Glycophorin A-based exclusion of red blood cells for flow cytometric analysis of platelet glycoprotein expression in citrated whole blood.

Objectives Analysis of platelet glycoprotein (GP) expression by flow cytometry is applied for diagnostic confirmation of GP-associated thrombocytopathies. While platelet-rich plasma may be used for distinct identification of target events, this strategy is not feasible for small sample volumes or for patients showing low platelet counts and/or giant platelets. However, also the use of whole blood (WB) is hampered by the difficulty to discriminate platelets from red blood cells (RBC) in such patients. To circumvent these limitations, we evaluated the feasibility of a RBC gating-out strategy. Methods In addition to platelet GPIb, GPIIa/IIIa, as well as P-selectin (CD62P), citrated whole blood (CWB) samples were stained for RBC-specific glycophorin A (CD235a). CD235a-negative platelet events were further discriminated by forward-/side-scatter characteristics and platelet GP expressions analyzed relative to that of a healthy control sample processed in parallel. Results Established reference intervals allowed for clear identification of decreased GPIIb/IIIa- or GPIb expression pattern in samples of patients with confirmed Glanzmann thrombasthenia or Bernard-Soulier syndrome, respectively. It could be shown that the analysis of 2,500 platelet events is sufficient for reliable GP expression analysis, rendering the proposed method applicable to samples with low platelet counts. Conclusions This study demonstrates the feasibility of CD235a-based exclusion of RBC for platelet GP expression analysis in CWB. In contrast to direct staining of platelet-specific antigens for target identification, this indirect gating out approach is generally applicable independent of any underlying platelet GP expression deficiency.

A case report on anti-Mia antibody in a multi-transfused patient from India.

GP.Mur antigen belongs to the MNSs system and the corresponding antibody is called as anti-Mia antibody. Anti-Mia antibody is a clinically significant antibody capable of causing haemolytic disease of the new born (HDFN) and intravascular haemolytic transfusion reactions. Literature on anti-Mia antibody from India is very limited. We report here a case of anti-Mia antibody in a multi-transfused patient from India.

Red blood cell antibody-induced anemia causes differential degrees of tissue hypoxia in kidney and brain.

Moderate anemia is associated with increased mortality and morbidity, including acute kidney injury (AKI), in surgical patients. A red blood cell (RBC)-specific antibody model was utilized to determine whether moderate subacute anemia could result in tissue hypoxia as a potential mechanism of injury. Cardiovascular and hypoxic cellular responses were measured in transgenic mice capable of expressing hypoxia-inducible factor-1α (HIF-1α)/luciferase activity in vivo. Antibody-mediated anemia was associated with mild intravascular hemolysis (6 h) and splenic RBC sequestration ( day 4), resulting in a nadir hemoglobin concentration of 89 ± 13 g/l on day 4. At this time point, renal tissue oxygen tension (PtO2) was decreased in anemic mice relative to controls (13.1 ± 4.3 vs. 20.8 ± 3.7 mmHg, P < 0.001). Renal tissue hypoxia was associated with an increase in HIF/luciferase expression in vivo ( P = 0.04) and a 20-fold relative increase in renal erythropoietin mRNA transcription ( P < 0.001) but no increase in renal blood flow ( P = 0.67). By contrast, brain PtO2 was maintained in anemic mice relative to controls (22.7 ± 5.2 vs. 23.4 ± 9.8 mmHg, P = 0.59) in part because of an increase in internal carotid artery blood flow (80%, P < 0.001) and preserved cerebrovascular reactivity. Despite these adaptive changes, an increase in brain HIF-dependent mRNA levels was observed (erythropoietin: P < 0.001; heme oxygenase-1: P = 0.01), providing evidence for subtle cerebral tissue hypoxia in anemic mice. These data demonstrate that moderate subacute anemia causes significant renal tissue hypoxia, whereas adaptive cerebrovascular responses limit the degree of cerebral tissue hypoxia. Further studies are required to assess whether hypoxia is a mechanism for acute kidney injury associated with anemia.

The MNS glycophorin variant GP.Mur affects differential erythroid expression of Rh/RhAG transcripts.

BACKGROUND: The band 3 macrocomplex (also known as the ankyrin-associated complex) on the red cell membrane comprises two interacting subcomplexes: a band 3/glycophorin A subcomplex, and a Rh/RhAG subcomplex. Glycophorin B (GPB) is a component of the Rh/RhAG subcomplex that is also structurally associated with glycophorin A (GPA). Expression of glycophorin B-A-B hybrid GP.Mur enhances band 3 expression and is associated with lower levels of Rh-associated glycoprotein (RhAG) and Rh polypeptides. The goal of this study was to determine whether GP.Mur influenced erythroid Rh/RhAG expression at the transcript level. MATERIALS AND METHODS: GP.Mur was serologically determined in healthy participants from Taitung County, Taiwan. RNA was extracted from the reticulocyte-enriched fraction of peripheral blood, followed by reverse transcription and quantitative PCR for RhAG, RhD and RhCcEe. RESULTS: Quantification by real-time PCR revealed significantly fewer RhAG and RhCcEe transcripts in the reticulocytes from subjects with homozygous GYP*Mur. Independent from GYP.Mur, both RhAG and RhD transcript levels were threefold or higher than that of RhCcEe. Also, in GYP.Mur and the control samples alike, direct quantitative associations were observed between the transcript levels of RhAG and RhD, but not between that of RhAG and RhCcEe. CONCLUSION: Erythroid RhD and RhCcEe were differentially expressed at the transcript levels, which could be related to their different degrees of interaction or sensitivity to RhAG. Further, the reduction or absence of glycophorin B in GYP.Mur erythroid cells affected transcript expressions of RhAG and RhCcEe. Thus, GPB and GP.Mur differentially influenced Rh/RhAG expressions prior to protein translation.

The forensic application of the Glycophorin A on the Amussat's sign with a brief review of the literature.

Even if the Amussat's sign is known since the mid-19th century, few studies have been made in order to assess its real occurrence. In particular, the histopathologic examination of the Amussat's sign lacks in the medicolegal literature. The review of the literature shows indeed a significant range of variability (from 1.1 % up to 25 %) regarding the macroscopic detection of the Amussat's sign. In this study, the authors report that the identification of a vital Amussat's sign is important and may require the immunohistochemical staining for the Glycophorin A (a marker of vital reaction). The victim was a 63-year-old man, who was found suspended from the staircase with a rope. Both the carotid arteries were opened in situ by using fine scissors with blunt tips. A horizontal lesion (length 4 mm) of the intima of the left common carotid artery was documented. A sample was obtained; then, a standard post-fixative histopathologic examination and immunohistochemical staining for the Glycophorin A were carried out. The standard histopathologic examination only revealed the intimal laceration with a poor hemorrhagic infiltration. However, the immunohistochemical staining for the Glycophorin A allowed the clear identification of the hemorrhagic infiltration, which was documented both in the intimal laceration and in the periadventitial soft tissues. The immunohistochemical staining for the Glycophorin A can identify the vitality of an Amussat's sign. When an Amussat's sign is documented, the Glycophorin A may therefore help the forensic pathologist to differentiate a hanging death from a postmortem suspension of the body.

Identification of aged bloodstains through mRNA profiling: Experiments results on selected markers of 30- and 50-year-old samples.

Remarkable progress has been made in recent years on the research of body fluid identification through messenger RNA(mRNA) profiling. In order to examine the viability of mRNA profiling as a method to identify aged bloodstains, this study tested two groups of bloodstain samples, dated 30 years and 50 years back respectively, on seven blood specific markers, i.e. HBB, HBA, GYPA, CD93, ALAS2, SPTB (91bp and 247bp primers), and PBGD. Test results indicate that HBA and HBB are the most stable markers in aged bloodstains, returning positive results in over 80% of the 50-year-old samples and over 90% of the 30-year-old samples. This finding proves mRNA profiling an effective way of identifying aged bloodstains.

MINDEC-An Enhanced Negative Depletion Strategy for Circulating Tumour Cell Enrichment.

Most current methods of circulating tumour cell (CTC) enrichment target the epithelial protein EpCAM, which is commonly expressed in adenocarcinoma cells. However, such methods will not recover the fraction of CTCs that have a non-epithelial phenotype due to epithelial-mesenchymal transition. For phenotype-independent CTC enrichment, we developed a new enhanced negative depletion strategy-termed MINDEC-that is based on multi-marker (CD45, CD16, CD19, CD163, and CD235a/GYPA) depletion of blood cells rather than targeted enrichment of CTCs. Here we validated the performance of MINDEC using epithelial and mesenchymal cancer cell lines, demonstrating a mean recovery of 82 ± 10%, high depletion (437 ± 350 residual white blood cells (WBCs)/mL peripheral blood), linearity between spiked and recovered cells (correlation coefficient: r = 0.995), and a low detection limit (≥1 cell recovered in all four replicates spiked with 3 cells). For clinical validation of this method, we enumerated CTCs in peripheral blood samples from patients with metastatic pancreatic cancer, detecting CTCs in 15 of 21 blood samples (71%) from 9 patients. The promising performance of the MINDEC enrichment strategy in our study encourages validation in larger clinical trials.

Self-association of human erythrocyte glycophorin A. Appearance of low mobility bands on sodium dodecyl sulfate gels.

We have examined the self-association of glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, using sodium dodecyl sulfate (SDS) polyacrylamide gels and circular dichroism. Pure glycophorin A has a tendency to form multiple bands on SDS gels at positions of higher apparent molecular weight than the PAS 1 and PAS 2 bands previously seen. These high molecular weight bands do not have mobilities corresponding to integral polymers of PAS 1 and PAS 2. Circular dichroism spectra of solutions giving rise to these bands or to PAS 1 and PAS 2 bands alone, indicate that these species all have essentially the same peptide conformation.

The erythrocyte membranes in beta-thalassemia. Lower sialic acid levels in glycophorin.

The sialic acids content of glycophorin of thalassemic erythrocyte membranes is about 25% lower than in glycophorin of normal erythrocyte membranes. Glycophorin extracted from old thalassemic erythrocytes separated by density centrifugation, has about half the sialic acids content found in glycophorin extracted from young thalassemic erythrocytes. Possible sialidase activty was sought in the plasma and erythrocyte membranes of thalassemic erythrocytes. No increased sialidase activity was detected in the plasma of the patients as compared to that of normal donors. Thus, other sites for sialidase activity, or other possibilities have to be explored to account for the increased sialic acid hydrolysis of glycophorin of the thalassemic erythrocytes.

Red cell membrane sialoglycoprotein beta in homozygous and heterozygous 4.1(-) hereditary elliptocytosis.

Sialoglycoprotein beta, a minor sialoglycoprotein of the red cell membrane, was studied in homozygous and heterozygous 4.1(-) hereditary elliptocytosis, a variety of hereditary elliptocytosis characterized by total or partial absence of protein 4.1. Erythrocytes were treated with the periodic acid-NaB3H4 procedure. Following polyacrylamide gel electrophoresis in the presence of SDS, labelled sialoglycoproteins were revealed by fluorography. (i) In the ghosts from the 4.1(-) homozygote, sialoglycoprotein beta was sharply decreased. It is not sure whether the residual material is sialoglycoprotein beta itself, or a distinct sialoglycoprotein migrating in the same place. In long exposure fluorograms, sialoglycoprotein gamma (a sialoglycoprotein related to sialoglycoprotein beta) also turned out to be reduced. In the homozygote's Triton-shells, sialoglycoprotein beta and gamma appeared completely absent. (ii) In the 4.1(-) heterozygote, sialoglycoprotein beta appeared slightly reduced, whereas sialoglycoprotein gamma appeared normal. Both of these proteins were extracted in seemingly normal amounts in the Triton-shells. These observations bring further support to the view that there is an interaction between skeletal membrane protein 4.1 and sialoglycoprotein beta, that is additional to other interactions between the former protein and the lipid bilayer and/or other transmembrane proteins.

Biochemical studies of membrane glycoproteins during red cell aging.

(1) There is a significant decrease of ATPase activities in red cell membranes with increasing age of the donor. (2) A comparison of blood groups A and O relates the higher ATPase activities of young donors to blood group A. (3) Membrane ATPase activities reveal an age-dependent decrease in young and old donors with blood group A. (4) There is an age-dependent decrease of the N-acetylneuraminic acid concentration of glycophorin. (5) The galactose content of glycophorin only increases in blood group A. (6) There are no age-dependent changes of the exposed galactose. (7) The experiments so far reveal an increase of IgG absorption to old glycophorin.

Characterization of Glycophorin A and band 3 from Tn polyagglutinable erythrocytes.

The 2 types of erythrocytes from a person with persistent mixed-field polyagglutinability (Tn abnormality) were separated from each other by preparative cell electrophoresis. Surface labelling using the galactose oxidase/NaB3H4 technique followed by polyacrylamide gel electrophoresis showed a strong labelling in the glycophorin A region of Tn positive erythrocytes indicating exposed galactosyl N-acetyl/galactosaminyl residues. Tn positive cell membranes were labelled by the galactose oxidase/NaB3H4 technique and solubilized in non-ionic detergent. After chromatography on Helix pomatia lectin-linked Sepharose, glycophorin A was immunoprecipitated from the sugar eluate using specific antiserum. Glycophorin A from Tn negative cells and normal red blood cells did not bind to Helix pomatia lectin but to Lens culinaris lectin-Sepharose. Glycophorin A and band 3 were isolated by preparative gel electrophoresis from normal cells and the two red cell populations of the Tn individual. Pronase treatment of labelled glycophorin A followed by gel filtration revealed a more efficient proteolysis in molecules isolated from Tn positive cells. Mild alkaline treatment of galactose oxidase/NaB3H4 or periodate/NaB3H4 labelled glycophorin A liberated 3 different oligosaccharides from Tn positive cells. No significant difference was found between the oligosaccharides of band 3 protein from normal and Tn positive cells and the amounts of glycophorin A were identical in both cell types when determined by radioimmunoassay.

Beta and beta-related sialoglycoproteins in the human red cell membrane.

Recent studies on beta and beta-related sialoglycoproteins (SGPs) in the human red cell membrane are reviewed. These SGPs are transmembrane glycoproteins with conserved cytoplasmic domains that interact with the membrane skeleton via protein 4.1. The inheritance of these SGPs is described, and the possible locations of Gerbich (Ge2 and Ge3) and Wb blood group antigens reviewed.

Membrane glycophorins of Dantu blood group erythrocytes.

Glycophorins of erythrocytes of two unrelated individuals who exhibit the Dantu blood group phenotype were studied. Immunoblots indicated that erythrocytes of each individual contained a complement of a normal alpha-glycophorin (glycophorin A) and a variant N-glycophorin. delta-Glycophorin (glycophorin B) was present in one donor's cells but not the other's; the s and N phenotypes of the latter's erythrocytes may derive from the variant glycophorin. The variant glycophorin is of a smaller size, does not bind to Lens culinaris lectin agarose, and lacks residues approximately 40-60 of alpha-glycophorin and its single asparagine-linked carbohydrate; it contains approximately 2 less O-glycosidically bound units whose structures are identical to those found in alpha-glycophorins. All these properties are characteristic of delta-glycophorin. The variant is related to alpha-glycophorin in the carboxyl-terminal region as shown by reaction with a specific antiserum. Sequence analyses of a mixture of chymotryptic peptides of a CNBr fragment of the variant glycophorin identified the sequence Val-His-Arg-Phe-Thr-Val-Pro-Glu-Ile-Thr-Leu-Ile-Ile that contains the junction point of delta- and alpha-glycophorins spanning residues 33-38/39 of delta-glycophorin and residues 71/72-77 of alpha-glycophorin. Sequence analysis of a mixture of CNBr fragments allowed us to conclude that the variant originates from delta-s- rather than delta-S-glycophorin. The quantity of the variant Dantu glycophorin when compared to alpha-glycophorin differed in the two individuals, the ratio being 2/1 in one individual's cells and 0.5/1 in the other's. This may reflect that the two donors belong to different varieties of Dantu phenotypes. Together, the evidence indicates that both donors' erythrocytes contain a (delta-alpha) variant glycophorin, whose amino terminus originates from delta-s-glycophorin and the carboxyl end from alpha-glycophorin with a junction point around residues 39 of delta- and 71 of alpha-glycophorins. The results suggest that the unique junction region may be characteristic of the Dantu phenotype.

Erythrocyte membrane topo-optical staining reflects glycoprotein conformational changes.

The study is concerned with the relationship of induced birefringence to conformational alterations of glycocalyx glycoproteins. Human erythrocytes washed with phosphate-buffered saline (PBS) and fixed with glutaraldehyde exhibit the most intense topo-optical staining, which is thought to reflect the lipid bound state of glycophorins. Incubations at reduced pH, in hypotonic media or in the presence of procaine resulted in a significant decline of induced anisotropy. The effects were not due to degradation of glycocalyx constituents. Erythrocytes subjected to treatments with procaine or at pH 6x2 suffered from loss of electrophoretic velocity, a finding indicative of rearrangement of cell surface glycoproteins. by example of the red blood cell the findings of this study confirm the premised suggestion of the sensible detection by topo-optical toluidine blue staining of the conformational state of glycoproteins of the glycocalyx.

Topo-optical investigations of the human erythrocyte glycocalyx-age related changes.

The conformational state of the glycocalyx of the intact and altered erythrocyte membrane was studied by means of the topo-optical toluidine blue reaction, i.e. induced membrane birefringence. High membrane anisotropy represents the normal glycocalyx structure and its decline represents their perturbation. The results show that the glycocalyx structure is changed during ageing of the erythrocytes in vivo as well as in vitro. During fluid preservation, in vitro ageing and vesiculation of cells in vitro, a subpopulation of cells showed a decline of membrane anisotropy, but other cells demonstrated abnormally high values. In the latter cases, there is usually a correlation to spherocytes. From this point of view, it is to be assumed that spherogenesis during cell ageing is induced by cell vesiculation. This leads to a remodelling of an intact plasmalemma. In contrast, the cell fractions which are probably non-vesiculating seem to be more or less damaged by membrane and/or plasmic hydrolases. This can be mimicked by neuraminidase and protease treatment of erythrocytes in vitro. Membrane lesions caused by freeze preservation of red blood cells are rare. The topo-optical results are interpreted according to the assumptions of the theory of membrane anisotropy, i.e. the formation of dye-stuff micelles at distinct, clustered, sialylated carbohydrate chains of the glycophorin A.

Transient reduction in erythrocyte membrane sialoglycoprotein beta associated with the presence of elliptocytes.

Erythrocyte membranes from an anaemic patient receiving gold therapy for rheumatoid arthritis had reduced beta-sialoglycoprotein (beta-SGP) content but normal expression of sialoglycoproteins alpha, delta and gamma. Elliptocytes were present in the peripheral blood. The serum of the patient contained anti-beta-SGP which did not appear to bind to her own cells. It reacted with all erythrocytes apart from beta-SGP deficient Leach phenotype cells. The antibody was inhibited by purified beta-SGP from normal red cells, bound to beta-SGP on immunoblots and also reacted with the abnormal beta-related-SGP in erythrocyte membranes of both the Gerbich type and Yus type of Gerbich negative. Two years later the patient was no longer anaemic, no elliptocytes were seen in her peripheral blood film and her erythrocyte membranes had normal beta-SGP content. Antibody was no longer present in her serum and antibody from the earlier sample now reacted with the patient's erythrocytes. Erythrocyte membrane beta-SGP is known to be important in the maintenance of normal cell shape. It is likely that the transient occurrence of elliptocytes in the patient resulted from the concurrent temporary reduction in beta-SGP content of her erythrocyte membranes.

Anomalous clustering of underglycosylated band 3 in erythrocytes and their precursor cells in congenital dyserythropoietic anemia type II.

Congenital dyserythropoietic anemia type II (CDA II or HEMPAS) is a genetic anemia caused by membrane abnormality. Our previous studies indicated that in HEMPAS, erythrocytes band 3 and band 4.5 are not glycosylated by polylactosaminoglycans. The present study was aimed at determining how such underglycosylated band 3 behaves in erythrocyte membranes. By using anti-band 3 antibodies, immunogold electron microscopy revealed that band 3s are clustered in HEMPAS erythrocyte membranes. By freeze-fracture electron microscopy, band 3s were also seen as lightly clumped intramembrane particles on a protoplasmic fracture face. Erythrocyte precursor cells stained by anti-band 3 antibodies showed that band 3s are present in the cytoplasmic area of the reticulocytes as scattered single particles. However, in young erythrocytes in which intracellular membranes are almost degenerated, band 3s were clustered in the cytoplasmic area of the cell. These observations suggest that band 3s cluster before they are incorporated into the plasma membranes of HEMPAS erythrocytes. In contrast to band 3, glycophorin A detected by anti-glycophorin A antibodies did not show a noticeable difference between normal and HEMPAS. Such a clustering of band 3 may cause abnormal localization of band 3-associated proteins and may thus result in the macroscopic membrane abnormality seen in HEMPAS erythrocytes.

Erythroid blast crisis in chronic myelogenous leukemia.

Blood or bone marrow specimens from 22 patients with chronic myelogenous leukemia in blast crisis were studied for the surface expression of glycophorin-A, a marker for early erythroid differentiation. The leukemic blasts were stained with rabbit anti-glycophorin-A antiserum. The glycophorin-A molecules detected by the rabbit antiserum were identified by polyacrylamide slab gel electrophoresis of the immunoprecipitates from the membrane lysates of surface-labeled blasts. Blasts expressing surface glycophorin-A were found in 9 of the 22 patients. In 4 patients, almost all blasts were glycophorin-A positive, and in 5 patients, less than half of the blast population expressed glycophorin-A. The present study shows that when glycophorin-A is used as a marker for erythroid blasts, involvement of the erythroid lineage during blast crisis of chronic myelogenous leukemia seems to occur more frequently than previously recognized.

Structural analysis of the Ss sialoglycoprotein specific for Henshaw blood group from human erythrocyte membranes.

The N-terminal structures of the MN and Ss erythrocyte membrane sialoglycoproteins (glycophorins A, B) from two Henshaw (He) blood-group heterozygotes were determined by manual sequencing of tryptic glycopeptides and various secondary fragments. No structural alteration of the MN glycoprotein could be detected. The He-specific portion of the Ss glycoprotein was found to exhibit the N-terminal sequence Trp-Ser+-Thr+-Ser+-Gly-(+ = glycosylation). Thus it differs at three positions from its normal counterpart which possesses 'N' activity and exhibits the N-terminal structure Leu-Ser+-Thr+-Thr+-Glu-. Analysis of the Ss glycoprotein from 15 He-negative erythrocyte samples did not reveal any of the three He-specific structural alterations. The presence of a glycine residue at the fifth position of the blood-group-M-active MN glycoprotein as well as in the He-specific Ss glycoprotein provides an explanation for the occurrence of antisera (anti-Me) reacting with the M and He antigens.