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B K Shenton

Publications and source records attributed to B K Shenton.

At least 55 records · Page 3Linked to original sources
PubMed1995

The flow cytometric crossmatch in liver transplantation.

The role of the cytotoxic crossmatch in liver transplantation is generally considered controversial. The development of the flow cytometric crossmatch has allowed the detection of lower levels of donor-directed IgG than is possible with the conventional crossmatch. This assay has been shown to be useful in renal transplantation. However, with the controversial role of the standard cytotoxic crossmatch, the flow cytometric crossmatch has not been used in liver transplantation. Twenty-seven human orthotopic liver allograft recipients were tested for donor-directed IgG using the flow cytometric crossmatch. Thirteen recipients were identified with either T or B lymphocyte-directed IgG. This group had a significantly increased incidence of clinical rejection (75%) as compared with the negative group (29%, P = 0.02, Fisher's exact test). The differences were greatest with B lymphocyte-directed IgG and the rejections were generally steroid sensitive. In this series, the flow cytometric crossmatch proved to be a better prognostic indicator of rejection than the conventional cytotoxic crossmatch. In addition, the association of a positive flow cytometric crossmatch with rejection indicates that the liver follows the same pattern seen in renal and cardiac grafts.

Adult↗
PubMed1995

The value of posttransplant monitoring of interleukin (IL)-2, IL-3, IL-4, IL-6, IL-8, and soluble CD23 in the plasma of renal allograft recipients.

Over the past few years, the central role of cytokines in the amplification of the immune response has been reported and several studies have examined the relationship between the plasma level of individual lymphokines during renal allograft rejection. The aim of the present investigation was to study simultaneously IL-2, IL-3, IL-4, IL-6, IL-8, and soluble CD23. Analysis of results has allowed both the prognostic value and any possible interrelationships between the measured cytokines to be determined. We studied 16 renal transplant recipients for the first 14 days after transplantation. Seven patients showed clinical evidence of acute allograft rejection and 5 showed excellent stable graft function with no signs of rejection. Primary nonfunction was seen in 4 patients. The plasma levels of each cytokine were measured by commercially available ELISA and immunoradiometric assay kits. As reported in previous studies, plasma IL-2 levels, whenever found at detectable levels, were predictive of impending graft rejection. Serial monitoring of IL-4 and IL-6 was more reliable for the differential diagnosis of rejection, particularly toward the end of the first week after transplantation. IL-3, IL-8, and soluble CD23 were not diagnostic or predictive of rejection, due to the occurrence of significantly high levels in transplant patients who showed no evidence of clinical rejection. While the value of cytokine monitoring has been shown in this study, it should be remembered that infection, although not seen in these studies, may have a profound affect on the results obtained.

Biomarkers↗
PubMed1995

The clinical significance of post-transplantation non-HLA antibodies in renal transplantation.

This study was undertaken to examine the clinical relevance of antibodies detected in the sera of patients following renal transplantation. The sera from 23 transplant recipients with acute rejection and 10 transplant recipients with diagnosed chronic rejection were tested against various epithelial, monocyte and endothelial cell lines (A549, HTB44, primary renal epithelial, U937 and Ea-hy 926). The test used for detecting binding antibodies was a simple, indirect immunofluorescence flow cytometric technique. The level of IgG antibodies directed against the test cell lines was examined in the sera of patients with mild or severe rejection and compared to those of patients showing no signs of rejection. Patients with chronic rejection were found to have increased levels of antibodies (IgG and IgM) when compared to patients with either end-stage renal failure or patients with stable post-transplant renal function. Antibodies detected by the present technique were directed against antigens found on all cell lines tested, and immunoblotting indicated that they were directed against non-HLA antigens. In conclusion, monitoring for the presence of such antibodies may provide a valuable prognostic indicator of graft rejection in renal transplant patients.

Antibodies↗
PubMed1995

Renal allograft rejection: the temporal relationship and predictive value of plasma TNF (alpha and beta), IFN-gamma and soluble ICAM-1.

Recently, close interactions have been described between the tumour necrosis factors alpha and beta (TNF-alpha and beta), interferon-gamma (INF-gamma) and intercellular adhesion molecule-1 (ICAM-1) in T-cell mediated immune activation. During the process of renal graft rejection, the properties of these cytokines to act as powerful stimulators of macrophages, to upregulate class II MHC expression and to stabilise cell-to-cell binding make them of great potential interest. The aim of the present study was to determine the plasma levels of each cytokine and soluble ICAM-1 in 16 renal allograft recipients. We examined plasmas of patients for the first 2 weeks after transplantation and correlated results with the clinical pattern of rejection. Our data suggest an immunopathologic involvement of TNF-alpha, TNF-beta and slCAM-1 in renal allograft rejection and showed that there was a significant elevation in plasma concentrations of these parameters 2 or 3 days prior to the diagnosis of clinical rejection. Rises in INF-gamma did not appear to be significant with regard to rejection as very high levels were found in patients showing no evidence of clinical rejection.

Enzyme-Linked Immunosorbent Assay↗
PubMed1995

A flow cytometric study of c-erbB-3 expression in breast cancer.

In order to assess the specificity of biotinylated anti-c-erbB-3 antibody, screening was performed on a series of tumour cell lines and lymphocytes. Staining was found to be consistent, with good reproducibility. Twenty-nine consecutive breast cancer samples were obtained from women treated with tamoxifen and undergoing elective mastectomy. Twenty-eight invasive ductal carcinomas and 1 DCIS were stained for c-erbB-3 expression: 2 were grade I (Bloom and Richardson), 15 grade II, and 11 grade III tumours, 1 being unclassified; 16 were axillary node positive and 10 node negative; in 2 cases no nodes were sampled. Tumours examined by flow cytometry were stained with cytokeratin FITC antibody and the cytokeratin-positive population gated. Using Mann-Whitney analysis no association was seen between c-erbB-3 expression and Bloom and Richardson grade or axillary node status. In the tumour samples c-erbB-3 expression was found to show as association with EGF-R (P = 0.021 r2 = 0.16), PgR (P = 0.02, r2 = 0.16), c-myc (P < 0.0001, r2 = 0.5), c-jun (P = 0.001, r2 = 0.4) and c-fos (P = 0.001, r2 = 0.5) but not with c-erbB-2 (P = 0.2, r2 = 0.06), ER (P = 0.4, r2 = 0.02) or p53 1801 (P = 0.05, r2 = 0.2). Expression of c-erbB-3 may not be an independent marker of prognosis, but it is associated with other markers of poor prognosis and early cellular events linked with aberrant growth and differentiation.

Breast Neoplasms↗
PubMed1995

The relationship between flow-cytometric and immunohistochemically detected c-erbB-2 expression, grade and DNA ploidy in breast cancer.

Quantification of c-erbB-2 and its relationship with other prognostic markers using flow cytometry has been examined. In this study a level for c-erbB-2 expression above which tumours are classified as positive by flow cytometry has been determined by employment of positive cut-off threshold levels. c-erbB-2 expression by both flow cytometry and immunohistochemistry was studied using the monoclonal antibody NCL-CBII. The relationship of c-erbB-2 quantification by flow cytometry was then compared with ploidy, axillary node status, tumour size and grade. Increased c-erbB-2 expression was seen using flow cytometry. Correlation between immunohistochemistry and flow-cytometry methods just failed to reach significance (P = 0.06). Immunohistochemistry revealed a significant relationship between c-erbB-2 expression and aneuploidy (P = 0.04). Cytokeratin-positive cells from 110 samples obtained from patients with breast cancer were assayed for DNA content and c-erbB-2 expression by flow cytometry. No correlation was seen between these parameters upon application of Mann Whitney analysis. However, examination of fluorescence thresholds showed a positive correlation between grade and c-erbB-2 expression at a level of more than 3200 molecules (P < or = 0.03). At the level of 3600 molecules significance was increased (P = 0.004). These levels equated with between 15% and 19% of the samples being classified as c-erbB-2-positive. Application of these cut-off points showed no correlation between c-erbB-2 expression and ploidy, tumour size or axillary node status. Comparison of ploidy and grade showed a significant association (P = 0.0015), increased grade correlating with aneuploidy.

Adult↗
PubMed1995

p53 expression measured by flow cytometry. A comparison of three monoclonal antibodies and the relationship with grade and DNA ploidy in breast cancer.

The aim of this study was to quantify p53 expression by flow cytometry. A panel of three monoclonal antibodies: NCL-p53-240, NCL-p53-1801 and NCL-p53-DO7, was tested on breast cell lines and primary breast cancers. The relationships between ploidy, tumour grade and p53 expression for each antibody, were examined. Methodology was assessed using a variety of breast cell lines. Staining patterns were confirmed and the quantification technique qualified. Cytokeratin-positive cells from 58 samples obtained from patients with breast cancer were assayed for DNA content and p53 expression. p53 quantification was performed using calibrated fluorescent beads on cytokeratin-positive cells. Bloom and Richardson grading revealed 20 grade I and 38 grade II/III breast cancers. Examination of fluorescence thresholds showed a positive correlation between grade and DO7 (P = 0.003) at a level of 8900 molecules, 240 (P = 0.005) at a level of 2900 molecules and 1801 (P = 0.005) at a level of 1850 molecules. These levels equated with 34% (DO7), 43% (240) and 43% (1801) of the samples being classified as p53-positive. Examination of ploidy revealed 23 diploid and 35 aneuploid breast cancers. Application of p53 threshold levels on diploid and aneuploid tumours showed correlation between aneuploidy and p53 expression for DO7 at a level of 9000 molecules, 240 at a level of 1900 molecules and 1801 at a level of 1800 molecules. These levels equated with 34% (DO7), 52% (240) and 52% (1801) of the samples being classified as p53-positive. We conclude that measurement of p53 by flow cytometry may be of clinical importance by indicating levels of positivity using fluorescence thresholds. p53 expression has been shown to correlate with both grade and ploidy. Flow-cytometric measurement of p53 may be a useful prognostic assay.

Aneuploidy↗
PubMed1995

Flow cytometric crossmatching in renal transplantation--the long-term outcome.

The association of a positive flow cytometric crossmatch between recipient IgG directed against donor T lymphocytes and poor outcome is well described in renal transplantation. Until now no long-term follow-up on such patients has been available. In this study, 117 renal transplant patients were followed up for a period of 5 years. Of these 21 were known to have donor T cell directed IgG and five had B lymphocyte directed IgG. Both groups of patients with these antibodies had a significantly poorer outcome at 5 years than did the group of patients without IgG (p < 0.0001, Handel Maenzel test). Patients with antibody detected preoperatively were tested again either at the time of graft failure or at 5 years post-transplantation. The sera were tested against stored donor cells and the intensity of surface IgG compared with the preoperative levels. In those recipients who lost their grafts the levels increased in 60% of cases, but those who retained their grafts also had an increase in levels of donor directed antibody in 50% of cases. The changing levels of antibody therefore appeared to have little relevance to outcome. However when IgG isotypes were considered, in those who experienced graft failure and also had a gamma 3 isotype, a rise in IgG was demonstrated in all cases. Conversely, successful grafts with gamma 3 had a decline in levels between preoperative and 5-year samples in three of the four cases (not significant).

Flow Cytometry↗
PubMed1995

Are fine-needle breast aspirates representative of the underlying solid tumour? A comparison of receptor levels, ploidy and the influence of cytokeratin gates.

Fifty-three solid and 33 fine-needle aspirate (FNA) samples (20 paired) of human breast carcinomas were examined by flow cytometry. Experiments were conducted to assess whether FNA samples were phenotypically representative of the solid tumour. Quantification of oestrogen receptor (ER), epidermal growth factor receptor (EGFR), c-erbB-2 receptor levels and ploidy were examined on the total and cytokeratin-positive cell populations. The absolute number of molecules of cytokeratin per cell expressed on the FNA (n = 33) and solid tumour (n = 53) samples showed no significant difference, but, on a proportional basis, there was a significant difference between the two samples (P = 0.004), with lower expression exhibited by the FNAs. Examination of paired data showed no significant difference in the percentage of cytokeratin-positive cells (P = 0.51) or in the number of cytokeratin molecules expressed (P = 0.25). While the correlation for ER expression between paired tumour and FNA samples in the absence of cytokeratin gating was P = 0.06, r2 = 0.18, clear correlation was shown when a cytokeratin gate was used (P = 0.005, r2 = 0.4). Repeating this experiment for EGFR, it was found that no correlation was seen between FNA and solid tumour (P = 0.2, r2 = 0.14) in ungated populations, but use of the cytokeratin gate improved the correlation (P = 0.05, r2 = 0.3). A similar finding was seen with c-erbB-2 expression (P = 0.2, r2 = 0.1) without cytokeratin gating and when it was employed (P = 0.05, r2 = 0.4). Ploidy data showed concordance in 18/20 cases. Three cases of aneuploidy were missed by FNA, and this was because of an insufficient number of cells for analysis. The presented data suggest that FNAs are representative of solid tumours and may be useful for measuring receptor levels on clinical material when cytokeratin gating is used. However, observation by light microscopy is still necessary to confirm the presence of tumour cells in FNAs subjected to flow cytometry.

Biopsy, Needle↗
PubMed1995

P53 and ploidy assessed by flow cytometry in bladder washings.

OBJECTIVE: To determine p53 expression in cells in bladder washings and to relate this to DNA content and clinical outcome. PATIENTS AND METHODS: Washings from 102 patients (41 with newly diagnosed superficial tumours [pTa and pT1], 49 with recurrent superficial tumours and 12 with carcinoma invading bladder muscle) were studied. In 39 cases, the primary bladder tumour was also analysed. The rates of tumour recurrence and progression were determined for the new superficial tumours and related to both p53 expression and DNA content. RESULTS: Cells positive for p53 were detected in 22 of 90 (24%) washings from patients with superficial bladder cancer. P53 expression correlated with tumour stage (P < 0.05), grade (P < 0.05) and abnormal DNA content (P < 0.05). The analysis of pure urothelial (cyto-keratin-positive) cells improved the detection of DNA abnormalities (P < 0.001). In 74% of cases where both washings and tumour were analysed, the results for DNA content agreed. Of 41 new superficial tumours, 27 (66%) recurred (11 were p53-positive, 16 were p53-negative, P = 0.221; 17 had abnormal DNA content, 10 were diploid, P = 0.069). Four patients progressed (one was p53-positive, P = 0.315 and all had abnormal DNA content, P = 0.072). CONCLUSION: P53-positive cells can be detected in washings using flow cytometry and were more commonly detected in association with aneuploid tumours. At short-term follow-up, flow cytometric analysis of DNA content in washings had greater predictive value than had p53 expression. Few washings contained aneuploid cells when the primary tumour contained diploid cells, although the collection of washings is a convenient way of sampling tumour cells.

Aged↗
PubMed1994

Flow cytometric method for the measurement of epidermal growth factor receptor and comparison with the radio-ligand binding assay.

A method for the purification, conjugation, and use of EGF-R antibody to quantify EGF receptors on tumour cells by flow cytometry is described. The quantification of both internal and external EGF receptors was determined by treatment with saponin, rendering the cells permeable to the EGF-R antibody. Using QCS bead standards, the number of EGF-R binding sites per cell was assessed. Results were compared with conventional EGF-R quantification using a radio-ligand binding assay. A high degree of correlation was found between the two methods. The flow cytometric method for EGF-R quantification described is simple, rapid, and reproducible. The assay may be of particular value in measuring EGF-R on urgent clinical samples or those that are too small (such as breast aspirates) for measurement by the radio-ligand binding assay. Advantages of this assay over the radio-ligand binding assay include reduction in use of radio-labelled iodine compounds, a decrease in analysis time, and reduced cost and quantity of material needed for assay. In addition, flow cytometry offers the possibility of selecting cell phenotypes by gating as well as live/dead cells by using multi-parameter flow cytometry.

Antibodies, Monoclonal↗
PubMed1994

Characterization of donor-directed antibody class in the post-transplant period using flow cytometry in renal transplantation.

Over the past few years there has been increasing awareness of the importance of humoral mechanisms in the rejection of renal transplants. In this study we have monitored the development of antibodies directed against donor T and B lymphocytes using the sensitive flow cytometric technique. Forty-two cadaveric renal transplants were studied both before and for a maximum of 14 days after transplantation. Donor cells were separated from spleen on the day of transplantation and stored in liquid nitrogen until required. The dual colour flow cytometric assay was used to detect IgG or IgM directed against donor T or B lymphocytes. Using AB sera as controls, results were expressed as relative median fluorescence (RMF) and then correlated with the clinical performance of the grafts. Significant associations were found between the incidence of donor-directed antibodies and the development of clinical rejection. The magnitude of the rise in antibody levels was also related to graft performance. In patients showing severe graft rejection, high levels of antibodies of the IgG class developed before the clinical diagnosis of rejection was made. The routine use of this test allows the prediction of impending severe rejection to be made and may have important implications for immunosuppressive therapy.

B-Lymphocytes↗
PubMed1994

The detection and relevance of pretransplant antibodies.

Over the last 25 years the importance of the humoral presensitization status of transplant recipients has increasingly been recognized. With greater understanding of the precise specificity of the pretransplant antibody status of recipients it is clear that the graft survival figures will improve. The development of new immunosuppressive regimens to reduce antibody levels both before and after transplantation may be critically important.

Antibodies↗
PubMed1994

The value of flow cytometric crossmatch in cardiac transplantation.

One of the major clinical problems in cardiac transplantation is that of moderate rejection of the graft, and over the past few years there is increasing evidence that humoral antibody may be important in graft prognosis. The sensitivity of the conventional cytotoxic crossmatch has been questioned, and an increased significance of there of the flow cytometric crossmatch (FCXM) to detect the presence of antibodies before transplantation has been reported. In this study we have examined the sera of 138 cardiac transplants (1988-1992) for the presence of donor-directed IgG and IgM antibodies using FCXM. Sera were collected immediately before transplantation and before the institution of immunosuppressive therapy. All pretransplant cytotoxic crossmatches were negative. After a minimum follow-up period of 3 months, the performance of the transplants was graded by endomyocardial biopsy: 1, no or mild evidence of rejection; 2, patients showing moderate rejection requiring increased immunosuppression. Of the 138 patients studied, 10 patients were excluded as they died within the first week of transplantation. Eight children were excluded since they were given prophylactic ATG (Merieux). A positive FCXM result was defined as showing values in excess of that found for the AB control sera. A significant association was found between the presence of both IgG to T and B cells and IgM to T cells and graft performance (P = 0.02 and 0.93, respectively). Indeed, IgM-directed T-cell antibodies were only found in patients with moderate rejecton. These two groups were mutually exclusive, so that the FCXM was able to identify the presence of moderate rejection in 55% of the patients. In conclusion, results show that pretransplant FCXM in cardiac transplantation provides a more sensitive assay of antibody status in recipients and has proved to be of prognostic value.

Adult↗
PubMed1994

Abnormalities in p53 and DNA content in transitional cell carcinoma of the bladder.

OBJECTIVE: To determine if transitional cell tumours of the bladder which contain mutations in the p53 gene have alterations in their DNA content. MATERIALS AND METHODS: In 33 transitional cell tumours of the bladder, DNA content was determined by flow cytometry and compared with expression of mutant p53 as assessed by immuno-histochemistry. RESULTS: Abnormal DNA content was associated with increased staining for p53 (P < 0.05), high tumour stage (P < 0.001) and increased histological grade (P < 0.01). Although positive staining for p53 was frequently associated with abnormal DNA content, a significant number of non-diploid tumours stained negatively for mutant p53. CONCLUSION: These data demonstrate that increased staining for p53 is associated with abnormalities in DNA content suggesting that mutation of the p53 gene is associated with an increased rate of chromosomal abnormalities and an increased rate of cell proliferation.

Carcinoma, Transitional Cell↗
PubMed1994

P53 expression, ploidy and progression in pT1 transitional cell carcinoma of the bladder.

OBJECTIVE: To investigate a potential role for overexpression of the p53 protein in the identification of pT1 bladder tumours destined to progress. PATIENTS AND METHODS: The protein expression of the tumour suppressor gene p53, nuclear ploidy and tumour grade were studied in 25 patients with pT1 bladder tumours. Follow-up data was available for 21 tumours over a 10 year period. p53 expression was determined by immuno-histochemistry on paraffin embedded sections and flow cytometry was performed on cell suspensions derived from the same blocks. RESULTS: Ten of 21 (48%) tumours progressed to muscle invasive disease. Nuclear staining for the protein was seen in 16 (64%) tumours and of these nine (56%) progressed. All tumours with abnormal DNA content and all high grade tumours demonstrated overexpression of p53. Significantly more patients with high levels of p53 expression (> 10% nuclear staining) progressed compared with tumours with less than 10% of nuclei staining (P = 0.007). However, grade was the most specific predictor of progression (100%), with all grade 3 tumours progressing and grade was also the most significant prognostic indicator in terms of survival (P = 0.025). CONCLUSION: Tumour grade is likely to remain as the most useful aid to management decisions in pT1 bladder tumours.

Aged↗