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An overview of laboratory and clinical aspects of leucocyte-depleted blood components
Pergamoll
0955.3886(93)EO805-P
Transfus.Sci. Vo1.15,No.1,PP.49-62, 1994 Copyright @ 1994ElsevierScienceLtd Printedin GreatBritain.All rightsreserved 09553886194$7.00 + 0.00
International Forum An Overview of Laboratory and Clinical Aspects of Leucocyte-depleted Blood Components M. J. Seghatchian,
PhD
U.K.: DISCUSSIONS ON LEUKOCYTE DEPLETION Editor’s Note In this issue of Transfusion Science the International Forum focuses on the U.K. and recent deliberations on leukocyte depletion. Dr Jerhard Seghatchian, one of our editors, has written an introduction to the topic and this is then followed by presentation of the abstracts of the British Blood Transfusion Society- Components Special Interest Group meeting which was held April, 1993 in Boumemouth. We hope that this format will give our readers particular insight into the U.K. concerns on this “hot” topic in Transfusion Medicine.
The provision of the safest and most efficacious blood components in a cost effective way and with consistency is one of the essential goals of a blood transfusion service. Since leucocytes are not clinically required in most patients in need of red cell and platelet concentrates and their removal not only improves the functional integrity of blood components during storage, but reportedly improves the clinical safety, there has been an ever increasing pressure to develop a new inventory to meet the urgent demands for leucodepleted blood and blood components. Nevertheless it should be emphasized that viable leucocytes do fulfil a very important bacteriostatic function, improving safety in the early stages of blood storage. Thus the removal of leucocytes from donor blood by in-line filters at collection sessions may prove to be a potentially harmful excess of zeal. On the other hand although many bacteria are effectively killed by phagocytic leucocytes, some microorganisms, such as S. mreus and Y. enterocolitica are capable of surviving phagocytosis to proliferate after leucocytes disintegrated, within a few days. Therefore further improvement in safety standards would be expected by removing both ingested/reappearing and non-engulfed bacteria. This is of particular relevance to the current concept of component production, as newer, large scale processing devices North London Blood Transfusion Service, Colindale Ave, London NW9 SBG, U.K
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such as haemapheresis and semi-automated buffy coat removal, do not remove sufficient leucocytes to prevent either delayed bacterial growth in cellular components or the potential adverse effects of leucocytes accompanying transfused blood components, without subsequent filtration. The consistency of filter performance is also a very important issue as a single result which is outside specifications (acceptable limits] may be sufficient to cause some unexpected major problems. This emphasizes the need for a stringent Quality Assurance system for leucocyte-depleted blood components with the use of appropriately agreed upon, and validated technologies to identify even minute deviations from set standards. Setting a standard protocol for leucodepletion is not problem-free either, as the filter efficiency is adversely affected if overloaded. An effective primary removal of the greater part of the leucocytes (i.e. by b&y-coat removal) will at least on a theoretical basis, help to reduce the number to be removed. On the other hand it is becoming apparent that efficient granulocyte depletion depends upon the platelet content of the red cell mass, prior to filtration (indirect adhesion] and the direct adherence of granulocytes to some filter materials. The overloading capacity and the cumulative load of leucocytes, the age and characteristic changes in the properties of cellular components of blood that are induced by processing, as well as, the flow rate and temperature during filtration would also alter the results. Therefore the characteristic property of each type of filter can only be presumed for a standard type of product and standardized protocol of filtration for each component. Hence standardization of several laboratory aspects of filtration is paramount in order to achieve consistent and reliable results. Despite these variabilities there is compelling evidence that filtration, using new generation, specific leucocyte depleting filters are the most effective way for reduction of leucocyte number by 3 to 6 log,,. 1. HISTORICAL BACKGROUND OF LEUCOCYTE DEPLETION It has been historically established’ that exposure of whole blood to PC, even for a short period (4-6 h) leads to platelet shape change and formation of clumps (10-50 urn in diameter] or so called microaggregates. Larger clumps (macroaggregate) consisting mainly of small clots, denatured proteins, aggregated platelets, leucocytes and trapped red cells are formed if the unit is centrifuged and the large subpopulation of platelets are not salvaged. It is therefore mandatory in the “Code of Federal Regulation” to use filters with a pore size ranging from 170430 urn to remove macroaggregates. The use of microaggregates filters with smaller pore size, has been more controversial as microaggregates appearance is proportional to the number of leucocytes and platelets left in the source blood and the length of storage. Since with blood component preparation using the buffy-coat removal protocol, more than 80% of the leucocytes are removed, questions arise about the validity of additional filtration which, itself, is not 100% efficient. In clinical practice, however, leucocyte depletion by filtration became popular when it became established that reduction of the leucocyte content results in a reaction-free transfusion in most patients with HLA antibodies, although the preparations were still immunogenic to a great number of patients.2 Recent evidence indicates that the content of leucocytes needs to be less than 5 x lo6 per transfusion to reduce appreciably immunogenicity.3 Another indication for leucocyte depletion is avoiding transmission of leucocyte-lodged viruses such as cytomegalovirus or HTLVl with blood components in immunocompromized recipients.4, 5 However there is no clear cut data to show how soon after collection the leucocytes have to be removed to minimize the release of viruses from degenerating leucocytes. Historically a new system for leucocyte depletion of red cell preparations by
International Forum
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filtration was introduced in 1962 by Greenwalt et a1.6 Since then the technique has advanced resulting in more effective filters and more practical procedures. Sterile docking devices are used thereby eliminating the risk of microbial contamination. Today, special bedside filters are available for use for patients with leucocyte antibodies and/or with chronic, transfusion-demanding diseases, with the aim of avoidance of alloimmunization. Paediatric microaggregate filters having smaller priming volumes have been developed and used in neonates undergoing extracorporeal membrane oxygenation and cardiopulmonary bypass. The practice of placing such an in-line filter in the extracorporeal system decreases the potential for microembolism in these patients during the procedure. Ideal leucocyte depletion filters, must fulfil some essential criteria of acceptability. These include: (4 safe passage of the cellular component and the soluble constituent, while achieving the specified level of leucocyte depletion; (ii] good flow rate and good capacity compatible with the normal requirements for the transfusion of fresh or stored blood and its cellular components, while remaining compact for ease of handling and minimizing product wastage; of complications and adverse (iii) highly effective in reducing the incidence reactions. 2. LABORATORY ASPECTS OF LEUCOCYTE DEPLETION The best means for achieving the specified level of leucocyte depletion, with consistency, are still evolving. To achieve optimal efficiency, attention should be focused on the potential variations of several influencing factors which apparently affect the processing efficiency. The most important parameter is the variation in the composition of cellular content of blood and blood collection/processing and storage conditions. It is known’ that varying numbers of leucocytes remain in the cellular components of blood, depending upon the length of time from collection to centrifugation (optimal 6-8 h); accuracy of rotor balancing; acceleration/deceleration time, handling prior to separation and the proximity of the interface to the top of the bag when separating platelet-rich plasma.‘, ’ Leucocyte reduced platelets can also be prepared by centrifuging pooled red cell contaminated PC, utilizing apheresis technology and automated buffy-coat removal methods, each producing products with differing properties.’ Nevertheless it should be remembered that any additional steps in processing may have some deleterious effect on the morphological and functional integrity of cellular components. Hence standardization of component collection, processing and storage conditions remains the most important issue in validation of filter efficiency. The proper assessment of the reasons for “filtration failure”, even in a well standardized and validated system as well as the use of an accurate and reliable method for counting low leucocyte numbers require special attention. In most cases the failure or shortcoming appears to be related to the characteristic adhesive properties of the cellular and hemeostatic components of the donor’s blood and/or the shortcomings in the final products rather than a deficiency in filter performance. For proper comparative analysis of filter efficiency, attention should be directed at reducing the influencing factors in the source product by using paired identical units, standardizing all the operational procedures and continuously monitoring relevant parameters in the individual product both during processing as well as the storage period. In this respect, for more efficient filters we may require more sensitive assays such as the phenotypic composition of the residual leucocyte cohorts [i.e. the percentage of antigen presenting cells (APC), and CD4 suppressor cells) and identification of variable membrane microversicles, produced during processing and storage. Currently,
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flow cytometry and PCR as well as molecular markers of the storage lesion are used for assessing changes in properties of post filtration products.‘,’ These, combined with quantitation of the cellular composition, cell-cell interactions and cell damage using new techniques and election microscopy will provide a better insight into the mechanism of action of leucocyte removal. Work in progress in the author’s laboratory, using an identical paired protocol system, reveal that there is a considerable change in the properties of the platelet concentrates subsequent to filtration. On average there is a 10 f 3% fall in platelet count in the filtered product and a loss of 20 mL total volume. In the majority of cases, the apparent mean platelet volume of the filtered platelet is smaller (- 0.3 fL) and there is a slight rise of pH (0.2) suggesting that large and more haemostatically effective platelets are preferentially removed by the filtration process. The filtered PC sometimes produces abnormal storage stability pattern in particular at the end of shelf lifesaa Different types of filters have a finite leucocyte removal capacity and the flow rate of PC through the filters must be meticulously adjusted in order to consistently achieve optimum leucocyte removal. On the other hand, non-filtered products, containing even a slightly higher leucocyte content (as low as 0.35 x 10’) show a larger reduction of the functional integrity as substantiated by an increased release of GpIb fragments in the platelet supematant, microvisculation measured by membranebound GpIIb/IIIa (microparticles) in the supematant, and release of vWF with reduced collagen binding activity. These deleterious effects might be associated with the reduced post-transfusion platelet response.8 The decrease in functional integrity is more likely to be a result of the release of leucocyte enzymes which have a high afflnity to platelet surface glycoproteins and certain adhesive proteins such as vWF.‘<’ Filtration of red cell products also appears to be associated with microvesiculation and increased release of haemoglobin. The storage stability pattern of post-filtered products at the end of storage also appears to be different. The significance of these findings in the clinical setting remains to be investigated. New potentials for the use of polyester filters stem from reports that these devices may remove small but significant quantities of engulfed and free bacteria from donor blood, opening a new area of research/development in filtration.g 3. MICROBIOLOGICAL
ASPECTS
OF LEUCOCYTE
DEPLETION
Post-transfusion sepsis, after the transfusion of platelet concentrate and the use of SAG-M red cells is steadily increasing.’ In most instances the contamination probably occurred at the time of venepuncture as a result of donor bacteraemia. Since leucocytes are known to prevent bacterial proliferation in contaminated units of blood it is warranted to relate the effect of leucocyte depletion and bacteriological safety. Evidence is accumulating’ that leucocytes play a critical role immediately after collection and a “holding” period of blood from 6-24 h at R.T. the period of 20-24 being optimal. Therefore in-line filter usage can only become significant with the use of new generation filters having not only the capacity for removal of leucocyte-lodged bacteria but also non-ingested bacteria directly. This is of particular relevance since leucocytes function poorly at the conventional conditions of red cell storage and they deteriorate and die after a few days, releasing the engulfed but still viable bacteria hence after this period it is likely to have little or no effect on the risk of posttransfusion sepsis. The per&operative blood transfusion can also contribute to post-operative infection by transmission and/or by an immunosuppressive effect, leading to decrease of resistance to micro organisms introduced during surgery.g This implies that multiple clinical studies in various disease categories are needed and the development of spe-
InternationalForum 53
cific guidelines for transfusion should address the question of post-operative infections. Microbiological results from most laboratory studies’ suggest that deliberately inoculated bacteria (1 organism/ml) exhibit a lag phase of growth for 24-48 h followed by rapid growth of 106 organisms/ml. The reported rates of bacterial contamination in platelets range between 0 to 4.9% for single donor apheresis platelets and O-10% in pooled random units.’ Since transfusion of the bacteriologically contaminated products in immunocompromized patients may lead to death, effective preventative strategies are needed. Some debatable issues include: avoidance of repeated use of same harvest site during collection; reduction of the number of homologous donor exposures (pooling process) and improved identification of microbiologically contaminated units (i.e. using polymerase chain reaction technologies similar to those available for the identification of HIV1 hence offering the potential benefits of identifying all heterogeneous types of bacteria on the basis of prokaryotic DNA) and shortening the storage time between collection and transfusion of platelets to 3 days particularly for use in immunocompromized patients. Large numbers of variables such as the nature of the anticoagulant and its ratio, the concentration of adhesive proteins such as fibronectin’ the temperature, complement activation and cell/cell/protein interactions may have a significant role in bacterial growth. To date, bacterial growth has not been enhanced in units that were deliberately contaminated, held and then filtered. 4. CLINICAL
ASPECTS AND COST EFFECTIVE STRATEGIES
It is now well established
that residual leucocytes in red cell and platelet concentrates contribute to some clinical side effects in certain groups of patients who are transfusion dependant. Leucocytes also have detrimental effects on patients undergoing extracorporeal circulation of the blood. The reported clinical benefits of the use of leucocyte filtered products in specific clinical settings and surgical areas are steadily increasing. Table 1 summarizes some of the potential clinical and practical benefits of leucocyte depletion. lGzo There has been a general criticism that until recently no proper comparative clinical trials and validation of filter performance have been made. This is partially due to the fact that blood components differ in their characteristic properties and a vast array of preparative and storage conditions are used {fresh, variably stored for different lengths of time; warm; cold; soft spin; hard spin; b&y-coat removed or
Table 1. Some Practical and Clinical Benefits of Leucocyte Depletion l
0 l l
0 l
0 0 0 l l l l l l
Preventing HLA Alloimmunization/Sensitization Reducing Platelet Refractoriness Preventing Transfusion-associated Cytomegalovirus (CMV, HTLVl ) Preventing Non-haemolytic Febrile Transfusion Reactions Reducing Transfusion-associated Thrombocytopenia Reducing Transfusion-associated lmmunomodulation Reducing Potential Reperfusion Injury Decreasing Usage of Platelets Reducing Need for HLA-matched Platelet Products Providing Alternative or Supplement to CMV Negative Blood Products Reducing the Overall Workload Reducing Biochemical Degradation During Storage Reducing Histamine Release and Reactions Associated With the Presence of Cytokines Reducing Vascular Occlusion and Depletion of Fibronectin Reducing Leucocyte-lodged and Some Free Bacterial Contamination
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present; high and low haematocrit) each of which may induce variability and influence the optimal performance and process efficiency of filters. Similarly, large differences exist in the adhesive properties of platelet products obtained from random donors, methods include single donor apheresis, the PRP method; single or pooled buffy-coat method involving storage at 22°C for variable lengths of time. The adhesive properties of such products containing different subpopulation of platelets are different and can influence the characteristic performance of filters, leading to inter- and intra- laboratory variability and loss of active subpopulations of platelet with leucocytes. Occasionally the overall poor function of some filters make any clinical comparison based on a well-standardized product a difficult task. Despite these uncertainties there is now ample clinical evidence that the prophylactic use of filtered red cells will prevent or delay non-haemolytic febrile transfusion reactions (NHFTR).” This is of particular relevance to patients who are transfusion dependant such as those with Beta-thalessaemia major, in whom prevention of HLA alloimmunization prior to bone marrow transplantation is indicated. Some other patient groups such as those with chronic aplastic anaemia, myelodysplasia, sickle cell disease and chronic renal failure who become unresponsive to erythopoitein are also at risk. There is now a general consensus that all newly diagnosed patients with severe aplastic anaemia (SAA] who are potential marrow transplant recipients, should receive leucodepleted cellular components3 Furthermore leucodepletion is also an acceptable alternative to cytomeglovirus antibody screened blood.4, 5 The prevention of platelet refractoriness” and immunomodulation12 are both affected by removal of leucocytes. Methods are now available which allow us to reliably achieve a residual leucocyte count of less than 5 x lo6 in a dose of approximately 3 x 10” platelets, but standardization remains an unresolved problem. There is also an urgent need to establish measures of clinical outcome, based on minimum red cell and platelet requirements, which would enable the cost-effective selection of patients who may suffer from the transfusion of allogeneic leucocytes.3 In general there are three distinct categories of disease in which the formation of HLA-alloantibodies is undesirable: (i) su;Fz;z;;a;hz;pT with platelet concentrates such as in aplastic anaemia, or (ii) heart, liver and ienal transplantation; (iii) patients requiring life-long red cell transfusion; While in the later it makes sense to start with standard leucocyte-reduced red cells and await febrile reactions, cardiac and liver transplant patients will benefit from filtered products. 14-17In kidney graft recipients the requirement for supportive therapy has enormously decreased due to a change in policy not to remove the non-functional kidney. Alternative strategies for reducing the adverse effects of leucocytes are also becoming established.3 For example, potential methods for prevention of graft versus host disease (GVHD) would be depletion of T-lymphocytes from the blood component before transfusion. This is attributed to the fact that both the incidence and severity of GVHD, after allogeneic BMT can be reduced if T-cells are eliminated from the donor marrow by a variety of techniques prior to grafting, including buffy-coat removal by centrifugation, direct and inverted centrifugation, washing, cotton and cellulose acetate filtration or freezing. These procedures reduce leucocytes by only 2-3 log,, leaving KY’-10’ lymphocytes behind, hence can only be considered as a first step in leucodepletion since less than 8 x lo4 lymphocyte per kg body weight appears to mediate a GVIYID.~In contrast, simple in vitro irradiation (with 1530 gy) accomplishes the desired goal inexpensively rapidly and with proven efficacy. W-ultraviolet light also abrogates GVHD in recipients with good preliminary results in minimizing
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Forum
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alloimmunization. Further work is nevertheless needed to determine clinical efficacy and side effects of this procedure. The need for pre-storage leucocyte-depleted cellular components (although debatable) currently presents an important inventory management issue in the U.S.A. An alternative strategy would be to put aside a specific number of group 0 negative packs of red cells to meet the needs of this patient population, though this may lead to excessive use of 0 negative red cells, deserving a special audit for successful inventory management. The rational comparison of benefits and costs is an area of major concern, 3 as the provision of leucodepleted blood components incurs costs ranging from materials to time of laboratory staff, while, at the same time provides benefits to patients and the health care service in general. SUMMARY Blood component therapy has enabled us to successfully support the increasing demands for treatment of haematological abnormalities. Continuous efforts are made to minimize or prevent immediate side effects of transfusion but the long-term effects of component therapy will remain unknown for some time. There is always a need for products with special qualities (i.e. no CMV, no HLA immunization etc.] however when considering adverse effects it is important to look for alternative strategies which produce minimal alteration in the cellular and fluid components of blood while maintaining optimal clinical efficacy. Continuous education of clinicians and laboratory scientists is needed so that the best possible products are provided reliably and with consistency. This review focused on some of the important debatable issues on the laboratory/microbiology aspects and clinical practice. It is clear that the presence of leucocytes is associated not only with reduced quality, based on the morphological and functional integrity of blood components but also reduced post-transfusion response in some specified cases. A stringent regular QA program is required for the assessment of the trend as both differences between filter batches and slight changes in laboratory operations contribute to the lack of reliability of the system in use. While until recently the main goal of leucocyte depletion for red cells was to prevent NHFTR in specified patients it appears that currently a more ambitious goal of avoiding other important leucocyte-induced transfusion complications are considered with some success, without leaving aside alternative strategies and their practical benefits. In respect to cost-effective transfusion strategies, in multiply-transfused patients, both induction of antibodies against HLA class I antigens and the development of leucocyte-associated virus infections could be prevented by using modem filters which lead to effective components with leucocytes below the threshold reported in the Edinburgh consensus meeting 3 whereas gamma-irradiation of blood components before transfusion, that decreases the mitogen induced stimulation of the lymphocytes is the best strategy for the prevention of post-transfusion GVHD. Many advances have been made in methods of detection of a low level of leucocytes, (Nageotte chamber, flow cytometry, PCR) and the storage lesion induced by the residual leucocytes,’ helping to better define product quality. A considerable degree of cooperation is also seen between clinicians, scientists and other disciplines and manufacturers of the diverse technologies. Acknowledgements I wish to express my sincere thanks to Pall Biomedical Ltd for supporting the symposium and to Vivian Sproule for typing the manuscript.
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REFERENCES 1. Hogman CF: Component preparation and quality assurance of stored blood, in Rock GA, Seghatchian MJ (eds): Quality Assurance in Transfusion Medicine, Vol. II. Florida, CRC Press, 1993, pp. 59-98. 2. Leikola J, MyllylH G: The clinical use of red cell components, in Summers SH, Smith DM, Granenko VA (eds): Hogman 40 Guidelines for Practice. Arlington VA, American Association of Blood Banks, 1990, p.1. 3. Royal College of Physicians of Edinburgh: Consensus Meeting on Leucocyte-depleted Blood Components. Edinburgh (March, 1993). 4. Gilbert GL, Hayes K, Hudson IL, et al.: Prevention of transfusion-acquired cytomegalovirus infection in infants by blood filtration to remove leucocytes. Lancet 1989;1228-1231. 5. Bowden RA, Sayers MI-I, Cays M, et al.: The role of blood product filtration in the prevention of transfusion associated cytomegalovirus (CMV) infection after marrow transplant (AABB, October 21-26 1989). Transfusion 29 (Suppl). 6. Greenwalt TJ, Gajewski M, McKenna JL: A new method for preparation of granulocyte-poor red cells by microaggregate filtration. A simplified method to minimize febrile transfusion reactions. VOXSang 1980, 39:282. 7. Seghatchian MJ, Brozovic B: An overview of current trends in platelet storage and clinical practice. Blood Coag Fibrin01 1992; 3:617-620. 8. Sloand EM, Klein HC: Effect of white cells on platelet during storage. Transfusion 1990; 30:333. 8a. Krailadsiri P, Seghatchian MJ: Effect of filtration, storage and platelet suspension media on platelet indices. Transfus Sci 1994; 15: in press. 9. Anderson KC: Bacterial contamination of platelets (Current Trends) Transfus Sci 1993; 14: 159-162. 10. Sirchia G, Wenz B, Rebulla P, et al.: Removal of white cells from red cells by transfusion through a new filter. Transfusion 1990; 30:30-33. 11. Sniecinski I, O’DoMell MR, Nowicki B, et al.: Prevention of refractoriness and HLAalloimmunisation using filtered blood products. Blood 1988; 71: 1402-1407. 12. Solheim BG: Immuno-modulation by transfusion and CMV transmission. BBTS, Aspects of Blood Filtration, 5th April 1990, Birmingham, England. 13. Hart S, Bareford D, Smith N, et al.: Post-transfusion thrombocytopenia: its duration in splenic and asplenic individuals. VOXSang 1990j 59:123-124. 14. Bando K, Pillai R, Cameron DE, et al.: Leukocyte depletion ameliorates, free radicalmediated lung injury after cardiopulmonary bypass. 1 Thorac Cardiovasc Surg 1990; 99: 873-877. 15. Pillai R, Bando K, Schueler S, et al.: Leukocyte depletion results in excellent heart-lung function after 12 hours of storage. Ann Thorac Surg 1990; 50:211-214. 16. Schueler S, Hatanaka M, Bando K, et al.: Twenty-four hour lung preservation with donor core-cooling and leucocyte depletion in a bilateral lung transplant model. Proc Am Co11 Surg (Surgical form) 1990; 2X1:405-407. 17. Bareford D, Chandler ST, Hawker RJ, et al.: Splenic platelet-sequestration following routine blood transfusion is reduced by filtered/washed blood products. Br f Haematol 1987; 67: 177-180. platelet 18. Van Marwijk Kooy M, Van Proojen HC, Moes M, et al.: Use of leucocyte-depleted concentrates for the prevention of refractoriness and primary HLA alloimmunisation: a prospective, randomised trial. Blood 1991; 77:201-205. 19. Saarinen UM, Kekomiiki R, Siimes MA, et al.: Effective prophylaxis against platelet refractoriness in multitransfused patients by use of leucocyte free blood components. Blood 1990; 75:512-517. 20. Sivakumaran M, Norfolk DR, Maior KA, et al.: A new method to study the efficiency of third generation blood filters. Br / Haematol 1991; 84:175-177.
0955.3886(93)EO805-P
Transfus.Sci. Vo1.15,No.1,PP.49-62, 1994 Copyright @ 1994ElsevierScienceLtd Printedin GreatBritain.All rightsreserved 09553886194$7.00 + 0.00
International Forum An Overview of Laboratory and Clinical Aspects of Leucocyte-depleted Blood Components M. J. Seghatchian,
PhD
U.K.: DISCUSSIONS ON LEUKOCYTE DEPLETION Editor’s Note In this issue of Transfusion Science the International Forum focuses on the U.K. and recent deliberations on leukocyte depletion. Dr Jerhard Seghatchian, one of our editors, has written an introduction to the topic and this is then followed by presentation of the abstracts of the British Blood Transfusion Society- Components Special Interest Group meeting which was held April, 1993 in Boumemouth. We hope that this format will give our readers particular insight into the U.K. concerns on this “hot” topic in Transfusion Medicine.
The provision of the safest and most efficacious blood components in a cost effective way and with consistency is one of the essential goals of a blood transfusion service. Since leucocytes are not clinically required in most patients in need of red cell and platelet concentrates and their removal not only improves the functional integrity of blood components during storage, but reportedly improves the clinical safety, there has been an ever increasing pressure to develop a new inventory to meet the urgent demands for leucodepleted blood and blood components. Nevertheless it should be emphasized that viable leucocytes do fulfil a very important bacteriostatic function, improving safety in the early stages of blood storage. Thus the removal of leucocytes from donor blood by in-line filters at collection sessions may prove to be a potentially harmful excess of zeal. On the other hand although many bacteria are effectively killed by phagocytic leucocytes, some microorganisms, such as S. mreus and Y. enterocolitica are capable of surviving phagocytosis to proliferate after leucocytes disintegrated, within a few days. Therefore further improvement in safety standards would be expected by removing both ingested/reappearing and non-engulfed bacteria. This is of particular relevance to the current concept of component production, as newer, large scale processing devices North London Blood Transfusion Service, Colindale Ave, London NW9 SBG, U.K
49
50
Trm~sfis. Sci.
Vol. 15, No. 1
such as haemapheresis and semi-automated buffy coat removal, do not remove sufficient leucocytes to prevent either delayed bacterial growth in cellular components or the potential adverse effects of leucocytes accompanying transfused blood components, without subsequent filtration. The consistency of filter performance is also a very important issue as a single result which is outside specifications (acceptable limits] may be sufficient to cause some unexpected major problems. This emphasizes the need for a stringent Quality Assurance system for leucocyte-depleted blood components with the use of appropriately agreed upon, and validated technologies to identify even minute deviations from set standards. Setting a standard protocol for leucodepletion is not problem-free either, as the filter efficiency is adversely affected if overloaded. An effective primary removal of the greater part of the leucocytes (i.e. by b&y-coat removal) will at least on a theoretical basis, help to reduce the number to be removed. On the other hand it is becoming apparent that efficient granulocyte depletion depends upon the platelet content of the red cell mass, prior to filtration (indirect adhesion] and the direct adherence of granulocytes to some filter materials. The overloading capacity and the cumulative load of leucocytes, the age and characteristic changes in the properties of cellular components of blood that are induced by processing, as well as, the flow rate and temperature during filtration would also alter the results. Therefore the characteristic property of each type of filter can only be presumed for a standard type of product and standardized protocol of filtration for each component. Hence standardization of several laboratory aspects of filtration is paramount in order to achieve consistent and reliable results. Despite these variabilities there is compelling evidence that filtration, using new generation, specific leucocyte depleting filters are the most effective way for reduction of leucocyte number by 3 to 6 log,,. 1. HISTORICAL BACKGROUND OF LEUCOCYTE DEPLETION It has been historically established’ that exposure of whole blood to PC, even for a short period (4-6 h) leads to platelet shape change and formation of clumps (10-50 urn in diameter] or so called microaggregates. Larger clumps (macroaggregate) consisting mainly of small clots, denatured proteins, aggregated platelets, leucocytes and trapped red cells are formed if the unit is centrifuged and the large subpopulation of platelets are not salvaged. It is therefore mandatory in the “Code of Federal Regulation” to use filters with a pore size ranging from 170430 urn to remove macroaggregates. The use of microaggregates filters with smaller pore size, has been more controversial as microaggregates appearance is proportional to the number of leucocytes and platelets left in the source blood and the length of storage. Since with blood component preparation using the buffy-coat removal protocol, more than 80% of the leucocytes are removed, questions arise about the validity of additional filtration which, itself, is not 100% efficient. In clinical practice, however, leucocyte depletion by filtration became popular when it became established that reduction of the leucocyte content results in a reaction-free transfusion in most patients with HLA antibodies, although the preparations were still immunogenic to a great number of patients.2 Recent evidence indicates that the content of leucocytes needs to be less than 5 x lo6 per transfusion to reduce appreciably immunogenicity.3 Another indication for leucocyte depletion is avoiding transmission of leucocyte-lodged viruses such as cytomegalovirus or HTLVl with blood components in immunocompromized recipients.4, 5 However there is no clear cut data to show how soon after collection the leucocytes have to be removed to minimize the release of viruses from degenerating leucocytes. Historically a new system for leucocyte depletion of red cell preparations by
International Forum
51
filtration was introduced in 1962 by Greenwalt et a1.6 Since then the technique has advanced resulting in more effective filters and more practical procedures. Sterile docking devices are used thereby eliminating the risk of microbial contamination. Today, special bedside filters are available for use for patients with leucocyte antibodies and/or with chronic, transfusion-demanding diseases, with the aim of avoidance of alloimmunization. Paediatric microaggregate filters having smaller priming volumes have been developed and used in neonates undergoing extracorporeal membrane oxygenation and cardiopulmonary bypass. The practice of placing such an in-line filter in the extracorporeal system decreases the potential for microembolism in these patients during the procedure. Ideal leucocyte depletion filters, must fulfil some essential criteria of acceptability. These include: (4 safe passage of the cellular component and the soluble constituent, while achieving the specified level of leucocyte depletion; (ii] good flow rate and good capacity compatible with the normal requirements for the transfusion of fresh or stored blood and its cellular components, while remaining compact for ease of handling and minimizing product wastage; of complications and adverse (iii) highly effective in reducing the incidence reactions. 2. LABORATORY ASPECTS OF LEUCOCYTE DEPLETION The best means for achieving the specified level of leucocyte depletion, with consistency, are still evolving. To achieve optimal efficiency, attention should be focused on the potential variations of several influencing factors which apparently affect the processing efficiency. The most important parameter is the variation in the composition of cellular content of blood and blood collection/processing and storage conditions. It is known’ that varying numbers of leucocytes remain in the cellular components of blood, depending upon the length of time from collection to centrifugation (optimal 6-8 h); accuracy of rotor balancing; acceleration/deceleration time, handling prior to separation and the proximity of the interface to the top of the bag when separating platelet-rich plasma.‘, ’ Leucocyte reduced platelets can also be prepared by centrifuging pooled red cell contaminated PC, utilizing apheresis technology and automated buffy-coat removal methods, each producing products with differing properties.’ Nevertheless it should be remembered that any additional steps in processing may have some deleterious effect on the morphological and functional integrity of cellular components. Hence standardization of component collection, processing and storage conditions remains the most important issue in validation of filter efficiency. The proper assessment of the reasons for “filtration failure”, even in a well standardized and validated system as well as the use of an accurate and reliable method for counting low leucocyte numbers require special attention. In most cases the failure or shortcoming appears to be related to the characteristic adhesive properties of the cellular and hemeostatic components of the donor’s blood and/or the shortcomings in the final products rather than a deficiency in filter performance. For proper comparative analysis of filter efficiency, attention should be directed at reducing the influencing factors in the source product by using paired identical units, standardizing all the operational procedures and continuously monitoring relevant parameters in the individual product both during processing as well as the storage period. In this respect, for more efficient filters we may require more sensitive assays such as the phenotypic composition of the residual leucocyte cohorts [i.e. the percentage of antigen presenting cells (APC), and CD4 suppressor cells) and identification of variable membrane microversicles, produced during processing and storage. Currently,
52
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flow cytometry and PCR as well as molecular markers of the storage lesion are used for assessing changes in properties of post filtration products.‘,’ These, combined with quantitation of the cellular composition, cell-cell interactions and cell damage using new techniques and election microscopy will provide a better insight into the mechanism of action of leucocyte removal. Work in progress in the author’s laboratory, using an identical paired protocol system, reveal that there is a considerable change in the properties of the platelet concentrates subsequent to filtration. On average there is a 10 f 3% fall in platelet count in the filtered product and a loss of 20 mL total volume. In the majority of cases, the apparent mean platelet volume of the filtered platelet is smaller (- 0.3 fL) and there is a slight rise of pH (0.2) suggesting that large and more haemostatically effective platelets are preferentially removed by the filtration process. The filtered PC sometimes produces abnormal storage stability pattern in particular at the end of shelf lifesaa Different types of filters have a finite leucocyte removal capacity and the flow rate of PC through the filters must be meticulously adjusted in order to consistently achieve optimum leucocyte removal. On the other hand, non-filtered products, containing even a slightly higher leucocyte content (as low as 0.35 x 10’) show a larger reduction of the functional integrity as substantiated by an increased release of GpIb fragments in the platelet supematant, microvisculation measured by membranebound GpIIb/IIIa (microparticles) in the supematant, and release of vWF with reduced collagen binding activity. These deleterious effects might be associated with the reduced post-transfusion platelet response.8 The decrease in functional integrity is more likely to be a result of the release of leucocyte enzymes which have a high afflnity to platelet surface glycoproteins and certain adhesive proteins such as vWF.‘<’ Filtration of red cell products also appears to be associated with microvesiculation and increased release of haemoglobin. The storage stability pattern of post-filtered products at the end of storage also appears to be different. The significance of these findings in the clinical setting remains to be investigated. New potentials for the use of polyester filters stem from reports that these devices may remove small but significant quantities of engulfed and free bacteria from donor blood, opening a new area of research/development in filtration.g 3. MICROBIOLOGICAL
ASPECTS
OF LEUCOCYTE
DEPLETION
Post-transfusion sepsis, after the transfusion of platelet concentrate and the use of SAG-M red cells is steadily increasing.’ In most instances the contamination probably occurred at the time of venepuncture as a result of donor bacteraemia. Since leucocytes are known to prevent bacterial proliferation in contaminated units of blood it is warranted to relate the effect of leucocyte depletion and bacteriological safety. Evidence is accumulating’ that leucocytes play a critical role immediately after collection and a “holding” period of blood from 6-24 h at R.T. the period of 20-24 being optimal. Therefore in-line filter usage can only become significant with the use of new generation filters having not only the capacity for removal of leucocyte-lodged bacteria but also non-ingested bacteria directly. This is of particular relevance since leucocytes function poorly at the conventional conditions of red cell storage and they deteriorate and die after a few days, releasing the engulfed but still viable bacteria hence after this period it is likely to have little or no effect on the risk of posttransfusion sepsis. The per&operative blood transfusion can also contribute to post-operative infection by transmission and/or by an immunosuppressive effect, leading to decrease of resistance to micro organisms introduced during surgery.g This implies that multiple clinical studies in various disease categories are needed and the development of spe-
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cific guidelines for transfusion should address the question of post-operative infections. Microbiological results from most laboratory studies’ suggest that deliberately inoculated bacteria (1 organism/ml) exhibit a lag phase of growth for 24-48 h followed by rapid growth of 106 organisms/ml. The reported rates of bacterial contamination in platelets range between 0 to 4.9% for single donor apheresis platelets and O-10% in pooled random units.’ Since transfusion of the bacteriologically contaminated products in immunocompromized patients may lead to death, effective preventative strategies are needed. Some debatable issues include: avoidance of repeated use of same harvest site during collection; reduction of the number of homologous donor exposures (pooling process) and improved identification of microbiologically contaminated units (i.e. using polymerase chain reaction technologies similar to those available for the identification of HIV1 hence offering the potential benefits of identifying all heterogeneous types of bacteria on the basis of prokaryotic DNA) and shortening the storage time between collection and transfusion of platelets to 3 days particularly for use in immunocompromized patients. Large numbers of variables such as the nature of the anticoagulant and its ratio, the concentration of adhesive proteins such as fibronectin’ the temperature, complement activation and cell/cell/protein interactions may have a significant role in bacterial growth. To date, bacterial growth has not been enhanced in units that were deliberately contaminated, held and then filtered. 4. CLINICAL
ASPECTS AND COST EFFECTIVE STRATEGIES
It is now well established
that residual leucocytes in red cell and platelet concentrates contribute to some clinical side effects in certain groups of patients who are transfusion dependant. Leucocytes also have detrimental effects on patients undergoing extracorporeal circulation of the blood. The reported clinical benefits of the use of leucocyte filtered products in specific clinical settings and surgical areas are steadily increasing. Table 1 summarizes some of the potential clinical and practical benefits of leucocyte depletion. lGzo There has been a general criticism that until recently no proper comparative clinical trials and validation of filter performance have been made. This is partially due to the fact that blood components differ in their characteristic properties and a vast array of preparative and storage conditions are used {fresh, variably stored for different lengths of time; warm; cold; soft spin; hard spin; b&y-coat removed or
Table 1. Some Practical and Clinical Benefits of Leucocyte Depletion l
0 l l
0 l
0 0 0 l l l l l l
Preventing HLA Alloimmunization/Sensitization Reducing Platelet Refractoriness Preventing Transfusion-associated Cytomegalovirus (CMV, HTLVl ) Preventing Non-haemolytic Febrile Transfusion Reactions Reducing Transfusion-associated Thrombocytopenia Reducing Transfusion-associated lmmunomodulation Reducing Potential Reperfusion Injury Decreasing Usage of Platelets Reducing Need for HLA-matched Platelet Products Providing Alternative or Supplement to CMV Negative Blood Products Reducing the Overall Workload Reducing Biochemical Degradation During Storage Reducing Histamine Release and Reactions Associated With the Presence of Cytokines Reducing Vascular Occlusion and Depletion of Fibronectin Reducing Leucocyte-lodged and Some Free Bacterial Contamination
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present; high and low haematocrit) each of which may induce variability and influence the optimal performance and process efficiency of filters. Similarly, large differences exist in the adhesive properties of platelet products obtained from random donors, methods include single donor apheresis, the PRP method; single or pooled buffy-coat method involving storage at 22°C for variable lengths of time. The adhesive properties of such products containing different subpopulation of platelets are different and can influence the characteristic performance of filters, leading to inter- and intra- laboratory variability and loss of active subpopulations of platelet with leucocytes. Occasionally the overall poor function of some filters make any clinical comparison based on a well-standardized product a difficult task. Despite these uncertainties there is now ample clinical evidence that the prophylactic use of filtered red cells will prevent or delay non-haemolytic febrile transfusion reactions (NHFTR).” This is of particular relevance to patients who are transfusion dependant such as those with Beta-thalessaemia major, in whom prevention of HLA alloimmunization prior to bone marrow transplantation is indicated. Some other patient groups such as those with chronic aplastic anaemia, myelodysplasia, sickle cell disease and chronic renal failure who become unresponsive to erythopoitein are also at risk. There is now a general consensus that all newly diagnosed patients with severe aplastic anaemia (SAA] who are potential marrow transplant recipients, should receive leucodepleted cellular components3 Furthermore leucodepletion is also an acceptable alternative to cytomeglovirus antibody screened blood.4, 5 The prevention of platelet refractoriness” and immunomodulation12 are both affected by removal of leucocytes. Methods are now available which allow us to reliably achieve a residual leucocyte count of less than 5 x lo6 in a dose of approximately 3 x 10” platelets, but standardization remains an unresolved problem. There is also an urgent need to establish measures of clinical outcome, based on minimum red cell and platelet requirements, which would enable the cost-effective selection of patients who may suffer from the transfusion of allogeneic leucocytes.3 In general there are three distinct categories of disease in which the formation of HLA-alloantibodies is undesirable: (i) su;Fz;z;;a;hz;pT with platelet concentrates such as in aplastic anaemia, or (ii) heart, liver and ienal transplantation; (iii) patients requiring life-long red cell transfusion; While in the later it makes sense to start with standard leucocyte-reduced red cells and await febrile reactions, cardiac and liver transplant patients will benefit from filtered products. 14-17In kidney graft recipients the requirement for supportive therapy has enormously decreased due to a change in policy not to remove the non-functional kidney. Alternative strategies for reducing the adverse effects of leucocytes are also becoming established.3 For example, potential methods for prevention of graft versus host disease (GVHD) would be depletion of T-lymphocytes from the blood component before transfusion. This is attributed to the fact that both the incidence and severity of GVHD, after allogeneic BMT can be reduced if T-cells are eliminated from the donor marrow by a variety of techniques prior to grafting, including buffy-coat removal by centrifugation, direct and inverted centrifugation, washing, cotton and cellulose acetate filtration or freezing. These procedures reduce leucocytes by only 2-3 log,, leaving KY’-10’ lymphocytes behind, hence can only be considered as a first step in leucodepletion since less than 8 x lo4 lymphocyte per kg body weight appears to mediate a GVIYID.~In contrast, simple in vitro irradiation (with 1530 gy) accomplishes the desired goal inexpensively rapidly and with proven efficacy. W-ultraviolet light also abrogates GVHD in recipients with good preliminary results in minimizing
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alloimmunization. Further work is nevertheless needed to determine clinical efficacy and side effects of this procedure. The need for pre-storage leucocyte-depleted cellular components (although debatable) currently presents an important inventory management issue in the U.S.A. An alternative strategy would be to put aside a specific number of group 0 negative packs of red cells to meet the needs of this patient population, though this may lead to excessive use of 0 negative red cells, deserving a special audit for successful inventory management. The rational comparison of benefits and costs is an area of major concern, 3 as the provision of leucodepleted blood components incurs costs ranging from materials to time of laboratory staff, while, at the same time provides benefits to patients and the health care service in general. SUMMARY Blood component therapy has enabled us to successfully support the increasing demands for treatment of haematological abnormalities. Continuous efforts are made to minimize or prevent immediate side effects of transfusion but the long-term effects of component therapy will remain unknown for some time. There is always a need for products with special qualities (i.e. no CMV, no HLA immunization etc.] however when considering adverse effects it is important to look for alternative strategies which produce minimal alteration in the cellular and fluid components of blood while maintaining optimal clinical efficacy. Continuous education of clinicians and laboratory scientists is needed so that the best possible products are provided reliably and with consistency. This review focused on some of the important debatable issues on the laboratory/microbiology aspects and clinical practice. It is clear that the presence of leucocytes is associated not only with reduced quality, based on the morphological and functional integrity of blood components but also reduced post-transfusion response in some specified cases. A stringent regular QA program is required for the assessment of the trend as both differences between filter batches and slight changes in laboratory operations contribute to the lack of reliability of the system in use. While until recently the main goal of leucocyte depletion for red cells was to prevent NHFTR in specified patients it appears that currently a more ambitious goal of avoiding other important leucocyte-induced transfusion complications are considered with some success, without leaving aside alternative strategies and their practical benefits. In respect to cost-effective transfusion strategies, in multiply-transfused patients, both induction of antibodies against HLA class I antigens and the development of leucocyte-associated virus infections could be prevented by using modem filters which lead to effective components with leucocytes below the threshold reported in the Edinburgh consensus meeting 3 whereas gamma-irradiation of blood components before transfusion, that decreases the mitogen induced stimulation of the lymphocytes is the best strategy for the prevention of post-transfusion GVHD. Many advances have been made in methods of detection of a low level of leucocytes, (Nageotte chamber, flow cytometry, PCR) and the storage lesion induced by the residual leucocytes,’ helping to better define product quality. A considerable degree of cooperation is also seen between clinicians, scientists and other disciplines and manufacturers of the diverse technologies. Acknowledgements I wish to express my sincere thanks to Pall Biomedical Ltd for supporting the symposium and to Vivian Sproule for typing the manuscript.
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REFERENCES 1. Hogman CF: Component preparation and quality assurance of stored blood, in Rock GA, Seghatchian MJ (eds): Quality Assurance in Transfusion Medicine, Vol. II. Florida, CRC Press, 1993, pp. 59-98. 2. Leikola J, MyllylH G: The clinical use of red cell components, in Summers SH, Smith DM, Granenko VA (eds): Hogman 40 Guidelines for Practice. Arlington VA, American Association of Blood Banks, 1990, p.1. 3. Royal College of Physicians of Edinburgh: Consensus Meeting on Leucocyte-depleted Blood Components. Edinburgh (March, 1993). 4. Gilbert GL, Hayes K, Hudson IL, et al.: Prevention of transfusion-acquired cytomegalovirus infection in infants by blood filtration to remove leucocytes. Lancet 1989;1228-1231. 5. Bowden RA, Sayers MI-I, Cays M, et al.: The role of blood product filtration in the prevention of transfusion associated cytomegalovirus (CMV) infection after marrow transplant (AABB, October 21-26 1989). Transfusion 29 (Suppl). 6. Greenwalt TJ, Gajewski M, McKenna JL: A new method for preparation of granulocyte-poor red cells by microaggregate filtration. A simplified method to minimize febrile transfusion reactions. VOXSang 1980, 39:282. 7. Seghatchian MJ, Brozovic B: An overview of current trends in platelet storage and clinical practice. Blood Coag Fibrin01 1992; 3:617-620. 8. Sloand EM, Klein HC: Effect of white cells on platelet during storage. Transfusion 1990; 30:333. 8a. Krailadsiri P, Seghatchian MJ: Effect of filtration, storage and platelet suspension media on platelet indices. Transfus Sci 1994; 15: in press. 9. Anderson KC: Bacterial contamination of platelets (Current Trends) Transfus Sci 1993; 14: 159-162. 10. Sirchia G, Wenz B, Rebulla P, et al.: Removal of white cells from red cells by transfusion through a new filter. Transfusion 1990; 30:30-33. 11. Sniecinski I, O’DoMell MR, Nowicki B, et al.: Prevention of refractoriness and HLAalloimmunisation using filtered blood products. Blood 1988; 71: 1402-1407. 12. Solheim BG: Immuno-modulation by transfusion and CMV transmission. BBTS, Aspects of Blood Filtration, 5th April 1990, Birmingham, England. 13. Hart S, Bareford D, Smith N, et al.: Post-transfusion thrombocytopenia: its duration in splenic and asplenic individuals. VOXSang 1990j 59:123-124. 14. Bando K, Pillai R, Cameron DE, et al.: Leukocyte depletion ameliorates, free radicalmediated lung injury after cardiopulmonary bypass. 1 Thorac Cardiovasc Surg 1990; 99: 873-877. 15. Pillai R, Bando K, Schueler S, et al.: Leukocyte depletion results in excellent heart-lung function after 12 hours of storage. Ann Thorac Surg 1990; 50:211-214. 16. Schueler S, Hatanaka M, Bando K, et al.: Twenty-four hour lung preservation with donor core-cooling and leucocyte depletion in a bilateral lung transplant model. Proc Am Co11 Surg (Surgical form) 1990; 2X1:405-407. 17. Bareford D, Chandler ST, Hawker RJ, et al.: Splenic platelet-sequestration following routine blood transfusion is reduced by filtered/washed blood products. Br f Haematol 1987; 67: 177-180. platelet 18. Van Marwijk Kooy M, Van Proojen HC, Moes M, et al.: Use of leucocyte-depleted concentrates for the prevention of refractoriness and primary HLA alloimmunisation: a prospective, randomised trial. Blood 1991; 77:201-205. 19. Saarinen UM, Kekomiiki R, Siimes MA, et al.: Effective prophylaxis against platelet refractoriness in multitransfused patients by use of leucocyte free blood components. Blood 1990; 75:512-517. 20. Sivakumaran M, Norfolk DR, Maior KA, et al.: A new method to study the efficiency of third generation blood filters. Br / Haematol 1991; 84:175-177.