Clinical Trial of Young Red Blood Cells Prepared by Apheresis

ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 16, No. 6 Copyright 1986, Institute for Clinical Science, Inc. Clinical Trial of Young Red Blood Cells Prepared by Apheresis PATRICIA PISCIOTTO, M.D.,* THOMAS KIRALY, S.B.B.,t LOIS PARADIS,f RAM M. KAKAIYA, M.D.,f LINDA RINK, R.N.,$ and HOWARD A. PEARSON, M.D.$ *Division Blood Bank and Hematology, The University o f Connecticut Health Center, Farmington, CT 06032 and tamerican Red Cross Blood Services, Connecticut Region, Farmington, CT 06032 and tdepartment o f Pediatrics, Yale-New Haven Hospital, New Haven, CT 06504 ABSTRACT Transfusion of young red blood cells (YRBC) with prolonged survival should result in increased intervals between transfusions and, therefore, decreased transfusion-associated iron loading. A prospective clinical trial comparing YRBC transfusions prepared by apheresis versus washed or frozen red cell transfusions was performed in five children with transfusion-dependent thalassemia. A total of 152 YRBC units, evaluated by reticulocyte enrichment and pyruvate kinase activity, were transfused. While a slightly longer interval between transfusions was observed during the time period of YRBC versus the time period after (30.0 ± 1.5 days versus 27.9 ± 1.1 days, respectively, p < 0.02), there was no associated decrease in mg of iron transfused per kg. The effectiveness of transfused YRBC units was less than predicted by in vitro and in vivo studies. Introduction Transfusion-induced hemochromatosis is of major concern in patients receiving chronic transfusion therapy, such as patients with thalassemia. Support with routine transfusions in these patients has resulted in the developm ent of iron o v erlo a d by th e end o f th eir first decade.9 Chelation therapy with deferoxamine,10 and splenectomy,5 to reduce transfusion requirements, provides two modalities utilized to slow the progression of iron loading and improve survival. Another approach to m inim ize the rate of iron accumulation associated with transfusions has been the use of young 473 0091-7370/86/1100-0473 $00.90 Institute for Clinical Science, Inc.

4 7 4 P IS C IO T T O, KIRALY, P A R A D IS, KAKAIYA, R IN K, A N D P E A R S O N red cell products with prolonged survival. It has been theorized that the use of young red blood cells could extend the in terval b etw een tran sfu sion s and, therefore, reduce red cell req u irem ents.4 Based on the age-dependent density of red cells, several investigators have developed techniques to harvest young red cells from hum an donors u sin g con tin u o u s-flo w -c e n tr ifu g a tion4,12 13 or centrifugation of units of blood.2,6 A prospective clinical trial of transfusing young red blood cells prepared by a three hour apheresis procedure was conducted by the p resen t authors in patients with thalassemia to evaluate the effects on transfusion-associated iron loading. Materials and Methods B l o o d P r o d u c t s Young red blood cell units were prepared from volunteer donors by continuous-flow -centrifugation as previously described.12 All units were frozen within 24 hours of collection using a high glycerol procedure.8 Young red cell enrichment was assessed on all units prior to freezing. The parameters evaluated were reticulocyte counts and pyruvate kinase activity, an age-dependent en zym e.14 Enzym e analysis was performed after removal of w hite c e lls.1 Comparisons were made with donors pre-donation whole blood samples. The last 100 units transfused were filtered* to remove a large portion of the contaminating white blood cells prior to processing. Units were stored at 80 C until use. Washed red cells prepared by the IBM 2991 or frozen thawed deglycerolized red cells prepared by standard procedure8 were transfused during the time periods prior to and following YRBC transfusions. * Imugard IG500 filter, Terumo Co., Tokyo, Japan P a t ie n t s Five children with thalassemia major were included in the clinical trial. Four children, between the ages of 10 and 17 years had undergone splenectom y at least three years prior to initiating young red cell transfusions. One child, age two years and three months, had an intact spleen. The children had been routinely transfused at three to five week intervals with either washed red cells or frozen red cells to maintain a pre-transfusion hemoglobin of 10.0 g per dl. The four older children were also receiving chelation therapy. C l in ic a l T r ia l Units of young red cells were transfused for a minimum of six months (one child) and a maximum of nine months (three children). Transfusion requirements (designated as ml red blood cell [RBC] per kg) during the interval of young red cell administration were compared w ith e q u iv a le n t tran sfu sion periods prior to and after initiation of young red cells. Pre-transfusion hem oglobin, body weight, and ml of blood transfused were recorded for each transfusion episode, as well as any adverse reactions or intercurrent illnesses. Hem atocrit determ inations and weights were performed on all young red cell units and units of blood transfused during the succeeding period. The amount of transfused iron for each transfusion episode was calculated from the volume transfused, multiplied by the measured hematocrit and 1.08 mg iron per ml red cells. For the preceding transfusion period, a hematocrit of 67.8 percent was used based on the average hematocrit obtained on washed red cells during the transfusion period following young red cell transfusions. Statistical analysis was performed using the Student t test.

Results During the clinical trial of young red cells, 152 units w ith a final average hematocrit of 77.3 percent and average weight of 141.9 g were transfused. Evaluation of young red cell enrichm ent showed a significant increase in product reticulocyte count and pyravate kinase (PK) activity as compared to donors predonation sam ples (table I). The final average hematocrit and weight of units transfused during the period following young red blood cells were 67.8 percent ± 7.6 and 285.9 g ± 42.4, respectively. Evaluating the children as a group (table II) showed that the ml RBC per kg transfused during the period of young red cell transfusions was not statistically different from the periods prior to or after young red cell transfusions. A higher mean pre-transfusion hemoglobin was m aintained during the period of young red cell transfusions being slightly but significantly different than the mean pre-transfusion hemoglobin maintained during the time period after young red cells (p < 0.02). The m ean interval betw een transfusions, during the time period o f young red cells, was also slightly increased. Looking at the individual children (table III), in one patient (patient #3) there was a trend toward maintenance of a higher mean pre-transfusion hemoglobin in association with an increased time interval b etw een transfusions and a reduced amount of transfused iron per kg per week during the trial of YRBC. Even though there was no statistical difference observed during the different transfusion periods, the data suggest an increased survival of young red cells in this patient. Patient # 5 appeared to have a similar trend w hen com paring the transfusion period of YRBC to the period following the clinical trial. There were no associated illnesses during any of the transfusion periods that could account C L IN IC A L T R IA L O F Y O U N G B L O O D C E L L S 4 7 5 T A B L E I Evaluation of Young Red Blood Cell (YRBC) Units Prepared for Transfusion N = 152 Donor reticulocyte count (%) 2.3 ± 0.9* Product reticulocyte count (%) 6.5 ± 2.5f P.K. activity (ratio to donor pre-donation blood sample) 1.38 ± 0.19t Product hematocrit (%)$ 77.3 ± 7.2 Product weight^ 141.9 ±23.4 *Mean ± S.D. fp < 0.001 compared to donor pre-donation blood sample. $Deglycerolized YRBC unit. for th e o b serv a tio n s in th e s e tw o patients. Patient # 4 developed pneumonia during the transfusion interval after the clinical trial of YRBC, necessitating an additional transfusion. This contributed to the lower mean pre-transfusion hem o globin and the increased transfused iron per kg per week observed during this time period. Patients # 1 and # 2 appeared to benefit the least during the clinical trial of YRBC. Patient # 1 was noted to have a slight but significant (p < 0.05) increase in the iron per kg per week during the interval of YRBC, as compared to the time interval prior to but not after the trial of YRBC. The patient experienced a five-day episode of fever associated with the developm ent of a rash during the transfusion period prior to YRBC. There w ere also two days of fever and sore throat associated with exposure to streptococcus during the time period of YRBC transfusions. Four of the five children receiving young red cell transfusions, which had not been filtered prior to freezing, experienced transfusion reactions. These consisted of chills and fever in three children, regardless of whether or not th ey r e ceiv ed p re-m ed ication w ith Tylenol and Benadryl. One child experienced headache and low back pain, not

47 6 PISCIOTTO, KIRALY, PARADIS, KAKAIYA, RINK, AND PEARSON T A B L E II Comparison of Transfusion History During Intervals of Young Red Blood Cell (YRBC) Transfusions versus Frozen or Washed Red Blood Cell Transfusions T im e P e r io d P re-y R B C YRBC T r a n s f u s i o n s T im e P e r io d P o st-y R B C Pre transfusion Hb (g/dl) 10.8 ± 0.6* 11.0 ± 0.6f 10.5 ± 0.7 Transfusion episodes 44 41 43 Units transfused 152 95 Transfusion reactions (non-hemolytic) 5 8 3 Interval between transfusions (days) 27.6 ± 2.2 30.0 ± 1.5t 27.9 ± 1.1 ml RBC per kg transfused 86.5 ± 18.1 87.6 ± 17.4 84.3 ± 13.9 ml iron per kg transfused 93.4 ± 19.6 94.6 ± 18.7 91.1 ± 14.9 *Mean ± S.D. fp < 0.02 compared to time period post-yrbc. associated with fever, on two separate occasions. The four children had previous histories of febrile transfusion reactions. The one child not experiencing reactions had no prior history of adverse reactions to transfusions. Pre-filtration of young red cell units resulted in the elimination of further transfusion reactions. Evaluation of the effects of prefiltration of 70 young red cell units showed a mean red cell recovery of 92.2 percent with an average of 79.7 percent of white cells removed. The product reticulocyte count, performed on the post filtered units, remained significantly increased compared to donors pre-donation sam- TABLE III Summary of Individual Patient Transfusion Data No. Age* (Y rs) S p le n ectom y RBC P ro d u c t PFf P re - Number Donor T r a n s fu s io n T r a n s f u s i o n s / E x p o s Hb ( g / d l ) W eeks u r e s T r a n s fu s e d ( m l/kg) mg I r o n / kg /W eek _ 1 2 3/12 No LPRBC$ 10.3 + _ 0. 5 10/39 101.44 2.83 + 0.30ÎÎ YRBCÿ 10.3 + 0.8 9/37 17 108.69 3.20 + 0.42 LP RBC 10.1 + 0.9 10/37.9 11 100.25 2.93 + 0.59 2 10 Yes LP RBC + 11.5 + 1.1 9/40 _ 89.24 2.53 + 0.62 YRBC 11.6 + 1.1 9/37.9 39 92.11 2.74 + 0.93 LPRBC 10.9 + 1.0 9/37 18 82.78 2.55 + 0.63 3 10 2/12 Yes LPRBC +/- 10.3 + 0.8 10/36 _ 101.77 3.23 + 0.88 YRBC 10.8 + 1.2 9/39 31 90.48 2.56 + 0.92 LPRBC 10.2 + 0.9 9/37 25 88.73 2.60 + 0.27 4 15 9/12 Yes FRBC** + /-M 10.6 + 0.9 6/23.9 _ 57.63 2.74 + 0.72 YRBC 10.2 + 0.8 5/23.1 23 60.62 2.92 + 0.41 FRBC 9.7 + 0.9 6/24.3 16 62.45 3.43 + 2.2 5 16 8/12 Yes LPRBC + 11.2 + 0.7 9/33.9 _ 82.24 2.68 + 0.48 YRBC 11.5 + 1.2 9/37.1 35 86.08 2.67 + 0.73 LPRBC 11.4 + 0.8 9/34.7 25 87.47 2.76 + 0.67 *Age at initiation of YRBC. fdesferrioxamine. $Leukocyte Poor Red Blood Cells. Mean ± S.D. ip < 0.05, compared to transfusion period with YRBC. $Young Red Blood Cells Frozen Red Blood Cells fetf/- non-compliant.

pies (6.6 percent ± 2.9 percent versus 2.3 p ercen t ± 1.0 p ercen t, resp ectively). There was no effect on the ratio of the product P.K. activity compared to pre-donation samples (1.39 ± 0.21). Discussion Young red cells collected by continuo u s-flo w -c e n tr ifu g a tio n have b een shown, by radiochromium technique, to have prolonged half-life in both asplenic thalassemia major patients (47.7 days) and normal eusplenic donors (46.7 days) as compared to the measured half-life of standard frozen cells in the same individuals.4,12 Prolonged red cell radiochromium half-lifes (40 to 43.9 days) have also been reported with young red cells harvested from units of fresh w hole blood or frozen red cells, although not to the same degree as observed with young red cells obtained by continuous-flowcentrifugation.2,6 Early studies support the concept that young red cells have a longer in vivo survival and, therefore, regular transfusions of young red cell products should result in a reduction in the rate of transfusion-associated iron loading. Propper et al. rep orted a 13-day increase in the interval between transfusions in two patients transfused with young red cells prepared by apheresis over an eight m onth trial p erio d.13 Cohen et al. in 1984 reported on a one year clinical trial of young red blood cells prepared from units of fresh whole blood in six children with thalassemia major. M aintaining the same pre-transfusion hem oglobin le v e l there was a mean reduction in red cell requirement of 15.8 percent. This, however, was not sufficient to result in an increased time interval between transfusions.3 Furthermore, Marcus et al, reporting on a randomized clinical trial of young red cells prepared from fresh units of whole blood, found neither a significant reduction in the rate CLINICAL TRIAL OF YOUNG BLOOD CELLS 47 7 o f hem oglobin fall or a reduction in blood consum ption in splenectom ized patients w ith transfusion-dependent thalassemia.7 Based on our previous paired autologous chromium survival studies12 showing a mean increase of 35 percent of the observed T50Cr of young versus frozen red cells, a similar increase in the time interval required betw een transfusion would have been predicted by us. Our present data suggest that while individual patients may show a trend towards increased survival of transfused YRBC, it is less than the predicted advantage. Survival studies performed in normal adult eusplenic subjects, however, may not truly reflect the survival in individual patients with transfusion-dependent thalassemia. Early in the clinical trial, several children with previous histories of febrile tran sfu sion rea ctio n s e x p e r ie n c e d adverse reactions to the transfusions. W hether reactions resulted from the transfusion of several units of YRBC with residual antigenic m aterial resulting from breakdown of large num bers of white cells or the release of pyrogenic material is presently under investigation. Prefiltration of units to remove a large portion of white cells prior to processing elim inated further transfusion reactions. Our studies have shown that YRBC of consistent quality, based on in vitro assays, can be produced on a regular basis by apheresis. The cost of this product is expensive, being approximately 2.5 times the cost of a standard frozen cell unit and 4.5 tim es the cost of a washed cell unit. Approxim ately 1.6 YRBC units were transfused for each unit of frozen or washed cells given, adding not only to the expense of the transfusion episode, but to a greater number of donor exposures. The trial conducted by the present authors using an apheresis m ethod of

4 7 8 pisciorro, k i r a l y, p a r a d i s, k a k a iy a, r i n k, a n d p e a r s o n processing young red cells showed no significant clinical benefit in terms of increased interval between transfusions or decreased transfusion-associated iron loading. This does not preclude that other techniques which could produce younger cohorts of young red cells would not show some clinical benefit. Whether or not the benefit gained would justify the expense and time required to produce young red cells remains questionable. Acknowledgments The authors wish to thank the pheresis nurses of the American Red Cross Blood Services, Connecticut Region. The work was supported in part by A m erican Red Cross funds and the C onnecticut Campaign against Cooley s anemia. References 1. B e u t l e r, E., W e s t, C., and B l u m e, K. G.: The removal of leukocytes and platelets from whole blood. J. Lab. Clin. Med. «8:328-334, 1976. 2. B r a c e y, A., K l e i n, H. G., C h a m b e r s, S., a n d C o r a s h, L.: Ex vivo s e l e c t i v e i s o l a t i o n o f y o u n g r e d c e l l s b l o o d c e l l s u s i n g t h e IBM-2991 c e l l w a s h e r. B lo o d 6i:1068-1071, 1983. 3. C o h e n, A. R., S c h m i d t, J. M., M a r t i n, M. B., B a r n s l e y, W., a n d S c h w a r t z, E.: C l i n i c a l t r i a l o f y o u n g r e d c e l l t r a n s f u s i o n s. J. P e d i a t r. 104:865-868, 1984. 4. C o r a s h, L., K l e i n, H., D e i s s e r o t h, A., S h a f e r, B., R o s e n, S., B e m a n, J., G r i f f i t h, P., a n d N i e n h u i s, A.: S e l e c t i v e i s o l a t i o n o f y o u n g e r y t h r o c y t e s f o r t r a n s f u s i o n s u p p o r t o f t h a l a s s e m i a m a j o r p a t i e n t s. B l o o d 57:599 606, 1981. 5. G r a z i a n o, J. H., P i o m e l l i, S., H i l g a r t n e r, M., G i a r d i n a, P., K a r p a t k i n, M., A n d r e w, A., L o J a c o n o, N., and S e a m a n, C.: Chelation therapy in B-thalassemia major. III. The role of splenectomy in achieving iron balance. J. Pediatr. 99:695-699, 1981. 6. G r a z i a n o, J. H., P i o m e l l i, S., S e a m a n, C., W a n g, T., C o h e n, A. R., K e l l e h e r, J. E, J r., and S c h w a r t z, E.: A simple technique for preparation of young red cells for transfusion from ordinary blood units. Blood 59:865-868, 1982. 7. M a r c u s, R. E., W o n k e, B., B a n t o c k, H. M., T h o m a s, M. J. G., P a r r y, E. S., T a i t e, H., a n d H u e h n s, E. R.: A p r o s p e c t i v e t r i a l o f y o u n g r e d c e lls i n 48 p a t i e n t s w i t h t r a n s f u s i o n - d e p e n d e n t t h a l a s s e m i a. B r i t. J. H a e m a t o l. 60:153-159, 1985. 8. M e r y m a n, H. T. and H o r n b l o w e r, M.: A method for freezing and washing red blood cells using a high glycerol concentration. Transfusion i2:145-146, 1972. 9. M o d e l l, B.: Total management of thalassemia major. Arch. Dis. Child. 52:489-500, 1977. 10. M o d e l l, B., L e t s k y, E. A., F l y n n, D. M., P e t o, R., and W e a t h e r a l l, D. J.: Survival and desferrioxamine in thalassemia major. Brit. J. M. 284:1081-1084, 1982. 11. P i o m e l l i, S., S e a m a n, C., R e i b m a n, J., T y t u n, A., G r a z i a n o, J., T a b a c h n i k, N., and C o r a s h, L. : Separation of younger cells with improved survival in vivo: An approach to chronic transfusion therapy. Proc. Natl. Acad. Sci. 75:3474-3478, 1978. 12. P i s c i o t t o, P., K ir a l y, T., R o s e n, D., P a r a d i s, L., K a k a iy a, R. M., and M o r s e, E. E.: Preparation of young red cells for transfusion using the Fenwal CS 3000 cell separator. Am. J. Hematol. i7:185-191, 1984. 13. P r o p p e r, R. D., B u t t o n, L. N., a n d N a t h a n, D. G.: N e w a p p r o a c h e s t o t h e t r a n s f u s i o n m a n a g e m e n t o f t h a l a s s e m i a. B lo o d 55:55-60, 1980. 14. S e a m a n, C., W y s s, S., and P i o m e l l i, S.: The decline in energetic metabolism with aging of the erythrocyte and its relationship to cell death. Am. J. Hematol. 8:31-42, 1980.