood transfusions have a b e n e f i c i a l e f f e c t o n k i d n e y g r a f t s u r v i v a l . T h e r e a s o n f o r t h i s is still n o t c l e a r . T h e r e m i g h t h a v e b e e n a d i f f e r e n c e in t h e s e v e r i t y of t h e d i s e a s e , o r a s e l e c t i o n of g o o d m a t c h e s t o (c h i g h r e s p o n d e r s ~, o r a i m m u n o l o g i c a l i m m u n o s u p p r e s s i v e effect. T h e i m p r o v e d clin i c a l o u t c o m e may e v e n h a v e b e e n d u e to a c o m b i n a t i o n of s o m e of t h e s e f a c t o r s . S o m e p r o s p e c t i v e s t u d i e s w e r e r e p o r t e d b u t m o s t of t h e s e r i e s a r e s m a l l , so far. E x p e r i m e n t a l e v i d e n c e f r o m t h e L e i d e n g r o u p (PERSIJN et al.) i n d i c a t e s s t r o n g l y a b e n e f i c i a l e f f e c t b y t r a n s f u s i o n in m o n k e y s . H o w e v e r , a n i n t e n s i v e t r a n s f u s i o n r e g i m e n gives in h u m a n s q u i t e a h i g h f r e q u e n c y of H L A a n t i b o d i e s , s o m e 40-50 % (JEANNET et al., MANNONI et al., SUMIDA et al.) b u t n o t so m a n y , a b o u t 10 %, o f t h e s e a n t i b o d i e s a r e b r o a d s p e c t r u m ones. So a f t e r all, i t m i g h t n o t b e t o o d i f f i c u l t to f i n d d o n o r s f o r m o s t of t h e p a t i e n t s . On t h e o t h e r h a n d , t h e r e w a s e v i d e n c e f r o m J a p a n (SUMIDA et al.) t h a t f r o z e n r e d cells h a v e a p o s i t i v e e f f e c t o n g r a f t s u r v i v a l b u t give a l o w e r f r e q u e n c y o f c y t o t o x i c a n t i b o d i e s , a f i n d i n g w h i c h , h o w e v e r , is n o t in a g r e e m e n t w i t h t h a t o f o t h e r s (TERASAKI et al., Los A n g e l e s , USA). M y c o n c l u s i o n f r o m t h e d i s c u s s i o n is t h a t t h e b e s t t r a n s f u s i o n r e g i m e n still h a s to b e p r o v e n . As r e p o r t e d b y TERASAKI et al. c y t o t o x i c c o l d a n t i b o d i e s to B cell a n t i g e n s a r e posit i v e l y c o r r e l a t e d to g r a f t s u r v i v a l , w h e r e a s c y t o t o x i c w a r m antib o d i e s a r e n e g a t i v e l y c o r r e l a t e d . Dr. TERASAKI s u g g e s t e d t h a t t h e
H O G M A N C.F.
14
cytotoxic cold antibodies might be enhancing antibodies which is an interesting possibility. On the other hand, it was also shown that a p p a r e n t non-specific effects by glutaraldehyde treated red blood cell and hemoglobin depress the response in MLR. The question was raised in the discussion w h e t h e r the t r a n s f u s i o n effect in kidney t r a n s p l a n t a t i o n might be (partly ?) of a non-specific nature. The situation in bone m a r r o w t r a n s p l a n t a t i o n is c o n t r a r y to that in kidney transplantation. It is quite clear that previous transfusions in bone m a r r o w t r a n s p l a n t e d aplastic anemia patients is negatively correlated to patient survival. I n a r e p o r t f r o m the international bone m a r r o w t r a n s p l a n t registry BORTIN & RIMM (Milwaukee, USA) showed that patients who h a d received less than 15 transfusions showed a high survival rate (86 %), whereas patients with m o r e than 15 transfusions had a very p o o r survival rate (30 %).
HAEMOPOIETIC STEM CELLS
The possible use of haemopoietic stem cells collected f r o m peripheral b l o o d seems to be a new topic of some particular interest for the b l o o d banks. F r o m the FLIEDNER group in Ulm (FRG) it was s h o w n that there are some 100-300 stem cells per ml peripheral blood. A 12 litre 4 h o u r leucapheresis can give a yield of 0.8 X l0 G stem cells as d e t e r m i n e d by the colony forming technique in culture (CFUc). Two leucaphereses seem to give sufficient stem ceils to repopulate an aplastic bone m a r r o w (K/JRBLING et al., Ulm, FRG). The injection of dextran sulphate in dogs increased the yield of CFUc by 60 times. So if it can be considered ethical to use dextran sulphate in humans, the yield of peripheral stem cells would be no problem. The effect of extensive leucapheresis was studied in dogs. When the leucapheresis was continued for 24 h o u r s a very large n u m b e r of stem cells could be collected. Interestingly enough, the n u m b e r of circulating stem cells was not completely abolished w h i c h indicates that a large n u m b e r of hemopoietic stem cells can be mobilized into the circulation. F u r t h e r m o r e , the exchange between the circulation and extra-vascular sites seems to be intense. Freeze storage with 10 % DMSO as cryoprotective agent was possible with a high yield (close to 100 %) of CFUc (KifRBLING et al_). The collection and freeze storage of peripheral stem cells for autologous or allogeneic t r a n s f u s i o n seems to be an interesting possibility; then we would have not only the red cells, the different white cells and platelets in the t h e r a p e u t i c p r o g r a m m e , b u t also
A D V A N C E S IN BLOOD T R A N S F U S I O N
15
the peripheral stem cells. Whether such therapy will be practical remains to be explored in the future.
R6sumd de la r6daction Cet article rdsume les donndes les plus m a r q u a n t e s rapport6es au cours du Congr6s c o n c e r n a n t la prdparation et l'utilisation des produits cellulaires du sang. Dans le dornaine des globules rouges, l'accent a 6t6 rnis sur la valeur prdservatrice de l'ad6nine et de la guanosine ainsi que sur la tendance nouvelle h dissocier les actions anticoagulantes et prdservatrices des solutions de conservation du sang. Ces systhrnes (SAG ou Circle Pack) offrent le double avantage d'une conservation prolong6e de 35 jours et de l'utilisation de la couche leuco-plaquettaire de chaque unit6 de sang pour la p r e p a r a t i o n ~ grande 6chelle de l'interfdron. Les am41iorations techniques de la congdlation op4rationnelle des globules rouges en particulier h - - 2 0 ° C , la possibilitd d'allonger le ddlai de p6remption des globules ddcongel6s et l'utilit6 d'61ever des taux supra-normaux le 2,3 DPG intra-cellulaire ont 6t6 parrni les points les plus i m p o r t a n t s q u ' o n t abordds les sdances consacrdes /t la cryobiologie transfusionnelle. Dans le dornaine des plaquettes et des cellules blanches, la simplification et l'arn41ioration des techniques de cong41ation des plaquettes restent l'obje&if des ann6es /t venir de m a r n e que l'6tude plus approfondie des pararnhtres irnrnunologiques de l'efficacit6 des transfusions leuco-plaquettaires, lesquelles posent toujours les probl6mes des ressources limitdes en donneurs et de l'immunisation des receveurs. La pr6paration h grande 4chelle de l'interf4ron est devenue une possibilit4 mais l'4valuation clinique de son efficacit6 reste /t faire. De rn6me, la possibilit6 de recueillir par cytophdr6se les cellules souches h~mato-poidtiques constitue a u j o u r d ' h u i un rnoyen dont les applications pratiques prornetteuses restent ~ prdciser. Enfin, s'il sernble qu'en rnati6re de t r a n s p l a n t a t i o n r6nale l'effet bdn6fique des transfusions sanguines soit plus que probable, le r6gime transfusionnel ideal reste ~ trouver et contraste a v e c la nocivit6 confirrnde des transfusions sanguines chez les futurs transplant6s de rnoelle. Docteur H~JGMAN, Blood Transfusion Service University Hospital, 75014 Uppsala, SUEDE.