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.     

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.     

The role of the elution in antibody investigations

BACKGROUND: The direct antiglobulin test (DAT) commonly detects immunoglobulin G (IgG) molecules or complement fragments on the red blood cell (RBC) surface. If IgG antibodies are present then elution procedures can be performed to identify the specificity of these antibodies. Our reference laboratory performs elutions on the RBCs of those patients who have received cellular blood products in the past 30 days and have either a newly identified positive DAT with anti-IgG or the agglutination strength is increased over a previous DAT and if ordered by a clinician regardless of transfusion history. This study questioned how frequently elutions contributed novel serologic information under our reference laboratory's current policy or whether elutions should be performed in more selective serologic conditions.
STUDY DESIGN AND METHODS: Recipients whose RBCs underwent eluate testing were identified from the blood bank's database and information about the antecedent DAT and antibody detection test and eluate was recorded.
RESULTS: In total 648 eluates were evaluated and 82 of 648 (12.7%) revealed a novel antibody not present in the serum (an informative eluate). In 2 of 82 informative eluates non–anti-A/B alloantibodies that were not present in the serum were detected: one example each of anti-D and anti-E. Both were associated with a microscopically positive antecedent DAT. The rate of an informative eluate was higher when the antibody detection test was negative.
CONCLUSION: The strength of the DAT does not indicate the likelihood of an informative eluate. Performing an eluate when the antibody detection test is positive has limited value.     

The evaluation of a positive direct antiglobulin test (autocontrol) in pretransfusion testing revisited

Direct antiglobulin tests (DATs) using anti-IgG were performed on 65,049 blood samples from prospective transfusion recipients; 3570 tests (5.49%) were positive. Using criteria published previously (primarily excluding patients not transfused within the preceding 14 days), 778 samples from other than neonatal patients were selected for further evaluation. Eluates that did not react were obtained on 518 (66.6%) of these samples. Warm-reactive autoantibodies were apparent in 192 eluates, while 16 contained drug-related antibodies, anti-A or anti-B from prior transfusion with ABO mismatched blood components, or anti-D passively acquired from immune serum globulin. Fifty-two eluates contained alloantibodies; however, in only six of these cases did the corresponding serum lack unexpected alloantibodies, as determined by routine pretransfusion studies. Three additional weakly reactive clinically significant alloantibodies were detected solely through additional serum tests performed on DAT-positive samples. On the basis of these findings, the DAT had a low predictive value when used to detect the early manifestations of an immune response to recently transfused red cells. Elimination of the autocontrol from routine pretransfusion testing, therefore, carries minimal risk to patients yet will undoubtedly contribute to the containment of health care costs. Moreover, the risk is lower than that associated with the elimination of the antiglobulin crossmatch.     

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.     

Resolution of red cell compatibility testing problems in patients receiving anti-lymphoblast or anti-thymocyte globulin

Passively acquired hemagglutinating antibodies may be detected in the serum of patients receiving horse anti-lymphoblast globulin (ALG) or anti-thymocyte globulin (ATG). Thirty-seven patients receiving ALG following renal transplantation were studied. Eight patients monitored daily all developed positive direct antiglobulin tests (DAT) and positive red cell antibody screening tests. Fifteen of 32 recipients developed red cell antibodies reactive at room temperature in saline, 4 of 32 in albumin at 37 degrees C, and 33 of 37 in the antiglobulin test. Horse globulin was detected on the red cells of all six recipients tested with rabbit anti-horse globulin. Ether eluates prepared from the red cells of 20 patients showed no specificity for common red cell antigens. Anti-human globulin (AHG), absorbed with ALG- coated red cells to remove the component in the AHG which was crossreacting with horse globulin, was used successfully for antibody screening and identification, direct antiglobulin testing, and/or the antiglobulin crossmatching of 27 ALG and ATG recipients, including five with red cell alloantibodies.     

An immediate hemolytic reaction induced by repeated administration of oxaliplatin

BACKGROUND: Platinum-based chemotherapy agents have been associated with potentially fatal acute immune-mediated hemolytic anemia. The target antigen, cause of the positive direct antiglobulin test (DAT) and mechanism of hemolysis have been the subject of controversy. CASE REPORT: We report a patient who developed a DAT-positive hemolytic episode after a red cell (RBC) transfusion was delivered during the infusion of her 17th cycle of oxaliplatin. Standard pretransfusion testing was uncomplicated; however, after infusion, the serum was no longer compatible with the transfused units and a strong (4+) panreactive IgG antibody was detected. RESULTS: The patient's serum from 10 days after the episode, only when therapeutic concentrations of oxaliplatin were added, reacted with all RBCs tested using the indirect antiglobulin test (IAT) (3+). The effect was retained with a purified IgG fraction and almost eliminated with IgG-depleted serum. Immunoprecipitation analysis revealed a band with the molecular weight of the Band 3 anion channel only in the presence of the patient's serum and oxaliplatin. CONCLUSION: Our investigations indicated that oxaliplatin interacted with both an IgG antibody and a RBC membrane epitope probably located on the Band 3 anion channel.     

The detection of cell-bound antibody on complement-coated human red cells

This study sought to elucidate the mechanism by which human red cells, in a variety of clinical settings, become coated in vivo with autologous complement components in the absence of anti-red cell autoantibodies demonstrable by standard methods. By means of a newly developed complement-fixing antibody consumption test, previously undetectable red cell-bound gammaG globulin could be detected and quantified. By this technique, the complement-coated red cells of 13 of 16 patients were shown to carry abnormally high numbers of gammaG molecules per cell, which were nevertheless below the level for detection by the direct antiglobulin test. Eluates were made from the red cells of seven of these patients and each eluate, when sufficiently concentrated, was capable of sensitizing normal human red cells (with gammaG antibodies) to give a positive indirect antiglobulin test with anti-gammaG serum. In the presence of fresh normal serum, six of the eluates so tested were capable of fixing complement to normal human red cells. The antibodies in the red cell eluates did not exhibit Rh specificity and did not react with nonprimate red cells. When studied by sucrose gradient ultracentrifugation, the gammaG antibodies to human red cells in these eluates sedimented in the 7S region. It is concluded that in many patients in whom direct antiglobulin tests reveal only cell-bound complement, the complement fixation is mediated in vivo by small quantities of "warm-reacting" erythrocyte autoantibodies of the gammaG class.     

Positive direct antiglobulin tests at birth without demonstrable maternal antibody

A healthy 38-year-old white woman had two abortions and three live children. Red cells from each of her living children at birth had a strongly positive direct antiglobulin test. Detailed studies on the third child showed that the cells were sensitized by IgG. Maternal serum, tested by a range of techniques against reagent red cells and the husband's cells, showed no unusual antibody. Maternal serum and eluates prepared from red cells of the third child did not react with fresh cells from the older siblings (aged 7 and 10 years). Follow-up on the third child showed that the direct antiglobulin test was positive at 2 months, weakly positive at 4 months, and negative at 8 months. Red cells collected at 8 months of age did not react with the stored eluate prepared from the baby's sensitized red cells at birth. The most likely explanation of these data is that the children inherited a paternal antigen that is only present as a red cell surface-active structure during fetal development.     

Red cell and platelet-bound IgG penicillin antibodies in a patient with thrombocytopenia

A patient was found to have a positive direct antiglobulin test and thrombocytopenia while on a moderate dose of intravenous penicillin. Serological evaluation of the patient's red cells demonstrated that the positive antiglobulin test was due to antipenicillin antibody. This antibody also was demonstrated in the patient's serum. The patient's platelets had increased quantities of IgG; an eluate from her platelets gave positive test results with platelets treated with penicillin but not normal platelets. Her serum also reacted only with penicillin- treated platelets. Multiple absorptions of her serum with red cells treated with penicillin reduced reactivity against both fresh red cells and platelets treated with penicillin. This patient demonstrated the coexistence of drug-induced immune phenomena directed against both red cells and platelets.     

Delayed hemolytic episodes due to anti-M

We observed three cases in which anti-M, undetectable in pretransfusion serum, was responsible for accelerated hemolysis of crossmatch- compatible red blood cells 5 to 15 days after transfusion. In each case, the direct antiglobulin test, which had been negative on pretransfusion testing, was weakly positive on the posttransfusion sample. Anti-M was identified in both the serum and eluate from the posttransfusion sample in two cases. In the third, anti-M could be identified only in the posttransfusion serum. Hemolysis was mild, and was not clinically suspected in any of these three patients until blood was requested for further transfusion. In one of the cases, the antibody was undetectable within a few weeks after the hemolytic episode. Destruction of transfused red blood cells by newly synthesized alloantibodies, particularly those of the Kidd system, is a familiar phenomenon to blood bankers. It is apparent from these studies that anti-M also can behave in this fashion.     

Two cases of "hrB-like" autoantibodies appearing as alloantibodies

Two cases are described in which autoantibodies mimicked alloantibodies. The direct antiglobulin test (DAT) on the red cells (RBCs) from both patients was negative when routine manual hexadimethrine bromide (Polybrene) and enzyme-linked antiglobulin techniques were used. The RBCs also did not react on direct bromelin and direct Polybrene tests. However, an "hrB-like" antibody was eluted from the RBCs of both patients. The sera from these patients reacted with all e+ hrB+ RBCs but not with e+ hrB-, e-, or their own RBCs. The antibody in the serum of one patient was not adsorbed by R2R2 RBCs. Serologic tests initially suggested (by direct testing and adsorption studies) that the serum antibodies were alloantibodies rather than autoantibodies. RBCs taken from one patient, 8 months after her sample was first referred to our laboratory, reacted with a serum sample from her first admission. An RBC sample taken from the other patient, initially typed e+ and hrB- but 1 month later typed e+ and hrB+ by using the same anti-hrB sera, was used to test the earlier samples.     

Delayed hemolytic transfusion reaction caused by anti-Jsb in a Js(a+b+) patient

A 39-year-old multiparous woman developed a mixed field positive direct antiglobulin test within 15 days of receiving four units of crossmatch compatible red blood cells. Anti-Jsb was demonstrable in both the serum and the eluate. The case was further complicated by the fact that the patient's pretransfusion red blood cells typed as Js(b+). Serologic studies demonstrated that this was a case of allo-anti-Jsb in a Js(b+) patient which provides evidence of heterogeniety of the Js locus.     

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.     

The gel test: some problems and solutions

The gel centrifugation test (GT) is a method of transfusion serology, based on the fact that, after centrifugation, unagglutinated red blood cells (RBC) pass easily through a gel, while agglutinated RBC do not. The introduction of the GT to our blood bank transfusion routine [strictly following the manufacturer's instructions (DiaMed ID Micro Typing System)] resulted in problems with the interpretation of the results. These were overcome after the introduction of modifications, which included: (1) the systematic use of 1% RBC suspensions; (2) the use of 50 microliters of 1% RBC suspensions and 25 microliters of serum in all tests; (3) the control of all negative indirect antiglobulin tests (IAT) and direct antiglobulin tests (DAT) by the addition of 50 microliters of a 1% IgG coated RBC suspension followed by centrifugation; and (4) the systematic use of saline-suspended RBC for ABO typing in patients with positive DAT.     

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.     

Hemolytic anemia associated with injection of fluorescein

A 49-year-old woman presented with a hemoglobin level of 9.5 g per dL (95 g/L), reticulocyte count of 6.7 percent (0.067), and hemoglobinuria. The next day, the hemoglobin had dropped to 5.8 g per dL (58 g/L), and total bilirubin was 8.8 mg per dL (150 mumol/L). The serum reacted 2+ with all red cells (RBCs). The direct antiglobulin test (DAT) was 3+ with anti-IgG and 1+ with anti-C3, but eluates prepared by two different methods did not react with untreated RBCs. The eluate reacted 2+ with amoxicillin-coated RBCs; amoxicillin had been listed in the patient's record as a previous medication. The patient denied recent ingestion of amoxicillin. Further investigation documented the injection of a dye, fluorescein sodium (AK-FLUOR-25%), for a ophthalmologic fluorescein angiographic study 2 days before admission. RBCs coated with AK-FLUOR reacted with the eluate. Controls consisting of normal serum, an eluate prepared from DAT-negative RBCs, and a serum known to contain anti-penicillin did not react with AK- FLUOR-coated RBCs. Nine days later, the DAT was negative and the serum did not react with untreated RBCs. In the presence of AK-FLUOR (1-in- 125) or amoxicillin (1 mg/mL), the serum reacted 2+ in the antiglobulin test. Antibodies to AK-FLUOR and amoxicillin appeared to react by two mechanisms, which is similar to results in recent reports of other drugs associated with hemolytic anemia. AK-FLUOR has not previously been reported to be associated with hemolytic anemia.     

Association of positive direct antiglobulin test (DAT) with nonreactive eluate and drug-induced immune hemolytic anemia (DIHA)

Background and Objectives: Drug-induced immune hemolytic anemia is a rare condition that occurs primarily because of drug-induced antibodies, either dependent or independent and positive direct antiglobulin test. Our aim was to evaluate the association of positive DAT with nonreactive eluate and DIHA. Materials and Methods: From 2014–2018, we evaluated 159 patients who presented positive DAT with a nonreactive eluate. Laboratory and clinical analyses were performed including HIV, HBV and HCV testing. All patients were exposed to the following drugs: Dipyrone in 63.5 %, Furosemide in 28.9 %, Metoclopramide in 34.6 % and Ondansetron in 41.5 %. Results: Results of DAT showed IgG in 125 (78.4 %) patients and C3d in 24 (15.1 %) with reactions varying from 1+ to 4+. HIV test was positive in 10 (16.1 %) patients, HBV was positive in 3 (4.7 %) and HCV was positive in, 1 (1.5 %). There was no clinical significance when the parameters of hemoglobin, hematocrit, reticulocytes and LDH were evaluated, only a slight increase in bilirubin, especially, in patients with positive DAT reacting 3+/4+ due to IgG and C3d sensitization. Clinical evaluations showed that all patients were asymptomatic. Conclusions: The association of drugs with positive DAT can be a challenge to transfusion services and immunohematology reference laboratories. There was no evidence of any case of severe hemolysis with clinical repercussion through the clinical and laboratory findings analyzed with the drugs associated with positive DAT. Dipyrone and Furosemide have already been associated with DIHA but there are no studies reporting the association of Metoclopramide and Ondansetron with DIHA.     

Matuhasi-Ogata phenomenon involving anti-ampicillin

A 47-year-old group A, Rh1Rh1 woman treated with intravenous ampicillin for chronic pyelonephritis received two units of blood and also received oral cephalexin. Three months after the transfusions she was noted to have allo-anti-E and anti-c, and a 2+ positive direct antiglobulin test. Anti-E and anti-c could be eluted from her cells, yet neither antigen could be demonstrated on the patient's circulating red blood cells. Also present in the serum and in the eluate was anti- ampicillin antibody. Studies of the patient's red blood cell eluates using ampicillin-treated R1R1 and untreated R2R2 cells demonstrated anti-E complexed with anti-ampicillin in a drug-related example of the Matuhasi-Ogata phenomenon. Artificially created mixtures of anti-E and drug antibody could reproduce the effect in vitro. No effect of cephalexin could be demonstrated. The variability of the Matuhasi-Ogata phenomenon is discussed with regard to the sequence of antibody attachment, and the possible relationship to cephalexin is discussed. Drug antibodies may be involved in the Matuhasi-Ogata phenomenon in cases where another red blood cell antibody cannot be shown to be present.     

Spontaneous agglutination of red cells with a positive direct antiglobulin test in various media

Red cells from 475 individuals (donors and patients) with a positive direct antiglobulin test (DAT) (66% were 1/2+ to 1+, 19% were 1 1/2+ to 2+, and 15% were 2 1/2+ or greater) were examined for spontaneous agglutination following incubation in various media. Twenty-three percent of the samples showed spontaneous agglutination in commercial Rh control solutions, 6 percent in 30 percent albumin, 3 percent in 10 percent albumin, 2 percent in 6 percent albumin, and 1 percent in saline. Cells suspended in serum were more prone to spontaneous agglutination. The strength of spontaneous agglutination varied; more than 50 percent of the samples reacted only 1+ or less, approximately 10 percent reacted 2+ or stronger. There was not a complete correlation with spontaneous agglutination and quantity of IgG on the cells, as determined by the antiglobulin test. Of those samples showing spontaneous agglutination, 50 percent were associated with a DAT strength of more than 2+, 27 percent with a DAT of 1 1/2+ to 2+, and 23 percent with a DAT of 1/2+ to 1+. Spontaneous agglutination occurred with the same frequency whether the cells were sensitized with both IgG and C3 or with IgG alone. Surprisingly, 7 percent of the samples sensitized with C3 but no detectable IgG also demonstrated spontaneous agglutination. Marked differences in reaction strength were seen with Rh control solutions from different manufacturers, and the degree of spontaneous agglutination was inconsistent with individual products.