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CE UPDATE- BANKING II German F. Leparc, MD

Leukocyte Reduction in Cellular Blood Components Downloaded from https://academic.oup.com/labmed/article/28/5/328/2503810 by guest on 27 September 2021

it is clear that some patients undergoing transfu­ ABSTRACT As we better understand the biologic effects of sion will benefit from the removal of leukocytes the presence of leukocytes in cellular blood components, we in their cellular blood components. must analyze the benefits, costs, and potential drawbacks ofleukodepletion procedures. Leukocyte removal can signifi­ Leukocyte Reduction Methods Methods for leukodepletion can be classified in cantly reduce the rate of febrile, nonhemolytic reactions as terms of removal rate in the following ways: well as the risk of cytomegalovirus . Leukodepletion • Low performance (<90%, 1 log reduction) can also prevent alloimmunization, bacterial growth, and • Intermediate performance (90%-99.9%, 1 to 3 storage lesions. We must address the timing of leukocyte log reduction) removal, quality assurance procedures, and specific indica­ • High performance (>99.9%) tions for each component to ensure that the appropriate Low-performance methods include saline wash, commitment of resources is made to leukocyte-reduction removal, and spin-cool filtration. procedures. Intermediate-performance methods, such as glycerolization-freeze-thaw-deglycerolization This is the second article in a four-part series on blood banking. Other articles discuss new technologies in transfusion medicine, transfusion in the immuno­ (a method to remove leukocytes by repeated compromised host, and marrow transplantation. On completion of this series, washings), differential centrifugation, and the the reader will be able to list three advantages and disadvantages of the solid phase early adhesion-based filters (modified cotton technique, the gel test, and affinity column technology; recognize different wool or cellulose acetate), failed to consistently leukocyte reduction methods and identify the indications for the use of leukocyte- yield components with residual leukocyte counts reduced blood components; recommend strategies and select the most clinically less than 5 X 106. High-performance methods appropriate blood component for immunosuppressed patients; and describe multiple advantages of DNA techniques compared with traditional include so-called third-generation filters that serologic methods for HLA typing. combine size retention, electrostatic attachment, and receptor-ligand interactions.6 When used From Florida Blood Leukocyte-reduced blood components (LRBCs) properly, these filters can deliver less than 6 Services, Tampa. are routinely available to physicians who order 5 X 10 leukocytes per unit transfused. Reprint requests to transfusions. As the biologic effects of collecting, In addition, manufacturers of Dr Leparc, Florida separating, storing, and transfusing blood or its equipment have developed -harvesting Blood Services, 3602 components become better understood, the list Spectrum Blvd, technologies that allow to be collected Tampa, FL 33612; of side effects attributed to the presence of leuko­ with minimal leukocyte contamination at levels ore-mail: gleparc@ cytes (white blood cells [WBCs]) continues to that consistently meet requirements for labeling as ix.netcom.com expand.1 Leukocytes have been perceived as an LRBC. potentially harmful to the viability of red blood cells2'3 and platelets2,4,5 intended for transfusion, Timing of Leukocyte Reduction and have been associated with adverse reactions Studies indicate that the timing of leukocyte in patients who are transfused (Table 1). To removal may be critical in achieving benefits confuse matters further, leukocytes may have linked with WBC depletion. During storage, the beneficial effects based on empiric observations release of (particularly in platelet com­ that have not been proven (Table 2). Nevertheless, ponents, because the leukocytes are metabolically active at the higher storage temperatures that platelets require) and proteases (responsible in

Downloaded from https://academic.oup.com/labmed/article-abstract/28/5/328/2503810 by guest LABORATORY MEDICINE VOLUME 28, NUMBER 5 MAY 1997 on 28 May 2018 part for the structural changes in cellular ele­ benefits of leukodepletion in the treatment of ments that are described generically as "storage certain patients. It does not appear necessary to lesions") begins within a few hours of collection.7 remove leukocytes in routine blood processing in By removing leukocytes within the first 24 hours view of the higher costs involved. of collection, the source of the noxious leukocytic Although no single indication has yielded suf­ byproducts is removed before significant levels ficient data to qualify as a requirement for leuko- build and cause measurable effects. reduction, some generally accepted indications Although no controlled human studies have for the use of LRBCs follow: been published supporting the claim, animal • Prevention of febrile, nonhemolytic transfusion

models support the proposed theoretic benefits Downloaded from https://academic.oup.com/labmed/article/28/5/328/2503810 by guest on 27 September 2021 reactions, particularly in multitransfused patients of early leukodepletion in the prevention of who have experienced this complication in past alloimmunization to HLA.8'9 transfusions. Taking into consideration that Moreover, some studies suggest that early removal of leukocytes may significantly decrease bacterial contamination in stored units.10"13 Language of Leukodepletion that enter the unit at collection time are phagocytized (ingested) by leukocytes soon after­ Leukocyte—A white blood or corpuscle ward. The dangers of contamination are averted by removing the bacteria-laden cells within 24 Leukocyte load—The amount of white blood cells present in the hours of collection. component to be processed. It is probably the most important element in the leukoreduction equation, because different leukocyte Quality Control Procedures depletion methods vary in their load capacity. On average, a unit Each institution must validate its leukocyte- of whole blood contains from 2 to 3 X 109 white blood cells. reduction procedures to ensure that the equip­ Components collected by apheresis procedures vary in their ment and method in use consistently meet the leukocyte load depending on the instrument, collection settings, residual-leukocyte counts and product-recovery and donor white count. The final product may contain rates claimed. Laboratory or bedside filters must from less than 1 X 106 to more than 5 X 109. be validated each time a new method or filtering device is implemented.14 A sampling plan should Leukodepletion—The process of removing white blood cells be developed to provide a level of confidence that Leukoreduction rate—The proportion of the total leukocyte load errors in the application of leukocyte-reduction that is effectively removed from the original component. It may be procedures are detected and corrective action is expressed as a percentage (eg, 99.5%) or as a logarithmic implemented. expression (eg, 3 logs). The two terms may be used interchangeably, Manual or automated procedures for counting and log reductions (ie, base 10 logarithmic expressions of leukore­ the number of residual WBCs must be estab­ 0 duction rates) can be converted readily to percentages as follows: U lished. The method in use must meet sensitivity u thresholds and linearity requirements to accu­ 1 log reduction = 90% rately count small numbers of leukocytes 2 log reduction = 99% 14 0 (<3 WBC/|xL) in the processed components. 3 log reduction = 99.9% H Methods for counting WBCs in very low con­ 4 log reduction = 99.99%, and so on centrations include the following: Recovery rate—The amount (expressed as a percentage) of cells • Manual counts using the Nageotte (Bright Line, intended for transfusion in the original component that are left u Hauser Scientific, Horsham, Pa) chamber (done after the leukocyte reduction process is completed. The recovery rate either on fresh samples or aliquots fixed with 3% should be as high as possible to maximize the benefits of transfusion. 15 paraformaldehyde for batch testing) Recommendations issued by the Food and Drug Administration's • Low cytometry (conventional or automated Center for Biologies Evaluation and Research (CBER)* state that volumetric cytometry16) methods used must "retain a minimum of 85% of the original • Polymerase chain reaction product."

Residual leukocyte count—The amount of white blood cells found in Indications for the Use of the component after leukoreduction. CBER recommendations state Leukocyte-Reduced Blood Components that cellular blood components labeled with the "leukocytes reduced" Consensus exists regarding the benefits of remov­ modifier must be prepared "by a method known to leave a residual ing "passenger" leukocytes in cellular blood leukocyte count of less than 5 X 106." components as well as the proven and potential •FDA memo.18

Downloaded from https://academic.oup.com/labmed/article-abstract/28/5/328/2503810 MAY 1997 VOLUME 28, NUMBER 5 LABORATORY MEDICINE 329 by guest on 28 May 2018 TABLE 1. ADVERSE REACTIONS ASSOCIATED WITH THE PRESENCE OF DONOR LEUKOCYTES residual leukocyte counts when used for this pur­ 6 Febrile, nonhemolytic transfusion reaction pose also should be less than 5 X 10 . Alloimmunization to leukocyte Given that the complications listed above are Graft vs host more likely to develop in patients requiring long- term supportive transfusion therapy, considera­ Transmission of bloodborne (cytomegalovirus, human tion should be given to the routine use of LRBC T-cell lymphotrophic -1) in patients with such chronic hematologic condi­ or immune modulation (associated with tions as hemoglobinopathies, fail­ release) ures (including bone marrow transplantation), as

"Storage lesion" in cellular blood components (associated with well as those with lymphoproliferative or myelo­ Downloaded from https://academic.oup.com/labmed/article/28/5/328/2503810 by guest on 27 September 2021 protease release) proliferative and solid tumors treated with myelosuppressive adjuvant . cytokine levels found in stored blood compo­ nents are proportional to the number of leuko­ Further studies are warranted regarding the cytes present, the American Association of Blood role of leukoreduction in the following: 17 Banks (AABB) Standards state, • Prevention of bacterial contamination of blood When intended for prevention of febrile components nonhemolytic transfusion reactions, the blood • Reduction of the immune-modulating effects of component should be prepared by a method known to reduce the leukocyte number in the final • The role, if any, of transfused leukocytes in graft component to less than 5 X 108. vs effect, as well as on solid tumor growth in patients with (Note that this amount is 2 logs above the maxi­ Test Your mum allowed by Food and Drug Administration Conclusion Knowledge [FDA] licensure requirements for LRBC.)18 Most laboratory professionals agree that leuko­ Look for the CE • Prevention of cytomegalovirus (CMV) transmis­ Update exam on cytes in red blood cells and platelets have a detri­ sion, because the effectiveness of leukoreduction Blood Banking (703) mental effect and that potential benefits may be has been supported in controlled studies, and in the July issue of gained by using leukodepleted products in cer­ Laboratory Medicine. leukoreduced components are considered equiva­ tain situations. No single indication has yielded Participants will earn lent to components prepared from CMV seroneg­ sufficient data to qualify as a requirement for 4 CIVILE credit hours. ative donors.19"21 American Association of Blood leukoreduction, however. It is important to mon­ Banks standards22 for leukocyte reduction are the itor the use of leukodepleted components as well same as those set by the FDA (ie, less than 5 X as their consistent quality in light of the costs 106) when CMV prevention is intended. involved. Although poststorage filtration offers • Reduction of the incidence of HLA alloimmu­ some logistic advantages, prestorage filtration at nization may be achieved in patients whose clini­ the laboratory prevents the harmful effects of cal condition or treatment will require repeated cytokines and protease release that have been transfusion episodes resulting in sensitization to associated with the leukocyte content in cellular HLA antigens.23"25 This prophylaxis may be of blood components.® particular clinical value when used for candidates for bone marrow transplantation. Recommended References TABLE 2. POTENTIAL BENEFICIAL EFFECTS OF LEUKOCYTES 1. Lane TA. Leukocyte reduction of cellular blood compo­ IN BLOOD COMPONENTS nents. Effectiveness, benefits, quality control, and costs. Arch Pathol Lab Med. 1994;118:392-404. Bacteriostatic/bactericidal effect 2. Hogman CF, Hedlund K, Akerblom O, et al. preservation in protein-poor media. I. Leukocyte enzymes Immunosuppression or immune modulation (in autoimmune as a cause of hemolysis. Transfusion. 1978;18:233-241. processes such as Crohn's disease,* or in solid 3. Brecher ME, Pineda AA, Torloni AS, et al. Prestorage transplantation) leukocyte depletion: effect on leukocyte and platelet metabo­ lites, erythrocyte lysis, metabolism, and in vivo survival. Graft vs leukemia effect Setnin Hematol. 1991;28(suppl 5):3-9. 4. Gottschall JL, Johnston VL, Rzad L, et al. Importance of Decreased incidence of miscarriage in habitual abortion* white blood cells in platelet storage. Vox Sang. 1984;47:101-107. * Peters WR, Fry RD, Fleshman JW, et al. Multiple blood transfusions 5. Andreu G. Early leukocyte depletion of cellular blood reduce the recurrence rate of Crohn's disease. Dis Colon Rectum. components reduces red blood cell and platelet storage 1989,32:749. f lesions. Setnin Hematol. 1991;28(suppl 5):22-25. Antin JH. Graft-vs-leukemia: no longer an epiphenomenon. Blood. 6. DeChristopher PJ. Leukocyte reduction filtration: technolo­ 1993,82:2273. 22 gies, benefits, applications, and limitations. Lab Med. * Unander. 1994;25:96-101.

Downloaded from https://academic.oup.com/labmed/article-abstract/28/5/328/2503810 by guest 330 LABORATORY MEDICINE VOLUME 28, NUMBER 5 MAY 1997 on 28 May 2018 7. Heddle NM, Klama L, Singer J, et al. The role of the plasma r ascp, from platelet concentrates in transfusion reactions. N Engl J Med. 1994;331:625-628. 8. Blajchman MA, Bardossy L, Carmen RA, et al. An animal model of allogeneic donor platelet refractoriness: the effect of the time of leukodepletion. Blood. 1992;79:1-5. 9. Novotny VMJ, van Doom R, Witvliet MD, Claas FHJ, Brand A. Occurrence of allogeneic HLA and non-HLA antibod­ workshops ies after transfusion of prestorage filtered platelets and red blood cells: a prospective study. Blood. 1995;85:1736. 10. Barrett BB, Andersen JW, Anderson KC. Strategies for the avoidance of bacterial contamination of blood components. Transfusion. 1993;33:228-233.

11. Hogman CF, Gong J, Hambraeus A, et al. The role of white • technologists Downloaded from https://academic.oup.com/labmed/article/28/5/328/2503810 by guest on 27 September 2021 cells in the transmission of Yersinia entewcolitica in blood com­ ponents. Transfusion. 1991;32:658-662. 12. Kim DM, Brecher ME, Bland LA, et al. Prestorage removal of Yersinia entewcolitica from red cells with white cell-reduction filters. Thws/usiow.l992;32:658-662. 13. Wenz B, Burns ER, Freundlich LF. Prevention of growth of columbus Yersinia enterocolitica in blood by polyester fiber filtration. adam's mark hotel September 2-6,1997 Transfusion. 1992;32:663-666. ascp-ams reception on September 2 14. Dumont LJ, Dzik WH, Rebulla P, Brandwein H, members five cross-training workshops of BEST working party of the ISBT. Practical guidelines for process control of white-cell reduced blood components: report 22 workshops of the biomedical excellence for safer transfusion (BEST) work­ ing party of the International Society of Blood Transfusion (ISBT). Transfusion. 1995;36:11-20. 15. Prati D, Brandwein H, Capelli C, et al. Multicenter evalua­ tion of the 3% paraformaldehyde method for white cell count­ ing in leukocyte-reduced red blood cells. Vox Sang. milwaukee isconsin 1996;70:241-245. 16. Dietz LJ, Dubrow RS, Manian BS, Sizto NL. Volumetric marriott hotel September 11-13,1997 capillary cytometry: a new method for absolute cell enumera­ ascp-ams reception on September 11 tion. Cytometry. 1996;23:177-186. 17. Klein HG, ed. Standards for Blood Banks and Transfusion six workshops Services. 17th ed. Bethesda, Md: American Association of Blood Banks; 1996. 18. FDA memo. Recommendations and licensure require­ ments for leukocyte-reduced blood products. Dated May 29, 1996. 19. Bowden RA, Cyas M, Schoch G, et al. Comparison of fil­ minneanolis innesota tered blood to seronegative blood products for prevention of cytomegalovirus infection after marrow transplantation. Blood. thunderbird hotel October 1-3,1997 1993;82:204. ascp-ams reception on October 1 20. Hillyer CD, Emmens RK, Zago-Novarreti M, et al. Methods for the reduction of transfusion transmitted six workshops cytomegalovirus infection: filtration vs the use of seronegative donor units. Transfusion. 1994;34:929-934. 21. Gilbert GL, Mayes K, Hudson IL, et al. Prevention of transfusion-acquired cytomegalovirus infection in infants by blood filtration to remove leukocytes. Lancet. 1989;1:1228-1231. sUwfe ISSOUPl 22. Unander AM, Lindholm A. Transfusion of leukocyte-rich erythrocyte concentrates: a successful treatment in selected cases holiday inn westport October 8-10,1997 of habitual abortion. Am J Obstet Gynecol. 1986; 154:516-520. ascp-ams reception on October 8 23. Saarinen UM, Kekomaki R, Siimes MA, et al. Effective prophylaxis against platelet refractoriness in multitransfused six workshops patients by use of leukocyte-free blood components. Blood. 1990;75:512-517. ASCP workshops for technologists and technicians 24. Oksanen K, Kekomaki R, Ruutu T, et al. Prevention of alloimmunization in patients with acute leukemia by use of provide practical tips, techniques and strategies that help white cell-reduced blood components—a randomized trial. you provide the best health care possible. Contact ASCP Transfusion. 1991;31:588-594. Customer Services for more information. 25. van Marwijk Kooy M, van Prooijen HC, Moes M, et al. Use of leukocyte-depleted concentrates for the prevention of Telephone: 800-621-4142 press 1, thenx260 refractoriness and primary HLA alloimmunization: a prospec­ (Illinois residents: 312-738-4890) tive, randomized trial. Blood. 1991;77:201-205. • Fax: 312-738-0102 * Email: [email protected]

.^Associate Read the workshop descriptions at http://www.ascp.org. Member ^V Section

Downloaded from https://academic.oup.com/labmed/article-abstract/28/5/328/2503810 MAY 1997 VOLUME 28, NUMBER 5 LABORATORY MEDICINE 331 by guest on 28 May 2018