Methodologic and genetic influence on immunohistochemical demonstration and semiquantitation of blood group antigen A in human ureter urothelium

In order to improve the accuracy and prognostic value of ABH blood group antigen loss in urothelial tumors, the effect of Lewis blood type and methodologic factors on detectability and distribution of blood group antigen A in human formalin-fixed, paraplast-embedded urothelium and endothelium was investigated by means of the Tween 20-modified indirect immunoperoxidase staining technique. Urothelium of Lea-b+ and Lea-b- individuals expressed significant higher amounts of blood group antigen A compared to urothelium of Lea+b- individuals. The expression on endothelial cells was related to vessel type and size, but not related to Lewis types. Compared to human anti-A, monoclonal anti-A demonstrated blood group antigen A with higher sensitivity and, due to reduced background staining, higher specificity. Consequently monoclonal anti-A detected blood group antigen A in the urothelium of Lea+b- individuals where human anti-A failed to stain, and different staining patterns became apparent. Both a two- to fourfold variation in the proportion between tissue section area and volume, and the volume of anti-A applied induced minor changes in sensitivity and specificity. The monoclonal anti-A method and knowledge about erythrocyte Lewis types might prove valuable in evaluating changes in blood group antigen-A expression in urothelial tumors.     

The role of immunoperoxidase staining in diagnostic cytology

A survey was made of the immune staining characteristics of 60 malignant neoplasms. Cytologically positive smears from each case were tested against a panel of six antibodies (alpha-antichymotrypsin, carcinoembryonic antigen, cytokeratin, desmin, vimentin and S-100 protein). The smears were decolorized and stained with polyclonal sera using the standard avidin-biotin immunoperoxidase procedure. In selected cases, the application of Diatex compound for partition of smears was necessary to obtain optimal results. Most staining reactions reflected the histogenesis of the neoplasms. However, more than one of five reactions was nonconclusive due to background staining, scanty cellularity or poor cytoplasmic preservation; furthermore, the 238 reactions scored as positive or negative included 33 unexpected positives and 15 unexpected negatives. In a series of 20 additional cases, selective immunoperoxidase staining was used in an attempt to solve specific diagnostic problems; the results in 13 of 15 cases with conclusive staining agreed with the cytologic impression. It is concluded that standard immunoperoxidase techniques can contribute to the solution of certain diagnostic problems in cytology; however, the results should be interpreted with caution and with full knowledge of the limitations of the technique.     

The occurrence of the feline A blood group antigen on lymphocytes

Sera from 300 cats were tested for the presence of anti-lymphocytic antibodies. One hundred and nineteen sera showed some activity with the majority (79) reacting only with lymphocytes from blood group A cats. Absorption of two such sera with A, AB and B erythrocytes and absorption of AB system reagents with lymphocytes from A and B blood group cats demonstrated that the A antigen is expressed on both erythrocytes and lymphocytes. Blood group and lymphocyte typing tests of foetuses indicated that the A antigen is present on these tissues as early as 46 days gestation. The erythrocytic B antigen could not be demonstrated on lymphocytes although a single antiserum, which reacted against lymphocytes from group B cats, was found. Several sera containing anti-lymphocytic antibodies which were not related to the AB type were also detected.     

The occurrence of the feline A blood group antigen on lymphocytes

Sera from 300 cats were tested for the presence of anti-lymphocytic antibodies. One hundred and nineteen sera showed some activity with the majority (79) reacting only with lymphocytes from blood group A cats. Absorption of two such sera with A, AB and B erythrocytes and absorption of AB system reagents with lymphocytes from A and B blood group cats demonstrated that the A antigen is expressed on both erythrocytes and lymphocytes. Blood group and lymphocyte typing tests of foetuses indicated that the A antigen is present on these tissues as early as 46 days gestation. The erythrocytic B antigen could not be demonstrated on lymphocytes although a single antiserum, which reacted against lymphocytes from group B cats, was found. Several sera containing anti-lymphocytic antibodies which were not related to the AB type were also detected.     

The indirect effect of hemoglobin on the electrophoretic mobility of human blood erythrocytes

Using the factor analysis, it was shown that the total content of hemoglobin in human blood plays a limited and indirect role in the regulation of average electrophoretic mobility of erythrocytes. In this case, it is not the only parameter governing this level. The statistical relationship between the total content of hemoglobin and erythrocyte mobility in electrical field is not stable and is determined by the dependence of both indices on their common factor of control of erythroid homeostasis.     

Human ferritin: effects of antigen source and fixation on leucocyte staining by immunoperoxidase technique

The localization of ferritin was studied in peripheral blood cells and variously fixed tissues with the antibodies against ferritins isolated from human heart and spleen. The unlabelled antibody enzyme method (PAP) was used to detect the binding sites of antibodies. In peripheral blood cell smears both antisera gave rise to strong staining of polymorphonuclear (PMN) cell cytoplasm, whereas the monocytes stained relatively weakly. There were no staining differences between the two antisera. In human spleen sections the spleen ferritin antiserum stained the PMN cells and sinusoidal lining cells, whereas the heart ferritin antiserum stained only PMN cells. Neither of the two antisera stained monocytes in the spleen sections. This finding was observed in specimens fixed in Bouin's fixative, Baker's fixative and neutral formalin. However, the immunoreactivity of ferritin was totally destroyed by some other fixatives (Carnoy's fixative, formol sucrose and glutaraldehyde). These results suggest that ferritin is more readily released from monocytes than from PMN cells, and that mature spleen macrophages contain antigenic determinants of ferritin that are recognized only by anti-spleen ferritin antiserum.     

Rapid diagnosis of cytomegalovirus infections by direct immunoperoxidase staining with human monoclonal antibody against an immediate-early antigen.

Direct immunoperoxidase technique using a horseradish peroxidase (HRP)-conjugated Fab' fragment of human monoclonal antibody (humab C7), designated HRP-C7, was evaluated as a rapid diagnosis of cytomegalovirus (CMV) infection. A total of 138 clinical specimens consisting of 124 urine samples and 14 oral swabs were examined for CMV by the direct HRP-C7 staining in comparison with conventional virus isolation. The number of CMV-positive samples by each method was 40 (29.0%) for the former and 37 (26.8%) for the latter, respectively. By HRP-C7 staining, CMV was identifiable within 24 hr after inoculation. By conventional isolation method, an average of 10.3 days had passed before cytopathic effect characteristic of CMV appeared in the cell culture. Some false-positive and false-negative cases were discussed in relation to toxicity of urine samples, storage of the samples, and amount of CMV in the sample. The sensitivity and specificity of HRP-C7 method against conventional isolation method were 89.2% and 93.1%, respectively. Thus, HRP-C7 staining is useful for a rapid diagnosis of CMV infections.     

Demonstration of fibronectin in human articular cartilage by an indirect immunoperoxidase technique

Summary Fresh frozen tissue sections of human articular cartilage was treated without and with human testicular hyaluronidase (2×10⁶ units/l) for 60 min at 37° C and stained by the indirect immunoperoxidase technique with rabbit antihuman fibronectin. The rabbit antihuman fibronectin was purified by affinity chromatography on human fibronectin-Sepharose. Fibronectin was only found on the acellular surface of the articular cartilage in tissue sections not treated with hyaluronidase. In this surface layer, probably identical to lamina splendens, the arrangement of fibronectin was as a membrane. No collagen was seen in this area by van Gieson staining. No staining for fibronectin was found in the cartilage matrix or in the chondrocytes. Treatment of the cartilage tissue with hyaluronidase resulted in visualization of high amount of fibronectin in the cartilage matrix, with the highest intensity around the chondrocytes. The staining of the acellular surface layer of the articular cartilage was identical with the results obtained without hyaluronidase treatment. These results indicate that articular cartilage is rich in fibronectin probably in complex with hyaluronic acid, and that the chondrocytes produce fibronectin in situ. It also demonstrates the steric hindrance of hyaluronic acid aggregates in diffusion of the antibody and the value of hyaluronidase treatment of tissue before demonstration of fibronectin.     

X-ray induction of micronuclei in human lymphocyte subpopulations differentiated by immunoperoxidase staining.

In this study we sought to confirm the radiosensitivity of human peripheral blood lymphocyte subpopulations using a micronucleus assay. Mononucleated cells isolated from peripheral blood were irradiated with X rays. After being cultured for 3 days, cells were fixed and stained using the immunoperoxidase staining technique. Lymphocyte subpopulations were characterized by means of the monoclonal antibodies Leu4 (CD3), Leu2a (CD8) and Leu19 (CD56). Dose-response curves were obtained by scoring the number of micronuclei in binucleated cells that reacted with a specific antibody and were then stained. The dose response of CD8+ (suppressor/cytotoxic) cells was quite similar to that of CD3+ (pan T) cells. In comparison, CD56+ (natural killer) cells were significantly less sensitive, although scorable binucleated CD56+ cells made up less than 4% of the total number of binucleated cells.     

Glycolipid- and glycoprotein-based blood group A antigen expression in human thrombocytes. A1/A2 difference

Total non-acid glycolipid fractions and total sodium dodecylsulphate (SDS) solubilized protein fractions were isolated from human thrombocytes obtained from single human donors having different blood group A₁/A₂ phenotypes. The blood group A glycolipid antigens were characterized by immunostaining of thin layer plates with different monoclonal anti-A antibodies. The glycoproteins carrying blood group A epitopes were identified by SDS-PAGE and Western blot analysis using a monoclonal anti-A antibody. Blood group A glycolipid antigens were found in both A₁ and A₂ thrombocytes but the A₂ individuals expressed at least ten times less A glycolipids compared to the A₁ individuals. Expression of A type 3/4 chain and small amounts of A type 1 chain glycolipids were seen in thrombocytes of both A₁ and A₂ individuals, while the type 2 chain A glycolipids appeared to be missing from the A₂ thrombocytes. Blood group A reactive glycoproteins were only found in thrombocytes of A₁ individuals and could not be detected in A₂ individuals or a blood group O individual. The major blood group A glycoprotein were found as a double band migrating in the 130 kDa region.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPTLC high performance thin layer chromatography - CBB Coomassie brilliant blue - GVH graft versus host Part of this work was presented at the Xth International Symposium on Glycoconjugates, Jerusalem, Israel. September, 1989.In the short hand designation for glycolipids, the letter indicate blood group determinant, the first numeral, the number of sugar residues, and the second numeral, the type of carbohydrate chain. Thus, A-6-1 means a hexaglycosylceramide with a blood group A determinant based on the type 1 carbohydrate chain.     

The blood group U antigen is not located on glycophorin B

Erythrocytes from twelve individuals with the S-s-U- phenotype and from ten with the S-S-U+ phenotype were analyzed and compared to control cells with S+/s+U+ determinants. No red cell abnormality was detected in S-s-U+ or S-s-U- carriers. Sialic acid content was similar (P greater than 0.05) for S-s-U+ and S-s-U- erythrocytes (74.6 +/- 7.14 and 71.4 +/- 8.53 nmol/10(9) red blood cells, respectively) but significantly less (P less than 0.001) than controls with 89.5 +/- 11.4 nmol/10(9) red blood cells, n = 16. Fluorographs of SDS-polyacrylamide gels showed no glycophorin B in membranes from S-s-U+ and S-s-U- erythrocytes labeled with NaB3H4. Glycophorins were extracted from red cell membranes in chloroform/methanol, labeled with 125I and analyzed by SDS-polyacrylamide gel electrophoresis. Periodic acid Schiff stain and autoradiographs of these gels also showed absence of glycophorin B in both S-s-U+ and S-s-U- cells. These findings suggested that the U antigen is not located on glycophorin B. This hypothesis was tested by determining blood group antigenicity on red cell membranes and on extracted sialoglycoproteins by the hemagglutination inhibition technique. Although U and S/s activities were detected in control red cell membranes, extracted glycoproteins demonstrated S/s activity but no U activity. Together the data indicate that both S-s-U+ and S-s-U- erythrocytes lack glycophorin B and that the U antigen is not located on glycophorin B. This deletion does not seem to affect the structure-function of the red cell.     

Solubilisation of human erythrocyte band 4.1 protein in the non-ionic detergent Tween 20

Extraction of spectrin-depleted erythrocyte membranes with the non-ionic detergent Tween 20, in a 0.1 M glycine-NaOH buffer (pH 9.8) leads to the solubilization of band 4.1 and the sialoglycoproteins. The comigration of band 4.1 with the sialoglycoproteins in gel filtration and detergent-free electrophoresis indicated that these proteins may be associated as complexes of high molecular weight. Although treatment of intact membranes with Tween 20 under the same conditions does not lead to direct solubilization of proteins, severe disruption of the membranes was observed under phase contrast microscopy. Suspension of the treated membranes in 5 mM phosphate buffer (pH 8.0) leads to the solubilization of band 4.1, spectrin, actin and the sialoglycoproteins. High molecular weight complexes of band 4.1 and the sialoglycoproteins were isolated from these extracts, suggesting a possible interaction between band 4.1 and sialoglycoproteins which may be important for linking the cytoskeleton to the membrane.     

Structural properties of the human MN blood group antigen receptor sites.

It is shown that the MN blood group antigen determinant of the major human erythrocyte membrane (MN) sialoglycoprotein is located on its N-terminal octaglycopeptide. The only analytically detectable difference between peptides from MM and NN cells are Ser/Leu and Gly/Glu polymorphisms at the first and fifth positions, respectively. Destruction of the antigens by removal of the N-terminal residues suggests that these amino acids represent a part of the receptor areas for various anti-M or -N reagents. Evidence is presented that the N-terminal structure of the Ss glycoprotein is identical with that of MN glycoprotein from NN red cells up to the fifth residue. This provides an explanation for the 'N' antigen on this molecule and direct support for the earlier proposal that the MNSs locus is represented by homologous genes.     

The influence of fixation on immunoperoxidase staining of plasmacells in paraffin sections of intestinal biopsy specimens

Summary The influence of different fixation methods on the results of immunoperoxydase staining of immunoglobulin and gastrin producing cells in gastric and duodenal mucosa was investigated. An indirect method was used on paraffin sections. It appeared that that fixatives containing sublimate gave the most consistent results, a sublimate-formaldehyde mixture being the best.