Leukocyte-poor blood components: issues and indications.

Critical Reviews in Clinical Laboratory Sciences

ISSN: 1040-8363 (Print) 1549-781X (Online) Journal homepage: http://www.tandfonline.com/loi/ilab20

Leukocyte-Poor Blood Components: Issues and Indications Benjamin Lichtiger & German Felix Leparc To cite this article: Benjamin Lichtiger & German Felix Leparc (1991) Leukocyte-Poor Blood Components: Issues and Indications, Critical Reviews in Clinical Laboratory Sciences, 28:5-6, 387-403 To link to this article: http://dx.doi.org/10.3109/10408369109106870

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Volume 28, Issue 5,6 (1991)

387

Leukocyte-Poor Blood Components: Issues and Indications Benjamin Lichtiger and German Felix Leparc

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ABSTRACT Leukocyte-poor blood components (LPBC) have now become part of the armamentarium of available transfusable blood components. Indications for the use of LPBC vary in accordance with the underlying clinical condition, as well as the intended objectives of the transfusion therapy. Technological advances have made it possible to prepare LPBC using rather simple procedures. However, any manipulation of blood components and the additional use of filters, washing, rinsing solutions, etc. inevitably result in additional costs to the patient, the health-care institution, or third-party payers. Requests for LPBC involve the preparation of RBC or platelets, leuko-depleted by at least one log. Transfusion of LPBC must be done in a logical fashion that meets the needs of the patient. Currently, LPBC is indicated for patients with a history of nonhemolytic febrile transfusion reactions to delay alloimmunization to HLA antigens and avoidance of cytomegalovirus (CMV) infection. Key Words: leukocyte-depleted blood components, leukocyte poor, febrile transfusion reactions, alloimmunization

1. INTRODUCTION Leukocyte-depleted blood components (LDBCs) have attained an increasing level of interest in light of a better understanding of the mechanisms involved and responsible for HLA and platelet antigen alloimmunization, transmission of cytomegalovirus (CMV) infectivity, graft vs. host disease (GVHD), etc. In view of the perceived significance of such factors in many disease processes, requests for transfusion of “leukocyte-poor” blood components have suddenly undergone an inflationary spiral, notwithstanding the cost in dollars and the negative impact on the overall cost of delivery of patient care services. The use of LDBCs is now considered by some almost as a modem panacea in the treatment of conditions requiring chronic transfusion therapy, without consideration as to whether or not reliable and reproducible clinical studies justify such an approach. There are many methods, techniques, and devices currently available to prepare LDBCs.

k&imh Lidtiger, M.D., W.D.,Professor and Chief of Transfusion Medicine and Laboratoy Immunology, Division of Laboratory Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, TX; German Felix hparc, M.D., Medical Director, Southwest Florida Blood Bank, Clinical Assistant Professor, Department of Internal Medicine, University of South Florida College of Medicine, and Clinical Assistant Professor, Department of Pathology and Laboratory Medicine, University of South Florida College of Medicine, Tampa, FL.


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Indeed, it is difficult at times to keep track of the names, brands, and manufacturers, let alone be conversant about the leukocyte removal efficiency, residual leukocytes, red blood cell recovery, and other technical aspects that are characteristic of each method or device. As a result, the hospital pathologist in charge of the transfusion service is often the one identified as responsible to recommend to hisher colleagues, clinicians, and surgeons the best possible procedure. At the same time, the pathologist must deal with the sales force of the different manufacturers of these devices in a coherent and critical manner. This is no easy task; any decision pro or con for a particular device may provoke the ire of other physicians in the clinicalhrgical services. Those same salespersons not only visit with the pathologists, but also aggressively market the ideas and devices for LDBCs to other practitioners and purchasing agents of health-care organizations. Thus, it is imperative that decisions to provide LDBCs be based on realistic and solid scientific data, with defined expectations that would justlfy the increased cost incurred by the use of such devices.

II. HISTORICAL BACKGROUND In 1926, Doan reported that compatible red blood cell (RBC) transfusions could lead to

severe reactions if there were concomitant leukocyte incompatibilities.' The observation he made came about from his elaborate studies on the clinical effect of leukoagglutinins. Doan noticed that when several of his patients were transfused with blood that was RBC and leukocyte compatible, no untoward reaction was seen. On the other hand, patients who had a history of severe reactions to transfusions of blood that was RBC compatible and not tested for leukocyte compatibility had severe reactions, which actually appeared to be life threatening to Doan. Thus, Doan concluded very early in his clinical experience that leukocytes were responsible for the severe transfusionreactionsoccurringin patients receivingcompatibleRBC transfusions. In 1954,Dausset reported on the clear association with the development of leukoagglutinins in patients who were administered multiple blood transfusions. The observations led him to conclude that the leukoagglutinins were reacting against defined moieties on the leukocytes, with deleterious effects on these cells.2 It is interesting to note that the observations reported by Doan and later by Dausset, analyzed from a retrospective vantage, start to lay the foundation for development of LDBCs as a desirable alternative. Unfortunately, the paucity of other reports on this potential need made this an issue that acquired its own impetus many years later. In 1957, Brittingham and Chaplin reported that otherwise unexplained febrile transfusion reactions were caused by alloimmunization to leukocytes present in donor bl00d.~Furthermore, when the same patients were then infused with the separated buffy coat, severe febrile reactions ensued. As a result of these observations, they went on to recommend that an inverted centrifugation technique be employed to remove the buffy coats from blood prior to transfusions intended for patients with known histories of febrile transfusion reactions (FTRs).3 Perkins et aL4reported on a study conducted on eight patients that developed FTRs when transfused with incompatible white cells. The investigation went on to determine the conditions under which FTRs occur, the extent to which the reactions are caused by leukocyte antibodies, and the appropriateness of routine testing for white cell antibodies, as well as how to prepare LDBC4 It is noteworthy to point out that Perkins et aL4 also reported that some patients failed to



389

develop FTRs in spite of having leukocyte antibodies. This was interpreted as probably due to a low number of leukocytes present in the units transfused. They further suggested that the transfusion blood components containing less than 10’ leukocytes were unlikely to result in F T R s . ~Since the publication of this r e ~ o r tLDBC ,~ transfusions achieved a special place in the hemotherapeutic armamentarium. The techniques for preparing LDBCs as well as the indication and use of this type of transfusion, have undergone numerous changes since then. Thus, clear experimental and clinical evidence show that the presence of leukocytes in blood was to be considered as a “contaminating” element and that the incidence of FTRs could be significantly decreased by reducing the load of leukocytes from a unit of b10od.i~ As a response to the newly defined need for providing blood components with a reduced leukocyte load (i.e., less than lo8 leukocytes per unit of blood). The suppliers to blood centers and transfusion services developed materials and methods to meet those needs. During the 1970s, the use of LDBCs was almost entirely reduced to the processing of RBC units, with the sole objective of reducing the incidence of FTRs. However, clinical observations indicated that platelet transfusions were also capable of inducing FTRs. The technology available at the time was not able to reduce the leukocyte load of platelet concentrates. Experimental data obtained on blood components processed with the available technology of the 1970s showed that LDBCs were mostly depleted of granulocytes, while still leaving most of the lymphocytes capable of producing other untoward effects not foreseen in the past.5 A case in point must be mentioned from the work reported by Class et al.5 who, in a mouse model, demonstrated the effect of contaminating leukocytes on the immunogenicity of platelet concentrates obtained from whole blood donations. The study showed that leukocytes with class II antigens are responsible for alloimmunization to transplantation antigens rather than the platelet antigens alone. Furthermore, they demonstrated that contaminating leukocytes with class 11 antigens below an undefined absolute number do not seem to induce alloimmunization. Class’ work5 opened up a completely new chapter in the philosophical approach to preparing LDBCs, as avoidance of FTRs became just one of the objectives, with many more goals, such as the prevention of alloimmunization, showing up on the horizon of potential benefits. Notwithstanding these, a new set of issues had to be dealt with as it became evident that remnants of relatively small numbers of lymphocytes could have long-term deleterious effects for specific patient populations who depend on the prolonged and continuing support of various transfusable blood components. Then, the issues to deal with included (1) when and to whom should LDBCs be administered, (2) what procedures should be employed to achieve the most effective leukocyte removal from the blood components intended for transfusion, and (3) how thorough should the leuko-depletion be and how much of the blood component loss would be acceptable for the component to remain a viable form of therapy?

111. ISSUES RELATED TO LEUKO-POOR PLATELET CONCENTRATES The issue of LDBCs is further complicated when one addresses the problem of ascertaining the therapeutic effectiveness of platelet transfusions. Alloimmunization and refractoriness to platelet transfusions are terms that require a clear understanding. The concepts associated

390



with these terms impinge rather frequently on the complex problems of whether or not a patient is a suitable candidate for LDBC therapy.

A. Alloimmunization to HLA and Platelet Antigens Class et al. demonstrated that alloimmunization to HLA antigens present on platelets (class I antigens) requires the presence of cells bearing class II antigens such as macrophages, B-lymphocytes, or dendritic cells. Both class I- and 11-bearing cells promote the development of antibodies that will result in refractoriness to platelet transfusions. As postulated by Class et al.,5 the effective removal of class II antigen-bearing cells, or at least their removal to an as yet undefined low number, abrogates the formation of HLA antibodies and thus delays the onset of alloimmunization.6 Therefore, transfusion of blood components depleted of white blood cells (i.e., antigen-presenting cells [APCs] bearing class I1 molecules) would not result in alloimmunization. Some clinical studies report that when the number of leukocytes per transfusion falls below 5 X lo6, alloimmunization is virtually eli~ninated.~ However, this kind of leukodepletion is seldom achieved with present technology. Currently, the most efficient methods of leuko-depletion consist of filtration of the blood components prior to transfusion. This is achieved with devices specially designed to remove 99.99% of leukocytes. (The types and characteristics of each filter are discussed later.) In most cases, 99.99% removal of leukocytes will leave a residual load of lo6 to lo7 cells, which most likely will delay but not eliminate the alloimmunization process. Unfortunately, a long-term prospective study of patients receiving LDBCs has not been published as yet. Another factor that must be considered is that 40 to 50% of all patients can be categorizedas nonresponders, and therefore not likely to be alloimmunizedto HLA antibodies.* B. Refractoriness to Platelet Transfusions This is characterized by the failure to achieve an adequate platelet count increment in peripheral blood after therapeutic doses of platelets have been transfused. The failure to produce meaningful increments after platelet transfusions should be observed in the absence of a series of clinical conditions, such as sepsis, splenomegaly, active hemorrhage, microangiopathies, disseminated intravascular coagulation, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, and others that are known to produce shortened survival of the transfused platelet^.^ Fever, generally believed to be responsible for lower posttransfusion platelet count increases, was found not to have such an effect by Bishop et al.9 Due to the complexity and interrelational nature of the processes responsible for platelet transfusion refractoriness, the role of LDBCs is not entirely clear or completely understood yet. The vast majority of cases of platelet transfusion refractoriness, however, are associated with HLA alloimmunization or immunization against platelet-specific antigens. The criteria for the determination of refractoriness to platelets may vary, but a commonly agreed threshold is a corrected count increment (CCI) of less than 7500 platelets per microliter 1 h after transfusion. CCI can be calculated as follows:1o CCI = platelet count after transfusion - platelet count before transfusion X bodv surface area (m2) absolute number of platelets transfused



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C. Graft Protective Effect (Nonspecific Immunosuppression) Blood transfusions have been found to cause nonspecific immunosuppression that was first recognized in kidney transplant recipients.’’ Since then, the search for the element in blood that causes the immunosuppressiveeffect has remained elusive. However, the studies done by Opelz and Terasaki suggest that the effect may be related to the presence of leukocytes. This is based on the outcomes observed in the recipients of leukocyte-depleted RBC components (mostly frozen RBCs), compared with those receiving RBCs or buffy coat concentrates before renal transplantation.I ’ The use of blood components containing leukocytes has also been reported as beneficial in patients with a history of repeated miscarriage.I3 The effects of using LDBCs in situations other than solid organ transplantation are as yet unknown. Tucker et al. l4 have reported on the potentially unfavorable effect of LDBCs for patients with acute myelogenous leukemia (AML). Tucker et al.I4 showed that AML patients receiving LDBCs had reduced duration of remission compared to patients receiving nonLDBCs. Therefore, the information currently available raises serious questions about the necessity of providing LDBCs to all patients, or at least to AML patient^.^.'^ D. Viral Agents and Leukocytes in Blood Components Due to the fact that many agents “travel” within leukocytes, either by incorporating into the cells’ genome or as cellular viral inclusions, it has been speculated that the removal of leukocytes in blood components may help reduce or eliminate the transmission of viral agents.19**0This has certainly not been the case for hepatitis viruses, or the human immunodeficiency virus. However, present evidence indicates that the risk may be eliminated or significantly reduced for the transmission of cytomegalovirus (CMV) and Epstein-Ban virus (EBV).1*-23This is based mostly on controlled studies using frozen RBCs as the leukocytepoor components.21-24 The role of leuko-depletion of blood components in the prevention and transmission of HTLV-I and -11 are not entirely clear at this time. Prospective studies to elucidate that issue are not possible, based on ethical principles. Retrospective analysis of a large number of leuko-depleted transfusions in long-term survivors may provide the necessary information to make a reasonable judgment. E. Leukocyte Load and Leukocyte Removal Perhaps one of the reasons the subject of leuko-depletion is poorly understood is the terminology and the quantitative nomenclature used when refemng to the leukocyte contents in blood components before and after leuko-depletion. Table 1 illustrates the figures that can be drawn for comparison in the evaluation of different methods or devices, assuming that the average unit of whole blood usually contains between 1 X lo8to 1 x lo9 leukocytes (Table 1). Thus, it is important to determine the amount of residual leukocytes present in the unit of blood or platelet concentrates. Reliance on the percentage of removal may be misleading; at times a donor may have peripheral blood leukocyte counts that may fluctuate between 4 x lo3 and 10 x lo3 cells per cubic millimeter. It becomes apparent that the percentage removal without expressing the number of residual leukocytes is of relative value. Notwithstanding this, another problem that will have to be eventually addressed is related to fragmentation of the leukocytes throughout storage and senescence. These will undoubtedly require selection of “fresh” blood products to remove intact leukocyte populations. Un-


392

Table 1 Relative Absolute Number, Expressed Exponentially, and Logs and Percentages for Defining Leuko-Depletion Efficiencies Reduction

Number of leukocytes


I I I I

x x x x

109 109 109 109

log

z

Residual number of leukocytes

90 99 99.9 99.99

1 x 108 I x 107 1 x 106 I x 105

doubtedly, demands for “fresh” blood products for leuko-depletion will place additional pressures on the supply of blood products.

F. Transfusion-Related Acute Lung Injury (TRALI) TRALI is characterized by acute respiratory distress that typically occurs within 4 h after a transfusion.25 Clinical manifestations include acute onset of respiratory failure, tachypnea, cyanosis, and chest rales on auscultation. Many cases of acute pulmonary edema as a consequence of transfusion have most likely been misdiagnosed as circulatory overload.25 The pathogenesis of TRALI is presumed to be due primarily to passive transfusion and reaction of the donor’s antibodies to leukocyte antibodies with the recipient’s granulocytes.As a result of this antibody activity, degranulation and release of proteolytic enzymes by the granulocytes leads to acute lung injury. Studies have shown that in 89% of the cases, TRALI is associated with granulocyte antibodies in the donor’s plasma that interact with the recipient’s granulocytes.z The reverse may occur in a minority of cases. Although TRALI is most likely a form of adult respiratory distress syndrome (ARDS), the mortality associated with TRALI is less than 10%. This outcome is in contrast to the overall 50 to 60% mortality associated with ARDS.25 Approximately 80% of patients with TRALI exhibited rapid resolution of pulmonary infiltrates and return of arterial blood gases to normal within 96 h after the initial respiratory insult.25

IV. METHODS FOR PREPARATION OF LEUKOCYTE-DEPLETED BLOOD COMPONENTS Numerous procedures have been developed to produce units of leuko-depleted RBCs and at the same time obtain a transfusable component with minimal loss of RBCs. The techniques’ principles are very similar, although the materials and methods used to accomplish these goals vary (Table 2).

A. Sedimentation Although the cellular blood components are in homogeneous suspension at the time the whole blood donation is collected, each of the elements will sediment into distinct layers, based on their corresponding densities. The major advantage of using a sedimentation procedure is that it requires no capital equipment and results in little red cell loss (approximately 5 % ) . A single sedimentation results in less than an 80% reduction in the total leukocyte


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Table 2 Methods of Preparation of LDBs (2) RBC recovery (%b)

WBC removal (56)

Ref.

Sedimentation

95

26, 21

Inverted centrifugation Saline wash Frozen-thawed RBC Spin-cool-filter Differential centrifugation and online filtration

89 80

80 (single); 95 (double) 80 93 98 80 91.4

90 94 90

28 29 30, 31 35, 36 31, 38

content in the original container.26 However, a second sedimentation procedure can be performed thereafter, resulting in the cumulative removal of approximately 95% of the leukocyte mass.27 A major drawback is that, under most circumstances, it is very timeconsuming and performed in an open system that affects the expiration time for the component prepared.

B. Inverted Centrifugation The centrifugation process duplicates the sedimentation phenomenon in an accelerated fashion. Although straight sedimentation and separation steps may be used, inverted centrifugation has gained better acceptance. The method consists of the centrifugation of a unit of blood (approximately 450 ml) placed in an inverted fashion in a standard blood bank centrifuge. This operation is performed for about 7 to 10 min at approximately 5500 x g. After centrifugation, the inverted bag of blood is carefully removed and hung in such a fashion that it allows drainage of the RBCs into another bag, leaving less than 30% of the red cells and accompanying buffy coat in the original bag.28 Tenczar reported in 1973 that, in his experience, inverted centrifugation was a simple and effective method that results in the greatest erythrocyte recovery and is best suited for clinical cases where prevention of FTRs is desired.” In this report, 219 units of blood were processed into leukocyte-depleted components using the inverted centrifugation approach. When compared with other contemporary methods, inverted centrifugation seemed to have a distinct edge in terms of RBC recovery and leukocyte removal, aside from clinical responses. The popularity of the method was short-lived, however, as automated equipment capable of performing leukocyte removal by saline was introduced into the market.

C. Saline Wash In 1978, Bryant et aLZ9published a method for preparing LDBCs with an IBM 2991 blood cell processor. The protocol designed by Bryant et al. was able to achieve leukocyte removal rates of up to 92% and RBC recovery of up to 82%. Later evaluations showed more realistic figures of 83 and 76%, respectively. Bryant’s communication opened the door for the introduction of semiautomation into an area that at the time required a heavy commitment of manpower. The introduction of the new method was very timely, as more and more patients seem to require LDBCs due to the advent of component therapy and chronic platelet transfusions. The practicality of the described methods allowed the extra flexibility

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of issuing a unit of blood, without any special manipulation, that could be leuko-depleted at a moment’s notice. Publication of the early results and aggressive marketing by the instrument’s manufacturer resulted in a proliferation of protocols, and some even recommended the routine use of saline-washed RBCs on all patients. The use of saline-washed RBCs required capital investment in an automated instrument and the use of a considerable amount of disposables or “software”. Drawbacks to this method include the likelihood of breakage of plastic bags, with the consequent loss of the unit of blood, and machine down time. However, at the time of release, this piece of equipment was a welcomed addition to transfusion medicine technology. Many washing units remain in operation, although used for different purposes.

D. Frozen-Thawed Red Blood Cells (FTRBCs) The development of methods for the cryopreservation of RBCs was developed with the specific objective of achieving long-term storage, measured in years after blood collection. Prolonged extension of the shelf life of RBC concentrates was seen as a practical way to deal with seasonal shortages, the long-term storage of units with rare phenotypes, and even for autologous blood collections for use in planned or speculative storage programs. Various techmques for effective and safe cryopreservation have been developed and are in use at centers with the sophistication and capacity to provide such services to their communities. One of the advantages noted very early in the developmental stages of this processing method was the rather accidental observation that FTRBCs were also leuko-depleted. This unexpected result was achieved through the repeated solution and fluid exchanges that take place during the freezing and thawing process. Likewise, the methods for cryopreservation of RBCs are not effective for preserving leukocytes, thus prompting the destruction of white blood cells (WBCs) during the process of freezing. Because of their properties, FTRBCs acquired a new and unforeseen indication: its use as a LDBC for patients with documented and repeated FTRs. In some services, FllU3Cs have been used routinely either as a prophylactic measure to avoid the development of FTRs or to transfuse them to patients who experienced them. Most users of frozen red blood for this purpose have documented an almost complete absence of FTRs after this use. However, the efficiency of producing FTRBCs is affected by many variables that must be taken into account, such as the cryoprotectiveagent (glycerol) concentration, type of cell washing equipment, and the kind of freeze-thaw protocol employed.14The best reported efficiency for leukocyte removal rate does not exceed 90 to 95% (i.e., 1 to 1.5 logs), with a RBC loss of about 20%. Crowley et al. clearly cautioned that any attempts to accurately enumerate the residual leukocytes of frozen-thawed blood components can lead to flawed results ,30 because during the process of freezing, thawing, and deglycerolyzing, leukocytes may fragment and not be entirely removed during the processing of the unit of blood. The cellular fragments are below the threshold sensitivity of any counting method. The remaining fragments also have antigenic properties and may induce in the recipient the same untoward effects as nondepleted blood c ~ r n p o n en t sIn . ~ ~addition, several publications have conclusively demonstrated that FTRBC units contain viable lymphocytes capable of inducing a GVH reaction in the recipient, as well as transmitting viral disease^.^^-^^ Due to the large capital expenditure needs, the logistical barriers that are an unavoidable part of developing and managing a program of FTRBCs, and the questions raised about its effectiveness as a LDBC, the use of FTRBCs is limited to the long-term storage of RBCs, homologous and autologous, for which it was originally designed.


Volume 28, Issue 5,6 (199 1)

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E. Spin-Cool-Filter Technique (SCFT) Bensinger et al. reported in 1983 that the efficiency of harvesting granulocytes from stored blood was a temperature-dependent event.35As the blood cools, microaggregates made up mostly of granulocyte, the large clump of leukocytes thus formed can then be easily trapped by filtration devices.36 It would then follow that this process would significantly decrease the number of free leukocytes, as the majority of leukocytes would be removed with the microaggregates. Initial publications showed SCFT to leave a residual of 5 X 10' or less leukocytes and result in minimal RBC loss (less than 6%).36 The same workers reported that the technique was simple and easy to master, required minimal, if any, preprocessing of the unit of blood, and no capital expenditure. Attempts to implement the system in the field showed it to be rather cumbersome and disruptive of normal laboratory activities. The expiration date of the components was also adversely affected, offering no advantages over washed or frozen-thawed RBCs. The number of residual leukocytes (about 5 x 10') was still rather large when compared with the effort involved. F. Differential Centrifugation and M i n e Filtration During Initial Processing This process draws on the concept of removing leukocytes as early as possible after blood collection. The aim is to trap leukocytes with a filtration device that is part of the collection system, the leukocyte depletion thus becoming part of the component preparation process. The process shifts the workload from the transfusion service to the blood center. It traps intact leukocytes, eliminating the possibility of allowing the cellular fragments formed due to the storage lesion of leukocytes to pass through the filtration system unscathed. The system configuration consists of an in-line, sterile, cellulose acetate depth filter connected to a multibag system designed for the preparation of various blood component^.^"^^ Once the blood is collected in the primary bag, it is centrifuged in standard fashion. The supernatant plasma is expressed into satellite bags and sealed off for further processing. An additive is then added to the RBCs in the primary bag. The RBCs are then routed through the in-line filter into the emptied additive bag. This last step can be performed either at room temperature or at 5°C. This process allows the leuko-depletion to be accomplished in a totally closed system without any break of sterility, resulting in a maximum storage time of up to 42 d. The efficacy of leukocyte removal is reported by the manufacturer (Cutter Biological Corp., Berkeley, CA) to be in the range of 91.4%, and about 2.5 x lo8 residual leukocyte residuals per unit of RBCs. RBC recovery is in the 90% range. This component may be effective in preventing FTRs in patients. However, there is no information in the literature about its effectiveness in avoiding or delaying alloimmunization to HLA and platelet antigens. The same manufacturer introduced another system to allow for a reduction of the leukocyte load of pooled platelet concentrates. The procedure calls for pooling up to six units of random donor platelets into a specially shaped bag, which is then subject to an additional centrifugation step in a customized cup at relatively low speed. Once retrieved from the centrifuge, a specially designed clamp is fitted on the pouch's top where the vast majority of leukocytes and residual RBCs are trapped. The bag containing the pooled platelets is then released for transfusion with the clamp Literature distributed by the manufacturer claims that this method allows for the removal of more than 90% of leukocytes and residual RBCs, and a loss of 10% of platelets. The method appears to be helpful in avoiding FTRs, but its effectiveness in the prevention of alloimmunization is not at all clear.

396



G. Laboratory and Bedside Filtration Filtration of blood components was recognized early as a viable method for the removal of not only microaggregates, but also of leukocyte^.^.^^ Throughout the years, the technological approaches to maximization of leukocyte removal have varied. A summary of the principles used and the performance characteristics of each type of filter follows. 1. Microaggregate Filters This type of filter was designed with the goal of removing cellular debris and precipitated protein clumps from stored blood components. The trapping of leukocytes in the filters was also observed and quickly added as a “bonus” to the use of this type of filter. Following its introduction into the market, a flurry of studies were published that reported performance comparisons, clinical data, and technical characteristics of each model. Basically, two types of filters can be described, based on their design: Screen fdters -Also known as “surface” filters, they are designed to remove microaggregates by simple sieving. Although some adsorption of smaller particles to the mesh does occur, the screen pore diameter will principally determine the size of the microaggregates filtered out. Some examples of screen filters are the (1) Pall@ microaggregate filter, which consists of a folded, woven polyester screen with an absolute pore diameter of 40 pm, and the (2) BiotesP microfilter, whose design includes a cascade of filters made of nylon mesh. In the sequence of filters, the pore size decreases from 200 pm, through 50 and 20 pm, to 10 pm. This type of mesh arrangement is intended to eliminate the “channeling” phenomenon seen in single-screen filters, in which the particles trapped initially create flow channels for smaller particles to go through, thus decreasing the efficiency of the filter. Depth filters - These filters are designed to present a huge foreign surface area to the blood component being filtered so that leukocyte removal occurs by adsorption of the cells to the various materials used as adsorbents. One of the first depth blood filters described was designed by Greenwalt et ale4’Its adsorbent element was nylon wool. After reports of success in preventing some FTRs, a number of new designs followed, each using a different adsorbent material. Clinical experience with these filters varied from study to study, and went through the whole spectrum of outcomes, from high efficiency to questionable value in preventing febrile reactions in the recipient. As our knowledge of the physiology and dosage needed to cause transfusion reactions increased, it became readily apparent that the microaggregate f‘iters did not meet the expectation of eliminating all transfusion reactions, and the search for more efficient ways to eliminate undesirable side effects continued. 2. Leukocyte-Depleting Filters (Table 3) As the efficiency of leukocyte removal increased, some authors proposed a new terminology for the designation of blood filters. Thus, the term “leukocyte-depleting” has been reserved for filters that consistently achieve a reduction of at least 1 log (i.e., 90%)in the number of postfiltration leukocytes. This is perhaps the fastest growing segment of filtering devices that are designed for either laboratory or bedside use. A description of the models currently available follows.

3. lmugard IG50025-20,41 This filter is manufactured and distributed by Terumo Corp., Piscataway, NJ. Its design is that of a classic depth filter, containing 22 g of cotton wool (obtained from Gossypium barbdense) in a plastic, pipe-like encasement. The material used has an adsorption capacity

IMMUGARD IG500 (Terumo Co.) SEPACELL R-500 (Asahi Medical Corp.) ERYPUR (Organon-Teknika Co.) MICROPOR L (Baxter Co.) PALL RCIOO, PLIM) (Pall CO.)

Name (manufacturer)

Table 3

Yes Yes Yes Yes

No No

Polyester fiber Cellulose acetate Cellulose acetate Polyester fiber Polyester fiber 99 94-97

90

95.9

99

94

WBC Priming removal required (%)

Cotton wool

Material composition

90 NA

NA

75-80

95

90

RBC

NA 87

72

NA

NA

90

Plat

Recovery

Yes

No

Laboratory or bedside Yes No

Laboratory

NA Yes

Yes

NA NA Yes

Yes

RBC Plat

Laboratory or bedside Yes (500 series) Yes Laboratory or bedside Yes

Laboratory

Site of filtration

Used for


65-68

64

53-63

57-62

48-56

Ref.

4

W

w


398


of 2.4 X lo9 W C s . Once this number is reached, the ability to remove leukocytes ceases due to saturation. The filter is for laboratory use, and requires a 30-min filtration time for a unit of RBCs at room temperature, or around 40 min for units at 4°C. Reports of leukocyte removal efficiency range from 90 to 95%. When the postfiltration contents of the filter are analyzed, the removed leukocyte mass is composed of both mononuclear cells and granulocytes. Use of the filter requires preliminary priming with normal saline solution (1 00 to 150 ml) before the packed cells are introduced. After the filtration, and in order to obtain acceptable RBC recovery, residual RBCs must be flushed out with additional saline (100 to 200 ml). The reported leukocyte removal rate per RBC unit is between 94 and 96%, with a RBC recovery rate of 90 to 95%. This filter has been also successfully used to remove leukocytes from platelet components, where it has been shown to remove 1 to 2 logs (i.e., 90 to 99%), with a platelet recovery rate of 90%.In v i m and in vivo studies on platelet functionality after filtration have shown no significant loss in hemostatic effectiveness in vivo, although in vitro studies have shown suboptiinal aggregation when normal saline was used as a flush solution. Normal in vitro aggregation was observed when the filter was flushed with 200 ml of plasma. Some studies seem to indicate that the rate of alloimmunization to HLA antigens is decreased in patients receiving LDBCs prepared using the Immugard IG-500 filter. The studies, however, do not assess the independent effect of the removal of leukocytes from the blood components. To date, studies on the efficacy of this filter in preventing CMV transmission have been published, indicating that the leuko-depletion obtained by using this filter may be sufficient to prevent infection with CMV."8-56

4. Sepaceii R-500 and 4C2324 These filters are manufactured by Asahi Medical Corp., Tokyo, Japan, and are distributed in the U.S. by Baxter Healthcare Corp., Fenwal Division, Deerfield, IL. Both filter types are depth filters made of fine, long fibers of densely packed polyester. The 500 series includes a laboratory filter as well as a bedside model (the 500-A). The removal rate for the bedside model is between 2 and 3 logs, and is reported to result in red cell losses of around 9%. This compares favorably with the laboratory models, which result in losses of 13 to 16%.The manufacturer states that the filter performs best with units that are less than 3 weeks old. The use of older units may result in slow infusion rates, clogging, and decreased leukocyte removal efficiency. The 500 series has been found unsuitable for use with platelet concentrates, since the filters remove a sizeable number of platelets. Performance with the 4C2324 type of filter has also been disappointing when used with platelet concentrates, since it removed only 14% of the l e u k o ~ y t e s . ~ ~ - ~ ~

5. Erypur This filter is manufactured by Organon-Teknika. It is a cellulose acetate-based filter, a medium that has been used for years in laboratory filters. The filter has a "dead space" of approximately 80 to 100 ml. It comes in laboratory and bedside models, a popular marketing strategy. When used for two units of RBCs, it removes somewhat less than 2 logs of leukocytes (i.e., between 95 and 99%). It is recommended that the filter be used with units that are less than 10 d old to obtain peak performance. An important drawback seems to be the sizeable decrease in red cell mass after filtration of the units (on the order of 20 to 25%). The loss of red cells is less in the more compact bedside models (Erypur b).53*63


399

6. Micropore L


This filter is distributed by Baxter Healthcare Corp., Fenwal Division, Deerfield, IL. It is a cellulose acetate-based filter that has been studied mostly as a leuko-depletion instrument for the transfusion of blood components delivering platelets. In a published study, this laboratory filter gave a leukocyte removal rate of 1 log (i.e., 90%), and a mean platelet recovery rate of 72%. In vitro studies have shown adequate aggregation function on filtered platelets.@

7. Pall@ RC and PL Series The filters are manufactured by Pall Corp. and are polyester fiber-based elements. There are versions for RBC filtration (RC-50and RC-100) or for platelets (PL series). Both filters can be used either as laboratory filters or as bedside filters. No priming is required for either model, due to the special treatment of fibers that render them “wettable”. The reported efficiency of leukocyte removal is around 99% (i.e., 2 logs), with high recovery rates for both RBCs and platelet^.^^-^

V. IMPACT OF LEUKOCYTE CONTAMINATION ON PLATELET STORAGE The presence of leukocytes in stored blood components has been associated with some “storage lesions” that are observed in units used for transfusion. The leukocytes possibly affect platelet concentrates in a number of ways: (1) by releasing proteolytic enzymes during the degranulation process known to occur during storage, (2) by directly decreasing the pH in the storage media via lactic acid and other metabolic byproducts, or (3) by increasing oxygen consumption and decreasing oxygen availability for platelets.69-71 The studies published so far have not conclusively proven a link between the leukocyte concentration in stored platelets and storage lesions. Moroff s studies suggest the presence of a large, young, metabolically active subpopulation of platelets as the most likely source of degradation in the storage media.70The studies of Gottschall et al. ,71 on the other hand, suggest that leukocytes and platelets interact in some as yet undefined way to promote increased glycolysis and lactate production. He goes on to hypothesize that lymphocytes, when present in large numbers, compete with platelets for available oxygen and increase anaerobic glycolysis by the latter cells, thus increasing lactate production and the rate at which pH falls. One must keep in mind that most leukocyte-poor blood components are prepared in the laboratory immediately before transfusion, or depleted at bedside during the transfusion process. Obviously, under these circumstances, the storage conditions are not affected. Further studies are necessary to evaluate the hue impact during storage of the presence of leukocytes in platelet components. The development of appropriate technology for the removal of leukocytes or the preparation of leukocyte-depleted platelet components without affecting the sterility of the component at a reasonable cost will be needed if leukocytes are found to have a deleterious effect on platelet storage. A. Leukocyte Contamination in Platelets Collected by Apheresis The presence of leukocytes in platelets collected by apheresis has raised some attention because of two reasons: (1) the potential deleterious storage effect of the leukocytes, as discussed above, and (2) the concern about the increased chances of alloimmunization due to the large number of APCs that may be found.

400



As a result, several parallel studies have been published comparing the performance of several cell separators in relation to platelet yields as well as the amount of leukocytes present in the components ~ r e p a r e d . ~ *When - ~ ~ selecting a cell separator, one should not consider leukocyte “contamination” as the sole factor since, as discussed above, the impact of the leukocyte presence has not been firmly established. However, issues such as the immunogenicity of apheresis components containing large numbers of leukocytes must be considered. No prospective studies comparing the long-term effects of using components with high numbers of leukocytes (e.g., greater than 1 X lo9) vs. low numbers (e.g., 1 x 106) have been published at this time.

VI. CONCLUSION A review of the current knowledge on the characteristics and uses of leukocyte-depleted blood components indicates that:

1. 2.

3.

4.

5.

6. 7.

8.

The routine use of LDBCs is not justified. The decision to transfuse a patient with LDBCs should be made taking into consideration the potential benefits and risks, as well as long-term plans for the patient’s care. The decision process should involve the attending physician as well as the transfusion medicine specialist. It must be recognized that current filtration technology has almost achieved a “stateof-the-art” status, and that no other methods should be considered if the aim is to provide LDBCs . The process used for the selection of filtration devices should be made taking into account ease of use, cost, whether the design is for laboratory or for bedside use, assessment of the efficiency of leukocyte removal and primary component recovery rates, and the clinical objectives pursued (i.e., prevention of alloimmunization vs. prevention of febrile reactions). The timing of the filtration is of importance, as leukocytes may fragment during storage due to the senescence processes that continue even after blood collection. The effectiveness of using LDBCs for preventing alloimmunization is at best questionable with the use of present technology. Prevention of transmissible diseases through the use of LDBCs (mainly CMV infection) is currently under intense study. No conclusive proof of the effectiveness of LDBCs in disease prevention has been presented as yet. The likelihood of a potential graft-vs.-tumor effect of blood transfusions in certain leukemic processes being abrogated by the transfusion of LDBC has not been totally ruled out. Thus, in the leukemic patient, this issue merits serious study.

The use and indications of LDBCs are still mired in controversy and is not as simple as the suppliers of filtering devices try to portray. We urge those responsible for decisions that affect the management of transfusion therapy for patients to carefully consider all the elements that come into play in order to achieve the best results for each situation.


401

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