Incidence of Red Cell Alloantibody among the Transfusion Recipients of Universiti Kebangsaan Malaysia Medical Centre

Red blood cell alloimmunization is a common complication among the transfusion recipients. In Malaysia, multiple ethnicity causes genetic heterogeneity among the population which in turn can cause a wide variation of antibody. The objective of this study was to analyse the red cell alloantibody detected during the pre-transfusion testing. This was a cross-sectional study done in the blood bank of Universiti Kebangsaan Malaysia Medical Centre during the period of January–December 2010. The data was retrieved from the hospital laboratory information system. A total of 24,263 patients’ blood samples were subjected for pre-transfusion testing. Antibody screening was done using an indirect antiglobulin test method. The positive samples were further identified for antibody specificity. Antibody screening tests were positive in 184 patients out of 24,263 samples with the incidence of 0.76 %. Autoantibodies and alloantibodies were detected in 39/184 (21.2 %) and 140/184 (76.1 %) of the patients respectively. In five patients (2.7 %) the antibody specificity remained undetermined. Total 161 alloantibodies were identified. The suspected Anti-Mia alloantibody was observed most frequently (49/161, 30.4 %) followed by anti-E (30/161, 18.6 %) and anti-D (22/161, 13.7 %). Anti-E and anti-c were the most common combination of multiple alloantibodies. In view of the high incidence of suspected Anti-Mia antibodies, more efforts are needed to look into the techniques for confirmation of the Anti-Mia antibodies. Besides that, we suggested that all multiply transfused patients should be phenotyped for the Rh system and to supply Rh phenotype specific blood in order to limit alloimmunization.     

Comparison of Solid-Phase Antibody Screening Tests with Pooled Red Cells in Blood Donors

Solid-phase systems are very sensitive for detection of erythrocyte alloantibodies in serum and suitable for large scale donor screening. Serum samples of 10,008 blood donors were screened by three solid-phase tests (Capture R Ready Screen, Solidscreen II, and Solidscreen II-Donor) with pooled red cells derived from two donors. The prevalence of antierythrocyte IgG and IgM antibodies in donor sera was 0.56% (IgG antibodies: 0.42%). The test efficiency for IgG antibodies was 1.40 with Capture R Ready Screen (test sensitivity 97.6%, test specificity 99.39%), 1.78 with Solidscreen II (95.1, 99.88%), and 1.95 with Solidscreen II-Donor (92.7, 99.98%). The IgG antibody titers differed significantly between all tests including a gel matrix test: Capture R > ID-Gel > Solidscreen II > Solidscreen II-Donor. Previously characterized antibodies that were not detected after long-term follow-up by any of the three solid-phase tests had a prevalence of 0.10%. All three solid-phase tests detected the alloantibodies, which were of higher titers and considered clinically relevant in blood components. The significant difference in antibody titers between the tests was not matched by a similar variance in the detection of donors with antibodies. Even with sensitive solid-phase tests, many antibodies may not be detected after long-term follow-up.     

Intravenous immunoglobulin given to lymphoma patients with recurrent haemolytic transfusion reactions after transfusion of compatible blood

Accelerated destruction of red cells after transfusion of compatible blood has been reported in both sickle cell disease (SCD) and non-SCD patients. We report three patients with lymphoma, all of whom had recurrent haemolytic transfusion reactions after receiving compatible red cell units. The direct antiglobulin test (DAT) was negative and there were no detectable red cell alloantibodies in either pre-transfusion or post-transfusion samples. As there was no evidence of red cell antibody-mediated haemolysis and response to oral steroids, a trial of intravenous immunoglobulin (IVIg) was given. Immediate cessation of haemolysis with sustained haemoglobin level was achieved in all cases. The response to IVIg in these cases suggests that IVIg should be tried when recurrent non-antibody mediated haemolytic transfusion reactions occur in patients with a lymphoid malignancy.     

Intravenous immunoglobulin given to lymphoma patients with recurrent haemolytic transfusion reactions after transfusion of compatible blood

Accelerated destruction of red cells after transfusion of compatible blood has been reported in both sickle cell disease (SCD) and non-SCD patients. We report three patients with lymphoma, all of whom had recurrent haemolytic transfusion reactions after receiving compatible red cell units. The direct antiglobulin test (DAT) was negative and there were no detectable red cell alloantibodies in either pre-transfusion or post-transfusion samples. As there was no evidence of red cell antibody-mediated haemolysis and response to oral steroids, a trial of intravenous immunoglobulin (IVIg) was given. Immediate cessation of haemolysis with sustained haemoglobin level was achieved in all cases. The response to IVIg in these cases suggests that IVIg should be tried when recurrent non-antibody mediated haemolytic transfusion reactions occur in patients with a lymphoid malignancy.     

Antibody elutions in Thai patients with a positive direct antiglobulin test

Background

The direct antiglobulin test is performed to determine whether an anaemic patient with evidence of haemolysis has autoimmune or alloimmune haemolytic anaemia.

Materials and methods

We determined the antibody specificity of eluted IgG antibodies from patients’ blood samples with a positive direct antiglobulin test. Overall, 134 Thai patients were included in this study. EDTA blood samples were obtained from recently transfused patients, patients with unexplained anaemia and patients who had serum antibodies detected during routine pre-transfusion tests from different hospital blood banks. These complicated samples were sent to the National Blood Centre of the Thai Red Cross Society for investigation and to find compatible blood components. Each blood sample underwent a direct antiglobulin test with the gel technique using polyspecific antihuman globulin and mononospecific anti-IgG and anti-C3d. Acid eluates were prepared from the samples for which the direct antiglobulin test was positive and the specificities of the eluted antibodies were determined by the gel technique.

Results

Of the samples tested, 101 showed a positive direct antiglobulin test result (75.4%) using polyspecific antihuman globulin sera whereas only 95 samples (70.9%) were positive with anti-IgG or anti-IgG and anti-C3d. Moreover, 54 of 95 eluates (56.8%) were positive for antibody screening and tested with the reagent panel cells. Twenty-one eluates had specific alloantibodies, which were concordant with the findings in the patients’ sera and all patients had a history of blood transfusion. Additionally, 33 eluates contained pan-agglutinins. Interestingly, alloantibodies could be determined using titration studies in 5 of 26 eluates with pan-agglutinins.

Conclusion

Although the direct antiglobulin test is not routinely performed in pre-transfusion screening, this test and elution studies would be useful in patients with a history of previous transfusions, and in those for whom compatible blood cannot be found.     

Delayed hemolytic transfusion reaction due to anti-S antibody in patient with anti-Jk<Superscript>a</Superscript> autoantibody and multiple alloantibodies

We describe the case of a 60-year-old woman with a delayed hemolytic transfusion reaction (DHTR). She had a history of an ulcerative colitis, blood transfusion because of rectal bleeding, and surgical removal of descendent and sigmoid colon. At admission, laboratory data showed Hb 6.3 g/dL, reticulocytes 120×10⁹/L, serum total bilirubin 1.2 mg/dL (direct bilirubin: 0.2 mg/dL). Pretransfusion antibody screening procedures were positive. A monospecific autoanti-Jk^a and three alloantibodies (anti-c, -E, -K) were identified by immunohematologic studies. The patient received two units of crossmatch compatible concentrated red blood cells. Six days later biochemical serum values showed Hb 6.2 g/dL, LDH 975 I.U./L and total bilirubin 2.95 mg/dL (direct 0.35 mg/dL). Crossmatches with red cell suspension of transfused blood units and a post-transfusion serum were repeatedly positive. Laboratory tests showed the presence of anti-S alloantobody in the serum and eluate. Moreover, pre-transfusion serum of the patient was retrospectively retested: anti-S was not detected. These data suggested a DHTR. The present case is unusual and interesting because of the association of a rare autoanti-Jk^a, non responsible for anemia, and four alloantibodies of which anti-S involved in a DHTR.     

A prospective study of the incidence of delayed haemolytic transfusion reactions following peri-operative blood transfusion

Delayed haemolytic transfusion reactions (DHTRs) are a recognized sequel of blood transfusion. The true incidence and importance of this complication have been difficult to estimate due to the lack of any prospective studies. We have carried out such a study by testing 530 patients who were transfused during cardiac surgery. 2% of the patients had new red cell alloantibodies detectable 1 week following transfusion. Despite this finding, and the fact that at the time the study was performed pre-transfusion antibody screening of recipients was not routine practice, no DHTRs were diagnosed on clinical or laboratory criteria. These results indicate that the reported incidences, based on retrospective recognition of DHTRs, are not a serious underestimate of the frequency of the complication.     

Detection of Irregular Red Cell Antibodies: More than 3 Years of Experience with a Gel Technique and Pooled Screening Cells

Background and objectives: The purpose of this study was to evaluate more than 3 years of experience with a gel technique in combination with pooled screening cells for the detection of irregular red cell antibodies. Materials and methods: Conventional serologic methods were used for blood typing, antibody screening and cross-matching until the end of 1992. We introduced the gel technique as a routine assay for antibody detection and identification in 1993. Results: After the tube technique had been abandoned, the number of false-positive antibody screening tests was reduced by 71%, positive antibody screening tests by 33%, enzyme agglutination by 100% and rouleaux reactions and cold-reacting antibodies by more than 50%. There was a 40% increase in first-time detection of clinically relevant antibodies. We saw no increase in delayed haemolytic transfusion reactions. Conclusions: For the detection of irregular red cell antibodies, pooled screening cells in combination with a gel technique are at least as efficient and safe as a conventional tube technique with unpooled test cells.     

Provision of phenotype-matched blood units: no need for pre-transfusion antibody screening.

BACKGROUND AND OBJECTIVES: The Hong Kong government is planning to introduce an electronic smart identity card for all seven million citizens in 2003. If the smart card contains the full red cell phenotype/genotype of the individual, it may be possible to transfuse phenotype-matched blood units without pre-transfusion antibody screening. We conducted a feasibility study. DESIGN AND METHODS: Red cell phenotype was determined for 407 donor blood units and 493 patients for whom an antibody screen had been ordered. The computer program selected phenotype-matched blood from the donor stock for the patients according to actual transfusion request. For patients with a positive antibody screen, full crossmatching was carried out with the computer-selected phenotype units. The frequencies of the various red cell phenotypes in the population were calculated from Red Cross data of antigen frequencies. The probabilities of finding at least one unit of phenotype-matched blood from a 300-unit hospital stock and a 4,000-unit Red Cross stock were determined for each phenotype. Cost analysis was performed. RESULTS: Ninety-two out of 493 patients received a total of 395 blood units. The required number of phenotype-matched blood units could be found for 92 patients using a 300-unit pool and for all patients using a 4,000-unit pool. We calculated that phenotype-matched blood could be provided for more than 98% of patients without antibody screening. The total cost of the project is US$ 98 million with potential savings of US$ 14 million per year. INTERPRETATION AND CONCLUSIONS: It is feasible and cost-effective to transfuse patients with phenotype-matched blood without antibody screening using a smart card system.     

The Antigen Duclos

An antibody is described which defines a new high frequency red cell antigen, Duclos, whose expression seems to require the presence of both U and Rh fundamental antigens. Apart from the antibody maker's own red cells the only nonreactive samples were from Rhnull U impaired individuals, one example of which was shown however to yield very slight amounts of antibody through absorption-elution tests. Rhmod U weak cells gave very depressed and Rhnull U positive or Rh common U negative cells moderately depressed reactions. The proposita's red cells had an apparently normal Rh-LW condition but their U antigen was significantly decreased. No further Duclos negative individual was found by screening 8,500 blood donors in the Paris area.     

Comparison of conventional tube test with diamed gel microcolumn assay for anti-D titration

Anti-D titration is the first step in the evaluation of the RhD-sensitized patient. Traditionally, anti-D titration has been performed by tube agglutination. Gel microcolumn assay is a method that has gained widespread usage throughout the world, mainly for ABO/Rh typing, unexpected antibody screening and direct antiglobulin tests. As gel assay has become widely used as a routine method to detect red blood cell alloantibodies, a critical anti-D titer needs to be established. Seventy-nine known blood samples with anti-D (titers 1-32) were titrated simultaneously by the conventional tube test and the gel microcolumn assay. Red blood cells (R0r phenotype) were used, with a final concentration of 3% for tube and 0.8% for gel. Serial twofold dilutions (2-2.048) were prepared for each technique, followed by reading in antiglobulin phase. Anti-D titration in the gel microcolumn assay showed significantly higher titers (mean 3.4-fold) than the conventional tube test in all samples studied. Based on these data, it was not possible to determine a critical titer for anti-D titration by the gel microcolumn assay.     

Patients with red cell autoantibodies: selection of blood for transfusion

The provision of blood for transfusing patients whose sera contain red cell autoantibodies requires considerable expertise. Over 8 years, 3888 samples from 2149 patients were examined; the varying clinical presentation necessitated a flexible investigative approach. The autoantibodies showed evidence of blood group specificity in 706 patients (32.9%), usually within the Rh system for warm reacting antibodies, whereas cold antibodies were mostly anti-I. Concomitant alloantibodies were detected by noting varying reaction strengths during antibody investigation and compatibility testing, and by absorption techniques using autologous or selected allogenous red cells. Alloantibodies were found in 294 patients (13.7%); the most frequent were anti-E and anti-K. Compatibility tests were performed on SAG-M donor blood of suitable ABO group, similar Rh genotype, Kell negative and lacking antigens to any alloantibodies detected. All units of blood were incompatible by at least one technique and were issued as 'not compatible but considered suitable'. A total of 7052 units was issued for 1685 patients; no haemolytic reactions were reported. It was concluded that blood can be safely given to patients with autoantibodies, even in serologically complex cases, providing adequate investigations are carried out.     

A comparison of two automated methods for the detection and identification of red blood cell alloantibodies

Background

The aim of this study was to compare the routine use of two automated systems (OrthoAutoVue Innova, microcolumn, and Immucor Galileo, solid phase) for the screening and identification of irregular red blood cell alloantibodies in samples, analysed in our Transfusion Service during 6 months of normal activity. The study focused particularly on an evaluation of the repeatability of the screening tests, the identification of antibody specificities and the identification of antibodies in samples showing discordant results.

Materials and methods

Overall 2,229 samples from potential blood donors (A), multiply transfused patients with blood disorders (DH), potential transfusion recipients (TS), and external cases (E) were studied. The protocols were carried out according to the manufacturers recommendations.

Results

The screening tests detected 78 samples that were positive with both systems, while 18 were positive only with Immucor and 11 only with Ortho (thus, overall, Immucor detected 96 positive samples and Ortho 89 positive samples). The use of the respective identification panels enabled us to identify the antibodies in 65 samples with Immucor and in 61 samples with the Ortho system; 74 antibodies were identified with Immucor (55 with a single specificity and 19 with mixed specificities) and 68 antibodies with Ortho (51 and 17, respectively). In the remaining cases (31 samples for Immucor and 28 for Ortho), the antibody specificity was not identified. The two systems were found to be essentially similar. The Immucor system revealed a greater number of antibodies, mainly because of its greater sensitivity at detecting anti-D antibodies.

Conclusions

Both systems showed a repeatability of over 85%, demonstrating that automation of immunohaematological tests is advantageous. The specificity of the antibody was identified in 68% of the samples. Furthermore, using the two systems led to the identification of ten new antibodies (6 anti-D, 2 anti-E, 1 anti Le^a, and 1 anti-Vel), which would not have been detected had only one of the two methods been used.     

Comparison of conventional tube test with diamed gel microcolumn assay for anti‐D titration

Summary Anti‐D titration is the first step in the evaluation of the RhD‐sensitized patient. Traditionally, anti‐D titration has been performed by tube agglutination. Gel microcolumn assay is a method that has gained widespread usage throughout the world, mainly for ABO/Rh typing, unexpected antibody screening and direct antiglobulin tests. As gel assay has become widely used as a routine method to detect red blood cell alloantibodies, a critical anti‐D titer needs to be established. Seventy‐nine known blood samples with anti‐D (titers 1–32) were titrated simultaneously by the conventional tube test and the gel microcolumn assay. Red blood cells (R₀r phenotype) were used, with a final concentration of 3% for tube and 0.8% for gel. Serial twofold dilutions (2–2.048) were prepared for each technique, followed by reading in antiglobulin phase. Anti‐D titration in the gel microcolumn assay showed significantly higher titers (mean 3.4‐fold) than the conventional tube test in all samples studied. Based on these data, it was not possible to determine a critical titer for anti‐D titration by the gel microcolumn assay.     

A prospective study to determine the frequency and clinical significance of alloimmunization post-transfusion

Summary. There is debate in the literature about the frequency and importance of delayed transfusion reactions. This uncertainty could reflect the endpoints used (clinical or serological) and the type of study (typically retrospective or case series). In this report we describe a prospective investigation to determine the frequency of alloimmunization post transfusion and whether the alloantibody production is a laboratory event or has clinical relevance.
A total of 2490 patients were transfused 11218 red cell concentrates. One or more blood samples were collected within 7 d post transfusion and screened for serological evidence of alloimmunization. If any antibody was detected the patient's post-transfusion sample was screened for biochemical evidence of haemolysis and the patient's chart reviewed for documentation of clinical signs of a transfusion reaction. Post transfusion alloimmunization occurred in 2.6% of the patients (95% CI 2.1–3.6%), who had no detectable alloantibody in pre-transfusion testing. For those 86 patients (3.5%) with alloantibodies detectable pretransfusion, 8.9% (95% CI 3.6–17.4%) developed additional aloantibodies. The most common alloantibodies detected were anti-Jk^a, anti-E and anti-K. Despite the high frequency of serological evidence of delayed transfusion reactions, only one patient (005%) had clinical evidence of a delayed haemolytic transfusion reaction (95% CI 0.0–0.27%). Serological evidence of a delayed transfusion reaction is common; however, these reactions rarely cause clinical symptoms.     

Micro-Column Affinity Test and Gel Test: Comparative Study of Two Techniques for Red Cell Antibody Screening

BACKGROUND AND OBJECTIVES: We describe the results of a comparative evaluation of a gel test (ID Micro Typing) and a micro-column affinity test (MCAT, Cellbind Screen) for red cell antibody screening and identification under routine conditions. MATERIALS AND METHODS: 3,000 serum samples of patients from the Mannheim University Hospital were tested in parallel by means of the gel test and the MCAT, using the low-ionic-strength-saline indirect antiglobulin test and the protein G affinity technique, respectively. Test cells used were the same in all tests. In addition, we performed titration studies with all detected antibodies as well as with 59 frozen sera containing antibodies of known specificity. RESULTS: A total of 154 antibodies (5.1%) were detected, 149 by gel test and 147 by MCAT. The overall sensitivity and specificity of the gel test was 96.8 and 96.5% and of the MCAT 95.5 and 97.2%. No significant differences between the gel test and MCAT were found when the titer scores of all 213 (fresh and frozen) antibodies were used to check the results. The mean scores for the gel test and the MCAT were 26.8 and 28.5, respectively. For anti-Fy(a) and anti-Kell, a significantly higher titration score could be obtained in the MCAT, whereas anti-Lu(a) showed a significantly higher score with the gel test. CONCLUSION: For the screening of unexpected red blood cell antibodies, the MCAT is as sensitive as the gel indirect antiglobulin test. The sensitivity and specificity of the two systems are more or less the same although it seems that IgM antibodies are better detected by the gel test.     

Red Blood Cell Antibody Screening with Groupamatic System

Abstract. The trypsin-polybren-citrate (TPC) technique is based on Lalezari's method and has been developed on the Groupamatic equipment to allow the screening of irregular alloantibodies which are not detectable on this machine by the present routine techniques. TPC screening has two main advantages: it gives more reliable results for Rh, Kell, Lewis and P antibodies than bromelin-methyl-cellulose, and it permits the screening of Duffy and Kidd antibodies. However, although the TPC technique contributes to an improved quality of the automated screening of blood donor samples, it should not be used as the only method when recipient samples are concerned.     

In vitro determination of red cell alloantibody significance using an assay of monocyte-macrophage interaction with sensitized erythrocytes

One hundred and forty-eight red cell alloantibodies, of specificities generally considered to be of clinical significance, were studied in vitro for their ability to induce phagocytosis of sensitized red cells by allogeneic mononuclear phagocytes. Results indicate that only 53% of the alloantibodies studied mediated significant phagocytosis in vitro. The percentages for each blood group system were as follows: Kell, 73%; Jka, 32%; Jkb, 67%; D, 75%; E, 60%; Fya, 62%; Yta, 25%; Ge, 22%; and Vel, 25%. Significant phagocytosis was independent of the strength of the indirect antiglobulin test. The percentage of anti-Jka and anti-Fya mediating significant phagocytosis was increased when fresh complement was added during the sensitization procedure and/or red cells homozygous for the antigen in question were used. The in vivo clinical significance or lack of significance was documented for nine alloantibodies; five caused haemolysis and four did not. Those causing in vivo haemolysis mediated in vitro phagocytosis by monocyte-macrophages whereas the antibodies that did not result in haemolysis showed no increased in vitro phagocytosis. Autologous monocytes were more reliable than random allogeneic monocytes in that phagocytosis was increased over that obtained using allogeneic monocyte-macrophages with two of four alloantibodies having documented clinical significance. The use of target red cells homozygous for the antigen in question, the addition of fresh complement in the antibody sensitization procedure, and use of autologous and allogeneic monocyte-macrophages appear necessary for optimal results. Since 47% of those alloantibodies generally considered to be clinically significant failed to mediate phagocytosis in vitro, the monocyte-macrophage assay should not be considered a predictive assay of a given alloantibody's in vivo significance or lack of significance until more extensive correlation of these assays with in vivo red blood cell survival is obtained.     

Prevalence and specificity of red-blood-cell antibodies in a multiethnic South and East Asian patient population and influence of using novel MUT+Mur+ kodecytes on its detection

Background and Objectives Appropriate screening for irregular red‐cell antibodies is essential for ensuring transfusion compatibility and for antenatal management of mothers at risk of haemolytic disease of the foetus and newborn. Screening for all relevant antibodies is, however, limited by screening cells that do not express antigens present in the patient and donor population. Technology to artificially incorporate antigens into red cells is currently available and may be an option for customizing screening cells. Materials and Methods We sought to identify retrospectively the changing patterns of alloantibody prevalence in our multiethnic population on change of screening cells. Antibody screening records of 143 501 patients tested from 2004 to 2010 were retrieved and divided into two groups: period‐1 (2004–2008) and period‐2 (2009–2010). During period‐1, standard screening cells were used while in period‐2, MUT+Mur+ KODE^? transformed red cells (kodecytes) were used. Results Four per cent of samples tested during period‐2 were positive on antibody screening compared to 3·2% in period‐1. Specific antibodies, excluding anti‐D, were identified in 1·66% and 1·52% of patients in period‐2 and ‐1, respectively. When confined to antibodies of clinical significance only, period‐2 showed higher alloantibody prevalence of 1·16% as compared to 0·66% in period‐1. Antibodies to glycophorin variants of MNS (vMNS) were more commonly detected while antibodies to Lewis antigens declined during period‐2. Conclusion Antibodies to vMNS antigens are common in South and East Asian populations and are often missed when using standard screening cells. Use of specifically engineered screening cells to express red‐cell antigens artificially is beneficial in detecting the diverse alloantibodies present in our population.