Comparison of flow cytometric assays with isotopic assays of (51)chromium-labeled cells for estimation of red cell clearance or survival in vivo.

BACKGROUND: A comparison was made between flow cytometric and conventional radioisotopic assays in the determination of the clearance or survival of small volumes of (51)chromium-labeled D+ red cells after injection into volunteers. STUDY DESIGN AND METHODS: Four clearance studies were performed using 4 mL of autologous D+ cells coated with anti-D at two concentrations (5 or 10 microg anti-D/mL red cells) transfused to two subjects at separate times. Five survival studies were carried out using 5 mL of frozen-thawed D+ cells transfused to five D- subjects with no detectable anti-D. Sequential blood samples were taken for gamma counting and flow cytometry. Several methods were used to stain the transfused red cells, and the data were analyzed by using three flow cytometers. RESULTS: The determination of red cell clearance or survival by radioactivity measurements gave results consistent with published data. However, none of the flow cytometric assays exhibited the necessary sensitivity or accuracy in quantitation of the rare events to provide reliable data for the calculation of the initial clearance rate, the red cell half-life, or the mean cell lifespan, although rough estimates of red cell clearance were obtained in some subjects. This inability to accurately enumerate rare fluorescence-labeled cells was due mainly to the presence of "background" events, which were a considerable problem in some samples, when the coating level of anti-D was less than 3000 molecules of IgG per cell. CONCLUSION: Flow cytometry may enable the crude estimation of the percentage of small volumes (<5 mL) of transfused D+ red cells, but in this study it was found that this method was not sufficiently accurate to determine the initial clearance rate, red cell half-life, or mean cell lifespan. If the proportion of transfused cells in the recipient is about 0.2 percent or less, the use of radioisotopes for labeling cells for quantitative in vivo red cell clearance or survival data should remain the method of choice.

Expression of RHD and RHCE gene products using retroviral transduction of K562 cells establishes the molecular basis of Rh blood group antigens.

Retroviral-mediated gene transfer using cDNA transcripts of the RHD and RHCE genes resulted in the isolation of K562 clones expressing D and G or c and E antigens, respectively. These results represent the first direct demonstration that the RHD gene encodes the D and G antigens and the RHCE gene encodes the c and E antigens. Both c and E antigens were expressed after transduction of K562 cells with a single cDNA, indicating that the c antigen does not arise by alternative splicing (exon skipping) of the product of the RHCE gene, as has been suggested.

Quantification of IgG anti-D bound to D-positive red cells infused into D-negative subjects after intramuscular injection of monoclonal anti-D.

A flow-cytometric method was developed to determine the number of molecules of IgG bound to D-positive red blood cells (RBC) when sensitized with low plasma concentrations of IgG anti-D in the presence of an excess of D-negative RBC. D-positive RBC were infused into 12 D-negative male volunteers 2 days after intramuscular injection of monoclonal anti-D (BRAD-3, IgG3 or BRAD-5, IgG1). Blood samples were taken immediately before, 3 min and 3 h after injection of the RBC, incubated for 1 h at 37 degrees C, washed, then incubated sequentially with FITC-conjugated anti-IgG, IgM monoclonal anti-D, biotin-conjugated anti-IgM, and R-phycoerythrin-conjugated streptavidin. To prepare a standard curve, O R1R2 RBC were incubated in duplicate with varying dilutions of BRAD-3 or BRAD-5, and RBC from one set were mixed with an excess of D-negative RBC (1:100) and labelled as above, while cell-bound IgG on the other set was quantified by ELISA. The test samples and standards were analyzed by flow cytometry and the mean channel (FITC) fluorescence was plotted against molecules IgG/cell, from which the sensitization level of D-positive RBC in the test samples was determined. The use of IgM anti-D enhanced the discrimination between D-positive and D-negative RBC, especially when fewer than about 3000 molecules IgG/cell were bound. The assay was sensitive to about 1000 molecules IgG/cell. The sensitization levels of the D-positive RBC in samples taken 3 h after injection were found to be in the range 1500-10,000 molecules IgG anti-D per cell.

Monoclonal antibodies recognizing epitopes on the extracellular face and intracellular N-terminus of the human erythrocyte anion transporter (band 3) and their application to the analysis of South East Asian ovalocytes.

This report describes the production and characterization of 13 rodent monoclonal antibodies to the human erythrocyte anion transport protein AE1 (syn. band 3). Eleven antibodies (4 murine and 7 rat) recognize epitopes dependent on the integrity of the third extracellular loop of the protein. Two antibodies (1 murine and 1 rat) recognize epitopes on the N-terminal cytoplasmic domain. Quantitative binding studies using radioiodinated IgG and Fab fragments of antibodies to extracellular epitopes on AE1 ranged from 77,000 to 313,000 (IgG) and from 241,000 to 772,000 (Fab) molecules bound at saturation. The results indicate that the epitopes recognized by different antibodies vary in their accessibility and suggest that there is heterogeneity in the organization of individual AE1 molecules in the red blood cell membrane. Quantitative binding studies on South East Asian ovalocytes using several antibodies to AE1 and an anti-Wrb show a marked reduction in the number of antibody molecules bound at saturation. These results are consistent with the existence of highly cooperative interactions between transmembrane domains of AE1 in normal erythrocytes and the disruption of these interactions in the variant AE1 found in South East Asian ovalocytes.

A novel form of congenital dyserythropoietic anemia associated with deficiency of erythroid CD44 and a unique blood group phenotype [In(a-b-), Co(a-b-)].

We have used a panel of well-characterized monoclonal antibodies (MoAbs) to examine the blood cells of a patient with a novel form of congenital dyserythropoietic anemia (CDA) characterized by intra-erythroblastic and intra-erythrocytic membranous inclusions. Twelve antibodies defining three nonoverlapping epitope groups on the extracellular domain of CD44 all failed to react with the red blood cells (RBCs) of the patient. A rabbit antibody to the cytoplasmic domain of CD44 from normal RBCs failed to react with the patient's RBC ghosts. In contrast, the patient's lymphocytes, granulocytes, and monocytes showed apparently normal CD44 expression. Bone marrow preparations stained with CD44 antibodies and visualized with 125I antimouse Ig (F(ab')2) followed by autoradiography showed positive staining of lymphocytes and myeloid cells but not of most orthotolidine-positive erythroblasts. The patient's RBCs also gave weaker than normal reactions with MoAbs of anti-LWab specificity while MoAbs to glycophorins A, B, and C, Rh polypeptides, CD47, CD55, CD58, CD59, acetylcholinesterase, and Lutheran and Kell glycoproteins all gave normal reactions. Agglutination tests with human blood grouping sera demonstrated that the RBCs of the patient have the unique phenotype In(a-b-), Co(a-b-) and that they also lack the high incidence RBC antigen AnWj. The phenotype In(a-b-) would be expected because these antigens are known to be expressed on CD44. There is also some evidence associating the AnWj antigen with CD44. However, the CO blood group locus is on chromosome 7p whereas that for CD44 is on chromosome 11p. Quantitative binding assays using 125I-labeled Fab fragments of CD44 antibodies did not show any evidence for reduced levels of CD44 on RBCs from the parents of the patient or from her unaffected sister. The parents and sister had the common Colton blood group phenotype [Co(a+b-)]. Neither deficiency of CD44 nor absence of Colton antigens are general features of CDA because erythrocytes from patients with CDA I, CDA II, CDA III, and two other unclassified CDAs had normal expression of CD44 and normal Colton blood group phenotypes. Further analysis of the defect(s) present in the patient's erythroid cells may provide useful information regarding membrane assembly and the regulation of differentiation in normal erythroid cells.

Antibodies to platelet glycoprotein V in polytransfused patients with haematological disease.

Polytransfused patients often develop platelet-reactive antibodies (PRAb). These give positive reactions in the platelet immunofluorescence test (PIFT) and may be either lymphocytotoxic (LCTAb) or platelet-specific antibodies (PSAb). The latter may be detected in the PIFT using chloroquine-treated platelets (Chl-PIFT) or by immunoblotting. Serial samples from 106 multiply transfused patients with bone marrow failure were screened by PIFT using a microplate method and flow-cytometric analysis. PSAb activity was confirmed by Chl-PIFT. In 45 (42%) of the patients studied PSAb were detected; 37 (35%) formed LCTAb and 19 (51%) had co-existent PSAb. Sera from 25 of 27 patients with a positive Chl-PIFT, retested by immunoblotting, recognised determinants of Mr 82-160 kD on whole platelets. A large group became sensitised to a component of Mr 105-115 kD reduced (99 kD non-reduced) with similar electrophoretic mobility to GPIIIa using a monoclonal anti-GPIIIa and two human polyclonal anti-HPA-1a sera; some also produced anti-GPIIb. The largest group recognised a determinant of Mr 80-83 kD, probably glycoprotein V (GPV). Three sera were immunoblotted against thrombin-treated platelets and the results confirmed GPV specificity.

Analysis of the structure and activity of A and A,B immunoglobulin A monoclonal antibodies.

A study of the serologic activity and molecular structures of three spleen-derived mouse IgA monoclonal human blood group-specific supernatants was undertaken; this was part of an evaluation of these monoclonals as blood typing reagents. The monoclonal antibodies were eluted through a precalibrated size-exclusion column, and fractions were analyzed by immunoblotting, heavy and light chain-specific enzyme-linked immunosorbent assay, and liquid- and solid-phase serologic tests. Results indicated that one of the supernatants (anti-A specificity) contained tetrameric and monomeric forms of IgA, while the other two (anti-A,B specificity) contained three higher polymeric forms (1000-4000 kDa) and one dimeric form. The tetrameric and polymeric forms showed red cell agglutinating activity, whereas the dimeric and monomeric forms did not. All forms contained heavy and light chains. The monomeric anti-A showed specific binding to appropriate red cells in a solid-phase assay, but the dimeric anti-A,B fractions did not. Purified fractions stored at 4 degrees C did not show any equilibration toward other forms, which indicated that the molecules are stable once secreted. The use of such antibodies as blood grouping reagents requires careful monitoring to ensure that high proportions of nonagglutinating molecular weight forms are not produced, as they could compromise the performance of the reagent by binding to red cell antigen in competition with the agglutinating forms.

New monoclonal antibodies in CD59: use for the analysis of peripheral blood cells from paroxysmal nocturnal haemoglobinuria (PNH) patients and for the quantitation of CD59 on normal and decay accelerating factor (DAF)-deficient erythrocytes.

CD59 is a widely expressed cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein which acts as an inhibitor of the assembly of the membrane attack complex of autologous complement. Four new monoclonal antibodies to CD59 (2/24, 1B2, BRIC 229, BRIC 257) are described. Competitive binding experiments using these antibodies, two known CD59 antibodies (MEM-43, YTH 53.1) and a previously described antibody LICR-LON-Fib75.1 demonstrated that all seven antibodies see related epitopes on human erythrocyte CD59. In common with other GPI-linked proteins, CD59 (as defined by antibody 2/24) was sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) on lymphocytes and monocytes but not on erythrocytes. Flow cytometric analysis using antibody 2/24 identified two populations (CD59 positive and CD59 deficient) of lymphocytes, monocytes and erythrocytes in peripheral blood from a patient with paroxysmal nocturnal haemoglobinuria (PNH). The abundance of CD59 on normal erythrocytes was determined as 21,000 copies/cell when radioiodinated BRIC 229 was used. Other CD59 antibodies gave values of 10,000 (IF5) and 15,000 (2/24) against the same target cells. Radioiodinated Fab fragments of BRIC 229 gave a value of 39,000 copies/cell. Erythrocytes from two individuals with a rare inherited deficiency of decay accelerating factor (DAF), known as the Inab phenotype, expressed normal levels of CD59.

New monoclonal antibodies in CD44 and CD58: their use to quantify CD44 and CD58 on normal human erythrocytes and to compare the distribution of CD44 and CD58 in human tissues.

The cell-surface glycoproteins CD44 and CD58 are involved in cell adhesion reactions. In this paper 12 monoclonal antibodies in CD44 and two in CD58 are described. Competitive binding assays using CD44 antibodies identified three distinct epitope groups. Antibodies in Group 1 and, with one exception (BRIC 214), antibodies in group 2, but not antibodies in Group 3, recognized epitopes that are sensitive to reduction and to trypsin or chymotrypsin treatment of intact erythrocytes, and so these epitopes probably reside on the N-terminal disulphide-bonded domain of CD44. Antibodies in CD44 did not inhibit the binding of CD58 antibodies to erythrocytes or vice versa. Quantitative binding studies using radioiodinated IgG measured 1888-5592 copies of CD44 and 1772-3290 copies of CD58 on normal erythrocytes. Similar measurements with radioiodinated Fab fragments gave values of 6508-10,450 (CD44) and 3457-7622 (CD58). Immunocytochemical studies indicated that CD44 is much more widely expressed in non-haemopoietic tissues than CD58. Comparison with previously described CD44 antibodies suggests that antibodies in our Group 1 encompass Hermes 2 and that those in Group 2 encompass Hermes 1. All the CD44 antibodies gave weakened reactions with Lu(a-b-) erythrocytes of the In(Lu) type by one or more methods. BRIC 214 and antibodies in epitope Group 3 were used to demonstrate that CD44 on these variant cells gives membrane-bound trypsin and chymotrypsin cleavage fragments of similar molecular weight to those obtained with normal erythrocytes.

Mechanism of cryoprecipitation of anti-blood group B murine monoclonal antibodies.

In our experience, some examples of mouse monoclonal antibodies of anti-B blood group specificity develop a precipitate when stored at 4 degrees C. This poses problems during the preparation of blood grouping reagents containing anti-B, and in the storage and use of such reagents. Here we show that this problem can be circumvented by alteration of the glycan moiety of the secreted immunoglobulin, either by glycosidase treatment of the partially purified immunoglobulin, or by the addition of glycan processing inhibitors to the hybridoma cell cultures. These findings have importance for the manufacture of monoclonal antibodies, and highlight a possible new role for carbohydrate in immunoglobulin interaction and immune complex formation.

Studies on human red-cell membrane glycophorin A and glycophorin B genes in glycophorin-deficient individuals.

1. Genomic DNA derived from individuals who lack glycophorin A (GPA), glycophorin B (GPB) or both of these proteins was subjected to Southern-blot analysis using GPA and GPB cDNA probes. 2. Bands on the Southern blots were assigned to the GPA gene, GPB gene or to a putative pseudogene. 3. Genomic DNA derived from an individual of the Mk phenotype was shown to have deletions in the GPA and GPB genes. The simplest model for the results obtained is that a single deletion spans the GPA and GPB genes in the individual studied.

Studies on the defect which causes absence of decay accelerating factor (DAF) from the peripheral blood cells of an individual with the Inab phenotype.

1. We have studied the peripheral blood cells of an individual with the Inab phenotype who is deficient in decay accelerating factor (DAF). 2. In contrast with the situation in paroxysmal nocturnal haemoglobinuria, membranes from peripheral blood cells of the Inab phenotype individual lack DAF, but retain the other glycosylphosphatidylinositol-linked proteins acetylcholinesterase and LFA-3. 3. Unlike normal Epstein-Barr-virus-transformed lymphoblastoid cell lines (EBV-LCL), DAF was not expressed on EBV-LCL derived from peripheral blood lymphocytes of the Inab individual. 4. No differences in the DAF gene of normal and Inab phenotype individuals could be detected by Southern blotting studies. 5. EBV-LCL derived from the Inab individual had a gross reduction in the level of DAF mRNA compared with normal EBV-LCL. 6. Our results suggest that the DAF gene in the Inab phenotype contains a mutation which affects the transcription or processing of DAF mRNA.

The Ina and Inb blood group antigens are located on a glycoprotein of 80,000 MW (the CDw44 glycoprotein) whose expression is influenced by the In(Lu) gene.

The Ina and Inb blood group antigens were found to be located on an erythrocyte membrane glycoprotein of 80,000 MW by immunoblotting with human anti-Ina and anti-Inb antibodies under non-reducing conditions. This glycoprotein is shown here to be identical to that defined by monoclonal antibodies to CDw44, and a new murine monoclonal antibody (BRIC 35) is added to this cluster. Experiments with endo-beta-galactosidase and Endo F preparations suggest that the glycoprotein contains one or more N-glycans but that these oligosaccharides do not contain extensive poly-N-acetyllactosaminyl sequences. Experiments using membranes prepared from sialidase-treated normal erythrocytes, from Tn erythrocytes and from Cad erythrocytes suggest that the glycoprotein does not contain a substantial content of O-glycans. The Inb antigen and the epitope defined by a murine monoclonal antibody (BRIC 35) show reduced expression on Lu(a-b-) erythrocytes which result from the effect of the dominant inhibitor gene In(Lu). Evidence is presented here that the Inb antigen is expressed on normal granulocytes and lymphocytes and on the haemopoietic cell lines HEL, K562 and HL-60, a lymphoblastoid cell line and lymphocytes from two patients with B-CLL.

Monoclonal antibodies that recognize different membrane proteins that are deficient in Rhnull human erythrocytes. One group of antibodies reacts with a variety of cells and tissues whereas the other group is erythroid-specific.

1. Rhnull human erythrocytes lack all of the antigens of the Rh and LW blood group systems and have abnormal shape and an increased osmotic fragility. In this paper two murine monoclonal antibodies raised against intact human erythrocytes were used to investigate further the abnormalities in these cells. BRIC 125 reacts weakly with Rhnull erythrocytes and BRIC 69 does not react at all. The results showed that BRIC 125 reacts with a component of Mr 47,000-52,000 which has a substantial content of N-glycans. In contrast, BRIC 69 reacted with a band of Mr 31,000 together with a very diffuse band of Mr 35,000-52,000. Treatment of BRIC 69 immunoprecipitates with endoglycosidase F/peptidyl-N-glycosidase F resulted in the loss of both BRIC 69 reactive components and the appearance of a new band of Mr similar to that of the Rh(D) polypeptide. 2. BRIC 125 had a broad reactivity with cells in peripheral blood, whereas the reactivity of BRIC 69 was confined to erythrocytes. BRIC 125, but not BRIC 69, reacted with human kidney tissue and bound to endothelium in peritubular capillaries, arteries and veins as well as the epithelial tissue of distal tubules. BRIC 125 stained haemopoietic cells, foetal hepatocytes and megakaryocytes in foetal liver and sinusoidal cells, hepatocytes and portal tracts in adult liver. In contrast, BRIC 69 reactivity was confined to haemopoietic cells in foetal liver. The BRIC 125 epitope has a wide tissue distribution, suggesting the occurrence of a related group of polypeptides which have a general functional role on cell surfaces. 3. Rhnull erythrocytes are deficient in at least four different membrane polypeptides.

Genetic variants of human red-cell membrane sialoglycoprotein beta. Study of the alterations occurring in the sialoglycoprotein-beta gene.

We have studied the DNA of individuals who express an altered sialoglycoprotein beta on their red cells by using Southern blotting with sialoglycoprotein-beta cDNA probes. Individuals of the Leach phenotype do not express any beta (sialoglycoprotein beta) or gamma (sialoglycoprotein gamma) on their red cells, and we show that about 7 kb of DNA, including the 3' end of the beta gene, is deleted in this DNA. Any protein product of this gene is likely to lack the membrane-associating domain of beta. We have also examined the DNA of two types of other individuals (Yus-type and Gerbich-type) who have red cells that lack beta and gamma, but contain abnormal sialoglycoproteins related to beta. These two types of DNA contain different internal deletions of about 6 kb in the beta gene. We suggest that these deletions result from the presence of two different sets of internal homology in the beta gene, and on this basis we propose structures for the abnormal Yus-type and Gerbich-type sialoglycoproteins which are consistent with the other evidence that is available. We provide evidence that beta and gamma are products of the same gene and suggest a possible mechanism for the origin of gamma based on leaky initiation of translation of beta mRNA.