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PubMed2000

Flow cytometric immunophenotyping of serous effusions and peritoneal washings: comparison with immunocytochemistry and morphological findings.

AIM: To evaluate immunophenotyping by means of flow cytometry as a complementary method for the detection of malignant cells in serous effusions and peritoneal washings. MATERIAL AND METHODS: Frozen samples of 49 fresh serous effusions and peritoneal washings were analysed by flow cytometry, using monoclonal antibodies against CD45, Ber-EP4, and N-cadherin. Results were compared with smear and cell block morphology, as well as immunocytochemistry on paraffin wax embedded cell blocks. RESULTS: Seventeen specimens were cytologically diagnosed as malignant, whereas 25 were interpreted as benign. The remaining seven specimens were diagnosed as indeterminate or suspicious for malignancy. Ber-EP4 positive cells were detected in 16 of the 17 cytologically malignant effusions, as well as in five of seven suspicious cases and five of 25 specimens with benign cytology. In the latter group, three specimens showed atypical or malignant cell groups that were missed in routine morphological evaluation. In two additional samples, obtained from patients with benign and borderline ovarian tumours, Ber-EP4 positive cells showed benign or mildly atypical features, and were interpreted as exfoliated benign or borderline malignant epithelial cells of tubal origin, or as endosalpingiosis. All five Ber-EP4 positive indeterminate specimens showed atypical or malignant cells on re-evaluation, and were Ber-EP4 positive in four of five cases using immunohistochemistry in cell block sections. Large numbers of CD45 positive and relatively few N-cadherin positive cells were detected in most specimens with the use of flow cytometry, when compared with morphological evaluation. CONCLUSIONS: Flow cytometry is a rapid and highly effective method for the evaluation of effusions and peritoneal washings. The detection of Ber-EP4 positive cells using flow cytometry is strongly indicative of the presence of carcinoma cells in effusions and peritoneal washings. Although false positives are relatively infrequent, all specimens should be carefully evaluated morphologically to prevent the diagnosis of benign epithelial clusters as malignant.

Adult↗
PubMed1993

Prenatal diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative magnetic cell sorting.

Fetal nucleated cells in the maternal circulation constitute a potential source of cells for the non-invasive prenatal diagnosis of fetal genetic abnormalities. We have investigated the use of the Magnetic Activated Cell Sorter (MACS) for enriching fetal nucleated erythrocytes. Mouse monoclonal antibodies specific for CD45 and CD32 were used to deplete leucocytes from maternal blood using MACS sorting, thus enriching for fetal nucleated erythrocytes which do not express either of these antigens. However, significant maternal contamination was present even after MACS enrichment preventing the accurate analysis of fetal cells by interphase fluorescence in situ hybridisation (FISH). To overcome this problem, we used simultaneous immunophenotyping of cells with the mouse antifetal haemoglobin antibody, UCH gamma, combined with FISH analysis using chromosome X and Y specific DNA probes. This approach enables selective FISH analysis of fetal cells within an excess of maternal cells. Furthermore, we have confirmed the potential of the method for clinical practice by a pilot prospective study of fetal sex in women referred for amniocentesis between 13 and 17 weeks of gestation.

Cell Separation↗
PubMed2005

Metachronous extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) and peripheral T-cell lymphoma unspecified: histologic, immunophenotypic, and molecular documentation.

BACKGROUND: Coexistence of B- and T-cell lymphoid malignancies has been reported sporadically. CASE REPORT: A 68-year-old woman developed a lymphoid neoplasm in the large intestine and a second lymphoid neoplasm in the esophagus, 24 months after the diagnosis of the first lymphoma. Immunophenotypic analyses were consistent with extranodal marginal zone B-cell mucosa-associated lymphoid tissue type (MALT type) and peripheral T-cell unspecified lymphomas in the large intestine and the esophagus, respectively. The molecular analysis confirmed the B-clonal genotype of the first lymphoma, and disclosed a biclonal genotype of the second one (composite T- and B-cell lymphoma). No evidence of Epstein-Barr virus (EBV) association was shown in either tumor. CONCLUSION: B- and T-cell neoplasms represent two distinct malignancies rather than progression of the same neoplastic clone.

Aged↗
PubMed1992

Detection of structural chromosome aberrations in immunophenotyped mitoses.

The recently developed MAC (morphology-antibody-chromosome) method allows simultaneous immunophenotype and karyotype analysis in the same cell. To date, application of this new method has been hampered by the poor quality of chromosome banding. In this paper, we describe a modified simultaneous immunofluorescence and Q-banding technique, as well as a new combination of immunohistochemical and fluorescent R-banding methods. By further modifying the MAC method, we were able not only to achieve unequivocal results with weakly expressed antigens but also to improve the quality of the banding techniques, so that even structural chromosome abnormalities were well defined.

Chromosome Aberrations↗
PubMed1995

Clarification of dubious karyotypes in Hodgkin's disease by simultaneous fluorescence immunophenotyping and interphase cytogenetics (FICTION).

Cytogenetic studies on Hodgkin's disease (HD) typically reveal very complex karyotypes with a variety of numerical and structural abnormalities. The confusing thing is that about 10% of cases contain relatively discrete chromosome aberrations, for example a simple trisomy or loss of one single chromosome. Whether these karyotypes really correspond to Hodgkin and Reed-Sternberg (HRS) cells is uncertain. They could, for example, represent early stages in the evolution of the karyotype of the pathognomonic HRS cells. On the other hand, they could be artificial events that occur during the cytogenetic procedure. In our experience, isolated loss of the Y chromosome is the most frequent finding of this type. This aberration is usually considered to be a preparation artifact. However, if one takes into account that in HD up to 50% of male cases with complex karyotypes also lack the Y chromosome, a possible relation to HRS cells must be considered. The technique of simultaneous fluorescence immunophenotyping and interphase cytogenetic analysis (referred to as FICTION) is a powerful tool for studying the nature of cytogenetically abnormal cells. With the FICTION technique we studied four cases of HD in which the chromosome analysis had shown only the loss of the Y chromosome. Our aim was to clarify whether these karyotypes corresponded to the CD30-positive HRS cells. In two cases we found that HRS cells actually lacked the Y chromosome. There was strong evidence, however, that the HRS cells additionally had other chromosome aberrations and thus could not correspond to the cytogenetically determined karyotypes.

Cytogenetics↗
PubMed1991

The use of the alkaline phosphatase-antialkaline phosphatase technique for immunophenotyping acute myeloid leukemia.

The leukemic cells from 31 cases of acute myeloid leukemia were immunophenotyped by the alkaline phosphatase-antialkaline phosphatase (APAAP) technique, using 7 monoclonal antibodies reactive with cells of myeloid origin. We found a good correlation between the results obtained using the APAAP method and indirect immunofluorescence. In most cases, we observed a slight degree of variation in the percentages of reacting cells when comparing the two methods. Nevertheless, taking 20% of cells being immunolabeled as a threshold for defining a case as positive, we found no discrepancies in the final classification of each case. The main advantages of the APAAP method are: (1) its use with routinely prepared peripheral or blood marrow samples, and (2) the possibility of correlating immunological characterization with morphology. Since the results with the APAAP method were comparable with those obtained using indirect immunofluorescence, we suggest that this former technique can complement, and sometimes substitute, other methods of immunological evaluation.

Alkaline Phosphatase↗
PubMed1996

Immunophenotypic analysis of HL-60 cells during basophilic differentiation.

Differentiation along specific myeloid lineages may be accompanied by characteristic cell surface marker changes. We have examined leukemic HL-60 cell changes under conditions which induce basophilic differentiation. An increased surface expression of CD35, CD11b, and decreased expression of CD15 was found by flow cytometry during the 5-day induction period. Further investigation revealed two cell populations after 5 days in vitro: (i) a CD35-positive population (61% of cells present) containing a significant number of CD15-negative cells, and (ii) a CD15-positive/CD35-negative population. The CD35-positive subset appears to account for the majority of the basophilic cells induced under these conditions, as measured by histamine content and metachromatic staining. In addition, this subset contains a small number of early monocytic cells (CD14 and CD23 positive). The expression of CD11b is variably found on the CD15-positive/ CD35-negative subset of induced cells. These results suggest that CD35 and CD15 surface immunophenotyping can be used to map steps involved in myeloid development. A role for CD35 and CD15 in early basophil differentiation is proposed.

Antigens, CD↗
PubMed1991

Does the immunophenotype of renal cell carcinoma correlate with its clinical stage?

The following comparative study is an analysis of the clinical data, morphology and immunophenotype of 93 patients who have been operated on for renal cell carcinoma. We were able to show a close link between the histological grade and the occurrence of distant metastases: 33% of the patients with grade III tumours versus 11.5% of the patients with grade I tumours had developed metastasis by the time of the surgery. Histological subtyping per se did not give prognostic hints. Immunohistochemistry has revealed an inconsistent reaction pattern for the cytokeratin marker K11 (18/22). For proper diagnosis a panel of cytokeratin markers should be employed. The reaction patterns of monoclonal antibodies against the epidermal growth factor receptor (EGFR) and against myelomonocytic antigens in normal renal tissue (positive for the tubulus system) and in renal carcinoma indicate that renal cell carcinoma derive from the tubulus system. The proliferation marker Ki-67 correlates well with the histological grading. Although only a limited number of snap-frozen tumours have been investigated, this study indicates that EGFR is expressed by normal and by malignant renal tissue and that Ki-67 may serve as a prognostic marker.

Aged↗
PubMedDate not supplied

Combined fine needle aspiration biopsy, and immunophenotypic and genotypic approach to posttransplantation lymphoproliferative disorders.

OBJECTIVE: To report our experience with a combined approach to posttransplant lymphoproliferative disorders (PT-LPDs) that utilizes fine needle aspiration biopsy. STUDY DESIGN: A review of the files in the Department of Pathology, Saint Louis University Health Sciences Center; from 1988 to 1996 identified six patients with a diagnosis of PT-LPD who underwent either percutaneous or radiologically guided fine needle aspiration biopsy (FNAB). In all cases, material was collected for cytomorphology, flow cytometric analysis and, in selected cases, DNA polymerase chain reaction (PCR). Subsequent evaluations and clinical outcomes were obtained from the medical record. RESULTS: The six transplant recipients (4 men and 2 women; 3 cardiac, 2 renal and 1 hepatic transplant) had an age range of 16-65 years. The aspirate material on these six patients had a polymorphic pattern of lymphoid cells with varying sizes. By flow cytometry, two were monoclonal, while four had a polyclonal pattern. DNA PCR analysis on two FNABs demonstrated a monoclonal rearrangement of the immunoglobulin heavy chain gene. CONCLUSION: FNAB provides cytomorphologic characterization of PT-LPDs in transplantation patients and sufficient material for successful use of flow cytometry immunophenotyping and DNA PCR analysis. FNAB, therefore, has an important role in the evaluation of organ transplantation patients and is a valuable tool for assessing and diagnosing PT-LPD.

Adolescent↗
PubMed1997

A prospective study of the immunophenotype and temporal changes in the histologic lesions of canine demodicosis.

Mural folliculitis is a consistent histologic lesion of canine demodicosis. The objective of this study was to describe the immunophenotype and to evaluate temporal changes in histologic lesions of demodicosis during the course of therapy. Five dogs with demodicosis were examined and biopsied biweekly for up to 14 weeks; three dogs were evaluated once only. Lymphocyte subsets infiltrating the lesions were quantified using immunohistochemistry to detect CD3, CD21, CD4, and CD8 antigens. Lymphocyte subsets in blood were analyzed from four dogs using flow cytometry. Mural folliculitis was always present during clinically active disease. In contrast, following resolution of clinical lesions, perifolliculitis and/or perifollicular granulomas were present but mural folliculitis was absent. Most lymphocytes infiltrating the follicular epithelium in lesions of mural folliculitis were CD3+ and CD8+; the ratio of CD4+ :CD8+ cells in this epithelium was 0.032. In contrast, the perifollicular dermis contained approximately equal numbers of CD4+ cells and CD8+ cells, with slightly fewer CD21+B cells. In peripheral blood, the ratio of CD4+:CD8+ lymphocytes was reduced and the percentage of CD8+ cells was increased in three of four dogs. These results indicate that mural folliculitis is a consistent lesion of clinically active canine demodicosis and is characterized by infiltration of the follicular epithelium by CD3+ CD8+ T lymphocytes. These lymphocytes are cytotoxic T cells, which may mediate the injury to the follicular epithelium in demodicosis. Alternatively, CD8+ T cells may play a role in resistance to Demodex canis infection or may represent a deleterious immune response in dogs that develop demodicosis.

Animals↗
PubMed2001

Immunoreactivity of p53, mdm2, and p21WAF1 in dedifferentiated liposarcoma: special emphasis on the distinct immunophenotype of the well-differentiated component.

Alteration of the p53/mdm2 pathway has been reported in the well-differentiated liposarcoma (WDLS)/dedifferentiated liposarcoma (DDLS) group. We investigated the immunoreactivity of p53, mdm2, and p21WAF1, along with the MIB-1-labeling index (MIB-1-LI) in 21 WDLS and 21 DDLS cases, to clarify the association of these markers with the morphologic changes and the biological factors responsible for the aggressiveness of DDLS. Within DDLS, p53 and p21WAF1 expression and mdm2 overexpression were significantly more prevalent in the dedifferentiated (DD) components than in the well-differentiated (WD) components. The mdm2 overexpression and p21WAF1 expression was significantly associated with sclerosing liposarcomas in both WDLS and the WD components of DDLS. There was no significant difference in the immunoreactivity of p53, mdm2, or p21WAF1 or MIB-1-LI between WDLS and the WD components of DDLS. An association was found between p53 expression and mdm2 overexpression in the WD group (comprising WDLS and WD components of DDLS) and in the DD group, significantly so in the WD group. Notably, this correlation was found in the subtype of sclerosing liposarcoma but not in that of lipoma-like liposarcoma. Within DDLS, the clinical outcome of the nonaccessible soft tissue (non-AST: comprising retroperitoneum and mediastinum) group was significantly worse than that of the accessible soft tissue (AST: comprising extremities, buttocks, axilla, and scrotum) group; however, the immunophenotypes of p53, mdm2, and p21WAF1 and the MIB-1-LI showed no correlation with survival in the AST group alone, in the non-AST group alone, or in the 2 together. This study suggests that the immunoreactivity of p53, mdm2, and p21WAF1 is associated with the morphologic changes, but not with the biological factors responsible for the aggressiveness of DDLS.

Adult↗
PubMedDate not supplied

Frequent detection and immunophenotyping of prostate-derived cell clusters in the peripheral blood of prostate cancer patients.

BACKGROUND: Recent scientific studies have failed to determine parameters for the assessment of prostate cancer aggressiveness. The present study deals with the detection of blood-borne cancer cells based on polymerase chain reaction (PCR) and cell enrichment methods. The contradictory results reported in the literature have called into question the clinical usefulness of this diagnostic method in the preoperative staging of clinically localized prostate cancer. METHODS: We established a combined method of density gradient centrifugation and immunomagnetic separation using epithelium-specific antibodies, i.e. cytokeratins, to isolate prostate-derived circulating cells from the peripheral blood of patients with prostate cancer. Isolated cells were characterized by DNA staining and immunocytochemistry using antibodies for the detection of prostate-specific antigen (PSA), proliferation-associated proteins (MIB-1, H1 and H3) and apoptosis-associated proteins (M30, c-FasR). RESULTS: We applied these methods to 68 prostate cancer patients and were able to isolate cell clusters in 98%. Immunophenotypic and morphological characterization of PSA-positive prostate-derived cell clusters found in the peripheral blood of prostate cancer patients showed two main populations: 1) in 35% of the investigated prostate cancer patients we detected rounded cell aggregates of probable cancer cells expressing proliferation-associated proteins and lacking apoptosis-associated protein expression; 2) in all cases there was a high frequency of circulating dysmorphic cell clusters positive for apoptosis-associated protein expression. CONCLUSION: Our results demonstrate the existence of at least two different types of blood-borne prostate-derived circulating cell clusters. Of these, only the less frequent, round, small cell clusters harbor features that are probably necessary for the cells to survive for metastatic spread.

Biomarkers, Tumor↗
PubMed2003

The multidrug resistance-associated protein 3 (MRP3) is associated with a poor outcome in childhood ALL and may account for the worse prognosis in male patients and T-cell immunophenotype.

The family of multidrug resistance-associated proteins (MRPs) belongs to the superfamily of adenosine triphosphate-binding-cassette (ABC) transporters, which have the ability to function as outward pumps for chemotherapeutic drugs and therefore might be involved in drug resistance. In this study the expression of the MRP2, MRP3, MRP4, MRP5, and SMRP genes was measured using TaqMan real-time polymerase chain reaction (PCR) in 103 children with previously untreated acute lymphoblastic leukemia (ALL) (precursor B-cell ALL [B-ALL], n = 71; T-cell ALL [T-ALL], n = 32). All 5 genes were expressed with a great variability. Only MRP3 expression was associated with a significantly worse prognosis (P =.008). The median expression of MRP3 was 10-fold higher in T-ALL than in precursor B-ALL (P <.001) and 4-fold higher in male patients than in female patients (P <.001). The prognostic impact of MRP3 was independent of immunophenotype or sex. Higher levels of MRP3 were found in patients with a poor in vivo response to prednisone, but this could not be confirmed in an independent case-control study (40 patients) for prednisone response. In healthy donors, the median expression of MRP4 was 4-fold higher in bone marrow and 8-fold higher in CD34+ stem cells compared with peripheral blood (P =.002). Our results suggest that MRP3 is involved in drug resistance in childhood ALL. It therefore represents an interesting target to overcome multidrug resistance. High levels of MRP3 could possibly be the reason for the poorer prognosis of male patients or patients who have T-ALL. Similar to other members of the family of ABC transporters, MRP4 seems to be a marker for immature stem cells.

Adolescent↗
PubMed2002

Immunophenotypic evaluation of the plasma cell compartment in multiple myeloma: a tool for comparing the efficacy of different treatment strategies and predicting outcome.

Multiparametric immunophenotyping can be a sensitive method for analyzing the plasma cell (PC) compartment in patients with multiple myeloma because it discriminates between myelomatous and normal PCs. Using this approach, we compared the efficacy of high-dose chemotherapy followed by autologous stem cell transplantation (ASCT) with that of conventional chemotherapy. We found that ASCT provided a significantly greater reduction in the level of residual tumor PCs and with better recovery of normal PCs. This profile of coexistence of normal PCs and myelomatous PCs resembled that observed in monoclonal gammopathy of undetermined significance. We also found that treatment-induced changes in the PC compartment correlated with disease outcome. Thus, patients in whom at least 30% of gated PCs had a normal phenotype after treatment had a significantly longer progression-free survival (60 +/- 6 months versus 34 +/- 12 months; P =.02).

Adult↗
PubMed2003

A subset of morphologically distinct mammary myoepithelial cells lacks corresponding immunophenotypic markers.

INTRODUCTION: Immunostaining for smooth muscle actin (SMA) is commonly used to elucidate mammary myoepithelial (ME) cells, whose presence or absence is a reliable criterion for differentiating in situ and invasive carcinomas. However, some morphologically distinct ME cells fail to stain for SMA. This study intended to assess whether these SMA-negative cells also lack the expression of other ME cell markers. METHODS: Hematoxylin/eosin and SMA immunostained sections from 175 breast cancer patients were examined. Three cases were found to harbor ducts that showed morphologically distinct ME cell layers, but showed no SMA immunostaining in at least one-third of the layer or the entire layer. Eight additional consecutive sections from each case were stained for SMA, using a black chromogen, and each was then re-stained for one of eight additional markers supposed to exclusively or preferentially stain ME cells, using a red chromogen. SMA-negative ME cells were re-examined for the expression of other markers. RESULTS: SMA-negative ME cells in two cases also failed to display immunoreactivity for other markers, including calponin, CD10, smooth muscle myosin heavy chain, protease inhibitor 5 (maspin), Wilms' tumor-1, and cytokeratins 5, 14, and 17 (CK5, CK14, and CK17). However, in one case SMA-negative ME cells displayed immunoreactivities for maspin, CK5, CK14, and CK17. The distribution of these ME cells is independent of ductal size, length, and architecture. CONCLUSIONS: A subset of morphologically identifiable ME cells lack the expression of nine corresponding immunophenotypic markers, suggesting that ME cells might also be subject to different normal and pathological alterations.

Biomarkers, Tumor↗
PubMed2000

Immunophenotypic changes of fetal cord blood hematopoietic progenitor cells during gestation.

We measured cell surface expression of CD34, HLA-DR, CD38, CD19, CD33, CD71, and CD45 antigens in the hematopoietic progenitor cells of fetal cord blood to investigate immunophenotypic changes at different gestational ages. These antigens were identified by flow cytometry in 11 fetuses (gestational age 19-24 wk, in 12 preterm (25-28 wk) and in ten newborn infants born at term. The frequency and number of CD34+ cells were higher in the blood of the 11 fetuses; in addition, a statistically significant inverse correlation between number of CD34+ cells and advancing gestational age was noted. The numbers of CD34+ CD19+, CD34+ CD33+, and CD34+ CD45+ coexpressing cells were significantly higher in the fetuses, whereas CD34+ CD38+ cells were more represented in the neonates at term. Gestational age was inversely correlated with the number of CD34+ CD19+ and CD34+ CD33+ coexpressing cells. A positive correlation between gestational age and CD34+ CD38+ cells was noted. The number of CD34- CD19+, CD34- CD38+, and CD34- CD45+ cells was higher in term infants; furthermore, a significant correlation between advancing gestational age and CD34- CD38+ or CD34- CD45+ cells was demonstrated. The proliferative capacity was also higher at lower gestational ages. These data suggest that the development and lineage commitment of fetal cord blood hematopoietic progenitor cells are very active during the last two trimesters of pregnancy. The most significant changes of hematopoietic cells maturation seem to occur within 25 wk of gestation.

Antigens, CD↗
PubMed2000

Flow cytometric immunophenotypic characterization of pediatric and adult minimally differentiated acute myeloid leukemia (AML-M0).

We reviewed the clinicopathologic and immunophenotypic profiles of 7 pediatric and 11 adult minimally differentiated acute myelogenous leukemias (AML-M0). We also compared and evaluated myeloperoxidase in leukemic blasts using standard cytochemical and polyclonal antibody immunohistochemical stains. No distinctive clinical findings were noted in either patient group; however, thrombocytopenia typically was more prominent in adults. Adult AML-M0 also was associated with an immature myeloid profile (CD34+, terminal deoxynucleotidyl transferase positive, CD13+, and CD33+), in contrast with pediatric AML-M0, which usually lacked terminal deoxynucleotidyl transferase or CD34 but expressed bright CD33 with weak or negative CD13. Coexpression of the T-cell-associated antigen CD7 was observed in both groups. Antibody immunohistochemical stains were more sensitive than cytochemical stains for detection of myeloperoxidase activity and a useful adjunct for establishing a diagnosis of myeloid leukemia in paraffin-embedded marrow tissues.

Adolescent↗
PubMed2004

Flow cytometric assessment of TCR-Vbeta expression in the evaluation of peripheral blood involvement by T-cell lymphoproliferative disorders: a comparison with conventional T-cell immunophenotyping and molecular genetic techniques.

Molecular genetic T-cell receptor (TCR) and flow cytometric analysis using antibodies to conventional T-cell antigens and TCR beta-chain variable region families (TCR-Vbeta) were performed in 65 peripheral blood specimens evaluated for potential involvement by a T-cell lymphoproliferative disorder (TCLPD). A normal or reactive conventional T-cell immunophenotype was present in 36 cases; TCR-Vbeta flow cytometric and molecular TCR analyses were negative for clonality in 32 and 27 of these cases, respectively. In the remaining normal and reactive cases, one or both methods seemed to detect dominant cell populations in settings with limited T-cell diversity. We identified 29 TCLPDs; all studied cases had clonal molecular TCR results; 23 TCLPDs had clonal TCR-Vbeta flow cytometric results; the remaining were suggestive of (n = 3) or negative (n = 3) for clonality. TCR-Vbeta flow cytometric analysis is a powerful clinical laboratory tool that can be used to aid in the rapid diagnosis of peripheral blood involvement by T-cell malignant neoplasms.

Antigens, Differentiation, T-Lymphocyte↗