A straightforward method for determination of the sevoflurane metabolite hexafluoroisopropanol in urinary occupational medical samples by headspace-gas chromatography mass spectrometry.

Biological monitoring of the unmodified sevoflurane and its metabolite hexafluoroisopropanol (HFIP) in urine samples was proposed to determine the individual exposure levels of the medical staff. In this study, a method for simultaneous determination of both compounds in urine using static headspace-gas chromatography-mass spectrometry (HS-GC-MS) was developed. The method is linear over a broad concentration range from 1 to 1000 µg/L (r2 > 0.999) and shows high precision. Limits of quantification (LOQ) are 0.6 µg/L for sevoflurane and 3 µg/L for HFIP, representing an excellent sensitivity without the necessity of analyte enrichment. The method was successfully applied in a German pilot-study to monitor both compounds in samples from medical personnel working in operating theatres. Urinary concentrations of HFIP ranged between < LOQ and 145 µg/L, while sevoflurane was below the LOD in all samples.

Evidence for gene recombination in FCGR3 gene variants.

BACKGROUND AND OBJECTIVES: The genes encoding the Fcgamma receptors (FcgammaR) IIIa and IIIb (FCGR3A and FCGR3B) are clustered on chromosome 1 band q23-24 and exhibit allelic polymorphism. We investigated the molecular basis of additional new FCGR3 genomic variation. MATERIALS AND METHODS: A segment shared by FCGR3A and FCGR3B containing the polymorphic nucleotide positions 141, 147, 227, 266, and 277 in exon 3 was cloned and sequenced from genomic DNA of 30 donors and 3 bacterial artificial chromosome (BAC) clones. A mixture consisting of isolated FCGR3B*2- and FCGR3A- plasmids was cloned and sequenced as well. Additionally, nucleotide databases were screened for clones with variant FCGR3 sequences. RESULTS: A total of 12 FCGR3 variants defined by the polymorphic positions were detected in whole blood genomic DNA from 23 of 24 FCGR3B*2 and/or FCGR3B*3 positive donors, the DNA from two of three BAC clones and in the DNA mixture of isolated FCGR3B*2- and FCGR3A- plasmids. CONCLUSION: Nucleotide exchanges of the variants were non-random and resulted from two alternative nucleotides present in one of the polymorphic position of the basic FCGR3 forms. Polymerase chain reaction (PCR) artefacts cannot be excluded as origin of new variants, but there is strong evidence that at least two variants are the result of a somatic recombination.

Fcgamma receptor-mediated immune phagocytosis depends on the class of FcgammaR and on the immunoglobulin-coated target cell.

BACKGROUND AND OBJECTIVES: Three human Fcgamma receptors (FcgammaR) are known to mediate immune phagocytosis. A variety of different phagocytic assays have been described, but their comparability is complicated by the use of different effector cells and different antibody-coated target cells. The aim of this study was to determine the influence of these variable components on the FcgammaR-mediated phagocytosis. MATERIALS AND METHODS: We sensitized human red blood cells (RBC) with polyclonal human anti-D (huaD), or with human monoclonal anti-D of the isotypes IgG1 (huIgG1) or IgG3 (huIgG3). Sheep RBC coated with rabbit immunoglobulin (RBC-RAS) were also used. Monocytes or polymorphonuclear neutrophils (PMN) were incubated with different FcgammaR-specific antibodies or their F(ab')2 fragments to determine the contribution of the different FcgammaRs on these effector cells in the phagocytic process of different antibody-coated target cells. RESULTS: huaD-RBC and huIgG1-RBC were preferentially ingested via the FcgammaRI on monocytes and, to a minor extent, also by the FcgammaRII. PMN ingested these target cells only after induction of the FcgammaRI by interferon-gamma (IFN-gamma). huIgG3-RBC extensively formed rosettes with monocytes but were seldom ingested. RAS-RBC phagocytosis was induced primarily via the FcgammaRI on monocytes and was mediated by the FcgammaRII on PMN. CONCLUSION: When performing phagocytosis assays with different effector and target cells, one has to take into account that phagocytosis is mediated by different FcgammaR, making comparability of these assays more difficult.

No association between dopamine D2 receptor gene (DRD2) and human intelligence.

Significantly diminished intellectual functioning, as indicated by appropriately administered IQ tests with scores below 70, is a frequent mental handicap leading to severe social disadvantages and serves as a paradigm for molecular genetic research of complex disorders and traits due to its multitude of known and unknown, genetic as well as environmental causes. Since the number of confounding variables is expected to be considerably reduced in the normal population at the opposite ends of the IQ distribution, we employed a contrast of extremes approach by comparing adults of high (N = 71) and average IQ (N = 78) in association studies to search for genes involved in the multigenic forms of familial mental retardation. The dopamine D2 receptor gene (DRD2) was chosen as a candidate gene for general cognitive ability (g) since it has been found to be associated with visuospatial ability which in turn is highly correlated with g. Confirming two similar studies in children, however, no significant differences were obtained. Given three negative studies, the DRD2 gene is unlikely to pay a major role in g.

HLA-antibody testing: the immune phagocytosis inhibition test is superior to the PRA-STAT and NIH lymphocytotoxic test with respect to specificity.

We compared the specificity and sensitivity of four different methods for the detection of antibodies specific for HLA antigens. The NIH version of the complement-dependent cytotoxic test (CDC) was used as the gold standard to which we compared two Fcgamma receptor (FcgammaR)-dependent immune phagocytosis inhibition tests (IPI) and one commercial enzyme-labelled immunosorbent assay (ELISA) with soluble HLA class I-antigen preparations bound to the plate (PRA-STAT). Both IPI tests are based on the fact that HLA-antibodies specifically bind to antigens on the monocyte surface via their Fab portion, and in so doing block a neighbouring FcgammaR with their Fc region. This blockade prevents phagocytosis of IgG-coated red blood cells (RBCs), which can be measured either microscopically (IPIm) or photometrically (IPIp). The four assays were used in blind tests on 20 human alloantisera or monoclonal antibodies with known HLA-antigen reactivities. Additionally, two monoclonal antibodies and one human serum were titrated to elucidate the sensitivity of each test. After all tests were completed, the identities of the samples were disclosed. Both IPI methods detected and identified all clinically relevant HLA class I and class II specific antibodies. In contrast, the CDC was not able to detect noncytotoxic HLA-antibodies and HLA class II specific antibodies; however, it detected clinically insignificant IgM lymphocytotoxins. The PRA-STAT assay enabled identification of all cytotoxic and noncytotoxic IgG antibodies with specificity for HLA-class I antigens. With respect to sensitivity, the CDC and the IPI methods were superior to the PRA-STAT. These facts demonstrate the advantage of IPI methods in the detection of clinically relevant HLA-antibodies.

Equal distribution of congenital blood cell chimerism in dizygotic triplets after in-vitro fertilization.

The special situation of multiple pregnancies following IVF has led to a growing interest in the assessment of embryonal development by means of molecular genetics. We report a case of congenital blood chimerism in dizygotic triplets (two boys, one girl) present in erythrocytes and leukocytes in both sexes. Routine pre-operative blood serology of the 6 year old female triplet revealed chimerism of the red cells. Flow cytometry of the erythrocytes and DNA analysis of the leukocytes demonstrated that all three children had the same proportions of male and female cells. Fluorescent in-situ hybridization (FISH) analyses revealed Y chromosomes in 84% of the girl's leukocytes and in 89/92% of the two boys' leukocytes. The true genetic lines were determined by analysing polymorphism of serum groups (glycoprotein, transferrin, protease inhibitor and plasminogen) secreted by non-haematopoetic tissue, by blood group typing of hair roots and by DNA analysis of endothelial cells. Evidently placental anastomoses allowed a reciprocal intra-uterine transfusion of blood stem cells in the triplets.

High incidence of maternal HLA A, B and C antibodies associated with a mild course of haemolytic disease of the newborn. Group for the Study of Protective Maternal HLA Antibodies in the Clinical Course of HDN.

Mild courses of haemolytic disease of the foetus or newborn (HDN) due to Rh (D) blood group antibodies are associated with and may therefore be ameliorated by maternal antibodies reacting with human leucocyte antigens (HLA) of the child, an observation drawn from our own earlier data (Neppert J, Kissel K. Lancet 1992;339:1481). This study (i) corroborates this association; (ii) reveals shortcomings in the published data; and (iii) examines the characteristics of HDN cases when these shortcomings have been rectified. Samples from 51 women with antibodies against their child's blood group antigens of the Rh system were analysed for HLA A, B, C and DR antibodies during parturition. The mothers were divided into two groups, either severe or mild, dependent upon the clinical course of the HDN, and the incidence of HLA antibody production was determined for each group. HLA A, B, C and/or DR antibodies were detected in 85.2% of those women whose children had a mild course of HDN prenatally or perinatally (n=27). This is statistically greater than the incidence of 50.0% (Fisher's exact test: p=0.014) found in the group of women whose children had a severe HDN either pre- or perinatally (n=24) and is greater than the 35% (n=20; p=0.0001) found in women without Rh or other irregular antibodies. HLA DR antibodies were detected in three cases. The findings support our hypothesis that maternal anti HLA A, B and C antibodies may protect against a potential severe HDN. We therefore assume that those women will benefit who have already had a child with a severe HDN and in whom HLA antibodies were not previously detected, if HLA antibody production is provoked by subcutaneously inoculating with the father's leucocytes before or at the beginning of the new pregnancy.

Immunoenzymatic detection of the new proliferation associated protein p100 by means of a cellular ELISA: specific detection of cells in cell cycle phases S, G2 and M.

In vitro proliferation assays are widely used in biomedical research. We describe the immunoenzymatic (ELISA) detection of a recently described proliferation associated protein (p100) by means of a new monoclonal mouse IgG1 antibody (Ki-S2). P100 is a 100 kDa nuclear protein that is specifically detected during the cell cycle phases S, G2 and M. Comparative studies on lectin-stimulated leukocytes using 3H-thymidine labelling and Ki-67 antibodies revealed a statistically significant positive correlation. Since p100 is absent in GO and G1 cells, its detection permits the precise and specific measurement of actual cell cycle events under culture conditions.

NA1/NA2 antisera inhibit FcgammaRI- but not FcgammaRII- mediated phagocytosis.

BACKGROUND AND OBJECTIVES: Alloantibodies against the granulocyte-specific NA antigens play an important role in alloimmune neonatal neutropenia. As the NA system is located on the FcgammaRIIIb, the influence of NA-specific antibodies on granulocyte function is of special interest. MATERIALS AND METHODS: We tested alloantisera specific for NA1 and NA2 for their ability to influence FcgammaR-mediated phagocytosis of polymorphonuclear neutrophils by use of different FcgammaR-specific targets. Red blood cells coated with human IgG anti-D served as specific targets for FcgammaRI-mediated phagocytosis while mouse IgG1 anti-glycophorin A was used to coat red blood cells (RBCs) to obtain FcgammaRII specific targets. To test for a hypothetical induction of phagocytosis by FcgammaRIIIb we used D-- RBCs coated with human monoclonal anti-D as target cells for unprimed neutrophils. RESULTS: Granulocyte phagocytosis was directly induced by FcgammaRI and FcgammaRII but not by FcgammaRIIIb. NA1 alloantisera significantly inhibited FcgammaRI-mediated phagocytosis of IFN-gamma-stimulated neutrophils if the corresponding antigen was expressed. Conversely, NA2 alloantisera inhibited FcgammaRI-mediated phagocytosis in NA2-positive individuals. There was no effect of NA1- and NA2-specific alloantibodies on FcgammaRII-mediated phagocytosis. CONCLUSION: NA-specific alloantisera inhibit the FcgammaRI-induced phagocytosis in primed neutrophils, but they do not significantly inhibit their FcgammaRIIa-specific phagocytosis of mIgG1-coated RBCs.

Unsatisfactory detection of an in vivo haemolytic anti-vel by the gel test.

BACKGROUND AND OBJECTIVES: A patient experienced a severe haemolytic transfusion reaction. Neither the haemolytic property nor the specificity of the causative antibody had been sufficiently recognised when performing a microcolumn gel test. MATERIALS AND METHODS: Subsequent to the transfusion reaction, the serological property and specificity of the causative antibody were analysed. Tube and gel test methods were compared, as were various reagent red cell specimens and their constituents. RESULTS: A haemolytic anti-Vel was detected in the tube test. In contrast, the particular commercial gel test kit used did not reveal the haemolytic property or specificity of the antibody. Our experiments suggest that this was apparently due to the presence of EDTA in the low ionic strength saline solution of the test kit. CONCLUSIONS: In rare cases life-treatening haemolytic activity of an irregular blood group antibody may be undetected by a commericial microcolum gel test kit in which EDTA is a constituent.

The NA"null" phenotype of a young man is caused by an Fc gammaRIIIB gene deficiency while the products of the neighboring Fc gammaRIIA and Fc gammaRIIIA genes are present.

A 27-year-old man with an allergy to house dust mites was found to lack the Fc gammaRIIIb on his neutrophils. Cell surface marker and PCR techniques were used to investigate possible reasons for this deficiency. Agglutination and immunofluorescence assays using the man's neutrophils together with NA1- and NA2-specific antibodies were negative, and there was no reaction with the Fc gammaRIII-specific mAb 3G8. Indirect immunofluorescence demonstrated the presence of the CD24 molecule, which, like the Fc gammaRIIIb, is anchored to the cell membrane by glycosylphosphatidylinositol. Thus a lack of the Fc gammaRIIIb cell membrane anchor was excluded. PCR analysis confirmed the absence of the NA1 and NA2 alleles. The individual was therefore typed as NA"null". The products of those genes located together with the Fc gammaRIIIB gene within a complex on chromosome 1 (q23-24) were examined. Fc gammaRII was demonstrated on monocytes and B cells with the use of Fc gammaRII-specific monoclonal antibodies. About 5% of the individual's peripheral blood monocytes were positive with the 3G8 antibody, indicating the presence of Fc gammaRIIIa. From these data we concluded that the Fc gammaRIIIb deficiency on the neutrophil cell surface of this individual is due to a lack of the Fc gammaRIIIB gene while excluding a lack of the Fc gammaRIIA and the Fc gammaRIIIA genes.

Improved ELISA proliferation assay (EPA) for the detection of in vitro cell proliferation by a new Ki-67-antigen directed monoclonal antibody (Ki-S3).

We describe a simplified and improved proliferation assay based on a conventional ELISA system for the in vitro measurement of cellular proliferation (ELISA proliferation assay = EPA). The assay is based on a new monoclonal antibody (Ki-S3) to the proliferation-specific Ki-67-antigen and is carried out in 96-well microtiter plates using conventional immunoenzymatic methods. Ki-S3 is an immunoprecipitating monoclonal mouse IgG1 antibody, which recognizes a formalin-resistant epitope of the Ki-67 antigen. It can be used to measure proliferating cells in the cell cycle phases G1, S, G2 and M. In phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) the absorbance values obtained with the EPA show a statistically significant correlation to the number of Ki-S3 positive cells in simultaneously immunostained cytospin slides (r = 0.88). A direct comparison with [3H]thymidine labeling reveals the test to be an equally sensitive method for monitoring cellular proliferation (r = 0.91). This assay is an improved ELISA proliferation assay, which is easy to perform, does not require time-consuming pretreatments and avoids the hazards of radioactive isotopes.

Rapid typing of the human Fc gamma receptor IIA polymorphism by polymerase chain reaction amplification with allele-specific primers.

BACKGROUND: The human Fc gamma receptor IIa (Fc gamma RIIa) is expressed in two polymorphic forms, Fc gamma RIIa-H131 and Fc gamma RIIa-R131, that differ by the replacement of histidine by arginine at position 131. This replacement is caused by a single-nucleotide exchange of A-->G. The resulting receptor forms differ in their binding to human IgG2 and mouse IgG1, which may lead to a different immunologic defense to bacterial polysaccharides and encapsulated bacteria. STUDY DESIGN AND METHODS: A rapid and easy polymerase chain reaction(PCR) method of genotyping the Fc gamma RIIa was developed. Allele-specific primers discriminate between the Fc gamma RIIa-H131 and the Fc gamma RIIa-R131 forms of the receptor. The results were compared with those obtained by another DNA-based genotyping method, in which PCR-amplified DNA was hybridized with allele-specific oligonucleotides, and with a functional phagocytosis assay using mouse IgG1-coated red cells as target antigens. RESULTS: The genotypes deduced from the PCR with allele-specific primers were in complete accordance with those obtained by the data from the hybridization of PCR-amplified DNA with allele-specific oligonucleotides. Furthermore, the Fc gamma RIIa genotypes of 28 individuals in all cases corresponded to the functional phenotypes. CONCLUSION: The use of PCR with allele-specific primers provides a rapid and easily performed method for the determination the Fc gamma RIIa polymorphism.

Transplant rejection associated with the presence of human leucocyte antigen antibodies detected by the Fc gamma R inhibition test but not by the lymphocytotoxicity test.

The unselected sera from 869 human leucocyte antigen (HLA) immunized patients awaiting a kidney transplant were analysed using the complement-dependent lymphocytotoxicity test (LCT) with peripheral mononuclear blood cells and the complement-independent immune phagocytosis inhibition test (IPI) with monocytes derived from between five and 10 donors. Sera from 659 patients were LCT and IPI negative when tested against this small panel. Seventy-nine patients had HLA immunoglobulin-G (IgG) antibodies, detectable by the IPI only. Sera from 117 patients had concordantly positive IPI and LCT reactivity with cells from certain donors and concordantly negative IPI and LCT reactivity with cells from other donors (no isolated IPI and no isolated LCT reactions). Fourteen patients had a mixed type of reactivity. Laboratory findings were interpreted along with the transplantation history of the respective patients. Group 1 comprised patients for whom negative results were obtained in both the LCT and the IPI; group 2 patients who were also LCT negative but IPI positive. These two groups showed a significantly different history with respect to the number of irreversible immunological transplant rejections. In group 1, 25.3% of the transplanted kidneys had been rejected whereas in group 2, 56.0% of the kidneys had been rejected (p = 5 x 10(-5)). The high incidence of rejections in the group showing only IPI reactions was comparable with that of group 4 comprising patients with concordant IPI and LCT reactions (59.4%). It is inferred from this retrospective study that renal allograft rejection is associated with the development of IPI reactive antibodies which are not detectable by the LCT. The presence of these antibodies prior to transplantation could be detrimental to the transplanted organ. This being the case, the incidence of transplant failures could be reduced by pretransplant screening using the IPI and by avoiding crossmatch positive donors identified by IPI, especially in patients waiting for a retransplantation.

Inhibition of monocyte and polymorphonuclear granulocyte immune phagocytosis by monoclonal antibodies specific for Fc gamma RI, II and III.

We tested two Fc gamma receptor I (Fc gamma RI); six Fc gamma RII; and six Fc gamma RIII-specific monoclonal antibodies (mAb) for their capacity to inhibit monocyte and polymorphonuclear granulocyte (PMN) immune phagocytosis which is mediated by Fc gamma R. We used human red blood cells (rbc) coated with hIgG1 or mIgG1 as Fc gamma RI- and Fc gamma RII-specific target cells, respectively. The Fc gamma RI-specific mAbs 22.2 and 32.2 did not inhibit Fc gamma RI- or Fc gamma RII-specific monocyte immune phagocytosis. The Fc gamma RII-specific mAbs IV.3, CIKM5, FLI8.2, FLI 8.26, 2E1, and 41H16 inhibited Fc gamma RII-specific monocyte immune phagocytosis in all Fc gamma RIIa high-responder (HR) individuals but did not inhibit Fc gamma RI-specific phagocytosis. Using PMN, FL18.2 and 2E1 only partially inhibited phagocytosis in HR individuals, but the Fc gamma RIII-specific mAbs 3G8, DJ130c. MFM-154. B88-9 and MG38 completely inhibited Fc gamma RII-specific phagocytosis if the corresponding antigen was available on the cell surface. In these cases phagocytosis inhibition may be explained by cross-linking of Fc gamma RII and Fc gamma RIII via one antibody molecule, with the Fab portion binding to Fc gamma RIII and the Fc portion binding to Fc gamma RII.