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PubMed1983

Correlation of immunophenotype to morphology in unfavourable non-Hodgkin lymphoma.

The relationship between immunological markers and histology according to the Kiel classification was studied in 40 adult non-Hodgkin lymphoma patients of Rappaport unfavourable histology. The membrane-associated and cytoplasmic Ig as well as receptors for sheep erythrocytes, Fc gamma and C3d receptors were analyzed on cryostate sections and in suspension. In some cases, a more precise immunophenotype was achieved by the use of monoclonal antibodies detecting different T and B cell antigens. Eighty-eight per cent of the lymphomas had B-cell, five per cent T cell and 7 per cent non-B/non-T cell phenotypes. All CBCC and CC lymphomas expressed monotypic Ig, but only 66 per cent of the CB lymphomas. Thus, morphology alone did not consistently predict immunophenotype in large-cell lymphomas. A simultaneous expression of multiple heavy chains and cytoplasmic Ig was found in some lymphomas, suggesting an intratumoral differentiation. The nodular or irregular tissue distribution patterns for Ig and C3d receptors found in histologically diffuse follicle derived lymphomas also suggest intratumoral variations in the marker expression, probably related to differentiation. The results suggest that lymphoma immunophenotype is important in obtaining a definite diagnosis in large-cell lymphomas and that it may lead to a better understanding of the differentiation of lymphomas of low-grade malignancy.

Adult↗

PubMed1989

Immunologic characterization of lymphocytic leukemias. Methodologic and immunophenotypic considerations.

Immunophenotyping has become an important adjunct to the routine morphologic and cytochemical evaluation of leukemias. The development and refinement of immunologic techniques applicable to the general clinical pathology laboratory and the commercial availability of lineage-specific monoclonal antibodies have enhanced this technology as a diagnostic tool. Lymphoid leukemias, in particular, can be accurately identified and subclassified according to their immunologic characteristics, as defined by lymphocyte differentiation antigens. Immunophenotypic classification has also been demonstrated to have prognostic significance for certain acute and chronic lymphoid leukemias. This review considers current immunotyping methods, characteristics of the antibody probes used, and the immunophenotypic characteristics of the acute and chronic lymphoid leukemias.

Antibodies, Monoclonal↗

PubMed2000

Central nervous system lymphomas: a histologic and immunophenotypic analysis.

OBJECTIVE: To observe the spectrum of non-Hodgkin's lymphomas involving the central nervous system including morphological subtypes and immunophenotypic status. SETTING: Retrospective analysis of eleven years (1986 to 1996) data from surgical pathology files of Department of Pathology. RESULTS: Forty-three cases of non-Hodgkin's lymphomas were diagnosed during the period of eleven years (from 1986 to 1996), all of which were diffuse types. A total of 1177 Central Nervous CNS biopsies were examined, out of which 937 cases were diagnosed as CNS neoplasms, the remaining were non-neoplastic in nature. Among 937 CNS neoplasms, 43 cases (4.6%) were reported as non-Hodgkin's lymphomas. As most of the cases were outside referrals, the primary or secondary nature of the lymphomatous process could not be assessed. Seventeen cases were intracranial, while 26 cases were spinal in location. Majority of the intracranial lymphomas were biopsied from the cerebrum (12 cases). Male to female ratio was 1:2. The median age for intracranial lymphomas was 50 years and for spinal lymphomas 29 years. There were 16 cases (37%) of diffuse large cell lymphomas; 7 cases (16%) of diffuse mixed small and large cell lymphomas; 3 cases (7%) of diffuse large cell immunoblastic lymphomas; 2 cases (4.6%) of lymphoblastic lymphomas and diffuse small non-cleaved cell lymphomas and one case of small lymphocytic lymphoma and diffuse small cleaved cell lymphoma. One case of T cell rich B cell lymphoma was also diagnosed in the thoracic spine as primary extranodal lymphoma. Eight cases were unclassifiable and in 2 cases the features were suggestive of lymphoma. Immunophenotypic analysis was performed in 20 cases, however, in 2 cases the results were inconclusive. Fifteen cases (83%) showed immunoreactivity for B cell markers and 3 cases showed T cell phenotype out of which one case was lymphoblastic lymphoma. CONCLUSION: CNS lymphomas were uncommon tumors and comprised 4.6% of the total CNS neoplasms in our study. Moreover, these CNS lymphomas accounted for 2.2% of the total non-Hodgkin's lymphomas, including both nodal and extranodal. There was a higher incidence of location of these lymphomas within the spinal cord than brain. Most of the lymphomas were of intermediate or high grade (75%) according to the working formulation. Immunophenotypical status revealed B-cell phenotype in 84% of the lymphomas, in which it was tested (JPMA 50:141, 2000).

Central Nervous System Neoplasms↗

PubMed2004

Neuroectodermal immunophenotype in uterine malignant mullerian tumors (MMT): comparative immunohistochemical analysis with embryonal uterine development.

Multidirectional differentiation of neoplastic cells in uterine MMT is still a subject of controversy. The present study was designed to assess the immunophenotype of 15 uterine MMT paying special attention to the markers of neural (neuroendocrine) differentiation. In addition, the same immunohistochemical study was performed on 20 human fetal specimens in order to establish possible relationships between the immunophenotype of MMT and the expression of the corresponding antigens in the fetal tissues of the embryonal female genital tract. Besides the typical immunohistochemical patterns in three cases the epithelial component showed simultaneous coexpression of vimentin and desmin. EMA and cytokeratin, whereas epithelial markers were coexpressed with vimentin in the sarcomatous component of one adenosarcoma. Moreover, both components were immunoreactive to the markers of neural differentiation (PGP 9.5, GFAP, HNK-1, N-CAM, HBA71). This aberrant expression was not correlated with morphological signs of neural differentiation at either light microscopy or ultrastructural levels. Regarding the analysis of fetal tissues, both epithelial and mesenchymal elements in the fetal genital tract expressed the above-mentioned neural markers at different dates of gestation. The intensity of this expression diminishes as the fetus matures and at the end of antenatal life the immunophenotype characteristic for adult life is established. Taking into consideration the capacity of uterine tissue to reproduce embryonal phenotype during neoplastic transformation, we studied this abnormal immunoprofile and its hypothetic value for the diagnosis and prognosis of MMT.

Aged↗

PubMed1990

Comparison of histologic nodal reactive patterns, cell suspension immunophenotypic data, and HIV status.

While cell suspension immunophenotypic studies are widely used as an aid in the diagnosis and classification of lymphomas and leukemias, much less attention has been directed toward interpretation of the results in reactive lymphoid proliferations. Cell suspension immunophenotypic data were therefore analyzed for 119 lymph nodes with reactive lymphoid proliferations which were divided into five major histologic categories: follicular hyperplasia, marked (FH,M), or moderate (FH); dermatopathic lymphadenopathy (DL); diffuse hyperplasia (DH); or "other." With the aid of a computer-assisted morphometer, the following were also measured and calculated: proportion of node occupied by follicles, mean relative follicle size, and mean follicle shape factor. Finally, in 57 cases, the influence of human immunodeficiency (HIV) status on the findings was analyzed. Although individual cases varied widely, cases of DL had significantly more CD3+ (T) cells, higher CD4:CD8 ratios, and fewer CD19+ (B) cells than other categories. Cases of FH,M had significantly lower CD4:CD8 ratios and more CD19+, CD10+, and transferrin receptor positive cells. Cases of FH,M and FH known to be HIV-negative had higher CD4:CD8 ratios than the HIV-positive cases. Peripheral blood CD4:CD8 ratios performed in 38 patients showed a strong correlation with nodal ratios. Morphometric data supported the correlation between follicular hyperplasia and increased proportions of CD19+, CD10+, and transferrin receptor-positive cells. Rare cases had CD5:CD2 or CD3 ratios of greater than 1 or "monoclonal" kappa to lambda ratios. CD4:CD8 ratios varied widely, but aberrant T cell phenotypes were not identified. These studies demonstrate that, although great variation exists, there are certain associations between types of reactive lymphoid hyperplasia and cell suspension immunophenotypic findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗

PubMed1989

Clinical relevance of immunophenotypic and immunogenotypic analysis in acute non-lymphoblastic leukaemia.

Molecular biology is playing an increasing role in defining pathology today, and has clinical relevance in the routine work-up of patients with malignant diseases, especially those of the blood and lymphatic system. The majority of acute non-lymphoblastic leukaemias (ANLL) express specific myeloid differentiation markers and are terminal deoxynucleotidyl transferase (Tdt) negative. Rarely, some cases are Tdt+ and express the T-cell marker CD7. Although uncommon in Tdt-ANLL, there appears to be a significant incidence of T-cell receptor beta chain (TCR beta) and/or immunoglobulin heavy chain (IgH) gene rearrangements in Tdt+ ANLLs. We have investigated the immunogenotype of 39 patients with ANLL, including 28 from whom immunophenotypic data were available. Thirty-seven of 39 cases had germline IgH and TCR beta genes. Two cases, one with a myeloid/CD7+ CD2+ immunophenotype, had non-germline IgH gene arrangements detectable on Hind III digests only. The possibility of Hind III polymorphisms, or of true somatic gene rearrangement of the IgH gene in these patients, is discussed. Two additional cases had a Tdt+ CD7+ immunophenotype with germline IgH and TCR beta chain genes. Our results confirm the infrequent occurrence of IgH and TCR beta gene rearrangements in ANLL. Expression of Tdt and/or CD7, often in association with IgH gene rearrangements, would appear to identify a subgroup of ANLL patients that respond poorly to standard ANLL therapy and have a poorer prognosis. The diagnostic and prognostic importance of multiparametric analysis in ANLL is emphasised.

Adolescent↗

PubMed1988

Clonal analysis of untreated non-Hodgkin's lymphoma utilizing immunoglobulin gene rearrangement and immunophenotype.

We have prospectively examined 66 consecutive initial diagnostic lymph node biopsies from unselected patients suspected of having malignant lymphoma for clonal immunophenotypic and immunogenotypic markers. By morphological and cell surface phenotypic criteria 52 had non-Hodgkin's lymphoma derived from the B-cell lineage and in these we compared surface immunoglobulin criteria for clonality with immunoglobulin gene rearrangement as detected by JH, C kappa, and C lambda gene probes. We found that the addition of BglII and HindIII double digests to the standard BamHI and EcoRI restriction enzymes made it possible to detect rearrangement in the vast majority of lymphomas and that rearrangement of both JH alleles is the rule. A rearranged heavy and/or light chain gene was detected in 47 of 52 (91%) tumors and the JH probe alone detected rearrangements in 87% of tumors when multiple restriction enzymes were used. In contrast, surface immunoglobulin, the standard clonal marker for monoclonal B-cell malignancy, was either undetectable or did not exhibit light chain restriction in 29 of 52 tumors as detected by flow cytometric analysis. Further, in 24 of these 29 tumor DNAs we could detect an Ig gene rearrangement. In follicular (nodular) lymphoma which often gives ambiguous immunophenotypic results by cell suspension techniques, monoclonal gene rearrangements were detected in 16 of 18 tumor DNAs. Monoclonal surface immunoglobulin was detected in only 8 of 18 of this subset of cases. The 52 tumors were also analyzed for potential oligoclonality. We found that the use of BglII, a restriction enzyme that closely spanned the JH region, increased the sensitivity of detecting rearrangements and facilitated the identification of isotype switch variants. In only a single (1 of 52) tumor DNA were more than two rearranged bands seen with JH, C kappa, and C lambda probes, suggesting a multiclonal origin. Additional cases thought to potentially represent oligoclonality by immunophenotypic criteria proved to be isotype switch variants. We conclude that Ig gene rearrangement is an extremely sensitive method for defining monoclonality in lymphoma cell populations, particularly if multiple restriction enzymes are used, and that the vast majority of the clonal diversity seen in initial diagnostic biopsies is the result of isotypic switch within a given clone rather than true oligoclonality.

B-Lymphocytes↗

PubMed1987

Immunophenotyping of acute myeloid leukemia using monoclonal antibodies and the alkaline phosphatase-antialkaline phosphatase technique.

The leukemic cells from 41 cases of acute myeloid leukemia (AML) and 17 cases of acute lymphocytic leukemia (ALL) were immunophenotyped by the alkaline phosphatase-antialkaline phosphatase (APAAP) immunocytochemical technique utilizing eight monoclonal antibodies (MoAb) reactive with cells of myeloid origin and seven MoAb reactive with lymphoid antigens. Ninety percent of the cases of AML reacted with one or more of the pan-myeloid MoAb, My7, My9, or 20.3. Reactivity of the myeloid panel of MoAb showed some correlation with the French-American-British (FAB) classification of AML. Five of six cases of acute promyelocytic leukemia (APL) were HLA-DR negative; the one HLA-DR-positive APL had a minor population of HLA-DR-negative promyelocytes. OKM5 and/or My4 reacted with 16 of 16 monocytic leukemias. No specific marker of early erythroid development was identified. AP3, a MoAb reactive with platelet glycoprotein (GPIIIa), was specific for acute megakaryoblastic leukemia. Immunocytochemistry was also helpful in classifying seven cases of AML with equivocal or negative routine cytochemistry. Two cases of AML had minor populations of blasts detected by the APAAP technique that were immunologically distinct from the major blast population; these minor populations emerged as the predominant cell type at relapse. Two cases of ALL expressed multiple myeloid and lymphoid antigens. Two other cases that morphologically were ALL reacted with only myeloid MoAb; one consisted entirely of immature basophils on ultrastructural examination. Immunophenotyping results using the APAAP technique were comparable with those obtained with flow cytometry. The APAAP technique is a reliable method for immunophenotyping leukemia that complements other methods of immunologic evaluation. The primary advantages of this method include its use with routinely prepared blood and bone marrow smears and the ability to correlate immunocytochemical reactions with morphology.

Alkaline Phosphatase↗

PubMed1987

Immunophenotyping of non-Hodgkin's lymphomas using a panel of antibodies on paraffin-embedded tissues.

The use of monoclonal and polyclonal antibodies for the immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue has been limited by the fact that most antigens on lymphoid cells are denatured by histologic fixation, dehydration, and embedment. In this article the authors have analyzed a small panel of antibodies which represent exceptions to this rule, in that they identify denaturation-resistant determinants on leukocyte antigens in paraffin-embedded tissue. Monoclonal antibodies L26 [corrected] and 4KB5 label preferentially B cells, monoclonal antibody UCHL1 stains predominantly T cells, and monoclonal antibody MAC 387 reacts with granulocytes and some macrophages. A polyclonal antiserum raised against purified CD3 (T3) antigen, a T-cell-specific molecule, was also employed. This antibody panel was used to immunophenotype routinely processed tissue biopsy specimens from 61 non-Hodgkin's lymphomas (all of which had been previously phenotyped in cryostat sections). The lineage of the neoplastic cells was correctly identified in 32 of 34 (94%) cases of B-cell lymphoma, in 19 of 19 (100%) cases of T-cell neoplasm, and in 2 of 4 (50%) cases of histiocytic malignancy. It is concluded that this combination of antibodies is helpful in immunophenotyping non-Hodgkin's lymphomas when only paraffin-embedded tissue sections are available, although additional reagents of higher specificity are required to improve the identification of lymphomas.

Antibodies, Monoclonal↗

PubMed2025

Well-differentiated systemic mastocytosis: Genetics, mast cell immunophenotypes, and KIT autophosphorylation.

BACKGROUND: Well-differentiated systemic mastocytosis (WDSM) is a rare myeloid neoplasm where the genetic etiology is often unknown. OBJECTIVE: We aimed to assess WDSM patients for novel KIT variants, mast cell (MC) aberrant immunophenotypes, and KIT autophosphorylation patterns. METHODS: Next-generation sequencing, MC immunophenotyping, and KIT autophosphorylation studies were performed. RESULTS: Among 454 SM patients, there were 432 with KIT p.D816V+ SM and 4 with KIT p.D816Y+ SM-notably, none of these patients had WDSM. Of the remaining patients, we identified 7 with WDSM (1.5%) and 2 relatives with mastocytosis in skin. Next-generation sequencing revealed that 6 of 9 subjects carried known or novel germline KIT variants corresponding to regions outside of codon 816. Three patients had germline KIT p.K509I; 2 had germline KIT p.A533D; 1 had two germline KIT variants p.F681L and p.M541L; and 3 had no KIT mutation. Intracellular expression of CD2 and CD25 and less robust expression of CD30 was observed in MCs from WDSM patients. By developing a novel transient transfection assay in 293T cells, we found that unlike KIT p.D816F/V/Y variants that exhibit nearly exclusive intracellular localization and strong ligand-independent autophosphorylation (class II), WDSM-associated KIT variants showed enhanced ligand-dependent autophosphorylation relative to wild type (class I). CONCLUSIONS: Our study doubles the number of KIT variants identified in WDSM patients. No KIT p.D816V+ SM patient had WDSM. Intracellular CD2 and CD25 expression was more robustly detected in MCs from WDSM patients compared to CD30.

Humans↗

PubMed1996

Nodular basal cell carcinoma in vivo vs in vitro. Establishment of pure cell cultures, cytomorphologic characteristics, ultrastructure, immunophenotype, biosynthetic activities, and generation of antisera.

BACKGROUND AND DESIGN: In this study we developed an in vitro model of nodular basal cell carcinoma (BCC). We obtained pure cultures of BCC cells and compared the morphologic characteristics, ultrastructure, immunophenotype, and behavior of cultured tumor cells with those of their in vivo counterparts. Tumors were excised from patients undergoing Mohs micrographic surgery. We established 69 primary cell cultures from 32 patients with nodular BCC. RESULTS: Three cell types grew in primary cultures: fibroblasts, normal-appearing keratinocytes, and cells with dual (spindle and epithelioid) morphologic characteristics. Contaminating fibroblasts were removed using 0.125% trypsin-0.02% edetic acid, and normal-appearing keratinocytes were cornified and eliminated by temporarily increasing the concentration of calcium in the growth medium. The cells with dual morphologic characteristics remained intact and exhibited relentless growth in pure cultures. That these seemingly immortal cell strains represent true nodular BCC was demonstrated by (1) their biphasic morphologic characteristics and very slow cell growth rate, (2) their capability for anchorage-independent growth in soft agar, (3) their ultrastructural similarities to freshly excised nodular BCC, (4) their ability to generate antibodies selectively labeling nodular BCC tumor nests in vivo, and (5) their immunophenotypic similarities to BCC in vivo on more than 20 different cell markers. CONCLUSIONS: This study provides a simple technique for establishing pure cell cultures of nodular BCC and describes extensively the in vitro parameters of tumor cell growth. The striking differences in behavior of cultured tumor cells in the presence or absence of normal-appearing keratinocytes suggest that normal human epidermal keratinocytes can suppress the growth of BCC cells.

Adult↗

PubMed2002

Immunophenotype of conjunctival melanomas: comparisons with uveal and cutaneous melanomas.

OBJECTIVE: To characterize the immunophenotypic expression pattern of conjunctival melanomas, with the use of standard melanoma markers as well as microphthalmia transcription factor and p75 neurotrophin receptor. DESIGN: Eleven conjunctival melanomas, including 1 caruncular melanoma, were immunolabeled with a panel of antibodies that included S100, tyrosinase, melan-A, HMB-45 and HMB-50 combination, microphthalmia transcription factor, and p75 neurotrophin receptor. The results were tabulated on the basis of intensity and pervasiveness of labeling and compared with a previous study of uveal melanomas. RESULTS: Immunolabeling with S100 was at significantly higher levels in conjunctival melanomas than in uveal melanomas. Tyrosinase, HMB-45 and HMB-50 combination, melan-A, and microphthalmia transcription factor were expressed at high levels in conjunctival melanomas, whereas p75 neurotrophin receptor was not expressed. CONCLUSIONS: Melanomas of the conjunctiva, including the caruncle, expressed S100, tyrosinase, melan-A, HMB-45 and HMB-50 combination, and microphthalmia transcription factor at high levels, suggesting that these are good markers for this melanoma subtype. Expression of S100 was significantly higher in conjunctival melanomas than in uveal melanomas. The immunophenotypic pattern of conjunctival melanomas is most similar to the epithelioid subtype of cutaneous melanomas.

Biomarkers, Tumor↗

PubMed2002

Immunophenotypic differences between uveal and cutaneous melanomas.

OBJECTIVE: To determine the immunophenotypic differences between uveal and cutaneous melanomas, employing standard melanoma markers as well as p75 neurotrophin receptor (p75NTR) and microphthalmia transcription factor (MITF). DESIGN: Fifteen uveal melanomas (5 spindle, 5 epithelioid, and 5 mixed uveal subtypes) were immunolabeled with a panel of antibodies that included S100, tyrosinase, melan-A, HMB-45 and HMB-50 combination, MITF, and p75NTR. The results were tabulated on the basis of intensity and pervasiveness of the labeling and compared with a prior study on cutaneous spindle and epithelioid melanomas. RESULTS: In contrast to its strong labeling of cutaneous melanomas, S100 immunolabeling of uveal melanomas was weak and variable. p75NTR, known to differentiate spindle from epithelioid melanomas of the skin, did not immunolabel uveal melanomas. HMB-45, HMB-50, tyrosinase, melan-A, and MITF immunolabeled all uveal melanomas strongly, irrespective of the histologic subtype, but not cutaneous melanomas. Microphthalmia transcription factor was especially clear in its labeling of uveal melanomas. CONCLUSIONS: Although cutaneous and uveal melanomas share many molecular markers in common, there are differences between the 2 types of melanoma. First, the level of expression of S100 differs between cutaneous and uveal melanomas. Second, while cutaneous melanomas can be further subdivided into spindle and epithelioid types based on their immunophenotype, the uveal melanomas cannot.

Antigens, Neoplasm↗

PubMed1997

Concomitant chronic lymphocytic leukemia and acute myeloid leukemia with an uncommon immunophenotype.

We report a case of simultaneous diagnosis of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML), in which the use of flow cytometry analysis allowed the demonstration of two different cell populations and the study of both immunophenotyping patterns with a large panel of monoclonal antibodies (MoAbs). CLL cells showed a typical immunophenotype with coexpression of B cell markers with CD5, CD23, CD43, and weak surface immunoglobulin light chain restriction expression, whereas the AML population had a very uncommon phenotype with expression of myeloid markers and CD56 and lack of expression of other natural killer (NK) antigens, CD34 and HLA-DR. After chemotherapeutic treatment of AML with two induction courses, the patient achieved complete remission of the AML with persistence of a CD19/CD5 positive population. After consolidation chemotherapy, this latter population was no longer detectable despite the presence of lymphoid nodules in a bone marrow biopsy. Six months after diagnosis, the patient relapsed with AML and died shortly afterwards.

Antibodies, Monoclonal↗

PubMed1998

IgM myeloma: case report with immunophenotypic profile.

Immunoglobulin (Ig)M myeloma is a distinct subtype of multiple myeloma (MM) displaying clinical and pathologic features of both MM and Waldenström's macroglobulinemia (WM). Although the immunophenotypic characteristics of classic MM and WM have been reported, the surface antigen expression of IgM myeloma has not been reported. We report a case of IgM myeloma and describe its immunophenotypic profile using flow cytometry. The cells showed a hybrid MM-WM phenotype, strongly expressing CD38 but lacking CD45 and DR, typical for plasma cells; however, pan-B cell antigens CD20 and FMC7 as well as weak monoclonal surface Ig also were positive, resembling B-cell lymphoproliferative malignancies. Discordant B-cell antigen expression was present, in that pan-B antigens CD19 and CD22 were absent. In addition, B-cell activation antigen CD23, early B-precursor antigen CD10, and pan-T antigen CD5 were not expressed. Although CD20 and weak surface Ig expression have been reported in MM, FMC7 positivity has not been seen. The data therefore suggest that IgM myeloma may have a unique phenotype with characteristics of both MM and WM.

Aged↗

PubMed1997

Sequential immunophenotypic analysis of mast cells in a case of systemic mast cell disease evolving to a mast cell leukemia.

The immunophenotypic characteristics of both bone marrow (BM) and peripheral blood (PB) mast cells (MC), from a patient suffering from an aggressive systemic mast cell disease (SMCD), were sequentially analyzed by flow cytometry using direct immunofluorescence. Analysis was carried out at diagnosis, during clinical response induced by interferon alfa-2h/prednisone therapy, and later at relapse. Our results show that together with the CD117 and IgE characteristic markers, at diagnosis BM MC showed strong expression of CD11c, CD13, CD29, CD33, CD44, CD45, CD63, and CD71, and they were also positive for CD2, CD22, CD25, and CD54 although at a lower level. PB MC displayed similar immunophenotypic characteristics although they had a lower expression of CD11c, CD25, CD33, CD63, CD69, and CD71 with a higher reactivity for CD117. Unlike BM MC, PB MC were weakly positive for CD41a and CD61. Sequential studies showed decreased numbers of both BM and PB MC during clinical response associated with a higher expression of the CD29 and CD54 adhesion molecules. In turn, clinical relapse was related to increased numbers of PB and BM MC together with lower CD2, CD11c, CD45, and and CD54 expression and a higher reactivity for the CD117 and CD25 antigens. CD2 had become negative at the last follow-up study. In addition, an increased proportion of S-phase MC was observed at relapse. These findings suggest that the assessment of the quantitative expression of cell-adhesion molecules and growth-factor receptors together with cell cycle studies of mast cells could be of value for monitoring therapy and predicting clinical outcome in aggressive SMCD.

Aged↗

PubMed1997

Analysis of variation in results of flow cytometric lymphocyte immunophenotyping in a multicenter study.

Fifty-five laboratories participated in a send-out study of four peripheral blood samples comparing a standard protocol vs. local protocols for flow cytometric lymphocyte immunophenotyping. The standard protocol included centrally provided reagents, instrument setup using triple-fluorescent microbeads and a three-color, whole-blood immunostaining technique based on fluorescein isothiocyanate and phycoerythrin-labeled monoclonal antibodies, erythrocyte lysis, washing, fixation, and identification of nucleated cells by the DNA/RNA stain LDS-751. Data analysis guidelines included lymphocyte selection using CD45,CD14-assisted "backgating" on forward (FSC) and sideward (SSC) light scatter and placement of fluorescence (FL) markers on the basis of the isotype control staining. Most (i.e., 77%) of the variation in results of percentage lymphocyte subset assessments using the standard protocol was explained by laboratory, sample, background FL, and the interaction between laboratory and sample. Purity and completeness of the FSC,SSC lymphogate, background FL, flow cytometer type, and flow cytometer setup (which were either partly or entirely determined by laboratory) contributed significantly to the variation. The effect of the leukocyte differential count on the variation in absolute numbers of lymphocyte subsets was particularly large in lymphopenic samples. The use of this standard protocol vs. local protocols did not reduce the interlaboratory variation. Instrument incompatibility with the standard protocol (e.g., incompatible filter combinations for LDS-751 detection) and lack of experience of many participants with three-color flow cytometry (in particular with the use of LDS-751) may have contributed to that result. We suggest that training and experience in a universally applicable standard protocol are critical for minimization of interlaboratory variation in flow cytometric immunophenotyping.

Antibodies, Monoclonal↗

PubMed1998

Simplified immunophenotypic analysis by laser scanning cytometry.

Immunophenotypic analysis of hematologic specimens is a useful laboratory adjunct to surgical pathology and cytology to confirm or further characterize diagnoses of leukemia or lymphoma. Laser scanning cytometry is a new laboratory technology that has been adapted to perform immunophenotypic analysis of hematologic specimens, with numerous advantages as compared with flow cytometry. In order to make full use of the laser scanning cytometer's capabilities, a new method of specimen preparation and means of performing the immunofluorescent reactions was developed. The technique described in this report, specific only to laser scanning cytometry, enables panels of up to 36 different antibodies to be used on specimens as small as 50,000 total cells. The laboratory methodology is simple, requires 85% less antibody than flow cytometric methods, and allows individual cell cytologic morphology to be correlated with objective physical and fluorescent measurements on a cell-by-cell basis. Other advantages are described in the text. Over the course of nine months in our community hospital, we have used this technique clinically to analyze 172 cases of suspected leukemia or lymphoma. The method has proven remarkably useful, particularly for extremely small specimens such as fine needle aspiration biopsies.

Antigens, CD↗