Three examples of Rh haemolytic disease of the newborn with a negative direct antiglobulin test

SUMMARY. Typically the serological diagnosis of alloimmune haemolytic disease of the newborn (HDN) includes a positive direct antiglobulin test on the infant's red cells, and the presence of an IgG red cell alloantibody in both maternal and cord sera. HDN with a negative direct antiglobulin test has been reported with anti-A and anti-B, but not with other red-cell alloantibodies.
In this report we describe four examples of HDN in infants whose red cells had a negative direct antiglobulin test. The first case was diagnosed retrospectively when the infant was admitted to hospital aged 3 weeks with severe anaemia and cardiac failure, and subsequently died. Maternal and infant sera were both shown to contain anti-C: however, the direct antiglobulin test on the infant's red cells was negative. Approximately 1 year later the mother of this infant gave birth to triplets: soon after birth one of the triplets required an exchange transfusion, one had hyperbili-rubinaemia, and the third was unaffected. Anti-C and anti-e were detectable in the maternal serum at this time. The most probable Rh genotypes of the two affected infants were R₁R₂ (CDe/cDE), while the Rh genotype of the unaffected infant was R₂R₂ (cDE/cDE). Anti-c was implicated as causing HDN in a fourth infant (from a different family) who was a hydropic stillborn. The direct antiglobulin test on fetal blood was negative and other causes of non-immune hydrops were excluded. These four infants provide evidence that the direct antiglobulin test may be negative in some severely affected and even fatal cases of HDN.     

Red cell sensitization due to anti-D in anti-lymphocyte globulin

Four Rh-positive patients with severe aplastic anemia received equine anti-lymphocyte globulin. Each developed a positive direct antiglobulin test. Anti-D was identified in eluates prepared from the patients' sensitized red cells. The administered lot of anti-lymphocyte globulin was found to contain anti-D. Red cell sensitization due to passively acquired Rh antibodies can result from the administration of anti- lymphocyte globulin.     

The significance of red cell bound complement components in development of standards and quality assurance for the anti-complement components of antiglobulin sera

Human red blood cells, sensitized with complement in vivo and by a variety of methods in vitro, (e.g. blood group antibody, low ionic strength, alternate pathway), were tested with a battery of anti-complement sera (anti-C3, -C3c, -C3d, -C4, -C4c). Red blood cells could be prepared by relatively simple methods to yield cells sensitized with C3 and C4, C3 but not C4, C4 but no C3, C3d with no C3c and C4d with no C4c. These cells are suitable for standardization and quality assurance of antiglobulin sera (AGS). Anti-C3d is necessary for optimal detection of sensitization of red blood cells by complement in vivo by the direct anti-globulin test (DAT). Anti-C3d may also be optimal for the indirect antiglobulin test (IAT) especially if incubation periods greater than one hour are employed. Potent anti-C4 and anti-C3 antisera made in the authors' laboratory resulted in numerous weakly positive antiglobulin tests when testing red blood cells from refrigerated clots (especially anti-C4) but red blood cells from refrigerated anticoagulated segments gave negative results. When red blood cells were incubated in normal serum at room temperature (as in the room temperature phase of a compatibility test), some positive results were again obtained with the potent anti-C4 and anti-C3 antisera. However, one commercial antiglobulin serum containing anti-complement antibodies that were at least as potent as any other commercial antiglobulin serum gave uniformly negative results under the above conditions. Anti-C4 antibodies may be omitted from antiglobulin sera without decreasing the efficacy of such antisera to be used in compatibility testing. Thus, positive results in the compatibility test due to detection of clinically insignificant cold antibodies in the IAT by the anti-complement antibodies in AGS, may be avoided if anti-C4 is omitted or is in low concentration and if the concentration of anti-C3d is carefully standardized. A higher concentration of anti-C3d could be used for compatibility tests if red blood cells from anticoagulated segments were used instead of those from clots and if a separate tube were used for the IAT at 37 C rather than using one tube for both room temperature and 37 C incubations.     

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.     

Red cell autoantibody production in utero: a case report

BACKGROUND: Autoantibody production by the fetus is thought to be extremely unlikely. Only one possible case of in utero autoantibody production against red cells by the fetus has previously been described. STUDY DESIGN AND METHODS: A case of apparent red cell IgG autoantibody production in utero is reported. RESULTS: This was established by a positive direct antiglobulin test in a newborn infant without evidence of maternal alloantibodies or autoantibodies. There was no evidence of clinically significant hemolysis at the infant's birth. After 6 weeks, his direct antiglobulin test remained strongly positive. The infant thrived without evidence of hemolysis, and after 6 months the direct antiglobulin test was negative. CONCLUSION: The production of autoantibodies to red cells in utero is possible, though rare. This did not result in apparent hemolysis in this patient.     

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.     

Unexpected limitations in the use of commercial antiglobulin reagents

The majority of antiglobulin sera used in blood banks in the USA are commercially prepared immune rabbit sera, designed to be reactive only with human red blood cells sensitized with immunoglobulins or complement components. Current manufacturing methods result in a product that gives reliable specific reactions provided that the test red blood cells are normal, particularly with respect to sialic acid levels. This report describes our findings of false positive antiglobulin tests caused by incompletely absorbed antiglobulin reagents when cells with low sialic acid levels were tested. An evaluation of nine commercially prepared antiglobulin reagents revealed, in many of them, the presence of anti-species antibody, anti-T, and anti-Tn. Blood bank personnel must be aware of these characteristics especially when testing either enzyme premodified or polyagglutinable cells. The use of incompletely absorbed reagents might account for positive direct antiglobulin tests that are encountered occasionally in apparently normal healthy individuals. Furthermore, recent protocols advocating the use of trypsinized cells for the evaluation of anti-C3d activity of antiglobulin sera are invalid if the reagent is inadequately absorbed of anti-species agglutinins.     

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.     

Tolmetin-induced hemolysis

A case of drug-associated immune hemolysis in a patient taking tolmetin for arthritic pain is described. Serologic tests showed that in the absence of tolmetin, the patient had a negative antibody screening test but a strongly positive direct antiglobulin test. An eluate prepared from the patient's red cells caused agglutination of all cells tested. However, addition of tolmetin revealed a high-titered tolmetin-dependent antibody in the patient's serum; the addition of tolmetin did not affect the results obtained with the eluate. within 3 months after the patient discontinued tolmetin, his hematocrit had increased to 45 percent, and his jaundice and bilirubinuria had disappeared. These results are similar to those described for zomepirac, another of the group of nonsteroidal anti-inflammatory medications.     

Erb, an allele to Era, and evidence for a third allele, Er

An antibody against a low-incidence antigen was detected in the serum of a woman whose newborn infant was found to have a positive direct antiglobulin test. The antibody failed to agglutinate 79 examples of red cells having low-incidence antigens and 16 examples of high-incidence antigen-negative red cells. The woman's serum reacted strongly with her husband's red cells in the antiglobulin test and with 5 of 6 Er(a-) cell samples from unrelated donors, suggesting that the antigen has an antithetical relationship to Era. The failure of the serum to react with one Er(a-) cell sample and with cells from the Er(a+) daughter of an Er(a-b+) mother gives evidence for a silent allele, Er. Four Er(b+) bloods were found among 605 random white donors, indicating a gene frequency for Erb of 0.0033.     

Anti-Ytb in pregnancy

Anti-Ytb was readily eluted from the cord red blood cells of the second child of a mother with both anti-Ytb and anti-Kell in her serum. The baby was Yt (a+b+) and Kell negative. The cord red blood cells were found to have a weakly positive direct antiglobulin test. Clinically evident hemolytic disease of the newborn was not detected.     

A Method for Preservation of Papainized and Rh-Sensitized Red Cells

Papainized red blood cells and red blood cells sensitized with Rh antibodies were stored at 4 C in Alsever's solution containing inosine. The papainized cells were used for detection of Rh antibodies. The cells sensitized with anti-D were used for the positive control of the antiglobulin reaction. When stored for no more than three weeks, the preserved cells gave reactions almost identical to those obtained with freshly prepared cells.     

Pre-transfusion testing problems caused by anti-lymphocyte globulin and their solution

Anti-lymphocyte globulin (ALG) is an antibody to human lymphocytes used to decrease T-cells in renal transplant patients. We recently encountered serologic problems in testing blood from patients treated with ALG. Thirty-nine patients undergoing acute kidney rejection developed positive direct and indirect antiglobulin tests following the administration of equine ALG. Sera from these patients reacted with all red cells (RBCs) tested using both polyspecific and monospecific anti-IgG anti-human sera. Eluates prepared from the patients' RBCs showed similar reactivity. The ALG panagglutinin did not react by manual hexadimethrine bromide (Polybrene) technique. The ALG panagglutinin could be neutralized by anti-human globulin. In our hands, these techniques were useful in distinguishing ALG panagglutinin from co-existing alloantibodies.     

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.     

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.     

An Evaluation of Commercial Antiglobulin Sera with Particular Reference to Their Anticomplement Properties

Ten commercial "broad-spectrum" antiglobulin sera, which had satisfied NIH standards, were evaluated and compared with six antiglobulin sera of varying specificities prepared in the authors' laboratories. The majority of the commercial antisera had adequate anti-IgG, although four were definitely weaker and failed to detect weak IgG antibodies. Most of the commercial antiglobulin sera had inadequate anticomplement, eight failing to react with cells strongly sensitized with complement, from patients with hemolytic anemia. Most of the commercial antiglobulin sera reacted poorly with cells weakly or moderately sensitized with complement-binding blood group antibodies, such as anti-Lewis. Evidence that adequate anticomplement activity should be present in routinely employed antiglobulin sera is discussed.     

An IgG Anti-IT Detected in a Caucasian American

An isoantibody was detected in the serum of a Caucasian with Hodgkin's disease. The antibody reacted strongly by the indirect antiglobulin test with all fetal cord cells but was negative or only weakly positive when tested with adult cells, including five examples of the rare adult i cells. Rhesus monkey and rabbit cells also failed to react. No autoagglutination was seen and the direct antiglobulin test was negative. The antibody sensitized cells optimally at 37 C, reacting with anti-IgG but not anti-IgM, anti-IgA or anti-complement antiglobulin reagents. It was not inhibited by human saliva, milk, or 2-mercaptoethanol. The specificity met the criteria set for anti-I^T. It is unique among antibodies of this specificity since it is an IgG isoantibody, reacting optimally at 37 C by the indirect antiglobulin test, showing a marked difference in reaction between cord and adult cells and occurring in a Caucasian. This contrasts with the only other anti-I^T antibodies described in the literature which are all cold autoagglutinins occurring in isolated populations (Melanesians and Yanomama Indians of Venezuela), showing only marginal differences between adult and cord cells.     

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.     

A delayed hemolytic transfusion reaction due to anti-Cob

A delayed hemolytic transfusion reaction precipitated by anti-Cob is described in a multiple transfused primigravida woman with sickle-cell disease. Sixteen days after the prophylactic transfusion of the first of 4 units of red cells, she experienced a fall in hemoglobin concentration accompanied by a newly positive antibody screen and direct antiglobulin test. Anti-Cob was identified both in the patient's serum and in an eluate prepared from her red cells.     

Survival of Kn(a+) McC(a+) red cells in a patient with anti- "Kna/McCa"

The in vivo survival of Kn(a+)McC(a+) red cells in a patient with anti- "Kna/McCa" was studied using 51Cr-labeled incompatible cells. The antibody was IgG4, demonstrable at 37 degrees C in the antiglobulin test, and did not bind complement. Survival of tagged cells was 82.2 percent after 24 hours. The patient was transfused with a total of 29 units of incompatible blood with no evidence of ill effects. The direct antiglobulin test became positive after transfusion and remained positive for 2 1/2 months of observation. The findings suggest that individuals with anti- "Kna/McCa" may be transfused with Kn(a+)McC(a+) red cells.