Evaluation of Three Procedures for the Preparation of Leukocyte-Poor and Leukocyte-Free Red Blood Cells for Transfusion

Abstract. Three methods of leukocyte deprivation of blood for transfusion have been evaluated. Filtration of relatively fresh packed red cells (up to 5 days old) through Erypur filters appears to be the method of choice for the preparation of leukocyte- free red cells, used to prevent the production of antileukocyte antibodies in nonimmunized, nontransfused patients undergoing repeated blood transfusions. Double dextran sedimentation plus saline washings also yields leukocyte-free red cells, although with a lower frequency, and represents an alternative to Erypur filtration, especially because of the lower cost and no need of special equipment. Imugard filtration appears to be a simple and effective way for preparing leukocyte-poor red blood cells. For all the three procedures, results are definitely better when buffy coat-free packed red cell units are processed. In a given hospital the choice of the technique(s) for the preparation of leukocyte-poor (free) red blood cells for transfusion depends on a number of factors, such as type of patients to be transfused (immunized or nonimmunized, small children or boys and adults), aim of the procedure (prevention of the febrile transfusion reaction or prevention of antileukocyte antibody production), continuous availability of materials, cost, length and easiness of the procedure; these three latter parameters are in turn related to a number of local situations and facilities.     

The Clinical Importance of Leukocyte Depletion in Regular Erythrocyte Transfusions

Abstract. Antilymphocyte, antigranulocyte and antiplatelet alloantibodies, T lymphocyte subsets, expression of HLA-DR antigens on T lymphocytes and NK cell function were determined in 11 homozygous β-thalassemic children multitransfused ab initio with Erypur-filtered leukocyte-free red cell units (group A) and in 13 similar children multitransfused with standard packed red cell units (group B). No antibodies were found in group A patients, whereas 69% of group B patients were immunized. The two groups did not differ significantly with regard to the other test results. Considered together, thalassemia patients showed a percentage of T4⁺ cells and a NK cell function that were significantly lower than those found in a reference group of 16 healthy male blood donors. Thalassemics moreover showed a higher than normal percentage of T3⁺, T4⁺ and T8⁺ cells expressing HLA-DR antigens.
The results indicate that leukocyte-free red cells should be the treatment of choice for prospective recipients of multiple transfusions, since they are capable of preventing (or delaying) the production of alloantibodies against leukocytes and platelets. From the data of the present study, it does not seem that the transfusion of leukocyte-free red cells is capable of preventing the abnormalities of some immunological tests that occur in some multitransfused patients. Further investigations, however, are needed to draw conclusions on this problem.     

Filter Columns for Preparation of Leukocyte-Poor Blood for Transfusion

Filter columns (Imugard filters) packed with cotton wool prepared from Gossypium barbadense cotton removed 95--100% of leukocytes from packed red cell suspensions. Recovery of red cells was about 95%. The efficacy was similar for ACD and heparinized blood. Blood was filtered below 10 degrees C within 30 min under pressures of less than 0.2 kg/cm2. The shape and functions of the red cells were not altered by filtration. No pyrogenic or toxic substances, and little particulate matter were eluted from the filters.     

Prevention of refractoriness and HLA-alloimmunization using filtered blood products

Depletion of leukocytes from all blood products may decrease the incidence of alloimmunization to HLA antigens present on the white cells and thus delay the onset of refractoriness to random donor platelet support. In order to test this hypothesis, 54 patients with hematologic malignancy or marrow aplasia were entered on a prospective randomized trial using cotton-wool filtration as a method of leukocyte depletion of red cell and platelet concentrates. Forty patients were considered evaluable; 20 patients received filtered products and 20 patients in the control group received standard unfiltered products. The filter was 99% efficient in removal of leukocytes (average number of WBC/platelet product, 6 X 10(6)). Platelet loss by this technique was 8%. Alloimmunization was assessed by detection of de novo formed lymphocytotoxic and platelet specific antibodies by microcytotoxicity test, Staph A protein radioimmunoassay, and solid phase red cell adherence test. In the group receiving filtered products, three of 20 (15%) patients developed lymphocytotoxic antibodies while ten of 20 (50%) patients in the control group developed cytotoxic antibodies (P = .01 by actuarial methods). Platelet antibodies were detected in seven of ten alloimmunized patients in the control group and three of three patients in the study group. Clinical evidence of refractoriness was seen in three of 20 patients in the filtered group and ten of 20 in the control group (P = .01 by actuarial methods). The cost of filtration was a fraction of the cost of a plateletpheresis product. Filtration appears to be an effective and economical method for reducing alloimmunization and clinical refractoriness to random donor platelets in patient receiving long-term transfusion support.     

Platelet transfusion reaction associated with interdonor HLA incompatibility

An HLA-compatible platelet transfusion was followed by chills, fever, and severe respiratory distress in a multitransfused patient with chronic lymphocytic leukemia. During the previous 7 days the patient had received blood products without incident, including 8 units of red blood cells (RBC), 24 units of pooled random donor platelet concentrates, and five HLA-compatible platelet pheresis products. The patient had no demonstrable RBC, HLA lymphocytotoxic, platelet or granulocyte antibodies. The platelet donor, a multiparous female, had no granulocyte or RBC antibodies but had lymphocytotoxic antibodies against HLA-A2 CREG (cross-reacting group A2, A28, A23, A24) which reacted not with lymphocytes of the patient but with lymphocytes of the donor whose RBC were transfused 24 h prior to the platelet transfusion reaction and whose HLA type is A23, A24; B44, B57. No RBC donors had HLA lymphocytotoxic, granulocyte, or platelet antibodies against the platelet donor. The patient received three subsequent platelet transfusions from the same donor after removal of the antibody-laden plasma with no adverse reaction. These data suggest an interdonor reaction caused by the presence of cells from the RBC donor received by the patient 24 h prior to the transfusion of donor lymphocytotoxic antibody to HLA-A2 CREG antigens.     

Red cell antibodies in patients with homozygous sickle cell disease: a comparison of patients in Jamaica and the United Kingdom

The transfusion history and frequency of red cell antibodies in patients with homozygous sickle cell (SS) disease have been compared in 190 subjects from the Jamaican cohort study and 37 patients attending a sickle cell clinic in Manchester, England. The proportion of patients transfused did not differ between the groups although the number of units transfused and the frequency of red cell antibodies were significantly greater in the Manchester group. Immune antibodies occurred in three Jamaicans (2.6% of those transfused) and 16 UK subjects (76% of those transfused). Multiple antibodies occurred in 10 (63%) UK subjects but in no Jamaicans. Indications for transfusion also differed between the groups, Jamaican patients typically receiving 1-2 units for acute anaemia or acute chest syndrome, whereas UK patients frequently had multiple transfusions in preoperative exchange or prophylaxis programmes. The greater red cell alloimmunization among UK patients probably reflects both the greater use of transfusion and the disparity between donor and recipient populations in the UK.     

Successful transfusion results using Rg(a+) blood in four patients with anti-Rga

4 patients with anti-Rga successfully transfused in 1979 and 1980 with Rg(a+) donor units are herein reported since the literature lacks information on transfusion results in patients with this alloantibody. The transfusions of both Rg(a+) whole blood and packed red blood cell units caused no discernible immediate or delayed transfusion reactions. Clinically, predicted hematocrit increases were attained and sustained and the laboratory findings showed no evidences of shortened survivals of donors' red blood cells.     

Prevention of non-hemolytic transfusion reactions with leucocyte-poor blood: a prospective study

Twenty-five patients with homozygous beta-thalassemia, who complained of occasional nonhemolytic transfusion reactions (NHTR), were alternately transfused eight times with normal packed red cells (PRC) and leucocyte-poor blood prepared by three methods: cotton wool filtration (CWF), buffy coat removal after dilution with saline and upright spin (BCR), and microaggregate filtration (MAF). On overall, CWF-treated RBC concentrates (CWF-RBC) contained 0.02 x 10(9) leucocytes; BCR-RBC contained 0.4 x 10(9), MAF-RBC 1.3 x 10(9) and PRC 1.7 x 10(9). The frequencies of NHTR were: CWF, 0.02; BCR, 0.04; MAF, 0.20; PRC, 0.28. Compared to PRC, both CWF and BCR significantly reduced the frequency of NHTR (p less than 0.01), whilst MAF did not (p greater than 0.1). On the other hand, in one patient CWF caused the two most severe NHTR recorded in this study, both of which were characterized by anaphylaxis.     

Preparation of Granulocyte-Poor Red Blood Cells by Microaggregate Filtration

Abstract. A simple, effective method for removing granulocytes from stored blood is described. Microaggregate filtration removes approximately 95% of the granulocytes from blood which has been stored for 2 weeks, centrifuged and filtered. The mean number of remaining leukocytes is 8 ± 3.7 × 10⁸/unit. The residual white cell population, which is composed almost entirely of lymphocytes, is substantially less than the average number of cells previously associated with febrile reactions. 45 patients were selected for the study. All had significant febrile transfusion reaction histories, and averaged one reaction for every 3.6 U of conventional red cell product transfused. Administration of 212 units of microaggregate filtered granulocyte poor red cells caused a 95% reduction in the incidence of fibrile reactions. The technique is inexpensive, easily incorporated into the routine of the clinical blood bank, and does not require 'open-system' processing. These considerations make microaggregate filtration a logical first choice method for the preparation of granulocyte-poor red blood cells.     

Liver function tests in sickle cell disease

We investigated the prevalence of positive viral hepatitis titres in sickle cell disease (SCD) and the relationship of abnormal liver function tests (LFTs) to transfusions and ferritin levels. Charts from 141 patients with SCD were reviewed and recent laboratory data on serum ferritin, hepatitis serology, units of packed red blood cells transfused and LFTs were collected. Hepatitis B core antibodies were positive in 14% of patients (12/86) and Hepatitis C viral antibody titres were positive in 16.5% (15/91). There was a relationship of positive serologies to transfusion for hepatitis C virus (HCV), but not for hepatitis B virus (HBV). Hepatitis C antibody negative (HCVAb-) patients had fewer packed red blood cells (pRBC) transfused than Hepatitis C antibody positive (HCVAb+) (6.4 vs. 20.3, P=0.08). Patients with ferritins < 500 ng/ml compared to those with > 1000 ng/ml also showed a difference in units transfused (P < 0.003). Steady state LFTs, with the exception of alkaline phosphatase, had no relationship to serum ferritin or hepatitis serologies. Males were twice as likely to have positive serology as females but more females had elevated ferritin levels. Paired analysis of LFTs in steady state and crisis failed to demonstrate deterioration during crisis. We conclude that: (1) there is a relationship of positive Hepatitis C serology, but not Hepatitis B serology, to transfusion; (2) ferritin levels correlate with transfusion number but not with LFTs; (3) in our population, LFTs in SCD are usually normal and do not increase in vaso-occlusive crises.     

Removal of Leukocytes from Whole Blood and Erythrocyte Suspensions by Filtration Through Cotton Wool

Abstract. 1,820 units of leukocyte- and platelet-poor erythrocyte suspensions were prepared by filtration through cotton wool. On the average more than 98% of the leukocytes and 90–95% of the platelets could be removed. The red cell recovery was 96%. 97% of the units given to polytransfused patients did not cause febrile reactions. Serological follow-up of future transplantation recipients indicated that immunization may be avoided by using erythrocyte suspensions of fresh, filtered blood.     

Use of leukocyte-depleted platelet concentrates for the prevention of refractoriness and primary HLA alloimmunization: a prospective, randomized trial

Compared with conventional transfusion regimes a strong reduction in HLA alloimmunization and refractoriness to platelet transfusions is obtained when both red blood cell concentrates (RBCs) and platelet concentrates (PCs) are depleted of leukocytes by filtration. Because most of the leukocyte contamination is introduced by transfusion of RBCs, filtration of RBCs appears rational, but uncertainty exists regarding the degree of leukocyte-depletion of PCs needed for the prevention of HLA alloimmunization and refractoriness. We conducted a prospective trial and randomized patients with acute leukemia to receive leukocyte-depleted PCs prepared either by centrifugation (mean leukocyte count 35 x 10(6)/PC of 6 U) or by filtration (mean leukocyte count less than 5 x 10(6)/PC of 6 U). Both groups received RBCs that were filtered after prior removal of the buffy coat. Clinical refractoriness occurred in 46% (12 of 26) of the evaluable patients that were transfused with centrifuged PCs and only in 11% (3 of 27) in the filtered group (P less than .005). De novo anti-HLA antibodies were detected in 42% (11 of 26) patients in the centrifuged group and only in 7% (2 of 27) of the patients receiving filtered PCs (P less than .004). In 8 of 11 alloimmunized patients in the centrifuged group antibodies were detected in the first 4 weeks of transfusion therapy while none of the patients in the filtered group became immunized against HLA antigens during that period. We conclude that for the prevention of HLA alloimmunization and refractoriness to platelet transfusions from random donors, both RBCs and PCs have to be leukocyte-depleted by filtration.     

Filtered Microaggregate-Free Erythrocyte Concentrates with 35-Day Shelf Life

Abstract. Packed erythrocytes which are white cell and microaggregate poor and virtually platelet free, with a long shelf life (35 days), can be prepared from a routinely used plastic pack system incorporating a cotton wool filter module. The filtered packed cells have improved physical and biochemical properties after prolonged storage, and with acceptable autologous erythrocytic survivals the system lends itself to routine clinical practice. It avoids the need for microaggregate filters at administration, at the same time reducing the immunogenicity of the transfused blood. Blood component harvesting is not compromised.     

Febrile Transfusion Reaction: What Blood Component Should Be Given Next?

Abstract: Reports of febrile, nonhemolytic transfusion reactions (FNHTR) occurring at hospitals served by a regional blood center supplying 99,658 units of blood during 1980 were analyzed to determine if leukocyte-poor red blood cells prepared by the inverted centrifu-gation technique (LP RBCs) were adequate to prevent subsequent reactions. FNHTR occurred following 0.5% of units transfused. The records of transfusions given to patients who had a FNHTR were reviewed in a subgroup of hospitals. Of 253 such patients, 161 received subsequent transfusions. 140 received red cells or LP RBCs without a reaction. The remaining 21 had a second reaction following transfusion of packed red cells. 12 of the 21 received further red cell transfusions. Only one experienced a third febrile reaction after receiving LP RBCs. We conclude that LP RBCs are adequate to prevent recurrence of FNHTR and question the need for costly saline-washed, leukocyte-poor red blood cells for this purpose.     

Red-blood-cell alloimmunization and number of red-blood-cell transfusions

Background Patients receiving red‐blood‐cells may form antibodies against the alloantigens expressed by red‐blood‐cells, with the risk of serious morbidity and the need for extensive phenotype‐matching in subsequent transfusions. The incidence of alloimmunization is considered variable for specific patient groups and for first time antibody formation. We therefore studied the cumulative incidence of the first formed alloantibody as a function of red‐blood‐cells exposure. Methods We performed a new‐user cohort among all previously non‐transfused non‐alloimmunized patients that received non‐extended matched (ABO and RhD) red‐blood‐cells transfusions from January 2005 to December 2009 in our university medical centre. Alloimmunization incidences were estimated by Kaplan–Meier survival‐analysis. Results A total of 3002 previously non‐transfused patients received 31 103 red‐blood‐cell units. A first time alloantibody forming event was experienced by 54 (1·8%) patients. The cumulative incidence of alloimmunization was 1·0% at 5 units, 2·4% at 10 units, 3·4% at 20 units and 6·5% at 40 units of red‐blood‐cells transfused. Conclusion The risk to develop a first red‐blood‐cells alloantibody increases up to the 40th transfusion and is similar for men and women. More data are needed to examine the risk after 40th transfusion.     

Phase I/II safety study of transfusion of prion‐filtered red cell concentrates in transfusion‐dependent patients

Background and Objectives Variant Creutzfeldt‐Jakob (vCJD) is a fatal transfusion transmissible prion infection. No test for vCJD in the donor population is currently available. Therefore, prion removal by filtration of red cell concentrate (RCC) is an attractive option for prevention. Materials and Methods Twenty patients were recruited with ethical permission, to receive clinically necessary transfusion containing one unit of pfRCC. Follow‐up at 24 hours, 6 weeks and 6 months was undertaken. A second pfRCC was administered to 6 patients with similar follow up. pfRCC were prepared using the CE marked P‐Capt device by the IBTS. Results In 20 transfused patients undergoing one exposure to a prion filtered unit, no attributable adverse events were noted. A subset of these (n = 6) underwent re‐exposure to a further filtered unit without incident. Conclusions This phase 1/11 clinical study provides encouraging data on safety of prion filtration which can be used to plan more extensive studies on the use of filtered blood in adults and children.     

Bedside filtration of blood products in the prevention of HLA alloimmunization--a prospective randomized study. Alloimmunisation Study Group

To test the efficacy of poststorage bedside leucodepletion of blood products in the prevention of primary HLA alloimmunization and its clinical sequelae, 172 patients with hematologic malignancy requiring intensive red blood cell and platelet support were randomized to receive either standard or filtered red blood cells and platelets. Quality control of bedside filtration was explored by sequential sampling downstream of the filter, but this did not predict the total number of leucocytes transfused. After exclusions, 123 evaluable patients were assessed every two weeks until the end of therapy. HLA antibodies developed in 21 of 56 (37.5%) nonfilter (NF) and 15 of 67 (22%) filter (F) patients (risk ratio estimate, 0.60 [95% confidence interval, 0.34 to 1.05]; P = .07). Patients with acute myeloid leukemia (AML; n = 53) had higher alloimmunization rates in both arms of the study, with a greater effect of filtration (62.5% NF and 31.0% F; P = .025). Bedside filtration did not affect the overall incidence of febrile transfusion reactions (FTRs; 37% NF and 34% F; P = .71) or of platelet refractoriness assessed in 50 patients (30% NF and 26% F), despite an association between broad HLA reactivity and both FTRs and refractoriness. However, FTRs were also seen in 28 patients without HLA antibodies. Five alloimmunized refractory patients (2 F and 3 NF) required HLA-selected platelets. This report, the first prospective study of bedside filtration, has failed to show clear clinical benefit. Methodological limitations may account in part for this failure, notably the difficulties in accurately assessing the number of leucocytes transfused.     

Red blood cell transfusions are associated with HLA class I but not H‐Y alloantibodies in children with sickle cell disease

Blood transfusions can induce alloantibodies to antigens on red blood cells (RBCs), white blood cells and platelets, with these alloantibodies affecting transfusion and transplantation. While transfusion‐related alloimmunization against RBC antigens and human leucocyte antigens (HLA) have been studied, transfusion‐related alloimmunization to minor histocompatibility antigens (mHA), such as H‐Y antigens, has not been clinically characterized. We conducted a cross‐sectional study of 114 children with sickle cell disease (SCD) and tested for antibodies to 5 H‐Y antigens and to HLA class I and class II. Few patients had H‐Y antibodies, with no significant differences in the prevalence of any H‐Y antibody observed among transfused females (7%), transfused males (6%) and never transfused females (4%). In contrast, HLA class I, but not HLA class II, antibodies were more prevalent among transfused than never transfused patients (class I: 33% vs. 13%, P = 0·046; class II: 7% vs. 8%, P = 0·67). Among transfused patients, RBC alloantibody history but not amount of transfusion exposure was associated with a high (>25%) HLA class I panel reactive antibody (Odds ratio 6·8, 95% confidence interval 2·1–22·3). These results are consistent with immunological responder and non‐responder phenotypes, wherein a subset of patients with SCD may be at higher risk for transfusion‐related alloimmunization.     

A prospective study to determine the safety of omitting the antiglobulin crossmatch from pretransfusion testing

Transfusion medicine laboratories routinely perform a series of pretransfusion serological tests including: ABO grouping, Rh typing, and investigation of the recipient's serum to detect antibodies against blood group antigens (antibody screen). As a final check, most laboratories also perform a crossmatch in which the recipient's serum is incubated with the donor's red cells followed by the addition of an antiglobulin reagent (antiglobulin crossmatch). The need for the antiglobulin crossmatch when the antibody screen is negative has been questioned because there are few antibodies that are detected by this test. Such antibodies are usually directed against low incidence antigens that are not expressed on the screening cells and in many cases the clinical importance of these antibodies is uncertain. For these reasons, we performed a prospective study in which patients requiring red cell transfusion had a group and screen performed. If the antibody screen was negative the antiglobulin crossmatch was omitted. Following the transfusion of the blood, the antiglobulin crossmatch was performed to look for any potential incompatibility. All patients were monitored both serologically and clinically. Over the 2-year interval of the study 9128 patients were entered. There were 8936 patients (97.9%) with a negative antibody screen and 26.9% (2404 patients) were transfused a total of 10,899 red cell concentrates. The antiglobulin crossmatch performed after the transfusion indicated that 168 red cell concentrates (1.5%) would have been incompatible if the antiglobulin crossmatch had been performed pretransfusion. These 168 red cell concentrates were transfused to 119 patients during 130 transfusion episodes (defined as all transfusions administered within 24 h). Of the 130 transfusion episodes, 79.2% (103/130) were false positive laboratory results. There were 27 transfusion episodes where the antiglobulin crossmatch on blood transfused was positive due to an IgG antibody. Even though these transfused red cell concentrates were designated incompatible by the antiglobulin crossmatch, none of the patients receiving this blood had clinical or serological evidence of haemolysis. We concluded that the antiglobulin phase of the crossmatch can be omitted from pretransfusion testing without putting patients at risk.     

Amount and Type of Leukocytes in ''Leukocyte-Free'' Red Cell and Platelet Concentrates

The exact number of leukocytes remaining in 'leukocyte-free' red cell and platelet concentrates cannot be measured by standard methods. We have therefore developed methods to harvest all the leukocytes from blood components. The leukocytes were then counted and identified using monoclonal antibodies. The leukocyte-free red cell concentrates were prepared by combining buffy coat removal and filtration through a Cellselect filter. The mean number of leukocytes per unit was 1.0 X 10(5). Most of the leukocytes were granulocytes and T cells. Only a few B cells or monocytes could be detected. Leukocyte-free platelets were prepared by filtering 4 units of PC through a cotton-wool (Imugard) filter. The mean number of leukocytes per PC unit was 0.4 X 10(5) of which 85-95% were T cells.