The efficacy of performing red cell elution studies in the pretransfusion testing of patients with positive direct antiglobulin tests

In order to evaluate the efficacy of performing red cell elutions in pretransfusion testing, the serologic records of 638 patients with positive direct antiglobulin tests (DAT) were reviewed. These patients were identified by routine antibody screening procedures that included an autologous control. DAT results on the red cells of these patients showed 279 with IgG and C3d sensitization, 319 with IgG alone, and 40 with C3d sensitization alone. Of 638 patients' red cell eluates, 401 demonstrated no reactivity, 154 demonstrated panagglutination, and 60 demonstrated passively acquired anti-A,B. Only 23 of 638 patients had alloantibody sensitization of their red cells. Of the 23, 19 had serum antibody corresponding to the specificity of antibody detected in the eluate. Thus, only four of 638 (0.6%) eluates gave results unavailable by serum testing alone. This study indicates that routine eluate investigation provides little useful information in assuring compatibility. Serum antibody testing and careful review of the clinical and transfusion history constitute appropriate pretransfusion testing in patients with positive direct antiglobulin tests. Eluate testing should be restricted to cases in which immune hemolysis is suspected clinically.     

Hypergammaglobulinemia can be associated with a positive direct antiglobulin test, a nonreactive eluate, and no evidence of hemolysis

To determine the cause of a positive direct antiglobulin test (DAT), blood banks routinely perform serologic tests on eluates prepared from DAT-positive red cells. Negative eluates traditionally have been suspected to be associated with drug reactions. This report confirms that the most frequent cause of a positive DAT and a nonreactive eluate is hypergammaglobulinemia. The results of 74 patient samples with positive DATs were analyzed retrospectively. Eluates prepared from the red cells of 54 patients (72.9%) reacted; eluates from 20 patients (27.1%) did not react. This latter group had identical serologic and clinical findings, suggesting that they made up a homogeneous group. In particular, the patients had a positive DAT, a negative indirect antiglobulin test, and a negative eluate; an increased serum concentration of IgG; and no evidence of hemolysis. In a subsequent study, DATs were performed prospectively on red cells from 44 consecutive patients with elevated serum IgG levels. The serum IgG concentration was highest in the three patients whose red cells had a positive DAT. The DAT also became positive in two patients treated with high-dose intravenous gammaglobulin (IV IgG). These studies indicate that a negative eluate from red cells with a positive DAT, a common serologic finding, is often caused by hypergammaglobulinemia. The authors postulate that IgG binds nonspecifically to the red cells because of the hypergammaglobulinemia.     

An autoanti-Kpb immunoglobulin M that simulates antigen suppression

BACKGROUND: Patients may present with an antibody against a blood group antigen, a negative direct antiglobulin test (DAT), and a null phenotype. Typically, this represents an alloantibody in a null individual. However, on occasion, the antibody disappears coincident with conversion to a positive red blood cell (RBC) phenotype. This has been called antigen loss, antigen suppression, or weakened antigenicity. Herein, a unique serologic profile that mimics this pattern, when in fact antigen loss did not occur, is described.
STUDY DESIGN AND METHODS: RBCs and serum were analyzed using a gel microtyping system and flow cytometry. Genomic DNA was amplified by polymerase chain reaction and sequenced.
RESULTS: Initially, an anti-Kp^b was detected in MTS gel, RBCs typed K−k−Kp(b−), and the DAT was negative for immunoglobulin G (IgG). Later, the anti-Kp^b disappeared and RBCs phenotyped as K−k+Kp(b+). Analysis of initial specimens by flow cytometry identified an immunoglobulin M (IgM) anti-Kp^b with a positive IgM-specific DAT; eluates contained an anti-Kp^b at immediate spin. Supporting the presence of the Kell glycoprotein, RBCs agglutinated with anti-Js^b. Sequencing showed homozygosity for Kp^b with no mutations surrounding the Kp^b polymorphism.
CONCLUSION: In antigen loss, antibody masking is excluded by a negative DAT. However, because typical DAT reagent does not detect IgM, such reasoning was inaccurate in the current case. In addition, an anti-Kp^b resulted in RBCs typing k−, even though no anti-k was detected. Overall, this case suggests that an IgM may mask adjacent epitopes and illustrates the potential to mistake a non-IgG autoantibody as antigen loss.     

Serologic findings in autoimmune hemolytic anemia associated with immunoglobulin M warm autoantibodies

BACKGROUND: Autoimmune hemolytic anemia (AIHA) associated with immunoglobulin M (IgM) warm autoantibodies is unusual but often severe, with more fatalities than other types of AIHA. Diagnosing this type of AIHA can be difficult because routine serologic data are not always informative.
STUDY DESIGN AND METHODS: Forty-nine cases of IgM warm AIHA in 25 years were studied by serologic methods.
RESULTS: Routine direct antiglobulin tests (DATs) detected red blood cell (RBC)-bound C3 in 90 percent of cases (65% had C3 but no immunoglobulin G [IgG] on their RBCs) and IgG in 24 percent. IgM was detected on 29 of 47 (62%) patients' RBCs; RBC-bound IgM was detected in 14 of 47 cases by a tube DAT method and in an additional 15 of 21 (71%) cases using fluorescein isothiocyanate anti-IgM and flow cytometry. Eighty-one percent of eluates from patients' RBCs reacted. Warm autoagglutinins were present in 94 percent of serum samples; untreated and enzyme-treated RBCs were hemolyzed at 37°C by 13 and 65 percent of serum samples, respectively. Most agglutinins were optimally reactive at 30 to 37°C. Patients' RBCs were spontaneously agglutinated in 78 percent of cases; washing with 37°C saline or treating RBCs with dithiothreitol resolved this problem. Clear specificity of autoantibody was defined in 35 percent of serum samples.
CONCLUSION: IgM warm AIHA can be confused with cold agglutinin syndrome and "mixed/combined"-type AIHA; a serologic workup by a specialist reference laboratory can help with the diagnosis.     

Is it necessary to add the eluate testing to the direct antiglobulin test to improve the detection of maternal erythrocyte alloantibodies?

BACKGROUNDThe screening of umbilical cord blood samples by the Direct Antiglobulin Test (DAT) is the reference tool for the identification of maternal erythrocyte alloantibodies present in erythrocytes; however, its diagnostic usefulness is controversial.OBJECTIVETo evaluate the diagnostic validity, safety, and efficiency of the eluate testing (detection of antibody in erythrocyte eluates by the Indirect Antiglobulin Test/IAT) in cord blood samples for detection of maternal erythrocyte alloantibodies in comparison with the DAT.MATERIALS AND METHODSEvaluation study of diagnostic tests. DAT and eluate testing were performed in 306 cord blood samples from neonates born to mothers admitted at Clínica Somer in Rionegro, Colombia; then, antibodies present in the eluates were identified with erythrocyte panels. Percentage of positive results by DAT and IAT were compared with the Pearson's chi-square test and the agreement between both assays with the Cohen's kappa coefficient. The diagnostic sensitivity, specificity, safety, and efficiency of the eluate testing were calculated, taking into account the use of DAT as an imperfect reference test.RESULTSThe DAT detected alloantibodies in 6.21% of samples and the eluate testing in 14.1 %; the strength of agreement between both tests was moderate (k = 0.56) due to 25 discrepancies. The eluate testing showed sensitivity and specificity of 98.83 % and 92.31 % respectively, and a negative predictive value of 99.9 %. The diagnostic efficiency was sufficient for detection of maternal erythrocyte alloantibodies. The antibodies identified in the erythrocyte eluates were anti-A or anti-B (79.5 %), anti-D (136%), anti-C (2,3%), and anti-Fya (2,3%).CONCLUSIONThe eluate testing in cord blood samples is a valid, safe, and efficient test for the diagnosis of maternal erythrocyte alloantibodies.     

Posttransplant immune-mediated hemolysis

BACKGROUND: Immune-mediated hemolysis is a well-recognized complication of transplantation, but few reports have drawn together the different mechanisms that could be involved. STUDY DESIGN AND METHODS: The clinical and laboratory records of three patients are used to illustrate different types and complexities of posttransplant immune-mediated RBC destruction. RESULTS: Patient 1 received bone marrow from an HLA-matched, unrelated donor. At 7 months after transplant, his Hb level fell to 50 g per L. The serum contained warm autoantibodies, and the DAT was strongly positive for IgG, IgM, and C3d; an eluate yielded IgG and IgM autoantibodies. Autoimmune hemolytic anemia was diagnosed. Patient 2, blood group A, experienced severe hemolysis 14 days after receiving a lung from a group O donor. The DAT was positive for IgG. Serum and RBC eluate contained anti-A produced by immunocompetent B cells in the transplanted lung-this was the passenger lymphocyte syndrome. Patient 3 experienced posttransplant hemolysis caused by two different immune mechanisms. Originally group A, D- with anti-C, -D, -E, she received a peripheral blood progenitor cell (PBPC) transplant from her HLA-identical group A, D+ son. Six months later, chimerism was evident; the remaining recipient marrow was still producing antibodies that destroyed D+ RBCs made by the transplant. Later, autoimmune hemolytic anemia also developed; the DAT became positive for IgG, and warm autoantibodies were eluted from D- RBCs. CONCLUSION: An understanding of the causes and circumstances under which posttransplant immune hemolysis arises is required for proper management. As more patients become long-term survivors of unrelated bone marrow and/or PBPC transplants, chimerism and complex serologic problems will become more common.     

False-positive eluate reactivity due to the low-ionic wash solution used with commercial acid-elution kits

BACKGROUND: During the use of commercial red cell (RBC) acid-elution kits for adsorption and elution (adsorption/elution) studies with anti- D, unexpected reactive eluates (anti-D) were obtained from D- RBCs. Such results were not obtained with a parallel xylene method or, historically, with heat and ether methods. STUDY DESIGN AND METHODS: Single-donor and commercial polyclonal anti-D samples were incubated with D+ and D- RBCs. Acid eluates were prepared by the manufacturers' directions. Variations in the wash step of the eluate preparation included the use of commercial kit wash solution versus phosphate- buffered saline versus solutions of various ionic strengths. RESULTS: Anti-D was eluted from 20 of 22 samples of D- RBCs after incubation with commercial polyclonal anti-D (titer 512) and from 2 of 3 samples of D- RBCs incubated with single-donor anti-D (titer 256). With a low- titer (16) single-donor anti-D, 0 of 4 eluates from D- RBCs reacted. When phosphate-buffered saline was substituted for the commercial wash solution, 0 of 11 D- RBC eluates reacted, as compared with 9 of 11 D- RBCs that yielded positive 1+(-)2+ eluates with the commercial wash solution. If the recommended initial phosphate-buffered saline wash was omitted before the use of the commercial wash solution, the eluate reactivity was stronger (2+(-)3+). When low-ionic-strength (< 0.03 M) saline was substituted, anti-D was eluted from D- RBCs. All last washes were nonreactive. Antiglobulin tests on all adsorbing D- were negative. CONCLUSION: Commercial wash solutions used for acid elution are at low ionic strength and commonly yield superior eluates, but in the presence of high-titer antibodies, false-positive eluates can result. It is our belief that the low-ionic-strength wash solution caused aggregation of IgG and nonspecific attachment of IgG on RBCs. Aggregates will contain IgG serum antibodies in proportion to the titer of the antibody. It is this nonspecifically bound antibody that is eluted from antigen- negative RBCs.     

Hemolytic anemia after kidney transplantation: a prospective analysis

BACKGROUND: Hemolysis may occur in 9% to 40% of patients after solid organ transplantation and be caused by the passenger lymphocyte syndrome (PLS). STUDY DESIGN AND METHODS: We have prospectively examined 217 kidney transplant recipients before (Day ?1) and after (up to Days +10, +20, and +30) surgery. ABO‐identical transplant was performed in 180 (82.9%) patients, while 37 (17.1%) individuals received ABO‐compatible nonidentical grafts. Direct antiglobulin tests (DATs) were performed by tube technique (polyspecific anti‐human globulin [IgG + C3d]), positive DAT samples were further tested by gel agglutination (monospecific anti‐IgG, ‐IgM, ‐IgA, or ‐C3), and eluates were prepared from DAT‐positive red blood cells (RBCs) by the dichloromethane elution test. RESULTS: We observed that 34 of 217 (15.7%) patients developed a positive DAT up to Day +30. The percentage of patients with positive DATs was significantly higher in those having ABO‐compatible nonidentical transplants compared to those that received ABO‐identical grafts (10/37 = 27.0% vs. 24/180 = 13.3%; p = 0.037). Specific RBC antibodies (anti‐A or anti‐B) were found in only 5 of 37 (13.5%) patients having ABO‐compatible nonidentical transplants who presented with clinical hemolysis. We found only three reactive eluates from 24 patients with positive DATs who received ABO‐identical transplants but had no hemolysis. CONCLUSIONS: Our data collected prospectively demonstrated that: 1) positive DATs occurred in 15.7% of all patients up to Day +30 after a kidney transplant; 2) the DAT positivity occurred up to Day +10 in 9.7% of all transplanted patients; 3) the majority of the transplant recipients with a positive DAT had a nonreactive RBC eluate; and 4) PLS was the cause of a positive DAT in 13.5% of patients submitted to ABO‐compatible nonidentical kidney transplants.     

Detection of multiple passively acquired alloantibodies following infusions of IV Rh immune globulin

BACKGROUND: Passively acquired blood group alloantibodies are detected regularly after infusions of IV Rh immune globulin (RhIG) for the treatment of immune thrombocytopenic purpura (ITP) in D+ patients. STUDY DESIGN AND METHODS: Blood samples from 16 D+ patients with ITP were tested after treatment with IV RhIG for the presence of passively acquired alloantibodies. Similar studies were conducted for three D- patients after injections of IM RhIG for Rh immunoprophyl-axis. Four production lots of IV RhIG and 2 lots of IM RhIG were tested for the presence of alloantibodies. RESULTS: All 16 D+ patients with ITP developed a positive DAT, as well as positive antibody detection test results, after infusions of IV RhIG. All postinfusion plasma samples contained anti-D, as well as one or more additional antibodies, usually anti-C, -E, -G, -V, or -Fy(a). Eluates from patients' RBCs with positive DAT results contained multiple passively acquired alloantibodies. Multiple alloantibodies were detected in samples of different production lots of IV RhIG or IM RhIG. No acute transfusion reactions were observed in five D+ patients with ITP who had been treated with IV RhIG and had been given serologically incompatible D+ RBCs. After injections of IM RhIG, the only passively acquired alloantibody detected was anti-D. CONCLUSION: Plasma samples from D+ patients with ITP treated with IV RhIG regularly contained anti-D and multiple other passively acquired Rh, Duffy, or Kidd system alloantibodies. Postinfusion RBC samples all had positive DAT results with eluates containing anti-D and multiple other Rh, Duffy, or Kidd system antibodies. The consistent detection of multiple passively acquired alloantibodies after IV RhIG, in contrast to the detection of anti-D only after IM RhIG, reflects the immediate effect of the entire (bolus) dose of RhIG by the IV route, the dose for treating ITP that is approximately 10 times the dose for Rh immunoprophylaxis, and the expected serologic incompatibility with recipients' D+ RBCs.     

The protein composition of red cell eluates

BACKGROUND: The protein composition of red cell (RBC) eluates has not been extensively studied. The purpose of this study was to determine the IgG content and protein composition of RBC eluates prepared by the acid and xylene elution methods. STUDY DESIGN AND METHODS: Six samples of group O R1R1 RBCs sensitized with anti-D in vitro, six nonsensitized samples of the same group O R1R1 RBCs, and six samples from patients with warm autoimmune hemolytic anemia (WAIHA) were studied. The eluate protein composition was determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, and immunoglobulin concentrations were estimated by an immunoblot technique using horseradish peroxidase- conjugated anti-IgG and 3,3' diaminobenzidine. RESULTS: The protein concentrations of the xylene eluates were significantly greater than those of the acid eluates (37.3 +/− 10.7 and 3.0 +/− 0.4 [SD], respectively; p < 0.005). In all samples, the proportion of IgG was less than 0.13 percent of the total protein content. The acid eluates of sensitized RBCs contained more IgG than the xylene eluates. The antibody titers of eluates from WAIHA RBCs were significantly lower than eluates of in vitro sensitized RBCs (p < 0.005). The estimated molecular weights of the Coomassie blue-stainable protein bands from xylene eluates were 97, 78, 63, 45, 31, 23, and 16 kDa, and those of bands from acid eluates were 97, 78, and 55 kDa. No periodic acid- Schiff reagent-stainable bands were detected. CONCLUSION: These data indicate that IgG represented only a small fraction of the proteins in the eluates and that the protein composition varies with the elution procedure.     

凝胶微柱法与试管法检测放散液中抗体的比较

目的比较凝胶微柱法和试管法检测红细胞释放液抗体的能力。方法将30例外周血和41例脐血红细胞用两种方法测定直接抗人球蛋白试验(DAT)并进行酸放散,将放散液与三种谱细胞、A1、B细胞反应检测抗体的特异性。71例中12例作为阴性对照样本(DAT均为阴性),其中6例为健康献血者外周血,6例为不发生新生儿溶血病(Heamolytic disease of thenewborn,HDN)的婴儿脐血样本。结果24例外周血放散液中有10例两种方法均阳性,12例均为阴性,2例自身免疫性溶血性贫血(AIHA)患者样本凝胶微柱法阳性而试管凝集法阴性,6例健康捐献者的样本DAT试验阴性且两种方法检测放散液均为阴性;41例脐血样本中33例放散液两种方法均反应,2例放散液在试管法中与A1和B细胞反应而同样的放散液做凝胶微柱法时一个和A1细胞反应,另一个只与B细胞反应。结论两种方法检测放散液结果基本是一致的,检测AIHA患者红细胞释放液抗体时用微柱法比较好,检测脐血细胞释放的同种血凝素时试管凝集法更好—些。     

Severe hemolytic anemia due to multiple red cell alloantibodies after an ABO-incompatible allogeneic bone marrow transplant

BACKGROUND: A patient who received an ABO-incompatible allogeneic bone marrow transplant experienced three episodes of immune hemolytic anemia due to multiple red cell (RBC) alloantibodies. CASE REPORT: A 41-year- old man with chronic myeloid leukemia received an ABO-incompatible bone marrow graft from his HLA-identical brother. Selective removal of RBCs from donor marrow before transfusion was performed by centrifugation using a continuous-flow blood cell separator. The patient was given group O Rh-positive RBCs and group A Rh-positive platelets. Prophylaxis for graft-versus-host disease consisted of cyclosporine and methotrexate. The patient experienced three hemolytic episodes, on Days 21, 35, and 160 which were due to different RBC alloantibodies (anti-K, anti-Jk(b), anti-M, IgG anti-A) produced by host lymphocytes surviving the conditioning regimen. RESULTS: The patient was group O, Jk(b-), and the marrow donor was group A, Jk(b+). After the first hemolytic episode (Day 21), immunohematologic studies showed group O RBCs and a positive direct antiglobulin test (IgG+, C3d+). Antibody screening test and eluate studies detected anti-M, anti-Jk(b), and anti-K. During the second hemolytic episode (Day 35), the patient's blood group showed a mixed population of group A and group O RBCs. The direct antiglobulin test was positive (IgG+, C3d+). Anti-M, anti-Jk(b), and IgG anti-A were detected in the serum. Eluates made from the recipient's RBCs showed the same specificity as serum antibodies. During the third hemolytic episode (Day 160), a mixture of group O and group A RBCs was still present, the direct antiglobulin test was positive (IgG+, C3d-), and anti-Jk(b) and IgG anti-A were observed in the serum and in an eluate made from the patient's RBCs. CONCLUSION: This is the first reported case of severe immune hemolytic anemia due to multiple RBC alloantibodies after an allogeneic bone marrow transplant. The time of appearance and the specificity of the antibodies strongly suggest that they were produced by residual recipient lymphoid cells.     

A red blood cell autoantibody with mimicking anti-E specificity

BACKGROUND: Uncommonly, antibodies that appear to exhibit antigenic specificity on red blood cell (RBC) panels fail to maintain specificity following alloadsorption (i.e., they mimic antigenic specificity). Understanding both the pitfalls and the proper pathways to establish the diagnosis and to interpret the clinical significance of these mimicking antibodies is important for patient management. CASE REPORT: A 68-year-old woman was admitted with dyspnea, anemia, bilateral pulmonary emboli, and metastatic ovarian cancer. Blood bank evaluation identified anti-E reactivity in the patient's plasma sample and a positive direct antiglobulin test (DAT). RESULTS: The DAT was positive for immunoglobulin G and negative for C3b. An eluate of the RBCs showed E-antigen specificity on a RBC antibody panel. Repeat serologic testing with RBC antibody panels with adsorbed patient plasma showed removal of apparent anti-E reactivity with either E-antigen-positive or E-antigen-negative RBC stroma. CONCLUSION: A mimicking autoantibody with apparent E-antigen specificity was identified in the plasma sample of a woman with newly diagnosed ovarian cancer. Despite their relative low frequency, mimicking antibodies, whether auto- or alloantibodies, may interfere with the timely issuance of compatible blood products and may confuse laboratory and clinical staff. Determining the clinical significance of the antibody, by taking into account the RBC phenotype of the patient and the antigen prevalence in the general population, guides the extent of workup required to best utilize resources while assuring patient safety.     

Immune hemolytic anemia due to cimetidine: the first example of a cimetidine antibody

BACKGROUND: Although there have been a few reports of immune hemolytic anemia (IHA) thought to be due to cimetidine, none of them provided proof (e.g., serologic detection of anti‐cimetidine and/or repeat of IHA upon drug rechallenge). One report used cimetidine as an example of how temporal associations of drug administration and hemolytic anemia are not proof of a cause‐effect relationship. STUDY DESIGN AND METHODS: A 63‐year‐old cancer patient developed IHA on two occasions after receiving cimetidine (with and without chemotherapy). Serologic methods included testing cimetidine‐treated red blood cells (RBCs) as well as testing untreated RBCs in the presence of cimetidine. RESULTS: The patient's direct antiglobulin test was positive (C3 only) and a serum antibody to cimetidine was detected by both testing methods. An eluate from the patient's RBCs was nonreactive. Cimetidine‐treated RBCs were optimally prepared at room temperature and needed to be tested on the day of preparation. CONCLUSIONS: This is the first reported case of IHA due to a cimetidine antibody where a drug‐dependent antibody was demonstrated. The patient had IHA after receiving cimetidine on two separate occasions.     

The evaluation of a positive direct antiglobulin test in pretransfusion testing

The results of serologic studies on 879 blood samples with a positive direct antiglobulin test (DAT) are presented. All blood samples were from patients who were either anemic, for reasons other than blood loss, recently transfused, or had serum antibodies detected during routine pretransfusion tests. Blood samples from only 81 of the patients included in this study had serologically reactive eluates (64 autoantibodies, three antibodies to penicillin and cephalothin treated red blood cells, three passively acquired anti-A antibodies, and 11 transfusion-induced alloantibodies). The eluted antibodies were also detected in the serum by routine pretransfusion tests in 13 of the patients whose red blood cells eluted autoantibodies, and in five of the patients whose red blood cells eluted transfusion-induced alloantibodies. All but one of the 11 transfusion-induced alloantibodies were detected within 14 days posttransfusion. Based on these findings, a cost-effective and safe approach to the management of blood samples with a positive DAT would be to restrict the preparation and testing of eluates to those samples from recently transfused patients. It is the contention of the authors that the incorporation of the DAT in pretransfusion testing should primarily serve to detect alloantibody formation before such antibodies are evident in the serum, and should not be used to screen patients for unsuspected autoimmune hemolytic anemia. Furthermore, the authors question the necessity for blood banks to routinely perform an autocontrol on all blood samples from prospective transfusion recipients.     

Tazobactam-induced haemolytic anaemia, possibly caused by non-immunological adsorption of IgG onto patient's red cells

A patient with pneumonia was treated with Tazocin (piperacillin/tazobactam). However, the expected haemoglobin (Hb) increment after transfusion was not achieved. Plasma bilirubin and lactate dehydrogenase were raised. The direct antiglobulin test (DAT) was positive (4+) for immunoglobulin G (IgG) only, but no RBC antibodies were demonstrable in the plasma or an eluate from the patient's RBCs. Drug-induced haemolysis was suspected. After discontinuing Tazocin administration, Hb and bilirubin levels returned to expected values. The patient's plasma gave a positive (3+) indirect antiglobulin reaction only with RBCs pretreated with tazobactam. However, random patient plasmas also gave weak (+/- to 1+) reactions, indicating non-immunological adsorption of IgG onto RBCs rather than specific anti-tazobactam antibodies. Subsequently, plasma samples with varying IgG levels (0.8-89.7 g L(-1)) were tested against RBCs pretreated with tazobactam. The amount of plasma IgG non-immunologically adsorbed onto the drug-coated RBCs was found to correlate directly with the plasma IgG level. The patient had a high plasma IgG level (41.6 g L(-1)) which explains why the antiglobulin test was stronger with the patient's plasma than with random plasma samples. Previous reports (Garratty & Arndt, (1998) British Journal of Haematology, 100, 777-783; Arndt & Garratty (2000) Transfusion, 40, 29S) suggested that non-immunological coating of RBCs with IgG may affect RBC survival; our results would support that suggestion. This is the first reported case of haemolytic anaemia associated with tazobactam.     

Clinical application of a flow cytometric direct antiglobulin test

BACKGROUND: The clinical application of flow cytometric direct antiglobulin test (FC-DAT) has rarely been evaluated for patients with various diseases including immune and nonimmune hemolytic anemia.
STUDY DESIGN AND METHODS: Blood samples from 380 patients with a variety of diseases were studied using the tube direct DAT and FC-DAT. The results of tube DAT and FC-DAT were compared. The predictive values of DAT for hemolysis were evaluated.
RESULTS: Of 57 patients with autoimmune hemolytic anemia (AIHA), 6 of the 17 with a negative tube DAT (immunoglobulin G [IgG]) had a positive FC-DAT (IgG) and 23 of the 36 patients with a negative tube DAT (complement 3d [C3d]) had a positive FC-DAT (C3d). In 57 patients with AIHA, the incidence of positive results of FC-DAT (IgG) and tube DAT (IgG) were similar (42 positive vs. 40 positive); but in 323 patients without AIHA, the incidence of positive FC-DATs (IgG) was higher than that of tube DAT (IgG; 47 positive vs. 9 positive). The higher incidence of positive FC-DAT (C3d) than that of tube DAT (C3d) was seen in patients with AIHA (42 positive vs. 21 positive) as well as in patients without AIHA (61 positive vs. 5 positive). Both DAT (IgG) and DAT (C3d) positive has highest positive predictive value for hemolysis, followed by DAT (IgG) alone positive and DAT (C3d) alone positive.
CONCLUSIONS: FC-DAT is a complementary test for diagnosing AIHA. There is a synergistic effect of the red blood cell–bound IgG and complement in predicting hemolysis.     

Hyperhemolysis syndrome in anemia of chronic disease

BACKGROUND: Occasional cases of delayed hemolytic transfusion reaction (DHTR) demonstrate severe and persistent hemolysis and are referred to as hyperhemolysis syndrome. This syndrome usually occurs in patients with sickle cell disease and possibly thalassemia who receive multiple transfusions. There are few such clinical reports in patients without hemoglobinopathies. CASE REPORT: A 67-year-old woman with anemia and a history of four previous transfusions was admitted with shortness of breath and a hematocrit (Hct) level of 27 percent. The patient was group O with a negative antibody screen. She received 1 unit of electronically cross-matched red blood cells (RBCs) and was discharged. Thirteen days later she returned to hospital with weakness and a Hct level of 23 percent. The antibody screen now demonstrated anti-K alloantibody. The direct antigloblulin test (DAT) was positive with both anti-immunoglobulin G and anti-complement (C3). Anti-K was recovered in the eluate. The previously transfused RBC unit was positive for presence of the K antigen. The patient's RBCs were negative for the presence of K antigen. Other laboratory data confirmed ongoing hemolysis, and a diagnosis of DHTR was made. She continued to display findings of active hemolysis for 9 more weeks requiring 19 units of RBCs. Thirty-four days after the original transfusion, her DAT remained positive and both the plasma sample and a RBC eluate demonstrated anti-K. CONCLUSION: The delayed hemolytic transfusion reaction with hyperhemolysis can occur among patients without hemoglobinopathies.     

Antibodies to co-trimoxazole (trimethoprim and/or sulfamethoxazole) related to the presence of the drug in a commercial low-ionic-strength solution

BACKGROUND: Drug‐dependent antibodies have been associated with approximately 10% of acquired immune hemolytic anemia cases. These antibodies are a rare cause of interference in pretransfusion red blood cell (RBC) serologic testing. The aim of this work was to report three cases of subjects developing antibodies against co‐trimoxazole, a combination of trimethoprim (TMP) and sulfamethoxazole (SMX). CASE REPORT AND METHODS: Blood samples of donor/patients were referred to our laboratory for the exploration of a positive antibody detection test. There was no recent history of drug taking. Antibody identification was performed by gel test using an indirect antiglobulin test, with reagent RBCs in low‐ionic‐strength solutions (LISS) containing co‐trimoxazole or not. RESULTS: All three sera showed positive reactions when RBCs were resuspended in LISS containing co‐trimoxazole, but negative reactions when RBCs were resuspended in LISS without antibiotic. We detected antibodies against co‐trimoxazole showing three different antibody patterns: anti‐TMP plus anti‐SMX, anti‐TMP alone, or anti‐SMX alone. Anti‐TMP showed an apparent anti‐Ku specificity in the two cases where it was present. Anti‐SMX showed an apparent anti‐H specificity in one of the two cases described. The drug‐dependent antibodies were not associated with acquired hemolytic anemia or other pathologies. CONCLUSION: Antibodies against co‐trimoxazole may only be detected when using a diluent for reagent RBCs containing the drug in question. Antibody pattern (anti‐TMP and/or anti‐SMX) may vary according to individuals' immune response. Drug‐dependent antibodies may react as antibodies against a high‐prevalence antigen, supporting the hypothesis of antibodies to drug and membrane components. Drug‐dependent antibodies such as anti‐co‐trimoxazole may be a serologic finding without clinical features.     

Evaluation of the manual hexadimethrine bromide (Polybrene) technique in the investigation of autoimmune hemolytic anemia

The use of the direct manual hexadimethrine bromide (Polybrene) test (DPT) in the investigation of patients for autoimmune hemolytic anemia (AIHA) was evaluated. Seventy-nine blood samples from 68 patients were tested. A direct antiglobulin test (DAT) using monospecific reagents and the DPT were performed, and a concentrated ether eluate was tested. The DAT was positive in 62 (78%) of 79 patients and negative in 17 (22%). There is a good correlation among DAT, eluate, and DPT in demonstrating the presence of immunoglobulin on the red cell surface. In contrast, the DPT does not detect C3d and is often negative in cases of AIHA in which C3d alone is demonstrated by the DAT. In DAT-negative cases, DPT results correlated with reactive eluates. However, in four cases of steroid-responsive, DAT-negative hemolytic anemia, the DPT supported the diagnosis of AIHA when the eluate did not react. The DPT is a useful additional screening test for the investigation of AIHA, but it is not recommended as a replacement for either eluate testing or the DAT.