A re-evaluation of the influence of maternal insulin-dependent diabetes on fetal nuchal translucency thickness and first-trimester maternal serum biochemical markers of aneuploidy.

OBJECTIVE: To evaluate the influence of maternal insulin-dependent diabetes mellitus (IDDM) on maternal serum free ß-hCG, pregnancy-associated plasma protein-A (PAPP-A) and fetal nuchal translucency (NT) thickness from 11 weeks to 13 weeks 6 days of gestation in a large cohort of women screened prospectively for chromosomal anomalies. METHODS: Information on maternal IDDM status, maternal serum biochemical marker levels and fetal NT were collected from the prenatal screening computer records. On total, the control group included 83,972 and the IDDM group included 489 pregnancies. The median-corrected free ß-hCG and PAPP-A, expressed as MoM, and fetal NT, expressed as delta values, in the IDDM and non-IDDM groups were compared. RESULTS: There were no significant differences between the IDDM and non-IDDM groups in median-corrected free ß-hCG (IDDM 1.01 MoM, non-IDDM 1.01 MoM; p = 0.970), or mean delta NT (IDDM 0.00 mm, non-IDDM 0.02 mm; p = 0.412). However, the median-corrected PAPP-A was significantly lower (IDDM 0.88 MoM, non-IDDM 1.03 MoM; p < 0.0001). CONCLUSIONS: In pregnancies with maternal IDDM, first-trimester screening for chromosomal defects does not require adjustments for the measured fetal NT and maternal serum free ß-hCG. However, for PAPP-A the 15% reduction is large enough to require correction in the calculation of risks for chromosomal defects.

Early vaginal bleeding has no impact on markers used in first trimester aneuploidy screening.

OBJECTIVE: To assess the impact of early vaginal bleeding on the levels of markers used in first trimester screening for aneuploidy. METHODS: A retrospective analysis was carried out on the free beta human chorionic gonadotrophin (beta-hCG) and pregnancy associated plasma protein-A (PAPP-A) levels and nuchal translucency thickness in 49 653 women with a normal singleton fetus who had first trimester combined screening for Down Syndrome in three centres. Median MoMs and the distribution of log MoMs of the two markers were compared in two groups-7470 women who self-reported vaginal bleeding and 42 183 women who reported no vaginal bleeding at any stage prior to the screening test. RESULTS: The overall median MoM free beta-hCG and that in the bleeding and non-bleeding group were 0.9854, 1.0012 and 0.9832, and for PAPP-A were 1.0407, 1.0413 and 1.037. There was no significant difference between the bleeding and non-bleeding group by median test (p = 0.080) or by t-test comparing log MoMs (p = 0.1305) for free beta-hCG and for PAPP-A with median test (p = 0.5071) or by t-test comparing log MoMs (p = 0.1740). For delta nuchal translucency (NT) there was also no significant difference between the bleeding and non-bleeding group (p = 0.055). CONCLUSION: Vaginal bleeding has little or no impact on first trimester marker levels and no correction is necessary.

Inhibition of humoral and cell-mediated immune responses in man by distinct suppressor cell systems.

Studies were designed to investigate whether the suppressor cell systems that regulate the humoral and cell-mediated immune responses belong to the same subsets of T cells or different subsets. Mitogen-activated suppressor cells were simultaneously assayed for their ability to inhibit (a) pokeweed mitogen-induced generation of plasma cells, (b) blastogenic response of lymphocytes to allogeneic cells, and (c) generation of killer cells in the cell-mediated lymphocytotoxicity assay. We found that suppressor cells that inhibited the generation of plasma cells were activated by concanavalin A (Con A) and were both radiation and prednisone sensitive. Suppressors that inhibited the blastogenic response in lymphocytes to allogenic cells were also activated by Con A but differed in that they were both radiation and prednisone resistant. In contrast, suppressors that inhibited the generation of the killer cells were activated with phytohemagglutinin and not Con A. These suppressors were prednisone and radiation resistant. These observations cannot be explained by differences at the pro-suppressor or suppressor activator levels as both T cell subsets are radiosensitive. Alternatively, heterogeneity of suppressor cell systems may explain these differences.

Use of anti-HLA antibodies to mask major histocompatibility complex gene products on tumor cells can enhance susceptibility of these cells to lysis by natural killer cells.

The role of major histocompatibility gene products (i.e., HLA molecules) in rendering tumor cells resistant to natural killer (NK) cell-mediated lysis was investigated by using mouse monoclonal antibodies to bind and mask HLA or non-HLA gene products on the cell membrane of human allogeneic tumor targets. Enhanced lysis of resistant lymphoid and certain other solid tumor cell lines was observed only when monoclonals used reacted to class I and II HLA molecules but not non-HLA molecules on tumor targets. Enhanced lysis was not due to antibody dependent cellular cytotoxicity or due to an effect of antibody on NK effectors. Of importance, normal autologous and allogeneic human lymphocytes could not be lysed by NK cells despite blast transformation with mitogens or masking of HLA membrane determinants on blasts with monoclonal antibodies. Enhanced lysis, in the presence of antibody to HLA antigens, was not due to increased NK cell binding to tumor targets, but a consequence of enhanced postbinding lysis. Studies using granules obtained from NK cells indicated that masking of HLA antigens did not enhance the susceptibility of tumor targets to cytolysins. Such observations would suggest that HLA antigens on tumor targets inhibit the triggering of effector cells (and release of cytolysins) after recognition and binding of NK cells to target cells.

Evidence demonstrating poor kidney graft survival when acute rejections are associated with IgG donor-specific lymphocytotoxin.

The current prospective investigation was conducted to determine whether development of IgG donor-specific lymphocytotoxins detected at the onset of acute rejections was predictive of a poor-prognosis acute rejection. Between January 1990 and August 1993, 206 kidney transplants were performed. Cadaver kidney recipients were managed with antilymphocyte globulin as induction therapy and all recipients (i.e., cadaver and living related donor kidneys) received triple immunosuppressive therapy, i.e., CsA, AZA, and prednisone. Rejections were treated with intravenous Solu-Medrol and OKT3. Presence of donor-specific IgG lymphocytotoxin was detected by using dithiothreitol-pretreated sera (obtained at onset of rejection) and frozen donor cells. In addition, percentage of panel reactive antibody was determined on this dithiothreitol-pretreated sera. Of the 82 patients with biopsy-proven acute rejections, 19 were found to have developed donor-specific IgG lymphocytotoxin and a marked increase in panel reactive antibody. One-year graft survival in this group was dismal (16%), despite OKT3 therapy. Over 90% of these patients lost their graft within 2 months of rejection diagnosis. In 63 recipients who had acute rejections without development of IgG anti-HLA antibody, 1-year graft survival was 72%. The majority of these patients lost their grafts from chronic rejection. No anti-HLA activity was found in patients who did not have rejection episodes. Based on this study, evidence indicates that assaying for IgG donor-specific antibody at time of rejection is a valuable tool for selecting a subset of patients with poor-prognosis acute rejections. Identifying this subset will become important as we enter an era of new immunosuppressive agents.

Racial disparities in renal transplant outcomes.

The purpose of our study was to evaluate the association of race and ethnicity with outcomes in the living related donor (LRD) renal transplant population, using multivariable adjustment for potential confounding variables. We prospectively analyzed 14,617 patients from the UNOS Renal Transplant Registry who underwent LRD renal transplantations in the United States between January 1, 1988 and December 31, 1996 using the Cox proportional hazards model. This model adjusts for the effects of potential genetic, social, and demographic confounding variables that may be associated with race or ethnicity long-term graft survival. Blacks were 1.8 times as likely as whites (P < 0.01, RR = 1.77) to suffer graft failure during the 9-year study period, which decreased minimally to 1.7 (P < 0.01, RR = 1.65) after controlling for potential confounding variables. Neither genotypic nor phenotypic HLA matching improved outcomes in blacks. Black renal transplant recipients had lower graft survival even after adjustment for matching and rejection, suggesting that non-HLA or socioeconomic mechanisms may contribute to racial differences in transplantation outcomes.

Comparison of a footpad analyser with a tetrapolar model for the determination of percent body fat in young men.

Since excess weight in adolescence predisposes to overweight and obesity in adulthood, a simple measure of excess adiposity in adolescents is important. Fast, inexpensive, bioelectric impedance analysers (BIA) which rely on two foot pad electrodes are now available to measure % total body fat (TBF), but are less well investigated than conventional tetrapolar models which require the subject to lie prone with four electrodes attached to hands and feet. The aim of this study was to compare the estimation of % TBF by a foot pad analyser and a tetrapolar model. Male students, n = 35, 17-19 years had height, weight, waist and hip circumferences measured and completed a questionnaire regarding age, ethnicity and time of last eating and drinking. Percent TBF was measured by a Tanita stand-on analyser (Tanita 105, Tanita Corporation, Japan) and a SEAC tetrapolar model (SEAC, SFB3, QUT, Australia). Mean age (+/- SD) of subjects = 18.2 +/- 0.6 years, BMI = 24.4 +/- 3.5 kg/m2, WHR = 0.81 +/- 0.04, % TBF, Tanita = 18.2 +/- 6.2 and SEAC = 20.4+/-4.8. Both measures of fat were correlated (r = 0.84, p<0.0001). A plot of the average versus the difference of the two analysers found the majority of differences were above zero, especially for measures of fat below 22%, indicating a negative bias for the Tanita. The limits of agreement are between -5.4 and 9% TBF. Information provided by this study will guide gymnasium operators and health professionals to comment on a relative degree of adiposity with greater confidence of data reliability.

The use of pronase-digested human leukocytes to improve specificity of the flow cytometric crossmatch.

Two-color fluorescence cytometry (FCXM) has recently been introduced to improve the detection of anti-HLA antibodies that react to donor cells, especially in recipients receiving kidney allografts. Although this assay system is highly sensitive, it lacks specificity. Between 70% and 90% of potential kidney recipients with a positive FCXM would have been denied transplant if such an assay had been used alone to detect antidonor antibodies. Lack of specificity is principally due to normal or irrelevant IgG in aggregates or immune complexes binding to Fc gamma R receptors on lymphocytes including B cells and a significant subset of T cells. To circumvent this problem, we digested Fc gamma R receptors on lymphocytes with pronase. We present data demonstrating that pronase digestion of lymphocytes does not alter HLA antigenicity. In addition, pronased lymphocytes allow one to use either single- or two-color FCXM. With single-color FCXM, one can quantitate antibody reactivity to lymphocytes via a cursor (on the fluorescence histogram) that separates lymphocytes that do not bind to antibodies. We present data demonstrating that this modification renders FCXM highly sensitive and specific. In addition, one can discriminate between IgG and IgM antibodies that react to lymphocytes.

Hyperacute renal allograft rejection from anti-HLA class 1 antibody to B cells--antibody detection by two color FCXM was possible only after using pronase-digested donor lymphocytes.

We present a report of a transplant recipient who lost her renal allograft from hyperacute rejection. This was secondary to a weak IgG anti-HLA class I antibody that was only reactive to donor B lymphocytes. This antibody was not detected in her pretransplant serum by the conventional complement-dependent cytotoxicity assays using donor blood lymphocytes. Pretransplant sera were analyzed retrospectively by two-color flow cytometric crossmatching (FCXM). It was difficult to determine if the recipient's serum contained an IgG antibody specific for HLA on donor B cells since IgG from control AB sera and pretransplant sera bound equally well to CD19 B cells. However, when donor lymphocytes were pretreated with pronase to digest the membrane receptor for Fc domain of IgG (Fc gamma R) on non-T-cells, control IgG in AB serum did not bind to B cells and, hence, it was easy to detect binding of IgG (in pretransplant sera) to HLA on B cells. This case underscores the importance of identifying weak anti-HLA class I antibodies reactive only to B cells. Moreover, it shows that the currently used two-color FCXM lacks the specificity to detect such antibodies.

One stop clinic for assessment of risk for fetal anomalies: a report of the first year of prospective screening for chromosomal anomalies in the first trimester.

OBJECTIVE: To evaluate the introduction of a one stop multidisciplinary clinic for screening for fetal chromosomal abnormalities in the first trimester by a combination of maternal serum biochemistry and ultrasonography providing a risk of chromosomal abnormalities within a one hour clinic visit. DESIGN: One year retrospective review of screening performance. POPULATION: All women attending for routine antenatal care. The population included 4,190 singleton pregnancies in women of all ages screened between 10 weeks and 3 days and 13 weeks and 6 days of gestation between the periods 1 June 1998 and 31 May 1999 in a district general hospital antenatal clinic. METHODS: All women booked into the clinic were offered screening by a combination of maternal serum free beta human chorionic gonadotrophin (hCG) and pregnancy associated plasma protein A (PAPP-A) and fetal nuchal translucency thickness. Women at increased risk of carrying a fetus with trisomy 21 or trisomy 18/13 (> or =1 in 300 at sampling) were offered counselling and an invasive diagnostic procedure. Follow up of the outcome of all pregnancies was carried out. MAIN OUTCOME MEASURES: The detection rate for trisomy 21, trisomy 18/13 and all aneuploides, false positive rate, uptake of screening, uptake of chorionic villus sampling in women identified at increased risk and fetal loss after chorionic villus sampling. RESULTS: Overall 97.6% of the women (4,088/4,190) accepted first trimester screening. The rate of detection of trisomy 21 was 86% (6/7), for trisomy 18/13 100% (9/9) and for all aneuploides 95% (18/19). Fetal death at presentation was found in 1.6% of pregnancies (69/4,088). Of women who accepted screening, 6.1% (257/4,088) presented too late for fetal nuchal translucency measurement and 6.5% of the women (271/4,088) presented too early. The false positive rate was 6.7% (253/3,762). Uptake of invasive testing was 83% (207/253). CONCLUSION: First trimester prenatal screening for chromosomal abnormalities using a combination of maternal serum biochemistry and fetal nuchal translucency thickness can achieve detection rates in excess of 90%. These services can be provided in a one stop multidisciplinary clinic.

An evaluation of squamous cell carcinoma antigen in patients with cervical squamous cell carcinoma.

In a prospective study, serum concentrations of squamous cell carcinoma antigen, a subfraction of tumor antigen (TA-4), were determined by radioimmunoassay from healthy donors, pregnant women, and subjects with various benign and malignant gynecologic diseases. Ninety-six percent of 99 healthy persons including all 52 female controls, the 15 pregnant patients, and all 23 subjects with benign gynecologic tumors, had squamous cell carcinoma antigen levels less than 2.0 ng/ml. Seven of 51 (14%) patients with cervical intraepithelial neoplasia and 16 of 24 (67%) patients with cervical squamous cell carcinoma had squamous cell carcinoma antigen levels greater than 2.0 ng/ml. Declining and rising levels of squamous cell carcinoma antigen, which were determined sequentially in nine cases of cervical carcinoma that were associated with elevated pretreatment levels of squamous cell carcinoma antigen, correlated with regression and progression of the disease. Serial serum levels of squamous cell carcinoma antigen provide a noninvasive means of monitoring the effects of individual therapy in patients with cervical squamous cell carcinoma.

Increased late hepatic artery thrombosis rate and decreased graft survival after liver transplants with zero cross-reactive group mismatches.

The use of broad-specificity cross-reactive groups (CREGs) at the A and B HLA loci has been proposed as a means to improve both access and outcome for renal transplantation but has not yet been studied for liver transplantation. We retrospectively analyzed our results for all adult liver transplantations performed at our center between 1989 and 1996 for which HLA typing and complete 3-year follow-up data were available. Two hundred eight transplantations were studied, with a mean follow-up of 66 +/- 2 months (range, 36 to 110 months); A and B loci were converted to CREGs by the method of Rodey. Thirteen percent of the patients with 0 CREG mismatches had hepatic artery thrombosis versus 2% for those with 1 or more mismatches (odds ratio, 6.7; 95% confidence interval, 2.6 to 17.3 by univariate analysis; odds ratio, 3.5; 95% confidence interval, 1.1 to 11.7 by logistic regression analysis). These events occurred significantly later in the 0-CREG mismatch group (21 +/- 7 v 4 +/- 2 months posttransplantation; P =.04 by Student's t-test). Graft survival rates were significantly lower for patients with 0 CREG mismatches (56% v 68% at end of study; P =.05 by Cox-Mantel test). The number of CREG mismatches had no effect on the frequency of rejection, steroid-resistant rejection, or infectious complications, including cytomegalovirus. Neither total nor individual A, B, or DR HLA matching had an effect on outcome. There appears to be little evidence that intentional CREG matching would improve outcomes for liver transplantation and, under some circumstances, could be deleterious.