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PubMed1989

Unilesional mycosis fungoides: clinical, microscopic and immunophenotypic features.

Seven cases are reviewed in which the histologic and immunohistologic features were those of mycosis fungoides, although the patients presented with solitary lesions which did not recur after local therapy (excision in six, radiotherapy in one), with follow-up of 10 months to 18 years. Immunophenotypic staining of paraffin-embedded tissue revealed a predominant T-lymphocyte proliferation in all cases, None of the four cases studied for BER-H2(Ki-1) were positive, in contrast to the "activated" types of spontaneously regressing lymphoid lesion, lymphomatoid papulosis and "Ki-1" lymphoma. Electron microscopy in one case demonstrated the highly convoluted nuclei of the T cells within the infiltrate. These seven cases represent examples of unilesional mycosis fungoides in which the disease has not recurred after therapy, despite features indistinguishable from those of typical generalised mycosis fungoides-type of cutaneous T-cell lymphoma.

Adult↗
PubMed1993

Immunophenotypic and molecular analysis of a case of lymphocytic interstitial pneumonia.

A case of lymphocytic interstitial pneumonia was studied immunophenotypically and with molecular methods in order to clarify its lymphocytic clonality. The patient, a 43 year old Japanese female, underwent lobectomy for a suspected malignant lymphoma as no clear diagnosis could be made from the biopsy specimen. An ill-demarcated, yellowish and elastic firm lesion measuring 60 x 35 x 20 mm in size was located in the peripheral part of the middle lobe of the right lung. Histologically, the alveolar, peribronchial-vascular and subpleural interstitia within the lesion were thickened markedly with severe cellular infiltration largely composed of small lymphocytes with germinal centers. Immunostaining revealed immunoglobulin (Ig) kappa and Ig lambda-bearing cells to be evenly distributed, suggestive of polyspecificity. Immunoglobulin gene analysis further demonstrated the unrearranged germ-line DNA but no rearranged band. These results strongly indicated a reactive process rather than a neoplastic nature for the lesion.

Adult↗
PubMed1994

Primary subcutaneous leiomyosarcoma exhibiting the characteristic immunophenotype positive for vimentin and desmin.

BACKGROUND: Primary subcutaneous leiomyosarcoma is a rare neoplasm composed of plump, elongated spindle cells arranged in interweaving fascicles. OBJECTIVE: Differential diagnosis might be facilitated by the use of immunohistochemistry. METHODS: A case of subcutaneous leiomyosarcoma was investigated histologically on paraffin-embedded tissue and immunohistochemistry was performed following standard procedures. RESULTS: The case reported exhibited light microscopic features and an immunophenotype characteristic of leiomyosarcoma. However, the intermediate filament desmin could only be found on a small number of tumor cells. CONCLUSION: Expression of these markers might vary considerably and immunoperoxidase stainings need to be carefully evaluated. Utilization of several antibodies directed against different desmin epitopes might be advantageous.

Aged↗
PubMed2006

Immunophenotypic patterns of T-cell activation in neuroinflammatory diseases.

OBJECTIVES: We aimed to gain insights into the pathogen-specific differences in early adaptive immune responses following central nervous system infections with Borrelia burgdorferi and viral pathogens by studying the immunophenotypic patterns of T-cell activation. Moreover, we wished to determine whether the expression of T-cell activation markers reflects disease activity in multiple sclerosis (MS). METHODS: Proportions of cerebrospinal fluid T-cells expressing the markers HLA-DR, CD25 and CD38 were determined in patients with MS (n = 40), acute viral meningomyeloradiculoneuritis (VID, n = 26), early neuroborreliosis (NB, n = 23) and non-inflammatory neurologic diseases (n = 51) by using flow cytometry. In relapsing-remitting MS, disease activity was assessed by clinical examination and magnetic resonance imaging. RESULTS: For each of the surface markers that were examined, significant differences in T cell proportions were found between patient groups. The proportion of HLA-DR+ T cells was higher and that of CD25+ T cells lower in NB compared with VID. These differences were attributable only to the early phase of the disease (< or = 6 days after symptom onset). Among MS patients, there was a trend for higher proportions of T cells expressing activation markers in patients with gadolinium-enhancing lesions. CONCLUSIONS: The decreased CD25 expression in NB may reflect immunomodulatory effects of B. burgdorferi facilitating persistent infection. Larger prospective studies of T-cell activation markers for ascertaining the association between cellular markers and clinical surrogates of disease activity in MS are warranted.

ADP-ribosyl Cyclase 1↗
PubMed1991

Pigmented purpuric eruptions: immunopathologic studies supportive of a common immunophenotype.

Traditionally, the pigmented purpuric eruptions (PPE) have been subdivided into four histologic categories, based upon variations in histologic pattern and clinical morphologies. In recent years, it has become apparent that the lesions behave similarly, and this family of eruptions has become grouped under the term PPE. The pathogenesis of these eruptions is largely unknown. In this study, we examined 13 examples of the histologic and clinical subtypes of the PPE with a panel of hematolymphoid immunophenotypic markers, in hopes of better understanding the pathogenesis of these curious eruptions. All lesions examined showed a predominantly T cell infiltrate composed of a mixture of OPD4 (CD4) positive and OPD4 negative cells. B cells were rare. Macrophages did not comprise a significant component of the infiltrate. The staining pattern was relatively constant, independent of the histologic pattern (i.e., Schamberg's vs. Gougerot-Blum, etc) of the PPE. Our results suggest that PPE are a group of related eruptions which may have a common pathogenesis. It seems likely that the vascular damage and erythrocyte extravasation are secondary to a localized cell-mediated immunologic event.

Adolescent↗
PubMed1995

Solitary reticulohistiocytoma in pregnancy: immunohistochemical and ultrastructural study of a case with unusual immunophenotype.

A case of a solitary reticulohistiocytoma during pregnancy is reported. The tumor arose on the right thigh of a 31-year-old woman during the 2nd month of gestation. Physical examination 6 months later revealed a 9 x 6 mm yellowish white papule. A biopsy showed a dermal proliferation of spindle to polygonal cells embedded in fibrous tissue with admixed neutrophils, eosinophils, and lymphocytes. Immunohistochemistry was positive for S-100, factor XIIIa, HLA-DR, KP-1, vimentin, and neuron-specific enolase. Ultrastructurally, the cells showed abundant lipid, lysozomes, and endoplasmic reticulum. No Birbeck granules could be identified. The results suggest an aberrant immunophenotype intermediate between indeterminate cells and dermal dendritic cells. The relationship of these lineages to reticulohistiocytoma and other dermal proliferative disorders is reviewed.

Adult↗
PubMed1995

Separate clones in concomitant multiple myeloma and a second B-cell neoplasm demonstrated by molecular and immunophenotypic analysis.

The occurrence of multiple myeloma (MM) and a second B-cell neoplasm in the same patient is a rare event. We present 2 such patients, and provide evidence to support the presence of separate clones in these coexisting neoplasms. In the first case, MM became evident 14 months after the diagnosis of chronic lymphocytic leukemia (CLL). In past reports, most occurrences of this association, when investigated, have been regarded to be biclonal disease processes; however, with few exceptions, most were documented by immunologic studies alone. To establish the clonality in our case of CLL with MM, we examined both immunophenotypic data obtained by standard two-color flow cytometric analysis, and patterns of immunoglobulin gene rearrangement, using standard Southern analysis and hybridization with 32P-labelled JH and JK probes. This provided evidence for the presence in this patient of two separate monoclonal populations of B cells, manifested as light chain restrictions and gene rearrangements which differed in blood (CLL) and bone marrow (MM) samples. In the second case, MM presented simultaneously with bone marrow lymphocytosis and abnormal peripheral lymphocytes. Clonality studies on blood were not done. Bone marrow B-cell gene rearrangement studies, however, revealed the presence of three bands in the JK blot of significantly different intensities, suggestive of two monoclonal populations. A monoclonal population of small cells with surface B markers and surface IgM was demonstrated by flow cytometry, while a second population of larger cells with intracytoplasmic IgG matching the patient's serum monoclonal protein was detected by immunofluorescence microscopy. The results in these 2 cases expand previous findings of the rare association of MM with a second B-cell neoplasm, and demonstrate the usefulness of molecular diagnostic investigation.

B-Lymphocytes↗
PubMed1996

The immunopathogenesis of retroviral diseases: no immunophenotypic alterations in T, B, and NK cell subsets in SIVmac239-challenged rhesus macaques protected by SIV delta nef vaccination.

Immunophenotype analysis was used to characterize circulating lymphocyte subset levels in both rhesus monkeys that were chronically infected with SIVmac239 and in those that had resisted SIVmac239 infection as a result of prior vaccination with an attenuated SIV strain. Alterations in T, NK, and B cell subsets were compared with those previously identified in humans chronically infected with HIV [8-11, 14, 22]. The well-known decrease in CD4+ cell levels was observed in the SIVmac239-infected animals. However, these animals had relatively little activation of circulating CD8+ T cells as compared with uninfected monkeys. This contrasts with chronically HIV-infected humans who have substantial activation of circulating CD8+ cells as evidenced by elevated HLA-DR and CD38 antigen expression on CD8+ cells as well as substantially increased percentages and numbers of total CD8+ cells. NK cells of the SIVmac239-infected animals, on the other hand, demonstrated the same changes recently described in HIV-infected humans, i.e., a decrease in circulating percentages and a decreased amount of FcRIII (CD16). B cell percentages were markedly increased in the SIVmac239-infected animals, a finding also noted in some children with HIV infection but not in HIV-infected adults. SIV delta nef-vaccinated/SIVmac239-challenged animals showed none of the immune alterations found in the SIVmac239-infected monkeys, providing further confirmation of lack of SIV disease in these vaccinated animals.

ADP-ribosyl Cyclase↗
PubMed1999

Morphologic and immunophenotypic characteristics of malignant lymphomas in SIV-infected rhesus macaques.

Eight cases of extranodal non-Hodgkin's lymphoma in simian immunodeficiency virus (SIV)-infected rhesus macaques, aged 4-9 years, were phenotypically and immunologically characterized, using the updated Kiel classification, in order to determine both the differences and the similarities between these types of lymphoma in immunodeficient rhesus macaques (Macaca mulatta) and man. The high-grade malignant tumors were of B-cell origin, with a predilection for extranodal growth in viscera and periorbital tissues. Immunophenotypical characterization showed that the monkey lymphomas were similar in many aspects to human immunodeficiency virus-associated lymphomas. The number of Ki67 positive cells varied from case to case and ranged from 50 to 90%. A serological screening for the simian equivalent of the Epstein-Barr virus (sEBV) by immunofluorescence assay revealed a prevalence of 92% of the sEBV antibodies in our cohort. The presence of Ebstein-Barr virus nuclear antigen (EBNA-2) could be demonstrated by immunohistochemistry in four out of eight cases. In situ hybridization revealed the presence of small EBV-encoded RNAs (EBER-1, EBER-2) in six of the eight cases. Further studies should define the precise role of herpesvirus infection for lymphomagenesis in SIV-induced immunodeficiency.

Animals↗
PubMed1994

Simultaneous immunophenotyping and FISH on fetal cells from maternal blood.

Current methods for the prenatal diagnosis of genetic disorders from fetal cells in the maternal circulation are restricted by maternal cell contamination and therefore must rely on the presence of features (such as Y-specific DNA sequences) absent from maternal cells. We have used density gradient centrifugation and magnetic-activated cell sorting to enrich maternal blood samples for fetal nucleated red cells. Mouse monoclonal antibodies for CD45 and CD32 were used to reduce the proportion of leukocytes. Unequivocal identification of fetal cells was achieved using an immunophenotypic test for fetal hemoglobin that allowed the simultaneous application of a diagnostic FISH analysis with chromosome-specific DNA probes. A positive diagnosis of female fetal sex (among other diagnoses) is thus possible even in the presence of an excess of maternal cells. The method, which appears to represent an advance over previous techniques, has considerable application in the development of noninvasive prenatal diagnosis from maternal blood.

Blood Cells↗
PubMed1995

Comparison of the effects of Ficoll-Hypaque separation and whole blood lysis on results of immunophenotypic analysis of blood and bone marrow samples from patients with hematologic malignancies.

We compared flow cytometric immunophenotyping results obtained by using the lysed whole blood method of sample preparation with those obtained by using Ficoll-Hypaque-separated cells on 44 consecutive specimens from patients with various hematologic malignancies. When the samples were analyzed as a group, seven antigens (CD2, CD3, CD5, CD11c, CD20, CD22, and CD34) demonstrated significantly different percentages of positively staining cells. When the samples were grouped by disease, results for patients with acute lymphocytic leukemia were discordant for CD22 and HLA-DR and results for patients with hairy cell leukemia were discordant for CD34. Most of the differences, however, were not with antigens critical to the evaluation of the malignancy. Additionally, the most frequent reason for differences in the percentage of positive cells was due to isotype control-based placement of the quadrant markers and not an actual discrepancy in staining. However, analysis of the CD34 antigen yielded eight instances in which staining of Ficoll-Hypaque-separated cells was essentially negative, but a clearly positive population was evident with the lysed preparation. This finding has important implications because of the prognostic significance of this antigen. Further studies are needed to determine the cause of this phenomenon.

Antibodies, Monoclonal↗
PubMed1995

Detection of CD4+ T cells harboring human immunodeficiency virus type 1 DNA by flow cytometry using simultaneous immunophenotyping and PCR-driven in situ hybridization: evidence of epitope masking of the CD4 cell surface molecule in vivo.

Human immunodeficiency virus type 1 (HIV-1) infection of T cells and cells of the monocyte/macrophage lineage requires a specific interaction between the CD4 antigen expressed on the cell surface and the HIV-1 external envelope glycoprotein (gp120). To study the association between HIV-1 infection and modulation of cell surface expression of the CD4 molecule in vivo, we examined the CD4+ T cells harboring proviral DNA obtained from HIV-1-infected individuals who had received no antiretroviral therapy for at least 90 days. Simultaneous immunophenotyping of CD4 cell surface expression and PCR-driven in situ hybridization for HIV-1 DNA were used to resolve the CD4+ T cells into distinct populations predicted upon the presence or absence of proviral DNA. Among the HIV-1-infected study subjects, the percentage of CD4+ T cells harboring proviral DNA ranged from 17.3 to 55.5%, with a mean of 40.5%. Cell surface fluorescent staining with anti-CD4 antibody directed against a non-gp120 binding site-related epitope (L120) or a conformation-dependent epitope of the gp120 binding site (Leu 3A) demonstrated either an equivalent or a 1.5- to 3-fold-lower cell surface staining intensity for the HIV-1 DNA-positive subpopulation relative to the HIV-1 DNA-negative subpopulation, respectively. These data suggest that masking or alteration of specific epitopes on the CD4 molecule occurs after viral infection.

CD4 Antigens↗
PubMed1999

Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes.

Immunophenotyping of blood lymphocyte subpopulations in neonates and young infants is hampered by the limited amount of blood that can be collected. Contamination of the flow cytometric "lympho-gate" by normoblasts and analysed erythrocytes, and therefore the underestimation of the relative frequencies of lymphocyte subpopulations, interferes with the precise calculation of absolute counts. A microassay was developed by adapting the lysed whole blood technique. Triple immunostaining in a single antibody staining step was used to reduce washing steps and cell loss. Introduction of a triple staining for CD71 (expressed by erythroid precursors), glycophorin A (GpA, expressed by all erythroid cells), and CD45 (expressed by all leucocytes) permitted the relative frequencies of normoblasts (CD71(+)/GpA+/CD45(-) population) and unlysed erythrocytes (CD71(-)/GpA+/CD45(-) population)to be identified and measured within the "lympho-gate" of neonatal cord blood samples. Particularly high frequencies were found (median: 31%) in cord blood samples from preterm neonates. These erythroid cells disappear rapidly by 1 week of age The relative frequencies of erythroid cells can be used to calculate correct lymphocyte subpopulation values. Using only 0.5-0.8 ml of blood, this micro- assay would also be suitable for rapid prenatal immunodiagnosis of congenital immunodeficiencies.

Antigens, CD↗
PubMed2005

Application of the FICTION technique for the simultaneous detection of immunophenotype and chromosomal abnormalities in routinely fixed, paraffin wax embedded bone marrow trephines.

The use of interphase fluorescence in situ hybridisation (FISH) to study cytogenetic abnormalities in routinely fixed paraffin wax embedded tissue has become commonplace over the past decade. However, very few studies have applied FISH to routinely fixed bone marrow trephines (BMTs). This may be because of the acid based decalcification methods that are commonly used during the processing of BMTs, which may adversely affect the suitability of the sample for FISH analysis. For the first time, this report describes the simultaneous application of FISH and immunofluorescent staining (the FICTION technique) to formalin fixed, EDTA decalcified and paraffin wax embedded BMTs. This technique allows the direct correlation of genetic abnormalities to immunophenotype, and therefore will be particularly useful for the identification of genetic abnormalities in specific tumour cells present in BMTs. The application of this to routine clinical practice will assist diagnosis and the detection of minimal residual disease.

Antigens, CD20↗
PubMed2005

KSHV/HHV-8 associated lymph node based lymphomas in HIV seronegative subjects. Report of two cases with anaplastic large cell morphology and plasmablastic immunophenotype.

BACKGROUND: Kaposi sarcoma associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) associated lymphomas, which often develop in human immunodeficiency virus (HIV) infected patients with advanced AIDS, present predominantly as primary effusion lymphoma (PEL) or, less frequently, as "solid" extracavitary based lymphomas, associated with serous effusions. These last lymphomas, also called "solid PEL", have been reported before the development of an effusion lymphoma and after resolution of PEL. Interestingly, KSHV/HHV-8 associated lymphomas that present as solid or extracavitary based lesions in HIV seropositive patients without serous effusions have been reported recently. METHODS/RESULTS: This paper provides evidence for the existence of a previously undescribed KSHV/HHV-8 associated lymphoma in HIV seronegative patients without serous effusions. These lymphomas exhibit a predilection for the lymph nodes and display anaplastic large cell morphology. These tumours were completely devoid of common cell type specific antigens, including epithelial and melanocytic cell markers. B and T cell associated antigens and other commonly used lymphoid markers were absent or weakly demonstrable in a fraction of the tumour cells. Conversely, immunohistochemical studies showed strong immunostaining with plasma cell reactive antibodies. CONCLUSIONS: Analysis of viral infection and immunohistological studies are of primary importance to define this lymph node based KSHV/HHV-8 associated lymphoma with anaplastic large cell morphology and plasmablastic immunophenotype occurring in HIV seronegative patients without serous effusions.

Aged↗
PubMed1994

Chronic prolymphocytoid leukaemia with an unusual immature immunophenotype.

A case of a 58 year old woman with a chronic lymphoproliferative disorder of unusual clinical presentation, disease course, and immunophenotype is presented. At diagnosis she had severe anaemia, moderate lymphocytosis with some cells having prolymphocytoid features and a normal platelet count. A clinical examination yielded negative results. Only anaemia related symptoms were found and the patient became blood transfusion dependent. Both the lymphocytosis and the proportion of prolymphocytoid cells rose insidiously and thrombocytopenia developed later during the course of the disease. Three years later, the patient had a white cell count of 269 x 10(9)/l almost exclusively of prolymphocytoid cells and the bone marrow was diffusely infiltrated. She was refractory to chemotherapy and the anaemia did not improve after treatment with cyclosporine. Lymphoid cells were positive for cytoplasmatic CD3, HLA-Dr, CD34, CD38, CD7, CD56, CD13, CD33 and CD65. Membrane alpha beta and gamma delta T cell receptors (TCRs) were not expressed and the beta chain TCR gene was in germline configuration. Other membrane T, B, natural killer, and myelomonocytic markers were negative. Karyotype analysis was tried several times but metaphases were not obtained, even after stimulation with T cell mitogens.

Anemia↗
PubMed1994

Polycythaemia rubra vera transforming to acute lymphoblastic leukaemia with a common immunophenotype.

Lymphoblastic transformation of polycythaemia rubra vera is an extremely rare phenomenon. A case of a 76 year old man with polycythaemia rubra vera who developed acute lymphoblastic leukaemia (ALL) 16 years after his initial diagnosis is reported. Membrane markers showed a CD10 positive (common ALL) immunophenotype. To our knowledge this association has not been previously recorded. The rare occurrence of ALL in polycythaemia rubra vera may indicate that in a minority of patients clonal expansion of an abnormal pluripotent haemopoietic stem cell is responsible for the polycythaemia rubra vera disease phenotype.

Aged↗
PubMed1998

Differentiation between actinic reticuloid and cutaneous T cell lymphoma by T cell receptor gamma gene rearrangement analysis and immunophenotyping.

AIMS: Differentiation between actinic reticuloid and cutaneous T cell lymphoma can be extremely difficult. Demonstration of clonal T cell receptor (TCR) gene rearrangements has been suggested as a potential diagnostic criterion, but the results obtained thus far have been conflicting. This study investigated whether TCR gamma gene rearrangement analysis, using polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) and immunohistochemistry, can serve as a diagnostic criterion. METHODS: PCR/DGGE was performed on skin, peripheral blood mononuclear cells, and/or lymph nodes of seven patients with actinic reticuloid, 11 patients with Sézary syndrome, and 15 patients with a benign form of erythroderma. The results of PCR/DGGE and Southern blot analysis of TCR beta gene rearrangements were compared. In addition, CD4:CD8 ratios in skin and peripheral blood samples were investigated. RESULTS: Clonal T cell populations were detected in 19 of 21 samples obtained from patients with Sézary syndrome but were not detected in any of the 12 samples from patients with actinic reticuloid. Clonal T cells were detected in the peripheral blood of only one of 15 patients with a benign form of erythroderma. PCR/DGGE and Southern blot analysis gave concordant results in 28 of 29 samples. Immunophenotypic analysis demonstrated increased proportions of CD8+ T cells in skin (seven of seven cases) and peripheral blood (four of seven cases) of patients with actinic reticuloid. CONCLUSION: The results of this study demonstrate that gene rearrangement analysis, in combination with immunohistochemistry, may be an important adjunct in differentiating between actinic reticuloid and cutaneous T cell lymphoma. In patients suspected of having actinic reticuloid, application of both techniques is recommended.

Adult↗