The use of pooled red cells and column techniques for routine red cell antibody detection

Summary. The object of antibody screening is to detect all clinically relevant antibodies. In order to do this effectively red cells are selected with an appropriate antigen profile. The introduction of column techniques for antibody screening by indirect antiglobulin testing (IAT) and two-stage enzyme testing (ETC) is perceived to lead to an increased sensitivity and an ability to detect red cell antibodies more easily than by traditional tube techniques because reactions in columns are more easily read and are stable. We evaluated the use of a column technology with pooled red cells for routine antenatal screening. The pooled cells used contained at least one cell with homozygous antigen expression for the majority of clinically significant antibodies known to be present, except for Kell. Pooled cell results were not as easy to read in gel columns when compared with single cell results due to weaker reactions which were often diffused throughout the gel in the column. We concluded that the use of pooled cells led to a decreased sensitivity which proved problematic for the interpretation of results. We used a two-cell and a three-cell pool and found that detection of known antibodies was reduced in IAT and ETC methods.     

Quantitation of red cell-bound IgG by an enzyme-linked antiglobulin test in human immunodeficiency virus-infected persons

Anemia, thrombocytopenia, and neutropenia have been observed in patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex. To investigate whether red cells (RBCs) of patients with human immunodeficiency virus infection were coated with IgG and/or complement (C3), blood samples of 239 patients were tested. The prevalence of a positive direct antiglobulin test on RBCs was 16.7 percent. By use of an enzyme-linked antiglobulin test (ELAT) to measure more accurately the number of IgG molecules per RBC in a group of 67 patients, 30 of the 67 individuals were observed to have increased numbers (mean, 155) compared to normal controls and to patients with hypergammaglobulinemia due to multiple myeloma or chronic liver disease. Hemoglobin level was correlated with the number of IgG molecules per RBC (p = 0.008), but no correlation could be demonstrated between those numbers and serum immunoglobulin (p = 0.10) or circulating immune complexes (p = 0.38). Our results with ELAT suggest that some AIDS patients may have specific binding of IgG on the surface of their RBCs, rather than nonspecific uptake; further clinical correlations are necessary to confirm these findings.     

固相微板法红细胞抗体检测技术的建立

目的　摸索建立一种简单实用的红细胞抗体检测技术。方法　采用一种新的固相红细胞方法 ,把筛选红细胞固定于 96孔U型板。分别用固相法和试管法 ,对多种抗血清、供血者、有免疫史的病人 3批标本检测 ,对结果加以比较。结果　对抗血清的效价测定 ,两种方法结果一致 ;在对供血者和病人的检测中 ,固相法检出了所有试管法能检出的抗体。结论　与试管法比较 ,固相法的灵敏度可以得到肯定 ,结果可靠 ,而且易制备 ,操作技巧容易掌握、普及。     

Cytolytic lymphocytic cells with complement receptor in human blood. Induction of cytolysis by IgG antibody but not by target cell-bound C3

Human blood lymphocytes were fractionated on glass bead columns charged with sheep erythrocyte (Es) membranes-bearing human C3b (7,000-10,000 molecules/Es). In the passaged cells the proportion of C receptor lymphocytes was strongly reduced, in parallel with the capacity to lyse chicken erythrocytes (Ec) in the presence of IgG-rabbit anti-Ec antibody. In other experiments, lymphocytes forming rosettes with Es bearing activated rabbit complement [C(ra)] from C6-deficient rabbits were removed by centrifugation through human serum albumin-gelatine mixtures. This procedure also depleted the lymphocyte preparations of antibody-dependent cytolytic effector cells. The results suggest that rations of antibody-dependent cytolytic effector cells. The result suggest that such effector cells have receptors for human C as well as for C(ra). Lymphocytes were not able to lyse erythrocytes bearing either human C3b (similar to 30,000 molecules/Ec) or activated C(ra) in the absence if IgG antierythrocyte antibodies. Under the same experimental conditions these target cells were efficiently lysed in the presence of small amounts of IgG antitarget cell antibodies. This suggests that the interaction between the cellular Fcreceptors and the Fc part of the inducing antibodies is of special significance for the triggering of the cell-mediated lytic reaction. However, although target cell-bound C did not trigger cytolysis, it seemed to potentiate antibody-dependent cytolysis, probably by enhancing effector cell-target cell contacts.     

The influence of Hymenolepis nana infection on antibody responses to sheep red blood cells in mice

The influence of Hymenolepis nana infection on the primary antibody responses to both sheep red blood cells (SRBC) and dinitrophenylated Ficoll-coated SRBC (DNP-coated SRBC) was examined in BALB/c mice. The in vitro cultures of spleen cells (Sp C) obtained from mice inoculated with either 100 or 1,000 eggs developed fewer plaque forming cells (PFC) than those of Sp C from non-infected mice, when the cells were exposed to SRBC. Thereafter, the number of PFC was gradually increased and almost reached the initial levels by day 21. On the other hand, the ability of Sp C obtained from mice infected with 10,000 eggs to produce PFC gradually dropped as the course of infection proceeded. However, H. nana infection did not reduce the capacity of Sp C to produce PFC to DNP-coated SRBC. Addition of T-cell enriched population from infected mice, which was obtained by passing through a nylon wool column, to anti-Thy-1.2 antibody treated non-infected Sp C significantly inhibited the production of PFC to SRBC. These results strongly suggest that H. nana infection impairs T-cell function in mice.     

A new method of antibody elution from red blood cells

A method of antibody elution from red blood cells using xylene is described. It can be completed in 15 minutes after cell washing and requires no special equipment. For warm antibodies, the eluates prepared were more potent than those prepared byRubin's ether method. It was as effective as Landsteiner and Miller's heat elution method in investigations of ABO hemolytic disease of the newborn. The clinical application of this method in the investigation of hemolytic disease of the newborn, delayed transfusion reaction and autoimmune hemolytic anemia was described.     

The effect of bumetanide on cation transport in human red blood cells.

Bumetanide, a sulfamyl-aminobenzoic acid derivative, is a new and highly effective diuretic agent. The present studies were designed to examine its effects on cation transport in human red cells. At a concentration of 10(-3) M, the drug inhibited both active and passive unidirectional sodium fluxes, as well as active potassium influx. It also caused a significant inhibition of glycolysis. The inhibition caused by bumetanide was less than that seen with ouabain alone, but a bumetanide effect was also present in ouabain-treated cells. Bumetanide had no effect on red cell Na-K adenosine triphosphatase activity and did not affect net transport of sodium in sodium-loaded cells. The data are consistent with a model in which the inhibition of monovalent cation movement in red cells by bumetanide is related to an effect of this compound in decreasing the permeability of the red cell membrane to sodium.     

Quantitation of red cell-bound immunoglobulin and complement using enzyme-linked antiglobulin consumption assay

Various techniques have been described for quantitating IgG or complement (C3) on red cells (RBCs). The techniques either are cumbersome, as the complement consumption test, or use radioactivity. This paper describes an antiglobulin consumption assay using an enzyme-linked immunosorbent method that can be used to quantitate IgG, IgM, and C3. With this technique RBCs from normal, healthy donors gave a mean value of 106 +/- 60 molecules of IgG per RBC, 4.5 +/- 3 molecules of IgM per RBC, and 37 +/- 28 molecules of C3 per RBC, respectively. The RBCs of hospital patients, particularly of those with infections or inflammatory conditions, contain increased amounts of nonspecifically bound immunoproteins. The availability of a common method to quantitate RBC-associated IgG, IgM, and C3 allows easy monitoring or study of the immune mechanism of autoimmune hemolytic anemia.     

流式细胞术检测抗体致敏红细胞方法的建立及初步应用

目的　建立应用流式细胞术 (FCM)测定被抗体致敏红细胞的方法。方法　用荧光素标记的绵羊抗人免疫球蛋白抗体 ,与致敏红细胞上的免疫球蛋白结合 ,以绵羊抗鼠IgG RITC标记的红细胞作同形对照 ,设定mark ,得测定管阳性细胞百分数。结果　Coombs试验在对比试验中 ,只能测出经 2 7稀释的抗D血清所致敏的红细胞 ,而FCM则能测出经 2 9稀释的抗D血清所致敏的红细胞。实际应用中 ,15例ABO新生儿溶血病标本 ,Coombs阳性率13 3% ,FCM阳性率 86 7% ,5例Rh新生儿溶血病标本 ,Coombs阳性率 4 0 % ,FCM为 10 0 %。结论　FCM技术比传统Coombs试验灵敏度高 ,定量更准确     

Number of random test cells for antibody detection

The sensitivity of antibody screening carried out by reacting a serum with random test cells depends on the probability of a given antigen being present in the cell panel. This probability (p) is defined by the formula p = 1-(1-f)n where f is the antigen phenotype frequency and n the number of reacted random cells.     

Storage-induced changes in human newborn red cells

Fetal red cells are well suited for intrauterine life; however, little is known about their response to postnatal environments. The purpose of this work was to investigate the metabolic and membrane changes affecting newborn red cells during their exposure to storage in citrate-phosphate-dextrose (CPD) and citrate-phosphate-dextrose-adenine (CPDA-1). The findings suggest that newborn red cells are affected more by storage than are adult cells. These accelerated storage changes in the red cells of newborns may be related indirectly to the rapid adenosine triphosphate (ATP) decline. As is the case with adult red cells, fetal cells withstand storage in CPDA-1 better than in CPD. The storage lesion in these cells was partly reversible, as in adult cells, by incubation with adenosine. It was therefore concluded that newborn red cells obtained from placentas and stored for several weeks in CPD or CPDA-1 media or other media that improve the metabolic profile of these cells may be acceptable for transfusion.