The Low Ionic Strength Reaction of Human Blood: Relationship between the Binding of Serum Immunoglobulin and Complement to Red Blood Cells and Surface Charge of the Cells

Using the sucrose haemolysis reaction of Hartmann & Jenkins (1966) as a basic model, the low ionic strength reaction (LISR) of human blood was studied to determine: (1) serum Ig uptake by RBC with saline elution and 125I-IgG uptake, and (2) complement fixation (CF) to RBC with lysis of PNH cells and C3H/C4 antiglobulin haemagglutination (AH) of normal cells. The saline eluates were found to contain IgG and IgM with traces of IgA; their pH optima for the uptake by RBC were 6.0 +/- 0.5, 5.5 +/- 0.5 and c 5.0 respectively. The ratio of bound IgG to IgM was linearly related to the uptake pH. Both C4 AH and lysis were found to be optimum at pH 6.0--7.5, whereas the maximum C3 AH was at pH 6.0 +/- 0.5. The LISR performed at a constant pH (6.1 +/- 0.1) showed that an increasing concentration of neuraminidase (VCN) used in pretreatment of RBC was associated with a decrease in both IgG uptake and CF activity. A maximum VCN effect reduced the Ig uptake to c 20% of normal and abolished almost all the CF activity. An impaired LISR to various degrees was also observed with RBC pretreated with ficin, papain, bromelin, trypsin or protamine, and RBC from two individuals of En(a-) type. Preincubation of serum at LIS with and without RBC resulted in respectively a 'complete' and partial consumption of C in the fluid phase. The latter was not enhanced or inhibited by the addition of VCN-treated RBC for preincubation. A hypothesis is proposed suggesting that in the LSR the Ig uptake by RBC is an electrostatic interaction of the oppositely charged RBC and Ig and the CF to RBC results from C activation by the cell-bound IgG and IgM. In addition, a pH-dependent inactivation of the cell-bound C3 in the LISR is demonstrated.     

Parthenocissus tricuspiduta Activity Directed against Human Red Blood Cells Coated with C3b

An extract of seeds from Parthenocissus tricuspidata strongly agglutinated red blood cells (RBC) coated with C3b. RBC coated with C3d, C4b or C4d and noncomplement-coated RBC were very weakly agglutinated. There was no correlation of reactivity with RBC groups.     

Automated Antibody Detection in a Low Ionic Strength (Glycine) Medium

Modifications were introduced into the low ionic strength system of Lalezari. Glycine was added in place of acidified glucose and the sensitisation and aggregation phases were segregated. In the automated glycine system a stable baseline is readily obtained as strong cell aggregation, with risk of cell adhesion to coil walls, is restricted to the last few turns of the aggregation phase. The automated glycine system is sensitive to a concentration of 0.0015 microgram/ml of anti-D diluted in saline and to 0.006 microgram/ml of anti-D in serum. Antibodies of a broad range of specificity are detectable.     

Inclusions in Red Blood Cells Containing Hb S or Hb C

To demonstrate and characterize red cell inclusions in 101 persons with Hb S or Hb C disorders three methods were used: (1) examination of unstained blood smears by dark field microscopy (DFM), (2) examination of blood smears after acid elution and staining (AE), and (3) measurement of membrane-associated denatured haemoglobin (MADH) in ghosts. The control group had inclusions in less than 5% of red cells by DFM and AE and the mean percentage of MADH per total cellular Hb was 0.030+/-0.016%. The highest percentages of red cells with inclusions and of MADH were present in clinically severe haemoglobin disorders, e.g. homozygous sickle cell disease (Hb SS) with less than 10% Hb F and Hb SOArab, with successively lower percentages in moderate to severe disorders, e.g. Hb SS-alpha thalassaemia, Hb-S-beta0 thalassaemia, Hb SC disease, and Hb SS with more than 10% Hb F, indicating agreement in results by three methods. In asymptomatic or mild disorders, e.g. Hb-S-beta+ thalassaemia, Hb CC, Hb AC and Hb AS, the results by AE and measurements of MADH were the same or similar to those in controls, while those by DFM were different. Of 56 patients with Hb SS or Hb SC, the group with functional asplenia had higher percentages of MADH and of red cells with inclusions than those with functioning spleens. Our study suggests that inclusions in sickling disorders may be due to denatured Hb S, with AE being the more accurate method for visualizing these inclusions, as results by this method correlate better with the amount of MADH than those by DFM.     

Complement-Independent Lysis of Human Red Blood Cells by Cold Hemagglutinins

Hemolysis mediated by human antibodies is generally ascribed to the attack of red blood cells (RBC) by complement. We here extend earlier in vitro observations which indicate that potent cold agglutinins can directly cause lysis of RBC without the participation of complement. We have noted that EDTA plasma taken from patients with cold agglutinin disease is frequently reddish if the plasma is not immediately separated from the cells at 37 degrees C. Moreover, eluates prepared in such cases from plasma or heat-inactivated serum (30 min at 56 degrees C) by absorption (at 4-20 degrees C) and elution (at 37 degrees C) are usually contaminated with hemoglobin, and a large number of RBC used for absorption is lost during the procedure. To characterize this phenomenon further, we examined the effect of different hemagglutinating antibodies in vitro on normal RBC in the absence of complement. Hemolysis (5-17%) of RBC only occurred after treating the cells with potent antibodies at low temperatures (0-20 degrees C). This hemolysis increased 2- to 3-fold when the RBC were treated with an enzyme and decreased with rising temperature. Unlike cells hemolyzed by complement activation, no C5b-9 complexes could be detected on RBC damaged by this mechanism.     

Preparation of Red Cells Coated with C4 and C3 Subcomponents and Production of Anti-C4d and Anti-C3d

Abstract. Methods of preparing red cells coated only with C4b, C3b or C3d have previously been described by others; the present observations supply further information about the optimal conditions for preparing such cells and also describe conditions for the preparation of cells carrying only C4d. In addition, the preparation of specific anti-C4d and anti-C3d sera is described.     

The Electrophoretie Mobility of Red Blood Cells of Normal Human Beings

Values obtained for the electrophoretic behavior of the red blood cells ofhealthy individuals is presented. The technic and instrument are described indetail and the necessary attention to meticulous care is emphasized. Thedata presented show that in an electric field the mobility of the red blood cellsof healthy persons is constant and reproducible. It was concluded that themethod is extremely sensitive and precise and that it may prove of value in thestudy of various disease states.

Submitted on July 15, 1960     

Lysis of P.N.H. Cells in Solutions of Low Ionic Strength

A test to detect P.N.H. red cells when exposed to fresh serum in a sucrose-veronal-albumin buffer solution of low ionic strength, pH 7.6, is described.
The serological findings suggest that the basis of the test is another example of the peculiar sensitivity of P.N.H. cells to complement in the presence of antibody.     

Preservation of Red Blood Cells: Content of Microaggregates and Di-2-Ethylhexylphthalate (DEHP) in Red Blood Cells Stored in Saline-Adenine-Glucose-Mannitol (SAGM) Medium

The content of microaggregates was determined in leukocyte-poor red blood cells (RBC) after 5 weeks of storage in citrate-phosphate-dextrose (CPD)-adenine plasma and in saline-adenine-glucose-mannitol (SAGM) medium. The size range was 3,600-200,000 fl, corresponding to a particle diameter of 19-73 micron, presuming a spherical particle shape. In comparison with CPD-adenine whole blood (100%), the total number of particles per unit of RBC was found to be 41.2% (CPD-adenine anticoagulant) and 25.4% (SAGM medium). The corresponding data for total particle volumes were 43.3 and 30.3%. The significant differences between the two categories of RBC could not be explained on the basis of differences concerning pH or cell composition. A majority of particles (50-62%) was observed in the lowest size range (19-29 microns), corresponding to only 18-26% of the total particle volume. The accumulation of di-2-ethylhexylphthalate (DEHP) was determined in two variations of RBC stored in SAGM medium and in RBC stored in CPD-adenine plasma. The quantities of DEHP per unit of RBC after 5 weeks were significantly smaller in SAGM medium (9.0 +/- 3.6 and 22.7 +/- 6.1 mg, respectively) compared to CPD-adenine plasma (30.1 +/- 9.5 mg).     

Diversity of Human Anti-D Monoclonal Antibodies Revealed by Reactions with Chimpanzee Red Blood Cells

Fifty-three human anti-D monoclonal antibodies (mAbs) revealed a striking diversity of reactions in tests with panels of chimpanzee red blood cells (RBCs) of various R-C-E-F blood group phenotypes (counterparts of the human Rh-Hr groups). The reactivities of these antibodies, which depended on the agglutination technique used, could be classified into four main types. These patterns of reactivity of anti-D mAbs with chimpanzee RBCs showed only limited correlation with types of reactions observed with human D variant RBCs. Primate red cells may, therefore, constitute an independent test system for subclassification of human monoclonal antibodies. Comparison of reactivities of human anti-D mAbs with chimpanzee and human D variant RBCs confirms the homology between the chimpanzee R^c, and the human D antigens. The chimpanzee R^c shares with human D the epitopes epD5, epD6/7 and epD8, but lacks epitopes epD1, epD2, epD3 and epD4 of the Rh mosaic, thus resembling the human D variants IVb and Vc.     

Antibody-dependent Cell-mediated Cytotoxicity (ADCC) of Penicillin-treated Human Red Blood Cells

S ummary . Penicillin-treated human red blood cells (RBC) were lysed by the cooperation of autologous nonsensitized peripheral blood mononuclear cells and human anti-penicillin serum. Using a rapid (3 h) assay of antibody-dependent cell-mediated cytotoxicity (ADGC), lysis was proportional to serum (anti-penicillin antibody) concentration, to incubation time and to the concentration of attacking cells, which were obtained from normal human peripheral blood by Ficoll-Hypaque separation. Incubation of these lymphoid effector cells on a nylon column prior to the tests depleted the number of phagocytic (latex positive) cells in the effluent; there was a concomitant drop in cytotoxic activity. Enrichment of mononuclear phagocytes in the attacking cell population by albumin gradient separation led to an increase in cytotoxicity. Granulocytes separated by Ficoll-Hypaque were not active in this system. Using specific antisera the antibody was found to be of the IgG1 sub-class. Anti-penicillin antibody activated the complement system in vitro , but failed to induce lysis of penicillin-treated RBC in the presence of complement without attacking cells.
These results suggest that ADCC may participate in the destruction of RBC in penicillin-induced haemolysis in vivo.     

Sialic Acid Deficiency of Human Red Blood Cells Associated with Persistent Red Cell, Leucocyte, and Platelet Polyagglutinability

S ummary . This study reports, for the first time, a persistent polyagglutinability involving all blood cells of a patient. The phenomenon is shown to be an immunological reaction due to an antibody present in sera of all normal adults, whereas the antigen, tentatively designated 'T_cr', is unique to the patient. It is shown that T_cr is related to T_n, an antigen previously described in association with red cell polyagglutinability. A partial deficiency of red cell sialic acid is also found to be associated with this abnormality. It is suggested that this sialic acid deficiency may be a manifestation of a primary underlying muco-protein abnormality.     

Agglutination of Red Blood Cells from Patients with Diabetes mellitus by a Polyclonal Human Antibody Specific for D-Glucose

We have previously described an antibody which will agglutinate red blood cells which had been incubated in vitro in D-glucose. This antibody is specific for the ring form of glucose, beta-D-glucopyranose. The current report demonstrates that without prior in vitro incubation with glucose, red blood cells from 36 of 38 patients with diabetes mellitus, and 7 of 70 patients not diagnosed as diabetic were agglutinated by this antibody. Strength of agglutination of red blood cells from diabetic patients correlated with both glucose (r = 0.61; p less than 0.001) and hemoglobin A1c levels (r = 0.50; p less than 0.01) in simultaneously obtained samples. This reactivity could be reversed by incubating red blood cells from diabetics for several hours in saline. This report suggests that red blood cells from diabetic patients have membrane-bound glucose that can be detected immunologically. Reversibility of the reaction and rapidity of in vitro glycosylation suggests short-term binding of glucose. To our knowledge, this is the first report documenting immunological detection of in vivo short-term reversible binding of glucose to cellular membranes.     

The Influence of Ionic Strength and Incubation Time on the Sensitivity of Methods Using Papainized Red Cells

The sensitivity of a combination of various papain methods with normal and low ionic strength (lis) of the serum-cell mixture for the detection of red cell antibodies was tested after varying periods of incubation of the mixture. The mixture was centrifuged before reading when the period of incubation was short. The maximum sensitivity without centrifugation was always reached after a period of 60 min. With a longer incubation the sensitivity decreased. A sensitivity that equalled the maximum sensitivity without centrifugation could be obtained after only 10 min of incubation, if it was followed by a centrifugation before reading. Lis consistently increased the sensitivity of the papain methods, especially after 45--60 min of incubation.     

In vitro Interactions of 51r in Human Red Blood Cells and Hemolysates1,2

Abstract. The intracellular distribution of radioactivity was studied in normal and sickle erythrocytes labeled with sodium ⁵¹ Cr chromate. Both types of cells had a higher fraction of ⁵¹ Cr bound to hemoglobin when labeled in the presence of ACD at a pH of 7.09 than when labeled in the presence of CPD at a pH of 5.96. Citrate at a pH of 5.96 or less entered the red blood cells and decreased the ⁵¹Cr binding hemoglobin. Binding of ⁵¹Cr to hemoglobin within the red cells was also reduced when the ATP and DPG levels in the red blood cells were elevated. Studies with hemoglobin solution showed that ⁵¹Cr binding to hemoglobin was influenced by 2,3-DPG, ATP and citrate.     

The Effects of Adriamycin (Doxorubicin HCL) on Human Red Blood Cells

We have studied the effects of adriamycin (doxorubicin HCl) on human red blood cells. The peroxidizing effect of adriamycin on the thiols of red cell constituents resulted in decreased glutathione stability, and oxidation of hemoglobin and membrane protein components 1, 2, and 3, forming large molecular weight complexes. Membrane lipids were also peroxidized. Adriamycin itself did not inhibit the enzymes of the reductions system (glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydro-genase, glutathione reductase, glutathione peroxidase, catalase, superoxide dismutase) of the red cells. Because adriamycin has the potential of inhibiting ATPase, including both Na-K-dependent ATPase and ouabain insensitive ATPase, at concentrations not inhibitory to other enzymes, the net sodium content increased, and potassium content decreased after incubation of red cells with adriamycin at high concentrations. The experimental results described with adriamycin may serve as a model for the possible mechanism of cardiotoxicity observed in its clinical use, and also explain the potential hemolyzing effect on red cells. There was greater oxidizing effect on glucose-6-phosphate dehydrogenase (G-6-PD) deficient than on normal erythrocytes. It is suggested that adriamycin be used with caution in individuals with G-6-PD deficient red cells.     

差速贴壁法体外培养人脐血内皮祖细胞的实验研究

目的 建立一种经济可靠、稳定、高纯度内皮祖细胞的培养方法.方法 采用Ficoll 密度梯度离心法从人脐血中分离单个核细胞,于包被人纤连蛋白的培养皿中贴壁培养,收集48 h后的悬浮细胞重新贴壁培养至第7天,利用免疫组化、免疫荧光及流式细胞术对培养的细胞进行鉴定.结果 培养的细胞呈短梭形、多角型、胞体小;可见到大量典型的内皮祖细胞克隆;vWF和flk-1免疫组化细胞阳性率>95%,免疫荧光Dil-ac-LDL和FITC UEA-1双染阳性的细胞阳性率>98%,流式分析CD133 细胞的百分率为(7.O±1.8)%.结论 差速贴壁法是一种经济可靠、稳定、高纯度内皮祖细胞的培养方法.     

差速贴壁法体外培养人脐血内皮祖细胞的实验研究

目的建立一种经济可靠、稳定、高纯度内皮祖细胞的培养方法。方法采用 Ficoll 密度梯度离心法从人脐血中分离单个核细胞,于包被人纤连蛋白的培养皿中贴壁培养,收集48 h后的悬浮细胞重新贴壁培养至第7天,利用免疫组化、免疫荧光及流式细胞术对培养的细胞进行鉴定。结果培养的细胞呈短梭形、多角型、胞体小;可见到大量典型的内皮祖细胞克隆;vWF 和 flk-1免疫组化细胞阳性率>95%,免疫荧光 Dil-ac-LDL 和 FITC-UEA-1双染阳性的细胞阳性率>98%,流式分析 CD_(133)~+细胞的百分率为(7.0±1.8)%。结论差速贴壁法是一种经济可靠、稳定、高纯度内皮祖细胞的培养方法。