Reactivity to sodium tetrachloropalladate (Na2[PdCl4]) compared to PdCl2 and NiCl2 in lymphocyte proliferation tests.

BACKGROUND: For patch testing, replacement of the commonly used palladium dichloride (PdCl2) by sodium tetrachloropalladate (Na2[PdCl4]) was recently demonstrated to improve test accuracy and show a significant correlation with nickel (Ni), supporting the concept of cross-reactivity between Pd and Ni. A promising alternative to metal allergy patch testing is the in vitro lymphocyte proliferation test (LTT). OBJECTIVES: The aim of this study was to test whether Na2[PdCl4] is also more sensitive for diagnosing Pd allergy with a standardized LTT. PATIENTS/METHODS: After determining optimal nontoxic and nonmitogenic concentrations for Na2[PdCl4], blood samples from 105 patients with clinical suspicion of metal allergy were tested with an LTT called memory lymphocyte immuno stimulation assay for Na2[PdCl4], PdCl2 and NiCl2. Reaction profiles were analysed for concordant positive reactions. RESULTS: Using the conventional cut-off of stimulation index > or = 3, 74.3% showed a positive reaction to NiCl2, 15.2% to PdCl2 and 28.6% to Na2[PdCl4]. All positive results to PdCl2 were covered by Na2[PdCl4]. From the 30 positive reactions to Na2[PdCl4], 26 (87%) were concordant for NiCl2 reactivity. CONCLUSION: In LTT, the use of Na2[PdCl4] results in more positive reactions in Pd allergy testing which are in concordance with positive reactions to PdCl2 and NiCl2.

European proficiency testing program for molecular detection and quantitation of hepatitis B virus DNA.

External quality control of hepatitis B virus (HBV) DNA detection remains an important issue. This study reports and compares the results obtained from two different proficiency panels for both the qualitative and quantitative assessment of HBV DNA. The panels were designed by the European Union Quality Control Concerted Action, prepared by Boston Biomedica, Inc., and distributed in May 1999 (panel 1) and February 2000 (panel 2). Each contained two negative samples and six positive samples with 10(3) to 10(7) copies/ml (panel 1) or 10(3) to 2 x 10(6) copies of HBV DNA per ml (panel 2). For panel 1, 42 laboratories submitted 20 qualitative (all in-house PCRs) and 37 quantitative (87% commercial assays) data sets. For panel 2, 51 laboratories submitted 25 qualitative (all in-house PCRs) and 47 quantitative (94% commercial assays) data sets. Five data sets (8.8%) in panel 1 and two data sets (2.8%) in panel 2 contained totals of six and two false-positives, respectively, corresponding to false-positive result rates of 5.3% for panel 1 and 1.4% for panel 2. The false-negative result rates of 10.5% for panel 1 and 17.4% for panel 2 were dependent on the detection levels of the assays employed as well as panel composition. In the qualitative analysis of all data sets, 47.4% (panel 1) and 51.4% (panel 2) had all samples correct. An adequate or better score (all correct or only the weak-positive sample missed) was obtained with 77.2% of the panel 1 samples and 68.1% of the panel 2 samples. In the quantitative analysis, 57.1% (panel 1) and 42.6% (panel 2) of the data sets achieved an adequate or better score (positive results within the acceptable range of the geometric mean +/- 0.5 log(10) of all positive results). These results demonstrate that while the qualitative performance of HBV detection has considerably improved compared to that of a previously published HBV proficiency study, the detection levels of many commercial quantitative assays are still too high to allow adequate quantitation of all relevant clinical samples.

Borderline and/or discordant Cobas Amplicor HCV Test version 2.0 results: clinical significance.

BACKGROUND: The Cobas Amplicor HCV Test Version 2.0 (Roche Diagnostics, Mannheim, Germany) allows highly sensitive detection of hepatitis C virus (HCV) RNA in patient samples (> or =100 copies/ml with 100% reproducibility) and yields reproducible, unambiguous results that correlate well with relevant serological and clinical parameters. Occasionally, however, results are borderline (defined by Roche Diagnostics as optical density 0.15-1.0) and/or discordant upon repetition. Such results are difficult to interpret: do they represent false-positives or reflect very low-level viremia (<100 copies/ml)? OBJECTIVE: To determine whether low-level viremia could be a plausible explanation for the borderline/discordant results observed with this test. STUDY DESIGN: (1) Analyse serial dilutions of two HCV standards and one HCV quantitated patient serum; and (2) correlate ambiguous results from 21 patients with available clinical and laboratory data. RESULTS: (1) All dilutions containing >100 copies/ml yielded 100% concordant positive results, whereas dilutions with <100 copies/ml yielded discordant results, often with borderline values. (2) All patients had either a confirmed HCV infection (n=14, seropositive, most undergoing therapy with interferon-alpha) or had a history of confirmed or suspected contact with HCV without confirmed HCV infection (seronegative): three needle-stick injuries, one newborn of an HCV seropositive mother, one woman with liver cirrhosis of unknown etiology, one iv drug abuser, and one nurse with a prior blood transfusion and "indeterminant HCV results" at a blood donation center. CONCLUSION: Based on our experience to date, borderline results and/or discordant replicates obtained with the Cobas Amplicor HCV Test Version 2.0 are indicative of very low level viremia (<100 copies/ml) due either to an HCV infection (patients will be seropositive) or to transient contact with the virus with or without subsequent HCV infection (patients will be seronegative and may remain so). Follow-up of such patients is mandatory.

Evaluation of SHARP signal system for enzymatic detection of amplified hepatitis B virus DNA.

The sensitivity, specificity, reproducibility, detection level, and quantification potential of the SHARP Signal System for enzymatic detection of amplified hepatitis B virus (HBV) DNA in clinical samples were evaluated by testing 104 samples in parallel in a SHARP PCR, an in-house HBV PCR, and a dot blot hybridization assay for semiquantification. SHARP PCR showed a sensitivity of 100%, a specificity of 92.3% (resolved, 100%), a reproducibility of 92.3% (all discrepant serum samples involved very low levels of HBV DNA), and a detection level of at least 3.5 pg/ml. Clinically relevant quantification of the amplified products was not feasible.

Detection of hepatitis B virus DNA in serum with nucleic acid probes labelled with 32P, biotin, alkaline phosphatase or sulphone.

To facilitate the clinical application of dot-blot hybridization for assaying hepatitis B virus (HBV) DNA, we compared the ability of nucleic acid probes labelled with 32P or with various non-radioactive markers to detect HBV DNA in patient serum. Cloned HBV DNA was hybridized with (1) 32P-labelled HBV DNA cloned in M13, (2) the 32P-labelled HBV RNA probe included in the HepProbe kit, (3) an alkaline phosphatase-labelled synthetic oligonucleotide of HBV, (4) biotin-labelled HBV DNA, and (5) sulphonated HBV DNA. Detection was either by autoradiography or an enzymatic colour reaction. The lowest level of detection of cloned HBV DNA was achieved with the 32P-labelled HBV RNA probe (0.3 pg HBV DNA, corresponding to 3 x 10(4) genomes in 50 microliters), followed by the 32P-labelled DNA probe (0.3-2 pg), sulphonated DNA (1-2 pg), biotin-labelled DNA (4 pg), and an alkaline phosphatase-labelled synthetic oligonucleotide (30 pg). Subsequently, sera from 159 patients with various constellations of HBsAg, HBeAg, and anti-HBe were tested with the most sensitive radioactive method (HepProbe) and the corresponding nonradioactive method (sulphonation). The overall concordance rate was 71% (r = 0.42). Compared with HepProbe results, sulphonation showed a sensitivity of 80% and a specificity of 67%. We conclude that radiolabelling (in particular 32P-labelling of HBV RNA) still allows the most sensitive and reliable detection of HBV DNA in patient serum using conventional dot-blot hybridization.

Serological responses to herpes simplex virus type 1 (HSV-1) analysed with enzyme-linked immunosorbent assay (ELISA) and western blot (WB).

Serological response of 56 patients to primary and recurrent herpes simplex virus type 1 (HSV-1) infection were studied by enzyme-linked immunosorbent assay (ELISA) and by Western blot analysis (WB). ELISA test showed a high sensitivity in detecting IgG, IgM, and IgA antibodies. From 27 patients with recurrent infection, 13 (48%) had IgM antibody. The percentage of patients positive for IgA was similar among those with primary (72.4%) or recurrent (81%) infection. The band pattern in WB alone did not allow to distinguish between patients with primary or recurrent infections. Furthermore, no correlation between the particular viral proteins and clinical manifestation could be determined. The kinetics of antibody response could be followed with both methods.

Evaluation of the commercially available HepProbe kit for detection of hepatitis B virus DNA in serum.

The commercially available HepProbe kit involving the use of a 32P-labeled RNA probe was evaluated for its sensitivity, specificity, and reproducibility in detecting hepatitis B virus (HBV) DNA in patient serum by dot blot hybridization. The level of detection was 0.3 pg, corresponding to 3 x 10(4) genomes in 50 microliters of serum. A total of 181 serum samples were tested; 53 (82%) of 65 patients positive for both hepatitis B surface antigen and hepatitis e antigen were positive for HBV DNA compared with only 12 of 74 (16%) hepatitis B surface antigen-positive but hepatitis e antigen-negative individuals. In addition, among all patients positive for HBV DNA, there was a statistically significant correlation between the concentration of HBV DNA in serum and the presence or absence of hepatitis e antigen. None of the 42 hepatitis B surface antigen- and hepatitis e antigen-negative patients tested was positive for HBV DNA. Reproducibility was 87%, with all discordant results representing borderline positives. The results indicate that HepProbe can be employed as a sensitive and reliable assay for HBV DNA in patient serum.

HLA-A,B,C,DR typing and 17-OHP determination for second trimester prenatal diagnosis of 21-hydroxylase deficient CAH.

In 18 families at risk for the HLA-linked, 21-hydroxylase deficient form of autosomal recessive congenital adrenal hyperplasia (CAH), prenatal diagnosis (PD) was performed using two methods: (1) HLA-A,B,C typing and in the latter 11 cases also DR typing of cultured amniotic fluid cells (AFC) using the standard microcytotoxicity assay, and (2) measurement of second trimester amniotic fluid 17-hydroxyprogesterone (17-OHP) concentration using gel chromatography and radioimmunoassay. The accuracy of the prenatal predictions was confirmed by postnatal HLA typing of umbilical cord blood lymphocytes and by clinical evaluation. In 16/18 families, both HLA typing of AFC and 17-OHP measurements proved informative for PD. The predictions of both methods were concordant in 14/16 families (88 per cent). In ten of these families, a normal fetus was predicted, and in four, an affected fetus; all pregnancies were carried to term and all predictions were confirmed postnatally. In 2/16 cases (12 per cent), however, the predictions were discordant: the prenatal HLA typing indicated an affected fetus, whereas the 17-OHP values predicted a normal fetus. Both pregnancies were continued and two healthy boys were delivered. The discordance proved to be due to a 'missed' HLA antigen in one case and to serologically cross-reactive HLA antigens in the second. Finally, in 2/18 cases, prenatal assessment of fetal genotype had to rely on HLA typing alone as 17-OHP measurement was not performed in one family and in the second family the 17-OHP values obtained were not informative due to inadvertent continuation of hormone therapy to the date of amniocentesis. In both cases, the HLA typing data accurately predicted a normal fetus. In conclusion, a combination of HLA typing of cultured AFC and 17-OHP measurements of amniotic fluid permits accurate prenatal diagnosis of CAH in most cases (88 per cent). In addition, the supplementary use of HLA-DR typing of AFC as presented here for the first time proved helpful in families with HLA-A,B homozygosity due to parental sharing of antigens and can be informative for identifying HLA-B/21-OH recombinant haplotypes.

HLA-D and -DR antigens on human amniotic fluid cells. II. Heterogeneous expression of HLA-DR and other cell surface markers.

To evaluate further the feasibility of HLA typing for prenatal diagnosis, we tested human amniotic fluid cells (AFC), known to express HLA-A, -B, and -C antigens, for the presence of HLA-DR antigens using type-specific antisera in the microcytotoxicity assay and a monoclonal antibody directed against the common HLA-DR structure (cDR) in indirect immunofluorescence. Prenatal typing of HLA-DR on AFC in the microcytotoxicity test was possible in only one out of eight families studied. The detected DR2 antigen was confirmed by postnatal typings of cord blood lymphocytes. Thereafter, 23 different AFC cultures were tested with monoclonal antibodies in indirect immunofluorescence. Only six cultures were partially positive (23-35% fluorescent cells) with the monoclonal cDR antibody while all AFC cultures demonstrated strong positive fluorescence (68-100%) with a monoclonal antibody against the common HLA-A, -B, and -C structure (cHLA). These data suggest that only a small subpopulation of AFC expresses class II (HLA-DR) antigens in contrast to the nearly ubiquitous expression of class I (HLA-A, -B, and -C) antigens. Furthermore, the heterogeneous expression of cell surface antigens within the various AFC cultures was substantiated with monoclonal antibodies directed toward cell surface antigens of the OKT, OKM, and Lyt series that have been found to be characteristic for subpopulations of lymphoid and hemapoetic cells. Thus, at present, HLA-DR typing is not reliable for prenatal diagnosis.

Primed lymphocyte typing predicts MLC reactivity between unrelated individuals.

The present study was carried out to evaluate the ability of primed lymphocyte typing (PLT) to predict mixed lymphocyte culture (MLC) reactivity between unrelated individuals. Eleven PLT cells generated against independent familial haplotypes, and one PLT cell generated against a homozygous typing cell (HTC), were tested for their response to cells of a random panel of 53 unrelated individuals. From Chi-square analysis of the response patterns of these 12 cells, nine "PLT specificities" could be defined. Most panel members (81.2%) types for 1 or 2 of these specificities; equal numbers (9.4%) types for 3 or none, respectively. Four of these 9 specificities were shown to be significantly correlated with HLA-Dw antigens defined by HTCs. Typing for these nine PLT specificities was found to be predictive of subsequent primary MLC reactivity between panel members: a) pairs of panel members sharing PLT specificities produced three-fold lower MLC results on the average than pairs of panel members disparate for PLT specificities (p less than 0.0001), and b) in MLC combinations, an increase in number of foreign PLT specificities presented by the stimulator cell was paralleled by a statistically significant increase in MLC reactivity. To the extent that low MLC reactivity is correlated with improved graft survival, the PLT method could have significant value in selecting histocompatible donors for organ transplantation.

HLA-D and -DR antigens on human amniotic fluid cells. I. Lack of expression of HLA-D.

Human amniotic fluid cells, known to express HLA-A, -B, and -C antigens, were tested for the presence of lymphocyte-stimulating antigens (LD or HLA-D) using modifications of the mixed lymphocyte culture (MLC) and primed lymphocyte typing (PLT) tests. Peripheral blood lymphocytes were co-cultured with various concentrations of allogeneic amniotic fluid cells, either growing as a monolayer culture in microtiter plates or suspended in medium following treatment with trypsin. The kinetics of such mixed lymphocyte amniotic fluid cell culture (MLAC) reactions were followed during days 3 to 8. Under none of these conditions did amniotic fluid cells significantly stimulate allogeneic lymphocytes, even after lymphocytes were specifically primed in the PLT assay to the HLA-D antigens segregating in the family of the amniotic fluid cell donor. Furthermore, in three-cell experiments, amniotic fluid cells failed to inhibit an ongoing MLC reaction, indicating that the absence of proliferative response to amniotic fluid cells is not due to active suppression. Taken together, these data strongly suggest that amniotic fluid cells either do not express HLA-D antigens or do not express them in a form that is detectable in either primary or secondary MLC.

Everything the pediatrician ever wanted to know about HLA but was afraid to ask.

Following a description of the genetic aspects of the human histocompatibility antigens system HLA and its principle typing methods, this paper reviews the relationship between HLA antigens, transplantation immunology and certain diseases. In particular, the role of the lymphocyte-defined antigens of the HLA-D system is emphasized on the basis of a special typing method, the PLT test, used in our laboratory. Aside from its necessity in bone marrow and kidney transplantation, HLA typing can be used as an additional diagnostic or prognostic tool for certain diseases. Among the pediatric age group, this includes rheumatic fever and other rheumatic diseases, insulin-dependent juvenile diabetes mellitus, some forms of Addison's disease, thyrotoxicosis, myasthenia gravis, celiac disease, and some complement deficiency disorders. Close linkage of the HLA system with steroid 21-hydroxylase deficiency has made it possible to diagnose this form of congenital adrenal hyperplasia in utero. This approach is illustrated in a large family at risk for this disorder.