Negative Direct Antiglobulin Test Research Articles

Opsonizing and agglutinating antibodies against Plasmodium falciparum schizont-infected erythrocytes in Gambian sera

The presence of an IgG antibody to autologous schizont-infected red blood cells (SIRBC) in the sera of Gambian children with acute malaria and a positive direct anti-globulin test (DAT) was demonstrated by two methods: IgG quantitation using 125I-labelled anti-IgG and phagocytosis of these SIRBC. A similar antibody could not be demonstrated in sera of children with acute malaria and a negative DAT. When a laboratory strain of parasite was used there was a more variable pattern of IgG uptake but the highest was from sera of immune adult Gambians and Gambian children with acute malaria and a positive DAT. The uptake of IgG by SIRBC may be a useful test to determine the specificities of antibodies against different isolates of P. falciparum.

Anti‐EnaFS detected in the serum of an MiVII homozygote

Anti-EnaFS was detected in the serum of a Caucasian woman, K.T., during her third pregnancy. She had not been transfused. At delivery, the infant's red cells (RBCs) had a negative direct antiglobulin test. The antibody was inhibited by isolated MN sialoglycoprotein (SGP). Unlike other En(a-) cells, K.T.'s EnaFS-negative RBCs were found to demonstrate normal sialic acid levels, and an MN SGP exhibiting normal sodium dodecylsulphate electrophoretic mobility and periodic acid-Schiff staining intensity. K.T., whose parents were consanguineous, appears to be the first known MiVII homozygote. In a three-generation family study, MiVII was shown to travel with MS. The Anek receptor of K.T.'s RBCs was found to be located on the trypsin-resistant and V8 protease-sensitive portion of the MN SGP (residues 40 to 57), whereas the Anek receptor of MiVI RBCs was shown to be associated with their abnormal Ss SGP. K.T.'s MN SGP appears to exhibit an amino acid sequence alteration in the region of the EnaFS determinant, which is located within the residues 46 to 56 of the normal MN SGP. These findings suggest that the MiVII and EnaFS receptors are the products of allelic genes.

Impaired reticuloendothelial function in patients treated with methyldopa.

In about 20 per cent of patients taking the antihypertensive agent methyldopa, IgG autoantibodies form against red cells, but most such patients do not have hemolysis. The reason for this is uncertain; it does not appear to be explained by known characteristics of the autoantibody. Since antibody-dependent reticuloendothelial function is an important determinant of cell clearance, we measured reticuloendothelial function in nine patients taking methyldopa. We did this by measuring the rate of clearance of radiolabeled autologous red cells sensitized with anti-D alloantibody. Five patients had a positive direct antiglobulin test, and four did not. Only one patient had laboratory evidence of hemolysis. The patients without hemolysis had significantly impaired reticuloendothelial clearance. In contrast, the patient with hemolysis did not have impaired reticuloendothelial function. This study suggests that in patients taking methyldopa who have a positive direct antiglobulin test the absence of hemolysis may be caused by an impairment in reticuloendothelial function. That the drug itself may be responsible for the impairment is suggested by the occurrence of abnormal reticuloendothelial function in patients taking the drug who have a negative direct antiglobulin test.

HEMOLYTIC TRANSFUSION REACTION (HTR) ASSOCIATED WITH INTRAVENOUS IMMUNE GLOBULIN (IV Ig)

High dose IV Ig has been tried in immune cytopenia(s) with few complications. We report a severe HTR due to IV Ig. A 10 yr-old B Rho(D) pos. boy who received a bone marrow transplant (BMT) from his O Rho(D) neg. sister was given IV Ig (C51911, GAMIMUNE, 5%, Cutter Lab.) for platelet alloimmunization. On day 9 post-BMT he developed a chill and hypotension after 120 ml of IV Ig and hemoglobinuria later. On day 10 he had a severe shaking chill after 100 ml of IV Ig and symptoms recurred after 2 ml of infusion. On days 10 to 12 he had hyperbilirubinemia (4.5 mg/dl), hemoglobinemia (98 mg/dl), hemoglobinuria (75 mg/dl) and no haptoglobin. Hb remained between 6-7 g/dl with 8 units of RBC. On day 13 urine became clear and Hb rose without transfusions (Tx). No Tx were given 48 hrs. pre-IV Ig. After BMT he was given O neg. deglycerolized-RBC and group B plasma and platelets. Pre-IV Ig he was B pos. and had a negative direct antiglobulin test (AGT) and antibody screen. Post-IV Ig direct and indirect AGT were positive (days 10-13); anti-B (1:4) was detected in his sera from day 12. Sera and eluate demonstrated antiglobulin reactivity with group B RBC. The IV Ig he received had 1:64 for anti-B and Anti-A. On day 16, patient was typed as O neg. without reticulocytosis, indicating massive intravascular hemolysis of autologous RBC, not a response to BMT. Since blood group antibodies are present in human immune serum globulin preparations, compatibility test should be done when using IV Ig to prevent HTR.

The direct antiglobulin test in P. falciparum malaria.

The direct antiglobulin test (DAT) was performed on 134 Gambian children with P. falciparum malaria. 52 children had a positive DAT and in 25 this was due to the adherence of C3 to their red cells whilst 13 had sensitization with IgG as well as C3. Sensitization with C4 alone or associated with IgG and/or C3 was only rarely found. The haemoglobin levels and reticulocyte counts were not significantly different in patients with a positive DAT from those with a negative DAT. The children with positive IgG DAT, however, were of a significantly older age group and had significantly higher parasitaemias at presentation than children with complement components only or with a negative DAT. In vitro assay of phagocytosis by peripheral blood monocytes (PBM) showed that PBM from malarious patients were less active in phagocytosis than normal male adult Caucasian PBM. Neither PBM from malarious patients nor normal adult Caucasians PBM showed phagocytosis of cells from DAT positive malarious children. The same in vitro assay was used to test for the presence of opsonizing anti-schizont antibodies in the sera of these children and in adult Gambians. This revealed the presence of a schizont specific opsonizing antibody in the sera of adult Gambians and in the sera of children with a positive DAT due to IgG but not in sera of patients with a negative IgG. These findings indicate that a positive IgG DAT in malaria does not necessarily lead to excess haemolysis of non-parasitized red cells. The presence of a schizont opsonizing antibody, leading to in vitro phagocytosis, in the sera of children with a positive DAT and in sera from adult Gambians indicates a relationship between the development of the positive DAT and acquisition of protective malarial immunity.

Detection of IgG sensitization of red cells with 125I staphylococcal protein A.

Most cases of immune hemolytic anemia are associated with a positive direct antiglobulin test. However, in some cases, the antiglobulin test is not sensitive enough to detect low levels of red-cell bound antibodies. This report describes a method using radiolabelled purified staphylococcal protein A which is capable of detecting IgG sensitization of red cells beyond the threshold of serologic techniques. It is less cumbersome than previously described methods and does not require antibody purification procedures. Its effectiveness was demonstrated for the detection of red-cell alloantibodies and in evaluation of patients with acquired hemolytic anemias associated with a negative direct antiglobulin test.

A fluorometric assay for red blood cell antibodies.

A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoresceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

ABO hemolytic disease of the newborn. A retrospective analysis of 254 cases.

ABO hemolytic disease of the newborn is the single most common cause of neonatal jaundice, with an incidence of 54.4 per 1,000 births; it occurs almost exclusively in infants of groups A or B having mothers of group O. Previous studies have shown a poor correlation between serologic tests on cord blood and clinical course in affected infants. In a retrospective analysis of 254 cases of ABO hemolytic disease of the newborn the relation of laboratory parameters to incidence and severity of jaundice was studied. Sixty-five per cent of the infants who had positive direct antiglobulin tests experienced jaundice, compared with approximately 35% of control infants or infants who had ABO hemolytic disease of the newborn with negative direct antiglobulin test results. Infants who had ABO hemolytic disease of the newborn with positive direct antiglobulin test results also had greater severity of jaundice than control infants or infants who had ABO hemolytic disease of the newborn with negative direct antiglobulin test results (P less than .0001). Thus, the direct antiglobulin test is a good screening test for ABO hemolytic disease of the newborn. Sex, race, gravidity, birth weight, and blood type of the infant did not have significant relationships to clinical outcome. The combined results of the direct antiglobulin test and the strength of reaction in a heat eluate may be of use in prognosis; although differences were not significant, infants who had stronger eluates had higher bilirubin values and were more likely to require therapy. Serologic analysis of cord blood can be useful in the early detection of infants having the risk of severe jaundice.

Positive direct antiglobulin (Coombs') test caused by cephalexin administration in humans.

Of 71 patients showing a negative direct antiglobulin (Coombs') test (DAT) before administration of cephalexin (CEX) three developed a positive DAT after therapy with this drug (4 percent). No case of immunohemolytic anemia was observed. The sera of the patients with a positive DAT reacted with cephalothin-coated red cells producing high titers, with penicillin cells normal titers and with CEX cells low titers. Since the red cell eluates of these patients did not give significant reactions with red cells sensitized with CEX, cephalothin and penicillin, a nonimmunological mechanism for the development of the positive DAT in these patients has to be assumed.

Positive Direct Antiglobulin (Coombs’) Test Caused by Cephalexin Administration in Humans

Of 71 patients showing a negative direct antiglobulin (Coombs’) test (DAT) before administration of cephalexin (CEX) three developed a positive DAT after therapy with this drug (4%). No case of immunohemolytic anemia was observed. The sera of the patients with a positive DAT reacted with cephalothin-coated red cells producing high titers, with penicillin cells normal titers and with CEX cells low titers. Since the red cell eluates of these patients did not give significant reactions with red cells sensitized with CEX, cephalothin and penicillin, a nonimmunological mechanism for the development of the positive DAT in these patients has to be assumed.

Autoimmune hemolytic anemia in Hodgkin's disease associated with anti-IT.

In this study, three cases of Hodgkin's disease with autoimmune hemolytic anemia are described. The IgG autoantibodies in each case were shown to have IT specificity. There is only one other case of an IgG anti‐IT described in the literature and that also was present in a patient with Hodgkin's disease. No other examples of anti‐IT were found in studies which included 50 cases of Hodgkin's disease with negative direct antiglobulin tests, three cases with positive direct antiglobulin test but no hemolysis, and 70 cases of autoimmune hemolytic anemia, either idiopathic or secondary to other diseases of the reticuloendothelial system. Fetal, cord and adult cells were tested with anti‐IT, anti‐I and anti‐i in an attempt to show the development of IT relative to i and I.

Reappraisal of the role of anti-i in haemolytic anaemia in infectious mononucleosis.

Summary. Current literature implies that haemolytic anaemia in infectious mononucleosis is regularly caused by the temporary production of high thermal amplitude cold agglutinins of anti‐i specificity. More recently, Capra et al (1969) suggested interaction between IgG anti‐i and anti‐IgG antibodies as the cause of haemolytic anaemia in infectious mononucleosis. A detailed serologic evaluation of three patients during moderate to severe haemolytic anaemia in infectious mononucleosis revealed high thermal amplitude anti‐i in only one. This patient's direct antiglobulin test (DAT) was negative using anti‐IgG but was 3 + using anti‐C3 and ‐C4. The serum antibody titre against cord or adult Oi cells was 512 at 4°C and 4 at 31°C. The anti‐i was inhibited by mercaptoethanol, and anti‐IgG was not found in the patient's serum. Patient 2 had a negative DAT and patient 3 had a 2 + DAT using anti‐C3 and anti‐C4. Anti‐i was present only in low titre at 4°C and was unreactive at 20°C. It was inhibited by mercaptoethanol. These patients' sera contained anti‐IgG antibodies. In none of our patients were warm autoantibodies detected. This data demonstrates that haemolytic anaemia in infectious mononucleosis is not necessarily associated with high thermal amplitude anti‐i. Further, since the anti‐i antibodies in patients 2 and 3 were of low titre, were inhibited by mercaptoethanol, and did not react at physiologic temperatures, the mechanism of haemolysis in these patients does not seem related to the interaction of anti‐IgG and IgG anti‐i antibodies. Further work is necessary to clarify the mechanism of haemolytic anaemia in infectious mononucleosis.

Red-cell antibodies in acquired hemolytic anemia with negative antiglobulin serum tests.

Abstract Of eight patients with acquired immunohemolytic anemia, six had persistently negative and two reversibly negative direct antiglobulin tests (Coombs tests). In five of the six, the red cell...

A-B Hemolytic Disease of the Newborn Analysis of 1480 Cord Blood Specimens, with Special Reference to the Direct Antiglobulin Test and to the Group O Mother

Abstract A study was made of oxalated umbilical vein blood of nearly every infant born at The Mount Sinai Hospital in a nine month period. A specimen of maternal blood was available for intragroup antibody screening and six cases of Rh-Hr hemolytic disease were eliminated from the data. The umbilical vein blood was tested, where possible, for: (1) group and Rh, (2) direct antiglobulin test, (3) hemoglobin, (4) reticulocyte count and examination of red cell morphology, (5) plasma bilirubin, and (6) osmotic fragility in 0.52 per cent NaCl. From the mothers’ blood groups, the infants were classified into group compatible and group incompatible, and the arithmetic means of the hemoglobin, reticulocyte count, and plasma bilirubin obtained for each class. A third class of infants, those with positive direct antiglobulin test, were analysed separately for comparison. 1. A weakly positive direct antiglobulin test was obtained on the umbilical vein blood of over 11 per cent of group incompatible infants but in none of the group compatible infants. 2. It appears that the weakly positive direct antiglobulin test detects an abnormal class of group incompatible infants, since their mean hemoglobin is low, their mean reticulocyte count is high, and their mean bilirubin is high, when these means are compared with those of the other group incompatible infants. 3. Thirty-eight of thirty-nine mothers of incompatible infants with positive direct antiglobulin test were group O. In comparison with the distribution of the blood groups of the mothers of other incompatible infants, this disproportion is of significance. 4. The mean reticulocyte count of incompatible infants with negative direct antiglobulin test is slightly (but with statistical significance) higher than the mean reticulocyte count of compatible infants. This difference was found to be associated almost entirely with group O mothers. 5. Thirty-one out of thirty-eight infants with positive direct antiglobulin test had increased osmotic fragility in hypotonic NaCl. Two of the negative cases appeared to have slight spherocytosis on blood smear.