ABH and Lewis antigens of tracheal glands. II. Lewis negative individuals

Immunocytochemical studies were performed on tracheal wall samples embedded in paraffin; the samples were taken at 23 autopsies. In all cases, the red cells had been typed in postmortem serological studies as being Le(a-b-). Blood-group antigens were demonstrated by the indirect immunoperoxidase technique, using monoclonal Anti-A, Anti-B, Anti-Lea and Anti-Leb; H was detected by UEA 1. The secretor characteristics could clearly be diagnosed from the ABH staining pattern of the mucous glands. In 11 cases, the lewis antigen labeling patterns were identical to the group of Lewis-positive individuals. It seems probable, from the statistical point of view, that these 11 individuals were, in fact, Lewis-positive and that the negative serology resulted from deterioration of the cadaver blood samples. The immunocytochemistry was quite different in the remaining 12 cases: (a) secretors (n = 9) were completely negative for Lea, Leb was equally negative in one case, but in the remainder it was detectable within mucous epithelia in minimal amounts and in an atypical granular distribution; (b) nonsecretors (n = 3) reversely exhibited complete negativity for Leb but a minimal staining for Lea. These findings are in harmony with the well established Lewis serology typing of secretions in Lewis negative individuals. Thus, a minimal Lewis antigen biosynthesis and secretion seem to occur in the absence of the Le gene: A alpha-4-L-fucosyltransferase of low activity might be the product of the allele le.     

Light-microscopic examination of ABH and Lewis antigens in human tracheal and epiglottic glands using the avidin-biotin-peroxidase complex technique

The localization of ABH and Lewis antigens was examined in formalin-fixed, paraffin-embedded human tracheal and epiglottic glands using monoclonal anti A, B, H, Lea and Leb antibodies. The mucous cells of the glands showed reactivity with antibodies corresponding to the respective ABO blood groups of the tissue donors. The mucous cells from one blood group A, Le(a-b-) individual showed no reactivity with any antibodies and those from another blood group A, Le(a-b-) individual showed reactivity only with anti A antibody. In individuals from blood group Le(a + b-) of all ABO groups, the mucous cells reacted exclusively with anti Lea. In blood group O, Le(a-b+) individuals, the mucous cells showed intense reaction with anti H and Leb antibodies and weak to moderate reactivity with anti Lea. In Le(a-b+) individuals of A1, B and A1B blood groups, the mucous cells showed strong reactivity with anti A and/or B antibodies, moderate with anti Leb, weak or no activity with anti Lea and absent with anti H. In blood group A2 Le(a-b+) individuals, the mucous cells stained with anti A were weakly stained or completely unstained with anti H antibody, but cells negative with anti A gave strong positive reactions with anti H antibody.     

Cytochemical detection of ABH antigens in human body fluids

The use of the peroxidase-anti-peroxidase (PAP) technique has been described previously for the detection of cellular antigens and in particular ABO antigens from tissue samples (Pedal and Hülle 1984; Pedal and Baedeker 1985; Pedal et al. 1985). In this survey, the PAP method has been employed to study the detection of ABO antigens in cells from body fluids of particular interest to forensic science, namely buccal cells and vaginal cells. Also tested, but in a limited number, were mixtures of body fluids and semen samples. No false reactions were obtained from buccal cells, all samples corresponding to the ABO blood type of the donor. Preliminary results from vaginal cells, vaginal/buccal cell mixtures, and semen were encouraging but must be treated with caution due to the limited number tested. Vaginal smears contaminated with semen showed varying degrees of nonspecificity.     

Immunocytochemical determination of ABH and MN antigens on dried blood traces in the nanoliter range.

The absorption-elution test and the mixed cell agglutination reaction are both ultimately based on the ability of indicator cells to agglutinate. This agglutination reaction requires a relatively large amount of antigenic epitopes, and, in addition, a relatively high volume of blood traces. The immunocytochemical demonstration of epitopes requires a lower volume, which, however must be fixed for investigation, thus possibly causing damage to the epitopes and thereby preventing detection. An immunocytochemical method is presented which permits ABH and MN antigen determination on dried blood traces of nanoliter quantities without special fixation. This method is based on immunocytochemical demonstration of antigens directly on the cell membrane in combination with the use of a coated glass slide that ensures maximum economy of epitopes.     

Immunochemical and chromatographic determination of ABH antigens in compact bone tissue

The identification of ABH antigens from compact bone tissue is known from many sources. The purpose of this study was to make a contribution to the localization of blood-group-active substances in compact bone tissue. A variety of preparation and identification methods were successfully used and compared. Samples were extracted from compact bone tissue, separated by HPTLC, and examined using the absorption-elution and PAP techniques. Additionally, the PAP technique was carried out on cryostat sections. Serologically active blood-group substances were consistently demonstrated in the organic components of the haversian canals.     

Evaluation of antisera for bloodstain grouping. I. ABH, MN, and Rh

Sixty-eight different commercially available blood grouping antisera and lectins with ABH, MN, and Rh D, C, E, c, and e specificities were serologically evaluated for their applicability to bloodstain antigen determination. The characteristics of the antisera were determined with red cells, with fresh bloodstains, and with series of aging bloodstains. The Rh antisera were tested under a variety of serological conditions and with bloodstains on various substrata. Additionally, studies on optimization of absorption-elution procedure variables were carried out, and some data on the storage characteristics of red cells and blood grouping antisera were gathered.     

Simultaneous identification and determination of species origin, ABH antigens and isoenzyme markers in the same bloodstain

A reliable procedure for the simultaneous identification of blood, determination of species origin and ABH antigens, and typing of isoenzyme markers from a sample of three 1-cm threads is described. Results obtained from known control as well as from casework bloodstains using this procedure were consistent with those obtained in parallel, conventional, individual tests under blind trial conditions.     

Detection of glycosphingolipids of ABH and Le antigens on thin-layer plates using the PAP method

Stroma from hemolyzed erythrocytes of blood groups 0, A1, B and A1B were obtained and subjected to butanol/phosphate buffer extraction. This extract was separated using HPTLC, and the ABH and Le substances were detected on the chromatogram using the PAP technique. The staining of the bands allowed specific demonstration of the serologically active glycosphingolipids present in the ABH and Le blood group substances. The antigens of the AB0 system showed a 3- to 12-band pattern. Each of the antigens Lea and Leb presented 3 bands. The slight differences in the levels of glycosphingolipids of equal chain lengths are probably due to differences in their chemical structures.     

The use of immunoenzyme analysis for determining ABH antigens in traces of saliva and sperm]

The authors suggest a simple sensitive technique for enzyme immunoassay (EIA) of ABH antigens in saliva and semen. A two-staged dot blot solid-phase EIA on nitrocellulose membranes was employed with anti-ABH monoclonal antibodies obtained in immunization of mice with human red cells. 4-chloro-1-naphthol substrate solution was used to visualize the peroxidase label. The results of analysis of salivary and spermatic samples obtained from donors of various groups evidence that this EIA variant may be useful in forensic medicine.     

Allele sharing in first-degree and unrelated pairs of individuals in the Ge F I AmpFlSTR Profiler Plus database

Eleven Italian forensic laboratories participated in a population study based on the AB Profiler Plus loci with proficiency testing. The validated database, including 1340 individuals, is available on-line. Tests for Hardy-Weinberg equilibrium, gametic unbalance, and heterogeneity of gene frequency were generally not significant. Gene frequencies at each locus were consistent with those of two previously published Italian studies, but different from a third. Individuals of each subsample were paired, and the total number of alleles shared across the nine loci was determined in each pair. The analysis was replicated over the total sample. In addition, two samples of mother-child pairs (N=315) and full-sib pairs (N=91) were subjected to allele sharing analysis. The resulting distributions were sufficiently distinct from the sample of unrelated pairs as to be of practical usefulness.     

Alleles responsible for ABO phenotype-genotype discrepancy and alleles in individuals with a weak expression of A or B antigens

ABO types obtained from evidentiary samples have been used effectively to obtain the initial information leading to the apprehension of culprits in Japanese criminal investigations. A simple ABO genotyping method using multiplex sequence-specific PCR and capillary electrophoresis was developed as a supplement to serological ABO typing. Limitations in predicting a phenotype based on genotype were evaluated using 1134 randomly selected Japanese peripheral blood samples. A concordance rate of 99.82% (1132/1134 samples) was found between genotypes and phenotypes defined as Groups A, B, AB, and O. Sequencing analysis revealed that one discrepant sample contained an O allele having a previously unreported point mutation at the primer binding site in exon 6, and another discrepant sample contained an O allele lacking the guanine deletion at nt 261 (the O301 allele). Therefore, the existence of such alleles must be given some consideration when predicting phenotype based on genotype.     

Distribution of A and B antigens in organs of blood group AB individuals: observations disclosed by a double immunoenzymatic labeling method

The localization of A and B antigens in the organs of blood group AB individuals has been studied using a double immunoenzymatic labeling method. Both A and B antigens were found in the various epithelial cells of these organs, but the epithelial cells could be classified into the following four types depending on the reaction pattern with anti-A and anti-B sera: type 1: cells that stained positive with both anti-A and anti-B sera; type 2: cells that stained positive with anti-A serum only; type 3: cells that stained positive with anti-B serum only; type 4: cells that were negative with both sera. The distribution of each of these epithelial cell types varied considerably, even in the same tissue and individual. Our results seem to suggest that a dissociation in the conversion to the A and B antigens occurs in the tissue of individuals belonging to blood group AB and that the degree of this dissociation varies from tissue to tissue and from cell to cell.     

A database of mitochondrial DNA hypervariable regions I and II sequences of individuals from Slovakia

In order to identify polymorphic positions and to determine their frequencies and the frequency of haplotypes in the human mitochondrial control region, two hypervariable regions (HV1 and HV2) of the mitochondrial DNA (mtDNA) of 374 unrelated individuals from Slovakia were amplified and sequenced. Sequence comparison led to the identification of 284 mitochondrial lineages as defined by 163 variable sites. Genetic diversity (GD) was estimated at 0.997 and the probability of two randomly selected individuals from population having identical mtDNA types (random match probability, RMP) for the both regions is 0.60%.     

Thymus and adrenal glands in elder abuse

Endogenous glucocorticoid-induced thymic involution is generally considered to be an important finding for determining child abuse. The present study investigated the weight of the thymus and the adrenal glands in elder abuse cases to identify a potential marker for elder abuse. There was no significant difference in the thymus and the adrenal weight between elder abuse and control cases. However, the elder abuse cases in which the duration of abuse was less than 3 months showed a significant increase in the adrenal weight in comparison to control cases. In such cases, histopathological findings showed a loss of intracellular light granules from the zona fasciculata, which might indicate a loss of cholesterol due to the overproduction of glucocorticoid. These results might imply that the elderly, who were maltreated for less than 3 months, were in the early phase of a long-term stress state during which stress-induced overproduction of glucocorticoid was observed in adrenal glands as indicated by Selye. Our results suggest that an increase in adrenal weight may be a potential marker for elder abuse of relatively short periods, especially less than a few months.     

Reliability of breath-alcohol analysis in individuals with gastroesophageal reflux disease.

Gastroesophageal reflux disease (GERD) is widespread in the population among all age groups and in both sexes. The reliability of breath alcohol analysis in subjects suffering from GERD is unknown. We investigated the relationship between breath-alcohol concentration (BrAC) and blood-alcohol concentration (BAC) in 5 male and 5 female subjects all suffering from severe gastroesophageal reflux disease and scheduled for antireflux surgery. Each subject served in two experiments in random order about 1-2 weeks apart. Both times they drank the same dose of ethanol (approximately 0.3 g/kg) as either beer, white wine, or vodka mixed with orange juice before venous blood and end-expired breath samples were obtained at 5-10 min intervals for 4 h. An attempt was made to provoke gastroesophageal reflux in one of the drinking experiments by applying an abdominal compression belt. Blood-ethanol concentration was determined by headspace gas chromatography and breath-ethanol was measured with an electrochemical instrument (Alcolmeter SD-400) or a quantitative infrared analyzer (Data-Master). During the absorption of alcohol, which occurred during the first 90 min after the start of drinking, BrAC (mg/210 L) tended to be the same or higher than venous BAC (mg/dL). In the post-peak phase, the BAC always exceeded BrAC. Four of the 10 subjects definitely experienced gastric reflux during the study although this did not result in widely deviant BrAC readings compared with BAC when sampling occurred at 5-min intervals. We conclude that the risk of alcohol erupting from the stomach into the mouth owing to gastric reflux and falsely increasing the result of an evidential breath-alcohol test is highly improbable.     

The detection of soluble ABH blood group substances in semen and saliva using monoclonal blood grouping reagents

Using an enzyme-linked immunosorbent assay (ELISA), this study investigated the use of monoclonal antibodies for detecting secreted ABH blood group substances in semen and saliva. The results demonstrated that the behavior of some monoclonals were unpredictable and often failed to detect the corresponding antigen in a number of the specimens tested. The suitability of the monoclonal reagents for detecting soluble blood group antigens could not be predicted by their behavior with red cell antigens. Consequently, care must be taken in the selection of monoclonal reagents for use in the detection of secreted blood group antigens.