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R R Tubbs

Publications and source records attributed to R R Tubbs.

At least 37 records · Page 2Linked to original sources
PubMed1999

The effect of fixation on detection of B-cell clonality by polymerase chain reaction.

It has been suggested that neutral buffered formalin (NBF)-fixed, paraffin-embedded, or fresh specimens might provide satisfactory DNA templates for polymerase chain reaction (PCR) assays used in establishing the clonality and presumptive B-cell lineage of lymphoma. The suitability of other fixatives used by hematopathologists, such as B5, is still undetermined. Thirty cases were identified from the files of the Cleveland Clinic Foundation, Cleveland Ohio, that showed abnormal immunoglobulin heavy chain (IgH) rearrangement by Southern blot analysis (SBA). Corresponding paraffin-embedded tissue samples fixed in NBF (21 cases), B5 (18 cases), Hollande's fixative (17 cases), zinc formalin (ZF) (5 cases), and Bouin's fixative (3 cases) were studied. With use of consensus primers against the framework 3 (FR3) and FR2 regions of the VH gene, paired against JH primer(s), PCR analysis was performed. bcl-2/IgH translocation was also studied. Ten reactive lymphoid samples were used as controls, and 40 cases were evaluated. Successful amplification of a clonal proliferation was manifested as one or two discrete narrow bands in the appropriate size range. The sensitivity of detecting clonality was 95, 94, 67, 80, and 0% for NBF, Hollande's fixative, B5, ZF, and Bouin's fixative, respectively. Although NBF and Hollande's fixative were 100% specific, consistent false-positive results were a major problem with B5-fixed tissue. Paraffin-embedded tissue, fixed in NBF, Hollande's fixative, and ZF solutions, may be used for DNA extraction and PCR assays for establishing B-cell clonality. The precipitating fixative B5 and Bouin's solution should not be used for this purpose until the issue of false-positive results is resolved.

B-Lymphocytes↗
PubMed1999

Detection of human papillomavirus in cervical carcinoma: comparison of peroxidase, Nanogold, and catalyzed reporter deposition (CARD)-Nanogold in situ hybridization.

We compared three in situ hybridization (ISH) methods for their applicability and sensitivity in detecting human papillomavirus (HPV) in 61 cases (1 Grade 1, 18 Grade 2, 42 Grade 3) of routinely processed squamous cell cervical carcinoma. A commercially available biotinylated probe for HPV-16/18 was applied to serial sections and detected by conventional streptavidin-biotin-peroxidase ISH, streptavidin-Nanogold-silver ISH, and catalyzed reporter deposition (CARD)-Nanogold-gold ISH. The latter method involved signal amplification by peroxidase-catalyzed deposition of biotinylated tyramides at the hybridization sites, followed by detection of accumulated biotin by streptavidin-Nanogold made visible by autometallography. The HPV-16/18 detection rates for the three methods were 39.3, 44.3, and 65.6%, respectively. In all of the three ISH methods, a punctate staining pattern (single or multiple intranuclear spots of variable size), presumably indicating viral integration, was highly predominant among the positive cases. Two of the cases identified as positive by streptavidin-biotinperoxidase ISH were rated negative with streptavidin-Nanogold-silver ISH, whereas six cases that were clearly negative with streptavidin-biotinperoxidase ISH became positively stained with streptavidin-Nanogold ISH. All of these discordant cases were positive by the highly sensitive CARD-Nanogold-gold ISH. In addition, the high detection sensitivity of CARD-Nanogold-gold ISH was confirmed by its ability to detect single copies of HPV-16 in SiHa cells. In general, we found that the intense black reaction product from Nanogold autometallography gave superior contrast to that obtained with the peroxidase system. After tyramide signal amplification, the staining was so clearly visible that preparations could be readily screened under low magnification. Our findings precisely demonstrated the need for improved sensitivity in the in situ detection of HPV. The CARD-Nanogold-gold technology looks promising as a highly sensitive method for routine ISH in molecular pathology.

Carcinoma, Squamous Cell↗
PubMed1998

Utility of cytokeratin subsets for distinguishing poorly differentiated synovial sarcoma from peripheral primitive neuroectodermal tumour.

AIMS: Poorly differentiated synovial sarcoma (PDSS) is a round cell sarcoma that may be difficult to distinguish from other round cell sarcomas, such as peripheral primitive neuroectodermal tumour (pPNET), on histological examination alone. Furthermore, these two tumours may show overlapping immunophenotypes, as some cases of PDSS express CD99, and, on the other hand, pPNET may express epithelial markers. The goal of this study was to determine the utility of cytokeratin (CK) subsets in distinguishing between these two lesions. METHODS AND RESULTS: We evaluated 13 cases of pPNET with RT-PCR detected EWS/FLI-1 fusion transcript and 21 cases of PDSS for the immunohistochemical expression of CK7 and CK19. All cases of PDSS had areas of recognizable monophasic or biphasic synovial sarcoma that expressed at least one epithelial marker. Thirteen of 21 (62%) PDSS stained with AE1/AE3. CK7 and 19 were expressed in 11 (52%) and nine (43%) cases, respectively. Although six of 19 (32%) PDSS demonstrated cytoplasmic staining for CD99, none showed a membranous pattern of immunoreactivity. In contrast, 12 of 13 (92%) pPNET showed strong membranous immunoreactivity for CD99. Four tumours (31%) showed focal staining with AE1/AE3, two of which (15%) stained for CK19. CK7 was not detected in any of the pPNETs. CONCLUSIONS: Although AE1/AE3 may be found in up to 31% of cases of pPNET, the expression of CK7 makes this diagnosis less likely.

12E7 Antigen↗
PubMed1998

Comparative analysis of interphase FISH and RT-PCR to detect bcr-abl translocation in chronic myelogenous leukemia and related disorders.

The t(9;22)(q34;q1 1) between the abl and bcr genes plays a pivotal role in the diagnosis and pathogenesis of chronic myelogenous leukemia (CML). Its detection is routinely accomplished by Southern blot analysis and karyotyping. Interphase fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) are emerging molecular techniques that offer viable alternatives. We analyzed 40 samples of peripheral blood and bone marrow (CML, 16; acute myelogenous leukemia, 6; acute lymphoblastic leukemia [ALL], 1; chronic lymphoblastic leukemia, 2; myelodysplasias, 4; myeloproliferative disorders, unclassified, 3; nonleukemic hematologic malignancies, 3; hypercellular bone marrow, 1; normal control samples, 2; and K562 cell line samples, 2) for the presence of bcr-abl fusion gene and its messenger RNA (mRNA) transcript by FISH and RT-PCR, respectively. We compared the results with results of Southern blot analysis and karyotyping when available. Cost analysis was performed. Thirty-three samples were evaluable by FISH; 14 of 14 evaluable CML samples and one ALL sample were positive for bcr-abl by FISH (100%). The other 15 evaluable samples were negative; 16 of 16 (100%) and 13 of 16 (81%) of CML cases were positive for bcr-abl mRNA by RT-PCR (chemiluminescent blot method) and RT-PCR (colorimetric method), respectively. The ALL sample was positive by both RT-PCR methods. All other samples were negative by RT-PCR (chemiluminescent blot method), and all but 1 case of myeloproliferative disorder tested negative by RT-PCR (colorimetric method). We conclude the utility of FISH and RT-PCR is associated with certain limitations, such as insufficient RNA for RT-PCR and the occasional absence of internal positive FISH control signals. However, each procedure offers (with a high concordance rate) a specific and cost-effective alternative to Southern blot analysis and karyotyping and improved turnaround time for the detection of bcr-abl fusion gene or its mRNA transcript.

Blotting, Southern↗
PubMed1998

Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare clonal disorder that consists of single or multiple mass lesions composed of cells with an abnormal Langerhans cell phenotype. Its etiology remains unknown, despite extensive searches for evidence of consistent cytogenetic abnormalities, gene rearrangements, or viral genomes. Similarly, the pathogenesis of the disease is enigmatic, although the altered expression of cytokines and cellular adhesion molecules, important for migration and homing of the activated normal Langerhans cell, may play an important role. The biologic behavior of LCH ranges from spontaneous remission to lethal dissemination, and such behavior cannot be predicted on the basis of histologic features. The presence and degree of organ dysfunction, together with the patient's age at diagnosis, remain the most reliable indicators of prognosis. Treatment of severe, refractory disease continues to be controversial and, in many cases, ineffectual. The revised classification scheme for LCH and related disorders recognizes the uncertain biological potential of LCH and its relation to other processes of dendritic and macrophage origin.

Diagnosis, Differential↗
PubMed1998

Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.

Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions.

Cells, Cultured↗
PubMed1998

A simplified method of CD34+ cell determination for peripheral blood progenitor cell transplantation and correlation with clinical engraftment.

Enumeration of CD34+ cells by flow cytometry is the recognized standard for quantitating progenitor cells for peripheral blood progenitor cell (PBPC) transplantation. Although many clinical studies have confirmed that the time to neutrophil and platelet engraftment is inversely proportional to the number of CD34+ cells infused, the minimum number of CD34+ cells necessary to acheive rapid engraftment has not been satisfactorily determined. The lack of a standardized method for quantitation of CD34+ cells by flow cytometry (FCM) is often cited as the reason for this ambiguity. This report describes an FCM method for CD34+ cell determination that is simple, highly reproducible, comparatively inexpensive, and validated by excellent correlation with clinical engraftment. Pheresis samples are stained and fixed within 4 hours of collection. Two hundred fifty thousand events are acquired as list mode data using a forward scatter threshold. The discrete CD34+ population is enumerated using a CD34-phycoerythrin FL2 vs. side scatter plot and Paint-A-Gate Pro software. The method was validated by excellent statistical correlation with clinical engraftment. Using this method, we determined the number of CD34+ progenitor cells necessary to achieve rapid engraftment to be 2 x 10(6)/kg.

Antigens, CD34↗
PubMed1998

Extension of useful reagent shelf life beyond manufacturers' recommendations. Cell Markers Committee of the College of American Pathologists.

OBJECTIVE: The College of American Pathologists Cell Markers Committee designed a study to evaluate the use of immunohistochemistry primary antibodies beyond manufacturers' recommended dates. METHODS: Pathologists were asked to save aliquots of primary antibodies during mid-1997 so that by spring 1998 the reagents would be "outdated" according to manufacturers' recommendations. Three tumors were immunostained both in mid-1997 and early 1998 (using outdated reagents in 1998). Two hundred twenty-one laboratories participated. PATIENT SAMPLES: Immunostained materials consisted of an angiomyolipoma immunostained for muscle-specific actin and HMB-45, a melanoma immunostained for S100 protein and HMB-45, and a large cell lymphoma immunostained for common leukocyte antigen and HMB-45. Blocks from the same tumor were used in each instance. MAIN OUTCOME MEASURE: We compared the immunostaining results as a percentage of laboratories indicating a positive or negative immunohistochemical result between the 1997 and 1998 time points. RESULTS: Only minor differences were identified for the 221 reporting laboratories in 1998 as compared with those in 1997. CONCLUSIONS: The data suggest review of the Health Care Financing Administration's ruling on extending the useful reagent shelf life beyond manufacturers recommendations. Similar studies using more inherently quantitative methodology are suggested.

Antibodies↗
PubMed1997

p53 overexpression is not an independent prognostic factor for patients with primary ovarian epithelial cancer.

BACKGROUND: The clinical significance of p53 overexpression in patients with ovarian carcinoma is uncertain. Previous studies have yielded conflicting results and have been hampered by small patient populations, failure to account for other well-known prognostic variables in multivariate analysis, and failure to account for the grade of p53 overexpression. The aim of this study was to investigate the independent prognostic significance of p53 overexpression in patients with primary ovarian epithelial cancer (POEC). METHODS: Tumors obtained from 221 patients with primary ovarian epithelial cancer (POEC) (Stages I-IV) were studied for p53 overexpression semiquantitatively by immunohistochemical techniques. The median duration of follow-up of surviving patients was 7 years. The presence or absence and degree of p53 overexpression were correlated with the clinicopathologic features of the study population and overall survival. Survival curves were constructed according to the Kaplan-Meier method, and differences in survival were assessed with the log rank test. The prognostic significance of p53 overexpression for survival was assessed in a multivariate analysis with the Cox proportional hazards model. RESULTS: One hundred seven tumors (48.4%) exhibited p53 overexpression. The overexpression was graded as mild in 16.7% of cases, moderate in 5.9%, and strong in 25.8%. p53 overexpression was associated with advanced stage (P = 0.04), higher grade (P = 0.0003), serous histology (P = 0.0018), and patient age > 61 years (P = 0.013). In univariate analysis, p53 overexpression was a significant prognostic factor (P = 0.049 for any degree of overexpression, P = 0.03 for strong overexpression). However, in multivariate analysis, after adjustment for stage and size of residual tumor following cytoreductive surgery, p53 overexpression did not retain statistical significance. Survival curves for patients with different stages and grades of tumor differentiation did not demonstrate a difference in survival among patients with no p53 overexpression, compared with those who demonstrated any degree of p53 overexpression or compared with those who demonstrated strong p53 overexpression. CONCLUSIONS: p53 overexpression is not an independent prognostic factor for patients with primary ovarian epithelial cancer.

Adenocarcinoma↗
PubMed1997

p53 and HER-2/neu overexpression in ovarian borderline tumors.

OBJECTIVE: To study p53 and HER-2/neu expression in borderline ovarian tumors (BLOT) by assessing their frequency, coexpression, and relationship to histologic type, FIGO stage, tumor recurrence, and survival. METHODS: Forty-two patients with confirmed BLOT (25 serous, 13 mucinous, and 4 seromucinous) were followed for 1.5 to 14 years (mean, 6.3). Thirty (71%) patients had FIGO stage I and 12 (29%) had FIGO stage III disease. Paraffin-embedded sections from the 42 BLOT, 5 normal ovaries, and 10 benign ovarian cystadenomas were stained using monoclonal antibodies against human p53 (DAKO-p53, DAKO, Denmark) and HER-2/neu (C-erB-2, Triton, Parkway, CA). Positive staining was semiquantitated depending on the number of positively stained tumor cells. p53 and HER-2/neu overexpressions were correlated to each other, to histologic subtype, stage, tumor recurrence, and survival. RESULTS: None of the patients had tumor recurrence. The 5- and 10-year disease-free survival was 100%. None of the normal ovaries or ovarian cystadenomas demonstrated overexpression of p53 or HER-2/neu. Ten (24%) BLOT demonstrated overexpression of p53 and 9 (21%) demonstrated overexpression of HER-2/neu. HER-2/neu overexpression was significantly greater in stage III than in stage I tumors (P = 0.0157). Seromucinous BLOT demonstrated significantly greater p53 overexpression compared with other histologic subtypes (P = 0.030). Coexpression of p53 and HER-2/neu occurred in 4 patients (9.5%). There was no significant correlation between the overexpression of p53 and HER-2/neu (P = 0.180) and no significant relationship between p53 and HER-2/neu overexpression and the presence of microinvasion, ability to perform optimal cytoreductive surgery, tumor recurrence, or survival. CONCLUSIONS: p53 and HER-2/neu overexpression occurred in about 20-25% of BLOT overall and more commonly in seromucinous and advanced stage tumors. Coexpression occurred in 9.5% of the cases with no significant correlation between the expression of p53 and HER-2/neu.

Adolescent↗
PubMed1997

Molecular regulation of intercellular adhesion molecule 1 (ICAM-1) expression in renal cell carcinoma.

Intercellular adhesion molecule-1 (ICAM-1) mediates two important functional aspects of tumor biology, namely enhancement of tumor metastasis and mediation of host defense mechanisms such as lymphocyte-mediated tumor cytotoxicity. Since ICAM-1 is expressed by most renal cell carcinomas (RCC), the regulation of ICAM-1 expression is important in understanding the biological behavior of RCC. We report an investigation on ICAM-1 expression and molecular regulation by cytokines and protein kinase C activator on RCC cell lines. Of the various cytokines, tumor necrosis factor alpha (TNF alpha), interferon-gamma (IFN gamma), and phorbol myristate acetate (PMA) strongly upregulated ICAM-1 protein expression on RCC. The kinetics of ICAM-1 message induction was studied by Northern analysis of total RNA extracted from RCC and normal kidney proximal tubular (NKPT) cells. Time course studies showed that ICAM-1 mRNA was upregulated by INF gamma, TNF alpha, and PMA, plateaued after 2 h, and remained increased for up to 24 h. Although ICAM-1 mRNA in NKPT cells was upregulated by these cytokines, their messages returned to basal levels after 24 h. ICAM-1 mRNA stability assays showed that both unstimulated and stimulated RCC cells had very stable ICAM-1 mRNA up to 24 h. In order to investigate whether increased gene transcription contributes to ICAM-1 upregulation, RCC cells were treated with TNF alpha, IFN gamma, or PMA with or without simultaneous addition of actinomycin D. ICAM-1 message induction-blocking studies suggested that primary upregulation of ICAM-1 mRNA may be caused by transcriptional upregulation. These results suggest that long-lasting ICAM-1 message upregulation in response to cytokines or PMA may be due to transcriptional upregulation in the early phase and stabilization of ICAM-1 message in the later phase (after 4 h). These observations suggest that RCC may lack the normal downregulatory mechanisms which control ICAM-1 expression and may explain the high frequency of ICAM-1 expression observed on primary human RCC.

Carcinoma, Renal Cell↗
PubMed1997

The use of amplified variable number of tandem repeats (VNTR) in the detection of chimerism following bone marrow transplantation. A comparison with restriction fragment length polymorphism (RFLP) by Southern blotting.

Chimerism analysis after allogeneic bone marrow transplantation (alloBMT) allows detection of early marrow engraftment, disease relapse, and graft rejection. Our objective was to do retrospective and prospective studies of chimerism analysis by restriction fragment length polymorphism (RFLP) by Southern blotting and variable number of tandem repeats (VNTR) by polymerase chain reaction (PCR) to compare and contrast the methods. The retrospective group comprised 46 samples from 26 patients previously analyzed by RFLP, while the prospective group contained 34 samples from 25 patients. Using four different VNTR primers (D1S80, D17S30, D1S111, and APO-B), the recipient and donor samples amplified by the PCR were screened for unique banding patterns. The VNTR primer with the unique banding pattern was used to detect chimerism in each sample. A total of 635 VNTR analyses were performed. Interpretation was blinded for previous RFLP results. A comparison between the VNTR and RFLP results and a cost analysis of the two procedures were done. A unique VNTR banding pattern was present in 49 of 51 patients (identical twins in one case). The VNTR analysis showed complete chimerism in 68 samples, mixed chimerism in 9, and recurrences in 2. This agreed with the RFLP results in 64 (80%) of 80 samples. Failure to detect 1% to 10% of recipient DNA accounted for 15 (VNTR, 8; RFLP, 7) discordances. Follow-up revealed all donor DNA in five cases, decreasing quantities of recipient DNA in two cases (six samples), and no additional studies available in four cases. In one case, VNTR detected a complete chimerism when the DNA was insufficient for RFLP analysis. The cost analysis revealed an approximately 50% savings with the use of VNTR; VNTR is a viable alternative to RFLP in the detection of chimerism after bone marrow transplantation and offers substantial cost savings, faster turnaround time, easier preparation of the DNA, smaller DNA requirements, and the elimination of radioisotopes and cumbersome restriction enzymes.

Blotting, Southern↗
PubMed1997

Defective granzyme B gene expression and lytic response in T lymphocytes infiltrating human renal cell carcinoma.

Granzyme B is a protein thought to play a pivotal role in the cytolytic functions of T cells. In view of this, the inducibility of this gene in freshly isolated T cells (T-TILs) infiltrating human renal cell carcinoma (RCC) in vitro was examined by using the reverse transcriptase-polymerase chain reaction (RT-PCR). A reduction in granzyme B messenger RNA (mRNA) expression in stimulated T-TILs from five of nine patients with RCC compared with autologous peripheral blood T cells was noted. The reduced expression was observed after multiple stimuli including anti-CD3 antibody, interleukin-2 (IL-2), and phytohemagglutinin (PHA). Because CD8+ T cells represent the predominant cytotoxic population, the ability of this cell population to express granzyme B mRNA after stimulation also was examined. When compared with CD8+ peripheral blood lymphocytes (T-PBLs) from patients with RCC and normal donors, the induction of granzyme B mRNA was reduced in CD8+ T-TILs. CD8+ T-TILs also had lower non-major histocompatibility complex (MHC)-restricted cytotoxic activity than did CD8+ T-PBLs against both Daudi cells and allogeneic RCC cell lines. These results show that in a subset of patients with RCC, depressed lytic activity of CD8+ TILs compared with CD8+ PBLs is present. Reduced granzyme B mRNA expression also was noted in selected patients.

Carcinoma, Renal Cell↗
PubMed1997

Quantitative comparison of immunohistochemical staining intensity in tissues fixed in formalin and Histochoice.

Formaldehyde fixatives have traditionally been used to preserve tissues as they impart excellent morphological preservation. Formaldehyde fixes tissue by cross linking, a process which can reduce the antigenicity of tissue and weakens the intensity of immunohistochemical stains. Preliminary studies have shown that Histochoice tissue fixative offers equal or greater staining intensity than neutral buffered formalin (NBF). This study compares these fixatives quantitatively and presents the results in unambiguous statistical terms. Tissue samples were collected, bisected, and fixed in NBF or Histochoice. The sections were stained with a total of 21 antibodies, and color images were collected. The hue, saturation, and value were determined for each positive pixel and an ANOVA performed. Small differences in hue were noted in 8 of 21 cases. Histochoice-fixed tissue gave a greater mean saturation than NBF with 57.1% of the antibodies tested. No significant difference in the saturation was detected in 28.6% of the cases; NBF gave higher mean saturation levels with only 14.3% of the antibodies. Histochoice-fixed tissue was found to give lower values in 66.7% of cases than those prepared with NBF, indicating darker staining. These results show that Histochoice produces staining intensity that is comparable, and in many cases superior, to formalin.

Analysis of Variance↗
PubMed1997

The use of isotypic control antibodies in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets by flow cytometry. Are they really necessary?

OBJECTIVE: Isotypic control reagents are defined as irrelevant antibodies of the same immunoglobulin class as the relevant reagent antibody in a flow cytometry panel. The use of the isotypic control antibody has been advocated as a necessary quality control measure in analysis of flow cytometry. The purpose of this study was to determine the necessity of an isotypic control antibody in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets. MATERIALS AND METHODS: We performed a prospective study of 46 consecutive patient samples received for lymphocyte subset analysis to determine the need for the isotypic control. For each sample, a sham buffer (autocontrol) and isotypic control reagent were stained for three-color immunofluorescence, processed, and identically analyzed with Attractors software. The Attractors software allowed independent, multiparametric, simultaneous gating; was able to identically and reproducibly process each list mode file; and yielded population data in spreadsheet form. RESULTS: Statistical analysis (Fisher's z test) revealed no difference between the CD3+ autocontrol and CD3+ isotypic control (correlation = 1, P < .0001) or between the CD3+, CD4+ autocontrol and the CD3+, CD4+ isotypic control (correlation = 1, P < .0001). The elimination of the isotypic control reagent resulted in a total cost savings of $3.36 per test. Additionally, the subtraction of isotypic background can artifactually depress population enumeration. CONCLUSIONS: The use of an isotypic control antibody is not necessary to analyze flow cytometric data that result in discrete cell populations, such as CD3+ and CD3+, CD4+ lymphocyte subsets. The elimination of this unnecessary quality control measure results in substantial cost savings.

Antibodies↗