Experimental 6 log,, white cell-reduction filters for red cells B.J. SADOFF,R.R. STROMBERG, K. MILLER,D. NGO,AND L.I. FRIEDMAN White cell (WBC) reduction of blood components has been receiving increased attention as a way of reducing transfusion-related complications such as WBC-associated HLA alloimmunization and transmission of cell-associated viral diseases. Currently available filters are limited to removing approximately 3 log,, (99.9%) of WBCs from red cells (RBCs). The performance of two experimental filters that were designed to remove 6 log,, WBCs from fresh RBCs during component preparation was evaluated. Both filters were able to meet this objective in less than 40 minutes with RBC losses of < 15 percent under nonoptimized conditions. Filtered RBCs showed storage parameters within the normal range over a 42-day period. The use of these filters, if combined with a sterile docking device or if incorporated into a collection set, should provide the means to supply highly WBC-reduced RBCs with a normal shelf life. TRANSFUSION 1992;32:129-133.
Abbrevlatlons: CMV = cytomegalovlrus; HIV = human lmmunodetlclency vlrus; HTLV1/11 = human 1-lymphotroplc vlrus type I or It; RBC(s) = red cell(s); WBC(s) = whlte cells.
THEREMOVAL OF WHITE CELLS (WBCs) from red cells (RBCs) by filtration has been recognized as important for the reduction of febrile transfusion reactions and WBCassociated HLA alloimmunization.' Viral infections that are specifically associated with WBCs, such as cytomegalovirus (CMV) and human T-lymphotropic virus type I or I1 (HTLV-MI), should also be reduced significantly, and may even be eliminated, if WBC reduction is extensive. 1-5 Recently, Rawal and coworkers6reported approximately 2 log,, reduction of viral titer in patients naturally infected with human immunodeficiency virus (HIV), achieved with a 3 log,, WBC-reduction filter. This group also demonstrated a 5.9 log,, reduction of HIV titer in seronegative RBCs inoculated with HIV,,infected HUT-78 cells. It has been suggested that perhaps the difference in results (2.0 vs. 5.9) is due to the altered immunologic and electrostatic properties of the HUT-78 cells versus the properties of naturally occurring WBCs. Similarly, Bruisten et aL7 reported that filtration through a polyester WBC filter decreased the infectivity of blood from HIV-seropositive individuals, as measured by coculture and polymerase chain reaction methods, by at least 2.5 log,,. Recently, Sekiguchi and coworkers* reported that HTLV-I, from seropositive blood, was significantly reduced after filtration through a 3 log,, WBC filter, as detected by polymerase chain reaction (Kobayashi M, et al. Written communication, December 1991). Currently available filters, such as the Cellselect (NPBI, Amstelveen, The Netherlands), the Leukotrap Red Cell
Storage System (Cutter Biological, Miles Inc., Berkeley, CA), the Erypur (Organon Teknika, Turnhout, Belgium), the Imugard IG-500 (Terumo Medical Corp., Tokyo, Japan), the RCl00 (Pall Biomedical Products Corp., Glen Cove, NY), and the Sepacell R-500 (Asahi Medical CO., Tokyo, Japan), are capable of removing 1 to 3 log,, (9099.9%) of WBCs from RBCS.~-'~However, on the basis of epidemiologic and clinical data, it has been hypothesized that a 6 log,, reduction of WBCs, platelets, and plasma would eliminate, or at least reduce, viral infectivity of RBC units.16 As part of a process designed to meet these reduction goals, we have collaborated with manufacturers in the development of filters that would remove at least 6 log,, of WBCs from an RBC unit. We chose to WBC-reduce the RBCs within 8 hours after phlebotomy so as to integrate the filtration process into normal component preparation activities. This should also reduce the number of WBCs that can subsequently release viral particles and/or other agents during storage.17s1* This article describes the evaluation of experimental filters that remove 6 log,, of WBCs from RBCs, in a rapid manner and with high RBC recovery. A limited study of RBC in vitro storage parameters indicates that filtration should not affect RBC shelf life. Materials and Methods RBC preparation Blood was collected from healthy donors in CPDA-1 (Baxter Healthcare Corp., Deerfield, IL) by the American Red Cross Blood Services, Washington, DC Region. Whole blood was cooled from 37°C to 20 to 24'C and centrifuged (Sorvall RQC, DuPont, Wilmington, DE) for 5 minutes at 1471 x g (2250 rpm) within 8 hours of phlebotomy. Platelet-rich plasma was
From the Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland. Received for publication on June 12, 1990; revision received July 2, 1991, and accepted August 5, 1991.
129
130
TRANSFUSION Vol. 32, No. 2-1992
SADOFF ET AL.
Table 1. Filter performance* with red cells (RBCs) suspended in different diluents
Number of RBC units filtered
Filter Sepacell 4120 Sepacell 4120 Sepacell 4120 RC600 RC600 RC600
* Fresh
ARC811
4
ARC811
Saline Adsol
54 56 57 54 56 55
Flow rate (mumin) 12 10 10 9 9
f2
0.5 -c 1 f 1
f
f
hours) with buffv coat log,; (WBC,MBC,.,)
RBCs (20-24°C. 5 8
t Log reduction
*
Diluent Saline Adsol
8 3 4 6 4
Percent hematocrit
=
5 of 8 were ~ 1 . 3 ~ 1 0 ~ .
removed. We used the remaining RBCs, with buffy coat, for all filtration studies. When Adsol was not used, we diluted the RBCs to a hematocrit of 54 to 57 percent (0.54-0.57) with saline (0.9% wt/vol, Baxter) or a newly developed, experimental resuspension solution, ARC8.I9 All filtration studies were carried out at room temperature.
Filters We itemized performance specifications (6 log,, WBC reduction from fresh RBCs in 6.2
3.2 1.7 8.5 1.5 14.0 1.1 14.0 f 2.2
Clogged7
Cloggedn
8.1 9.3
f ? f ?
5 Below sensitivity of the assay. 11 Experimental storage s~lution.’~ 7 Presumed to be due to ARC8-induced swelling of RBCs.
final 0.55-mL concentrated pellet. When an average of one cell is counted per chamber (0.9 pL, 6 cells/6 total chambers counted), the uncertainty is 2.5 cells (+ 1 SD). An average of one cell per chamber can be extrapolated to an average of 1390 WBCs remaining per RBC unit. In the event that a total of fewer than 6 cells were seen in six chambers, we have recorded the results as the log,, reduction equivalent to that which would be calculated from an average of 1 WBC per chamber, with the realization that the results are beyond the sensitivity of the assay. As a result, we may be underestimating the level of WBC reduction achieved. RBC loss was calculated from prefiltration and postfiltration RBC weight and hematocrit. The average flow rate was taken as the volume filtered divided by the total filtration time, including RBC priming.
In vitro storage studies We undertook a limited examination of in vitro, postfiltration storage characteristics to assure ourselves that the filtration process was doing no damage to the RBCs. In each case, an entire unit was filtered on the day of collection into a 600-mL polyvinylchloride bag (PL-146, Baxter). Postfiltration samples represented Day 0, and the units were stored at 4°C for 42 days. We treated control units similarly but did not filter them. Aliquots (approx. 3 mL) were taken every 7 days and assayed. We measured plasma and total hemoglobin with Drabkin’s reagent as described elsewherez1 and calculated the percentage of hemolysis. RBC ATP and 2,3 DPG were measured according to accepted methods (Technical Bulletins 336-UV and 35UV, Sigma Chemical Co.) using a spectrophotometer (Lambda 48 UV-Vis, Perkin-Elmer, Oak Brook, IL). We scored the morphologic index as described by Usry et aLz2to indicate the condition of the RBCs, on the following basis: smooth disc, 1.0; crenated disc, 0.8; crenated discoid, 0.6; crenated spheroid, 0.4; crenated sphere, 0.2; and smooth sphere, 0.0. We counted 100 cells for each sample, and the total of the morphology score formed the morphologic index. Extracellular sodium and potassium were measured by flame photometry (Model 943, Instrumentation Laboratories, Lexington, MA).
Results The results of WBC reduction obtained with the Sepacell 4120 and RC600 filters are shown in Table 1. Each gave an average 6 log,, reduction for fresh RBCs diluted to a hema-
TRANSFUSION 1992-Vol.
131
6 LOG,, WBC FILTRATION
32, No. 2
Table 2. In vitm storage of red cells in ARC8* (n =3) Sample Control
Sepacell 4120
Day
Percentage of hemolysis
ATP (@/g Hb')
(pM/g Hb')
0 7 14 21 28 35 42
0.03 0.06 0.15 0.17 0.18 0.26 0.38
f 0.3
13.9 8.8 4.0 1.7 1.8 1.5 2.2
1.8 2.4 f 0.5 f 0.1 2 0.1 f 0.3 f 1.7
164.2 f 2.3 158.6 f 3.2 152.5 f 3.0 146.2 f 5.5 144.1 -e 4.1 125.2 f 2.0 133.5 f 1.8
1.7 12.9 19.0 27.2 30.2 41.5 37.1
f 0.1 f 0.3 f 1.2
0.08 0.07
4.2 4.9 5.0 4.4 4.4 4.1 4.0
0
0.02 0.04 0.25 0.27 0.32 0.32 0.35
0.00 0.00 rt 0.08 f 0.06 f 0.03 f 0.03 f 0.03
4.3 4.1 4.8 4.4 3.6 3.7 3.1
f 0.2 f 0.1
12.1 7.5 3.9 2.8 1.9 1.4 1.9
f f f f
0.5 0.4 0.5 0.3 t 0.3 f 0.0 f 0.1
165.8 158.3 149.9 146.7 140.1 140.1 136.6
1.8 11.7 16.6 21.0 24.7 28.4 32.6
f 0.1 f 0.6
7 14 21 28 35 42
f 0.01
f 0.02 f 0.09 f
0.07
f 0.06 f f f rt
t 0.5
f 0.2 f 0.3 f 0.4 f 0.3 t 0.9
2
0.5
f 0.5 f 0.0 2 0.4
f 0.2
2,3 DPG f f
Extracellular Na+ Extracellular K+ (mM) (mM)
f 1.6 f 0.8 f
1.7
f 1.1 f 1.4 f 1.6
f 1.9
3.1 1.2 1.1 f 1.4 f 2 f
f
0.3
f 0.6
0.7 1.2 f 1.2 f f
PH
Morphology score %
0.01 0.02 0.01 t 0.04 f 0.03 f 0.04 f 0.03
99.4 88.6 87.4 84.2 83.2 75.5 79.1
7.21 f 0.04 6.95 f 0.04 6.85 f 0.02 6.76 f 0.01 6.71 f 0.00 6.62 f 0.01 6.55 f 0.02
99.5 88.7 84.8 82.6 79.2 78.3 77.1
7.09 6.91 6.80 6.75 6.69 6.70 6.55
f
f f
f 0.2 f
0.7
f 0.9 f f f f
2.2 1.8 2.6 1.5
0.1 0.4 ? 0.3 f 0.6 f 1.1 f 0.3 f 0.5 f f
' Hemoglobin.
Table 3. In vitro storage of red cells in Adsol (n = 3) Sample Control
Sepacell 4120
Pall RC600
Percentage of Day hernolysis
ATP (@/g Hb*)
2,3 DPG ( p M g Hb')
Extracellular Na+ Extracellular K+ (mM) (mM)
0 7 14 21 28 35 42
0.06 rt 0.02 0.10 2 0.02 0.15 2 0.06 0.18 rt 0.05 0.24 2 0.09 0.20 2 0.06 0.38 2 0.09
4.3 4.4 4.9 4.7 4.6 4.4 3.7
0.5 f 0.3 f 0.4 f 0.2 f 0.3 f 0.6 f 0.5
13.5 7.9 3.5 1.9 2.0 1.4 1.4
f f
0.6 0.6 f 1.2 f 0.5 f 0.4 f 0.1 f 0.4
152.7 146.4 141.0 135.6 131.8 137.8 121.9
f
3.7 f 1.9 f 5.1 f 4.3 f 5.0 f 1.6 f 1.6
1.8 19.4 26.7 32.6 38.3 34.0 45.3
0 7 14 21 28 35 42
0.09 0.12 0.14 0.22 0.23 0.32 0.34
f
0.01 0.02 0.02 f 0.03 f 0.03 f 0.07 2 0.07
5.3 f 0.5 4.4 f 1.0 4.9 f 0.3 4.9 rt 0.7 4.5 f 0.2 3.9 f 0.4 3.2 f 0.5
12.7 6.9 2.5 2.8 2.3 1.6 2.1
f
1.6 1.0 rt 0.2 f 0.2 f 0.2 f 0.2 f 0.4
154.2 147.2 144.4 137.4 135.3 129.9 129.4
f 2.8 f 2.5
f 3.0 f 3.0 f 3.6
2.3 14.3 19.6 25.4 30.0 32.8 36.9
0 7 14 21 28 35 42
0.22 0.30 0.43 0.53 0.56 0.82 0.84
f 0.13
4.9 f 0.2 4.9 f 0.6 5.1 f 0.2 5.0 f 0.4 4.5 f 0.3 3.9 f 0.2 3.6 f 0.2
15.8 8.5 2.7 2.5 2.2 1.5 2.9
f 1.2 f 1.7 f 0.9 t 0.2 f 0.2
154.9 f 1.4 142.9 f 4.0 139.3 f 3.4 134.8 f 2.4 131.0 f 3.0 124.9 f 2.7 122.1 2 3.0
2.4 17.1 24.5 30.9 35.8 39.1 42.7
-t f
0.13 f 0.20 t 0.15 f 0.17 f 0.09 f 0.07 f
f
f
0.1 f 0.1 f
-c 3.7 f 1.7
Morphology PH
score%
f f f f
0.3 3.1 2.9 0.6 f 1.3 f 1.4 f 0.5
7.29 7.05 6.90 6.84 6.77 6.59 6.65
rt
f f f f
0.4 0.8 0.4 2.4 f 2.5 f 2.8 f 2.4
7.24 7.14 6.95 6.75 6.68 6.68 6.53
f f
f 0.7 f 3.1 f 2.7 f 3.3
7.25 7.14 6.93 6.74 6.67 6.70 6.63
f 0.04 99.0 f 0.6 f 0.02 89.6 2 4.4 f 0.09 85.2 f 4.5 f 0.03 80.6 f 2.8
f
2.7
f 2.1 f
1.3
0.02 f 0.00 f 0.05 f 0.07 f 0.07 f 0.02 2 0.06
99.3 84.6 82.1 79.4 77.1 81.4 74.1
0.2 1.9 f 4.6 f 2.7 f 3.1 f 1.6 t 3.6
0.01 0.01 f 0.01 0.04 f 0.01 f 0.00 f 0.01
99.2 92.2 86.9 83.3 81.2 79.5 79.1
f
*
f f f
f f
0.1 1.0 1.1 rt 0.5 f 1.6 f 0.6 t 1.5 f 5
0.05 78.8 f 4.7 0.02 79.1 f 2.5 0.07 77.2 f 2.7
' Hemoglobin. tocrit between 54 (0.54) and 57 (0.57) percent. Filtration was completed within 40 minutes. Performance did not appear to be affected by the diluents, Adsol or saline, or by the manufacturers' filtkr sterilization process (data not shown). Prefiltration dilution with ARC8 did affect the performance of the RC600. This was possibly due to the swelling of the RBCs,I9 which may block the filter. Tables 2 and 3 show the percentaRe of hemolysis: the ATP, 2,3 DPG, extracellular sodium, a d extracellhar -potassium levels; pH; and morphologic score for ARC& and *Adsol-diluted RBCs stored after filtration. No attempt was made to compare the RC600 and Sepacell 4120 filters, as the sample size was small and comparisons of experimental products would not be appropriate. None of the measured values Were appre.. ciably affected by filtration.
Discussion
We report here the Derformance of two newlv developed eq-&mental fi& capable of removing a; least 6 log,, of WBCs from fresh RBC units in less than 40 minutes. RBC loss under nonoptimized operating conditions was less than 15 Percent. Both filters gave an average 6 lojz,,-, reduction. The averagenumber of WBCs remaining after filtration of fresh ma with a hematocrit of 55 percent (approx. 0.55) was less than 1400 per RBC unit. Sirchia and reported the efficiencies of commercially available filters using RBCs stored for
132
TRANSFUSION
SADOFF ET AL.
1 to 14 days at 4°C after component preparation. They reported the average residual WBC load in saline-adenine-glucose-mannitolunits filtered with the Sepacell WOO and the RClOO as being 2 x lo6 (range, 0-6 x lo6) and 0 x lo6 (range, 0-17 x 106). Similar results have been reported by other^.^-^' Such results correspond to approximately 3 log,, WBC reduction, respectively. Although there are differences between these data and our studies in testing methods and starting components, we believe that the Sepacell 4120 and the RC600 provide at least 2.5 to 3 log,, better reduction than any currently available filter. Our investigation of in vitro storage parameters showed no significant difference in the filtered and unfiltered (control) RBC units. A number of i n v e ~ t i g a t o r sfound ~-~~ improved storage in RBC units that were WBC-filtered. Hogman et aLZ*originally identified what they called “hemolytic enzymes,” which leaked from the WBCs during storage. In studies in which they added WBCs to RBCs that had been buffy coat-depleted, they found hemolysis and potassium leakage proportional to the number of WBCs added. They also found that the presence of either an enzyme inhibitor (Caminomethyl-cyclohexancarbonic acid) or natural inhibitors present in plasma reduced the amount of hemolysis and potassium leakage. Advances in WBC filtration and sterile docking have made it possible to filter fresh RBCs prior to storage with maintenance of their shelf life. Angue31 and LovricZ9and their coworkers found what they describe as marked differences between ATP, potassium, and other storage parameters in filtered and unfiltered RBC units, while Riedner30and P i e t e r s and ~ ~ ~their coworkers found more subtle differences, including differences in lactate dehydrogenase, elastase, and P-thrombogl~bulin~~ and in the rate of hemolysis, glucose consumption, and pH.32 The lack of a difference between control and filtered RBCs in our studies may be due to the small number tested. The fact that these filters showed no detrimental in vitro effect indicates that they provide RBCs of equal quality to those provided by currently available filters. The development and implementation of more efficient WBC-reduction filters for RBC and platelet components would not only help eliminate febrile transfusion reaction^'^.*^ and WBC-associated HLA alloimmunization’ but may reduce or even prevent transfusion-associated transmission of WBC-associated viral diseases, such as CMV and HTLV-HI. The preparation of WBCreduced RBCs at the time of component preparation (possibly with the use of a sterile docking device32), rather than at the bedside, may also produce a more consistent RBC. The incorporation of a filter into the collection set at the time of manufacturing may improve overall staff productivity. The technology, therefore, is available with which the blood banking community can
Vol. 32, No. 2-1992
supply a highly WBC-reduced RBC component with a shelf life that is expected to be normal. Acknowledgments The cooperation of Drs. D. Pall and T. Gsell of Pall Biomedical Products Corporation and of Messrs. K. Ohno and S. Oka of Asahi Medical Co., Ltd., and their respective staff members is appreciated.
References 1. Rebulla P, Bertolini F, Parravicini A, Sirchia G. Leukocyte-poor
blood components: a purer and safer transfusion product for recipients? Transfus Med Rev 1990;4(Suppl 1):19-23. 2. Gilbert GL, Hayes K, Hudson IL, James J. Prevention of transfusion-acquired cytomegalovirus infection in infants by blood filtration to remove leucocytes. Neonatal Cytomegalovirus Infection Study Group. Lancet 1989;1:1228-31. 3. Murphy MF, Grint PCA, Hardiman AE, Lister TA, Waters AH. Use of leucocyte-poor blood components to prevent primary cytomegalovirus (CMV) infection in patients with acute leukaemia (letter). Br J Haematol 1988;70253-4. 4. DiNubile MJ. Screening HIV-infected recipients of blood transfusions for CMV infection (letter). N Engl J Med 1990,323:1282-3. 5. De Graan-Hentzen YCE, Gratama JW, Mudde GC, et al. Prevention of primary cytomegalovirus infection in patients with hematologic malignancies by intensive white cell depletion of blood products. Transfusion 1989;29:757-60. 6. Rawal BD, Busch MP, Endow R, et al. Reduction of human immunodeficiency virus-infected cells from donor blood by leukocyte filtration. Transfusion 1989;29:460-2. 7. Bruisten SM, Tersmette T, Wester MR, Vos AH, Koppleman MH, Huisman J. Efficiency of white cell filtration and a freezethaw procedure for removal of HIV-infected cells from blood. Transfusion 1990;30833-7. 8. Sekiguchi S, Takahashi T, Kobayashi M, Abe H, Ikeda H. Leukocyte depletion of blood and its effect on viral transmission (abstract). Presented at British Blood Transfusion Society Conference on Aspects of Blood Filtration, Birmingham, UK, April 1990. 9. Wester MR, Prins HK, Huisman JG. A new radioimmunoassay for the detection of small amounts of white cells and platelets in red cell concentrates: implications for blood transfusion. Transfusion 1990;30117-25. 10. Davey RJ, Carmen RA, Simon TL, et al. Preparation of white cell-depleted red cells for 42-day storage using an integral in-line filter. Transfusion 1989;29496-9. 11. Pikul FJ, Farrar RP, Boris MB, et al. Effectiveness of two synthetic fiber filters for removing white cells from AS-1 red cells. Transfusion 1989;29:590-5. 12. Mijovic V, Brozovic B, Hughes ASB, Davies TD. Leukocytedepleted blood: a comparison of filtration techniques. Transfusion 1983;23:30-2. 13. Reesink HW, Veldman H, Henrichs HJ, Prins HK, Loos JA. Removal of leukocytes from blood by fibre filtration. A comparison study on the performance of two commercially available filters. Vox Sang 198242281-8. 14. Sirchia G, Wenz B, Rebulla P, Parravicini A, Carnelli V, Bertolini F. Removal of white cells from red cells by transfusion through a new filter. Transfusion 1990;30:30-3. 15. Sirchia G, Rebulla P, Parravicini A, Carnelli G, Gianotti A, Bertolini F. Leukocyte depletion of red cell units at the bedside by transfusion through a new filter. Transfusion 1987;27:402-5. 16. Wagner SJ, Friedman LI, Dodd RY. Approaches to the reduction of viral infectivity in cellular blood components and single donor plasma. Transfus Med Rev 1991;5:18-32. 17. Andreu G, Dewailly J, Leberre C, et al. Prevention of HLA immunization with leukocyte-poor packed red cells and platelet concentrates obtained by filtration. Blood 1988;72:964-9.
TRANSFUSION 1992-Vol. 32. No. 2
6 LOG,, WBC FILTRATION
18. Lindcna J, Papastavrou S, Seidel JW. Efficacy and safety of a polyestcr lcukocyte removal filter for whole blood and red cell conccntratc filtration. J Clin Chem Clin Biochem 1989;27:331-6. 19. Meryman HT, Hornblower M, Keegan T, et al. Refrigerated storage of washed red cells. Vox Sang 1991;60:88-98. 20. Sadoff BJ, Dooley DC, Kapoor V, Law P, Friedman LI, Shornberg RR. Methods for measuring a 6 log,, white cell depletion in red cells. Transfusion 1991;31:150-5. 21. Moore GL, Ledford ME, Merydith A. A micromodification of
the Drabkin hemoglobin assay for measuring plasma hemoglobin in the range of 5 to u)oo mg/dl. Biochem Med 1981;26167-73. 22. Usry RT, Moore GL, Manalo FW.Morphology of stored, rejuvenated human erythrocytes. Vox Sang 1975;28:176-83. 23. EWck M, Wagner M, Knuppel W, et al. Preparation of white celldcpleted blood: comparison of two bedside filter systems. Transfusion 1990;30:26-9. 24. Bodensteiner DC. A flow cytometric technique to accurately measure post-filtration white blood cell counts. Transfusion 1989;29:651-3.
25. Bodensteiner DC. Leukocyte depletion filters: a comparison of efficiency. Am J Hematol 1990;35:184-6. 26. Reverberi R, Menini C. Clinical efficacy of five filters specific for leukocyte removal. Vox Sang 1990.58188-91. 27. Steneker I, Biewenga J. Histologic and immunohistochemical studies on the preparation of white cell-poor red cell concentrates: the filtration process using three different polyester filters. Transfusion 1991;31:40-6. 28. Hogman CF, Hedlund K, Akerblom 0, Venge P. Red blood cell preservation in protein-poor media. 1. Leukocyte enzymes as a cause of hemolysis. Transfusion 1978;18:233-41.
133
29. Lovric VA. Schuller M, Raftos J, Wisdom L. Filtered microag-
pscaate-free erythrocyte concentrates with 35-day shelf life. Vox -Sang 1981;41:6-10. 30. Riedner C. Heim MU. MemDcl W. Wilmanns W. Possibilitv to improve piesewation of whde blood by leukocyte-depletioibefore storage. Vox Sang 1990,5978-82. 31. Angue M, Chatelain P, Fiabane S, Domy M, Guignier F, Richaud P. [Viability of human red blood cells preserved for 35 days after leukocyte depletion (invitro study).] (Eng abstract). Rev Fr Transfus Hemobiol 1989;3227-36. 32. Pietersz RNI, Reesink HW. de Korte D, Dekker WJA, van den Ende A, Loos JA. Storage of leukocyte-poor red cell concentrates: filtration in a closcd system using a sterile connection device. Vox Sang 1989;51:29-36. Bernard J. Sadoff, MS. Chemical Engineer, Product Development Laboratory, The Jerome H. Holland Laboratory for the Biomedical Sciences. Robert R. Shomberg, PhD, Scientist, Product Development Laboratory, The Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. [Reprint requests] Karen M. Miller, Bs, Research Assistant, Product Development Laboratory, The Jerome H. Holland Laboratory for the Biomedical Sciences. Dung P. Ngo, BS, Medical Technologist, Product Development Laboratory, The Jerome H. Holland Laboratory for the Biomedical Sciences. Leonard I. Friedman, ScD, Head, Product Development Laboratory, The Jerome H. Holland Laboratory for the Biomedical Sciences.
JEAN JULLIARD PRIZE The 13th Jean Julliard Prize, which was established by the International Society of Blood Transfusion in memory of its first Secretary General, will be awarded during the 22nd International Congress in Sao Paulo, Brazil, October 9-13, 1992. The Prize is reserved for scientists under 40 years of age, in recognition of recently completed work on blood transfusion or related subjects. In order to qualify, candidates should forward six (6) copies of their submission, including a Curriculum Vitae, to the Secretary General, before April 1, 1992. The Prize, 3000 Swiss Francs, will be awarded during the Congress. Full regulations are available on request from the Secretary General.
Abbrevlatlons: CMV = cytomegalovlrus; HIV = human lmmunodetlclency vlrus; HTLV1/11 = human 1-lymphotroplc vlrus type I or It; RBC(s) = red cell(s); WBC(s) = whlte cells.
THEREMOVAL OF WHITE CELLS (WBCs) from red cells (RBCs) by filtration has been recognized as important for the reduction of febrile transfusion reactions and WBCassociated HLA alloimmunization.' Viral infections that are specifically associated with WBCs, such as cytomegalovirus (CMV) and human T-lymphotropic virus type I or I1 (HTLV-MI), should also be reduced significantly, and may even be eliminated, if WBC reduction is extensive. 1-5 Recently, Rawal and coworkers6reported approximately 2 log,, reduction of viral titer in patients naturally infected with human immunodeficiency virus (HIV), achieved with a 3 log,, WBC-reduction filter. This group also demonstrated a 5.9 log,, reduction of HIV titer in seronegative RBCs inoculated with HIV,,infected HUT-78 cells. It has been suggested that perhaps the difference in results (2.0 vs. 5.9) is due to the altered immunologic and electrostatic properties of the HUT-78 cells versus the properties of naturally occurring WBCs. Similarly, Bruisten et aL7 reported that filtration through a polyester WBC filter decreased the infectivity of blood from HIV-seropositive individuals, as measured by coculture and polymerase chain reaction methods, by at least 2.5 log,,. Recently, Sekiguchi and coworkers* reported that HTLV-I, from seropositive blood, was significantly reduced after filtration through a 3 log,, WBC filter, as detected by polymerase chain reaction (Kobayashi M, et al. Written communication, December 1991). Currently available filters, such as the Cellselect (NPBI, Amstelveen, The Netherlands), the Leukotrap Red Cell
Storage System (Cutter Biological, Miles Inc., Berkeley, CA), the Erypur (Organon Teknika, Turnhout, Belgium), the Imugard IG-500 (Terumo Medical Corp., Tokyo, Japan), the RCl00 (Pall Biomedical Products Corp., Glen Cove, NY), and the Sepacell R-500 (Asahi Medical CO., Tokyo, Japan), are capable of removing 1 to 3 log,, (9099.9%) of WBCs from RBCS.~-'~However, on the basis of epidemiologic and clinical data, it has been hypothesized that a 6 log,, reduction of WBCs, platelets, and plasma would eliminate, or at least reduce, viral infectivity of RBC units.16 As part of a process designed to meet these reduction goals, we have collaborated with manufacturers in the development of filters that would remove at least 6 log,, of WBCs from an RBC unit. We chose to WBC-reduce the RBCs within 8 hours after phlebotomy so as to integrate the filtration process into normal component preparation activities. This should also reduce the number of WBCs that can subsequently release viral particles and/or other agents during storage.17s1* This article describes the evaluation of experimental filters that remove 6 log,, of WBCs from RBCs, in a rapid manner and with high RBC recovery. A limited study of RBC in vitro storage parameters indicates that filtration should not affect RBC shelf life. Materials and Methods RBC preparation Blood was collected from healthy donors in CPDA-1 (Baxter Healthcare Corp., Deerfield, IL) by the American Red Cross Blood Services, Washington, DC Region. Whole blood was cooled from 37°C to 20 to 24'C and centrifuged (Sorvall RQC, DuPont, Wilmington, DE) for 5 minutes at 1471 x g (2250 rpm) within 8 hours of phlebotomy. Platelet-rich plasma was
From the Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland. Received for publication on June 12, 1990; revision received July 2, 1991, and accepted August 5, 1991.
129
130
TRANSFUSION Vol. 32, No. 2-1992
SADOFF ET AL.
Table 1. Filter performance* with red cells (RBCs) suspended in different diluents
Number of RBC units filtered
Filter Sepacell 4120 Sepacell 4120 Sepacell 4120 RC600 RC600 RC600
* Fresh
ARC811
4
ARC811
Saline Adsol
54 56 57 54 56 55
Flow rate (mumin) 12 10 10 9 9
f2
0.5 -c 1 f 1
f
f
hours) with buffv coat log,; (WBC,MBC,.,)
RBCs (20-24°C. 5 8
t Log reduction
*
Diluent Saline Adsol
8 3 4 6 4
Percent hematocrit
=
5 of 8 were ~ 1 . 3 ~ 1 0 ~ .
removed. We used the remaining RBCs, with buffy coat, for all filtration studies. When Adsol was not used, we diluted the RBCs to a hematocrit of 54 to 57 percent (0.54-0.57) with saline (0.9% wt/vol, Baxter) or a newly developed, experimental resuspension solution, ARC8.I9 All filtration studies were carried out at room temperature.
Filters We itemized performance specifications (6 log,, WBC reduction from fresh RBCs in 6.2
3.2 1.7 8.5 1.5 14.0 1.1 14.0 f 2.2
Clogged7
Cloggedn
8.1 9.3
f ? f ?
5 Below sensitivity of the assay. 11 Experimental storage s~lution.’~ 7 Presumed to be due to ARC8-induced swelling of RBCs.
final 0.55-mL concentrated pellet. When an average of one cell is counted per chamber (0.9 pL, 6 cells/6 total chambers counted), the uncertainty is 2.5 cells (+ 1 SD). An average of one cell per chamber can be extrapolated to an average of 1390 WBCs remaining per RBC unit. In the event that a total of fewer than 6 cells were seen in six chambers, we have recorded the results as the log,, reduction equivalent to that which would be calculated from an average of 1 WBC per chamber, with the realization that the results are beyond the sensitivity of the assay. As a result, we may be underestimating the level of WBC reduction achieved. RBC loss was calculated from prefiltration and postfiltration RBC weight and hematocrit. The average flow rate was taken as the volume filtered divided by the total filtration time, including RBC priming.
In vitro storage studies We undertook a limited examination of in vitro, postfiltration storage characteristics to assure ourselves that the filtration process was doing no damage to the RBCs. In each case, an entire unit was filtered on the day of collection into a 600-mL polyvinylchloride bag (PL-146, Baxter). Postfiltration samples represented Day 0, and the units were stored at 4°C for 42 days. We treated control units similarly but did not filter them. Aliquots (approx. 3 mL) were taken every 7 days and assayed. We measured plasma and total hemoglobin with Drabkin’s reagent as described elsewherez1 and calculated the percentage of hemolysis. RBC ATP and 2,3 DPG were measured according to accepted methods (Technical Bulletins 336-UV and 35UV, Sigma Chemical Co.) using a spectrophotometer (Lambda 48 UV-Vis, Perkin-Elmer, Oak Brook, IL). We scored the morphologic index as described by Usry et aLz2to indicate the condition of the RBCs, on the following basis: smooth disc, 1.0; crenated disc, 0.8; crenated discoid, 0.6; crenated spheroid, 0.4; crenated sphere, 0.2; and smooth sphere, 0.0. We counted 100 cells for each sample, and the total of the morphology score formed the morphologic index. Extracellular sodium and potassium were measured by flame photometry (Model 943, Instrumentation Laboratories, Lexington, MA).
Results The results of WBC reduction obtained with the Sepacell 4120 and RC600 filters are shown in Table 1. Each gave an average 6 log,, reduction for fresh RBCs diluted to a hema-
TRANSFUSION 1992-Vol.
131
6 LOG,, WBC FILTRATION
32, No. 2
Table 2. In vitm storage of red cells in ARC8* (n =3) Sample Control
Sepacell 4120
Day
Percentage of hemolysis
ATP (@/g Hb')
(pM/g Hb')
0 7 14 21 28 35 42
0.03 0.06 0.15 0.17 0.18 0.26 0.38
f 0.3
13.9 8.8 4.0 1.7 1.8 1.5 2.2
1.8 2.4 f 0.5 f 0.1 2 0.1 f 0.3 f 1.7
164.2 f 2.3 158.6 f 3.2 152.5 f 3.0 146.2 f 5.5 144.1 -e 4.1 125.2 f 2.0 133.5 f 1.8
1.7 12.9 19.0 27.2 30.2 41.5 37.1
f 0.1 f 0.3 f 1.2
0.08 0.07
4.2 4.9 5.0 4.4 4.4 4.1 4.0
0
0.02 0.04 0.25 0.27 0.32 0.32 0.35
0.00 0.00 rt 0.08 f 0.06 f 0.03 f 0.03 f 0.03
4.3 4.1 4.8 4.4 3.6 3.7 3.1
f 0.2 f 0.1
12.1 7.5 3.9 2.8 1.9 1.4 1.9
f f f f
0.5 0.4 0.5 0.3 t 0.3 f 0.0 f 0.1
165.8 158.3 149.9 146.7 140.1 140.1 136.6
1.8 11.7 16.6 21.0 24.7 28.4 32.6
f 0.1 f 0.6
7 14 21 28 35 42
f 0.01
f 0.02 f 0.09 f
0.07
f 0.06 f f f rt
t 0.5
f 0.2 f 0.3 f 0.4 f 0.3 t 0.9
2
0.5
f 0.5 f 0.0 2 0.4
f 0.2
2,3 DPG f f
Extracellular Na+ Extracellular K+ (mM) (mM)
f 1.6 f 0.8 f
1.7
f 1.1 f 1.4 f 1.6
f 1.9
3.1 1.2 1.1 f 1.4 f 2 f
f
0.3
f 0.6
0.7 1.2 f 1.2 f f
PH
Morphology score %
0.01 0.02 0.01 t 0.04 f 0.03 f 0.04 f 0.03
99.4 88.6 87.4 84.2 83.2 75.5 79.1
7.21 f 0.04 6.95 f 0.04 6.85 f 0.02 6.76 f 0.01 6.71 f 0.00 6.62 f 0.01 6.55 f 0.02
99.5 88.7 84.8 82.6 79.2 78.3 77.1
7.09 6.91 6.80 6.75 6.69 6.70 6.55
f
f f
f 0.2 f
0.7
f 0.9 f f f f
2.2 1.8 2.6 1.5
0.1 0.4 ? 0.3 f 0.6 f 1.1 f 0.3 f 0.5 f f
' Hemoglobin.
Table 3. In vitro storage of red cells in Adsol (n = 3) Sample Control
Sepacell 4120
Pall RC600
Percentage of Day hernolysis
ATP (@/g Hb*)
2,3 DPG ( p M g Hb')
Extracellular Na+ Extracellular K+ (mM) (mM)
0 7 14 21 28 35 42
0.06 rt 0.02 0.10 2 0.02 0.15 2 0.06 0.18 rt 0.05 0.24 2 0.09 0.20 2 0.06 0.38 2 0.09
4.3 4.4 4.9 4.7 4.6 4.4 3.7
0.5 f 0.3 f 0.4 f 0.2 f 0.3 f 0.6 f 0.5
13.5 7.9 3.5 1.9 2.0 1.4 1.4
f f
0.6 0.6 f 1.2 f 0.5 f 0.4 f 0.1 f 0.4
152.7 146.4 141.0 135.6 131.8 137.8 121.9
f
3.7 f 1.9 f 5.1 f 4.3 f 5.0 f 1.6 f 1.6
1.8 19.4 26.7 32.6 38.3 34.0 45.3
0 7 14 21 28 35 42
0.09 0.12 0.14 0.22 0.23 0.32 0.34
f
0.01 0.02 0.02 f 0.03 f 0.03 f 0.07 2 0.07
5.3 f 0.5 4.4 f 1.0 4.9 f 0.3 4.9 rt 0.7 4.5 f 0.2 3.9 f 0.4 3.2 f 0.5
12.7 6.9 2.5 2.8 2.3 1.6 2.1
f
1.6 1.0 rt 0.2 f 0.2 f 0.2 f 0.2 f 0.4
154.2 147.2 144.4 137.4 135.3 129.9 129.4
f 2.8 f 2.5
f 3.0 f 3.0 f 3.6
2.3 14.3 19.6 25.4 30.0 32.8 36.9
0 7 14 21 28 35 42
0.22 0.30 0.43 0.53 0.56 0.82 0.84
f 0.13
4.9 f 0.2 4.9 f 0.6 5.1 f 0.2 5.0 f 0.4 4.5 f 0.3 3.9 f 0.2 3.6 f 0.2
15.8 8.5 2.7 2.5 2.2 1.5 2.9
f 1.2 f 1.7 f 0.9 t 0.2 f 0.2
154.9 f 1.4 142.9 f 4.0 139.3 f 3.4 134.8 f 2.4 131.0 f 3.0 124.9 f 2.7 122.1 2 3.0
2.4 17.1 24.5 30.9 35.8 39.1 42.7
-t f
0.13 f 0.20 t 0.15 f 0.17 f 0.09 f 0.07 f
f
f
0.1 f 0.1 f
-c 3.7 f 1.7
Morphology PH
score%
f f f f
0.3 3.1 2.9 0.6 f 1.3 f 1.4 f 0.5
7.29 7.05 6.90 6.84 6.77 6.59 6.65
rt
f f f f
0.4 0.8 0.4 2.4 f 2.5 f 2.8 f 2.4
7.24 7.14 6.95 6.75 6.68 6.68 6.53
f f
f 0.7 f 3.1 f 2.7 f 3.3
7.25 7.14 6.93 6.74 6.67 6.70 6.63
f 0.04 99.0 f 0.6 f 0.02 89.6 2 4.4 f 0.09 85.2 f 4.5 f 0.03 80.6 f 2.8
f
2.7
f 2.1 f
1.3
0.02 f 0.00 f 0.05 f 0.07 f 0.07 f 0.02 2 0.06
99.3 84.6 82.1 79.4 77.1 81.4 74.1
0.2 1.9 f 4.6 f 2.7 f 3.1 f 1.6 t 3.6
0.01 0.01 f 0.01 0.04 f 0.01 f 0.00 f 0.01
99.2 92.2 86.9 83.3 81.2 79.5 79.1
f
*
f f f
f f
0.1 1.0 1.1 rt 0.5 f 1.6 f 0.6 t 1.5 f 5
0.05 78.8 f 4.7 0.02 79.1 f 2.5 0.07 77.2 f 2.7
' Hemoglobin. tocrit between 54 (0.54) and 57 (0.57) percent. Filtration was completed within 40 minutes. Performance did not appear to be affected by the diluents, Adsol or saline, or by the manufacturers' filtkr sterilization process (data not shown). Prefiltration dilution with ARC8 did affect the performance of the RC600. This was possibly due to the swelling of the RBCs,I9 which may block the filter. Tables 2 and 3 show the percentaRe of hemolysis: the ATP, 2,3 DPG, extracellular sodium, a d extracellhar -potassium levels; pH; and morphologic score for ARC& and *Adsol-diluted RBCs stored after filtration. No attempt was made to compare the RC600 and Sepacell 4120 filters, as the sample size was small and comparisons of experimental products would not be appropriate. None of the measured values Were appre.. ciably affected by filtration.
Discussion
We report here the Derformance of two newlv developed eq-&mental fi& capable of removing a; least 6 log,, of WBCs from fresh RBC units in less than 40 minutes. RBC loss under nonoptimized operating conditions was less than 15 Percent. Both filters gave an average 6 lojz,,-, reduction. The averagenumber of WBCs remaining after filtration of fresh ma with a hematocrit of 55 percent (approx. 0.55) was less than 1400 per RBC unit. Sirchia and reported the efficiencies of commercially available filters using RBCs stored for
132
TRANSFUSION
SADOFF ET AL.
1 to 14 days at 4°C after component preparation. They reported the average residual WBC load in saline-adenine-glucose-mannitolunits filtered with the Sepacell WOO and the RClOO as being 2 x lo6 (range, 0-6 x lo6) and 0 x lo6 (range, 0-17 x 106). Similar results have been reported by other^.^-^' Such results correspond to approximately 3 log,, WBC reduction, respectively. Although there are differences between these data and our studies in testing methods and starting components, we believe that the Sepacell 4120 and the RC600 provide at least 2.5 to 3 log,, better reduction than any currently available filter. Our investigation of in vitro storage parameters showed no significant difference in the filtered and unfiltered (control) RBC units. A number of i n v e ~ t i g a t o r sfound ~-~~ improved storage in RBC units that were WBC-filtered. Hogman et aLZ*originally identified what they called “hemolytic enzymes,” which leaked from the WBCs during storage. In studies in which they added WBCs to RBCs that had been buffy coat-depleted, they found hemolysis and potassium leakage proportional to the number of WBCs added. They also found that the presence of either an enzyme inhibitor (Caminomethyl-cyclohexancarbonic acid) or natural inhibitors present in plasma reduced the amount of hemolysis and potassium leakage. Advances in WBC filtration and sterile docking have made it possible to filter fresh RBCs prior to storage with maintenance of their shelf life. Angue31 and LovricZ9and their coworkers found what they describe as marked differences between ATP, potassium, and other storage parameters in filtered and unfiltered RBC units, while Riedner30and P i e t e r s and ~ ~ ~their coworkers found more subtle differences, including differences in lactate dehydrogenase, elastase, and P-thrombogl~bulin~~ and in the rate of hemolysis, glucose consumption, and pH.32 The lack of a difference between control and filtered RBCs in our studies may be due to the small number tested. The fact that these filters showed no detrimental in vitro effect indicates that they provide RBCs of equal quality to those provided by currently available filters. The development and implementation of more efficient WBC-reduction filters for RBC and platelet components would not only help eliminate febrile transfusion reaction^'^.*^ and WBC-associated HLA alloimmunization’ but may reduce or even prevent transfusion-associated transmission of WBC-associated viral diseases, such as CMV and HTLV-HI. The preparation of WBCreduced RBCs at the time of component preparation (possibly with the use of a sterile docking device32), rather than at the bedside, may also produce a more consistent RBC. The incorporation of a filter into the collection set at the time of manufacturing may improve overall staff productivity. The technology, therefore, is available with which the blood banking community can
Vol. 32, No. 2-1992
supply a highly WBC-reduced RBC component with a shelf life that is expected to be normal. Acknowledgments The cooperation of Drs. D. Pall and T. Gsell of Pall Biomedical Products Corporation and of Messrs. K. Ohno and S. Oka of Asahi Medical Co., Ltd., and their respective staff members is appreciated.
References 1. Rebulla P, Bertolini F, Parravicini A, Sirchia G. Leukocyte-poor
blood components: a purer and safer transfusion product for recipients? Transfus Med Rev 1990;4(Suppl 1):19-23. 2. Gilbert GL, Hayes K, Hudson IL, James J. Prevention of transfusion-acquired cytomegalovirus infection in infants by blood filtration to remove leucocytes. Neonatal Cytomegalovirus Infection Study Group. Lancet 1989;1:1228-31. 3. Murphy MF, Grint PCA, Hardiman AE, Lister TA, Waters AH. Use of leucocyte-poor blood components to prevent primary cytomegalovirus (CMV) infection in patients with acute leukaemia (letter). Br J Haematol 1988;70253-4. 4. DiNubile MJ. Screening HIV-infected recipients of blood transfusions for CMV infection (letter). N Engl J Med 1990,323:1282-3. 5. De Graan-Hentzen YCE, Gratama JW, Mudde GC, et al. Prevention of primary cytomegalovirus infection in patients with hematologic malignancies by intensive white cell depletion of blood products. Transfusion 1989;29:757-60. 6. Rawal BD, Busch MP, Endow R, et al. Reduction of human immunodeficiency virus-infected cells from donor blood by leukocyte filtration. Transfusion 1989;29:460-2. 7. Bruisten SM, Tersmette T, Wester MR, Vos AH, Koppleman MH, Huisman J. Efficiency of white cell filtration and a freezethaw procedure for removal of HIV-infected cells from blood. Transfusion 1990;30833-7. 8. Sekiguchi S, Takahashi T, Kobayashi M, Abe H, Ikeda H. Leukocyte depletion of blood and its effect on viral transmission (abstract). Presented at British Blood Transfusion Society Conference on Aspects of Blood Filtration, Birmingham, UK, April 1990. 9. Wester MR, Prins HK, Huisman JG. A new radioimmunoassay for the detection of small amounts of white cells and platelets in red cell concentrates: implications for blood transfusion. Transfusion 1990;30117-25. 10. Davey RJ, Carmen RA, Simon TL, et al. Preparation of white cell-depleted red cells for 42-day storage using an integral in-line filter. Transfusion 1989;29496-9. 11. Pikul FJ, Farrar RP, Boris MB, et al. Effectiveness of two synthetic fiber filters for removing white cells from AS-1 red cells. Transfusion 1989;29:590-5. 12. Mijovic V, Brozovic B, Hughes ASB, Davies TD. Leukocytedepleted blood: a comparison of filtration techniques. Transfusion 1983;23:30-2. 13. Reesink HW, Veldman H, Henrichs HJ, Prins HK, Loos JA. Removal of leukocytes from blood by fibre filtration. A comparison study on the performance of two commercially available filters. Vox Sang 198242281-8. 14. Sirchia G, Wenz B, Rebulla P, Parravicini A, Carnelli V, Bertolini F. Removal of white cells from red cells by transfusion through a new filter. Transfusion 1990;30:30-3. 15. Sirchia G, Rebulla P, Parravicini A, Carnelli G, Gianotti A, Bertolini F. Leukocyte depletion of red cell units at the bedside by transfusion through a new filter. Transfusion 1987;27:402-5. 16. Wagner SJ, Friedman LI, Dodd RY. Approaches to the reduction of viral infectivity in cellular blood components and single donor plasma. Transfus Med Rev 1991;5:18-32. 17. Andreu G, Dewailly J, Leberre C, et al. Prevention of HLA immunization with leukocyte-poor packed red cells and platelet concentrates obtained by filtration. Blood 1988;72:964-9.
TRANSFUSION 1992-Vol. 32. No. 2
6 LOG,, WBC FILTRATION
18. Lindcna J, Papastavrou S, Seidel JW. Efficacy and safety of a polyestcr lcukocyte removal filter for whole blood and red cell conccntratc filtration. J Clin Chem Clin Biochem 1989;27:331-6. 19. Meryman HT, Hornblower M, Keegan T, et al. Refrigerated storage of washed red cells. Vox Sang 1991;60:88-98. 20. Sadoff BJ, Dooley DC, Kapoor V, Law P, Friedman LI, Shornberg RR. Methods for measuring a 6 log,, white cell depletion in red cells. Transfusion 1991;31:150-5. 21. Moore GL, Ledford ME, Merydith A. A micromodification of
the Drabkin hemoglobin assay for measuring plasma hemoglobin in the range of 5 to u)oo mg/dl. Biochem Med 1981;26167-73. 22. Usry RT, Moore GL, Manalo FW.Morphology of stored, rejuvenated human erythrocytes. Vox Sang 1975;28:176-83. 23. EWck M, Wagner M, Knuppel W, et al. Preparation of white celldcpleted blood: comparison of two bedside filter systems. Transfusion 1990;30:26-9. 24. Bodensteiner DC. A flow cytometric technique to accurately measure post-filtration white blood cell counts. Transfusion 1989;29:651-3.
25. Bodensteiner DC. Leukocyte depletion filters: a comparison of efficiency. Am J Hematol 1990;35:184-6. 26. Reverberi R, Menini C. Clinical efficacy of five filters specific for leukocyte removal. Vox Sang 1990.58188-91. 27. Steneker I, Biewenga J. Histologic and immunohistochemical studies on the preparation of white cell-poor red cell concentrates: the filtration process using three different polyester filters. Transfusion 1991;31:40-6. 28. Hogman CF, Hedlund K, Akerblom 0, Venge P. Red blood cell preservation in protein-poor media. 1. Leukocyte enzymes as a cause of hemolysis. Transfusion 1978;18:233-41.
133
29. Lovric VA. Schuller M, Raftos J, Wisdom L. Filtered microag-
pscaate-free erythrocyte concentrates with 35-day shelf life. Vox -Sang 1981;41:6-10. 30. Riedner C. Heim MU. MemDcl W. Wilmanns W. Possibilitv to improve piesewation of whde blood by leukocyte-depletioibefore storage. Vox Sang 1990,5978-82. 31. Angue M, Chatelain P, Fiabane S, Domy M, Guignier F, Richaud P. [Viability of human red blood cells preserved for 35 days after leukocyte depletion (invitro study).] (Eng abstract). Rev Fr Transfus Hemobiol 1989;3227-36. 32. Pietersz RNI, Reesink HW. de Korte D, Dekker WJA, van den Ende A, Loos JA. Storage of leukocyte-poor red cell concentrates: filtration in a closcd system using a sterile connection device. Vox Sang 1989;51:29-36. Bernard J. Sadoff, MS. Chemical Engineer, Product Development Laboratory, The Jerome H. Holland Laboratory for the Biomedical Sciences. Robert R. Shomberg, PhD, Scientist, Product Development Laboratory, The Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. [Reprint requests] Karen M. Miller, Bs, Research Assistant, Product Development Laboratory, The Jerome H. Holland Laboratory for the Biomedical Sciences. Dung P. Ngo, BS, Medical Technologist, Product Development Laboratory, The Jerome H. Holland Laboratory for the Biomedical Sciences. Leonard I. Friedman, ScD, Head, Product Development Laboratory, The Jerome H. Holland Laboratory for the Biomedical Sciences.
JEAN JULLIARD PRIZE The 13th Jean Julliard Prize, which was established by the International Society of Blood Transfusion in memory of its first Secretary General, will be awarded during the 22nd International Congress in Sao Paulo, Brazil, October 9-13, 1992. The Prize is reserved for scientists under 40 years of age, in recognition of recently completed work on blood transfusion or related subjects. In order to qualify, candidates should forward six (6) copies of their submission, including a Curriculum Vitae, to the Secretary General, before April 1, 1992. The Prize, 3000 Swiss Francs, will be awarded during the Congress. Full regulations are available on request from the Secretary General.
