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PubMed1999

Impact of immunophenotyping on management of acute leukemias.

BACKGROUND AND OBJECTIVE: The diagnosis of acute leukemias (AL) requires a multiparametric approach in order to apply risk-adapted therapeutic protocols and appreciate the potential outcome of any given patient. Blast cells immunophenotyping is a key test in this issue, yet the information provided by immunophenotyping has become staggering, and it may be difficult to identify relevant characteristics clearly. This manuscript provides a critical review of the literature regarding the importance of immunophenotyping in acute leukemia diagnosis and management. DATA SOURCES AND METHODS The information given here is based on the experience of the authors, on their literature files and on additional material retrieved through articles and reviews covered by the Institute for Scientific Information (ISI) and the Medline database. Studies with proper definition of the patients and sufficient information regarding follow-up were considered. RESULTS: Immunophenotyping allows an early confirmation of AL diagnosis and establishes lineage assignment. Adequate and comprehensive panels of monoclonal antibodies also allow detection of aberrant immunophenotypic profiles of prognostic value or of use in detecting minimal residual disease. A number of unusual immunophenotypic features are also associated with prognosis. The development of new antibodies, new insights in the functional properties of differentiation antigens, and the quantimetric approach of immunophenotyping will keep this field changing. Moreover, as therapeutic protocols evolve, some earlier results need to be reconsidered. INTERPRETATION AND CONCLUSIONS: Immunophenotyping, together with cytologic, karyotypic and molecular approaches, retains a crucial place in the diagnosis and management of acute leukemias. It remains a rather specialized approach and should be interpreted in a multidisciplinary perspective, considering for each patient the idiosyncrasies possibly relevant to prognosis.

Acute Disease↗

PubMed2003

Detection of minimal residual disease in unselected patients with acute myeloid leukemia using multiparameter flow cytometry for definition of leukemia-associated immunophenotypes and determination of their frequencies in normal bone marrow.

BACKGROUND AND OBJECTIVES: Detection of minimal residual disease (MRD) by multiparameter flow cytometry is an emerging prognostic factor in patients with acute myeloid leukemia (AML). The present analysis aimed at improving the applicability of this approach to more patients with AML. DESIGN AND METHODS: Bone marrow samples from unselected patients with AML at diagnosis and from healthy volunteers were immunophenotyped applying triple-stainings of 31 antigens. Leukemia-associated immunophenotypes were defined by gating on populations displaying an aberrant or infrequent immunophenotype and by applying Boolean algebra. The combination of gates obtained was applied to list mode data files containing measurements of normal bone marrow samples. Dilution experiments of AML samples in normal bone marrow were performed to test the linearity of measurements. RESULTS: At least one aberrant/infrequent immunophenotype was identified (median, 2; range, 1-5) in all of 68 analyzed AML patients. The median frequencies of cells displaying an aberrant/infrequent immunophenotype within normal bone marrow ranged from 0.00% to 1.20% (median, 0.07%). Limiting this analysis to only the most sensitive aberrant/infrequent immunophenotype per patient resulted in frequencies of cells displaying an aberrant/infrequent immunophenotype within normal bone marrow ranging from 0.00% to 0.43% (median, 0.05%). Serial dilution experiments confirmed the linearity of measurements (R>0.90 in all cases analyzed). INTERPRETATION AND CONCLUSIONS: The application of multiparameter flow cytometry to identify cells displaying an aberrant/infrequent immunophenotype and to quantify MRD is feasible in unselected patients with AML.

Acute Disease↗

PubMed1993

Immunophenotypic evaluation of the bone marrow in non-Hodgkin's lymphoma.

Immunophenotypic evaluations of the bone marrow (BM) are reported on 69 aspirates from 58 patients who had non-Hodgkin's lymphoma or chronic lymphocytic leukemia involving the BM. Using flow cytometry and immunofluorescence microscopy on density gradient isolated BM mononuclear cells, the neoplasm could be identified and characterized in 59 aspirates (86%) from 49 patients (84%). Using International Working Formulation guidelines the neoplasms were classified on the basis of prior or subsequent histopathology of lymph node, spleen, skin, or other soft tissue site, or by evaluation of peripheral blood in chronic lymphocytic leukemia. In nine cases the lymphoma could not be completely classified according to International Working Formulation guidelines because only BM was available for evaluation. The neoplasm in the BM was identified and characterized immunophenotypically in all 29 cases of chronic lymphocytic leukemia/well-differentiated lymphocytic lymphoma (WDLL) (100%), in 11 of 12 cases of low-grade lymphoma other than WDLL (92%), in 11 of 15 cases of intermediate-grade lymphoma (73%), and in two of four cases of high-grade lymphoma (50%). Six of the nine cases not classified by International Working Formulation guidelines could be characterized immunophenotypically. In 10 cases immunophenotypic studies revealed negative findings, although the concurrent core biopsy specimens were positive. In two cases immunophenotypic studies with positive findings accompanied a negative core biopsy specimen. A panel of immunohistochemical reagents reactive with fixative/paraffin-resistant antigens was used for a retrospective evaluation of the 69 core biopsy specimens. When compared with the immunophenotypic data obtained from the marrow aspirates these results proved to be only moderately reliable in B-lineage neoplasms and unreliable in T-cell neoplasms. Thus, immunophenotyping of aspirated marrow by flow cytometry was found to be the most reliable method for determining the antigenic profiles of BM-based lymphomas.

Antigens, CD↗

PubMed1999

Immunophenotypic analysis of peripheral blood and bone marrow in the staging of B-cell malignant lymphoma.

This study evaluated the contributing roles of flow cytometric immunophenotyping of blood and bone marrow and immunohistochemical paraffin section staining of bone marrow biopsies in the staging of B-cell malignant lymphoma. Flow immunophenotyping was performed on a marrow specimen in 175 cases; a corresponding blood specimen was also immunophenotyped in 135 of these cases. Morphologic marrow involvement by lymphoma was found in 59 cases; flow immunophenotyping identified 54 cases with a monoclonal B-cell process: morphology-positive/flow-positive (n = 49), morphology-positive/flow-negative (n = 10), morphology-negative/flow-positive (n = 5), and morphology-negative/flow-negative (n = 111). The 10 morphology-positive/flow-negative cases included 5 follicular and 5 large-cell lymphomas with minimal marrow involvement. All 5 morphology-negative/flow-positive cases were from patients with large-cell lymphomas and bulky clinical disease. Because the blood contained the same B-cell clone in 2 of 2 morphology-negative/flow-positive cases studied, blood contamination of marrow may account for these findings. Blood flow cytometric immunophenotyping studies were positive in 32 cases; 30 had marrow involvement by morphology and were from patients with follicular, mantle cell, lymphoplasmacytic, small lymphocytic, or marginal zone lymphomas. From our results, we conclude that (1) bone marrow flow cytometric immunophenotyping is not a cost-effective replacement for good morphologic evaluation in lymphoma staging and that (2) a positive peripheral blood flow cytometric immunophenotyping study when performed in low-grade lymphomas correlates with marrow involvement.

Adult↗

PubMedDate not supplied

[Immunophenotyping of leukemic cells in the diagnosis of hairy cell leukemia].

INTRODUCTION: It is established that immunophenotyping constitutes a useful method in the diagnosis of hairy cell leukaemia. However, no single marker is specific for hairy cell leukaemia. Two-color-flow cytometry can aid in the diagnosis by showing coexpression of CD11c, HC2 or CD25 with pan B cell markers. Recently, Matutes E. et al. [17] proposed a scoring system of immunophenotypic markers, which could be used to evaluate the diagnosis of hairy cell leukaemia. AIM OF THE STUDY: The aim of the study was to: a) confirm previous observations of immunophenotypic characteristics of hairy cell leukaemia; b) identify antibody combinations of two-color immunofluorescence staining that are most useful in the diagnosis of hairy cell leukaemia; c) examine the value of a scoring system of immunophenotypic markers in the diagnosis of hairy cell leukaemia. METHODS: We analyzed peripheral blood of 46 patients with hairy cell leukaemia using indirect immunofluorescence flow cytometry (EPICS-Coulter) with an extended panel of monoclonal antibodies: CD19, CD2O, CD22, CD24, CD10, HLA-DR, CD11c, CD25, HC2, Slg, kappa and lambda light chains. The diagnosis of hairy cell leukaemia in all patients was made using conventional criteria based on cell morphology, TRAP cytochemistry and bone marrow histology. One fourth of patients were also analyzed using two-color flow cytometry with three antibody combinations as follows: CD19 + CD11c, CD19 + CD25 and CD19 + HC2. RESULTS: Our results showed that hairy cells of our patients had a uniform and unique immunophenotype with expression of the following markers: CD19, CD22, CD2O, CD11c and HLA-DR in 100% of patients, CD24 in 93%, CD25 in 88%, Slg in 82%, HC2 in 67%, CD1O in 50%, kappa light chains in 38% and lambda light chains in 35% of patients (Table 1). The level of detectable circulating hairy cells varied widely, from 2% to 93% of total lymphocytes, and 12 patients (26%) with less than 5% of detectable hairy cells were excluded from analysis. Two-color cytometry showed that antibody combination CD19 + CD11c was coexpressed in 100% of patients, CD19 + CD25 in 78% of patients and CD19 + HC2 in 57% of patients (Table 2). Only patients with 5% or more double colored hairy cells for one antibody combination, were included in the analysis. On the basis of our results of in immunophenotyping of hairy cell leukaemia patients and results of other authors (17), we made our scoring system which considers the reactivity with the following markers: CD19, CD11c, CD25 and HC2. Each marker gives 1 point if positive and 0 point if negative. Score 4 had 83% of patients, score 3 had 14% of patients, score 2 had 3% of patients and no patient had score 1 or 0 (Table 3). CONCLUSION: Our results demonstrated that immunophenotyping with a selective panel of monoclonal antibodies could significantly increase the accuracy in diagnosis of hairy cell leukaemia. Two-color flow cytometry with antibody combination CD19 + CD11c showed coexpression of hairy cells in 100% of our patients. The scoring system for hairy cell leukaemia used in our patients showed that high scores 3 and 4 had 97% of patients.

Antigens, CD↗

PubMed2005

Myeloid enzymes profile related to the immunophenotypic characteristics of blast cells from patients with acute myeloid leukemia (AML) at diagnosis.

The purpose of this study was to assess the possible relationship between the cytochemical enzyme profile and immunophenotypic characteristics of distinct acute myeloid leukemia (AML) subtypes in discrete stages of leukemic cells maturation. As the proportion of leukemic blast cells is critical for exact cytochemical analysis, study was restricted to the evaluation of 48 adult and pediatric patients with newly diagnosed AMLs with 80% or more blasts in analyzed samples. The cytochemical investigation of myeloperoxidase (MPO), Sudan black B (SBB), chloroacetate esterase (CAE), alpha-naphthyl butyrate esterase (ANBE), alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AP) in peripheral blood and/or bone marrow was performed. The immunophenotype was examined for the maturation dependent myeloid antigens CD13, CD33, CD11b, CD14, CD15, CD65, CD36, cytoplasmic MPO, non-lineage associated CD34 and HLA-DR antigens, lymphoid- associated antigens CD7, CD4, CD38 as well as natural killer cell associated marker CD56. Flow cytometry by double marker staining and visualization of pathologic cells in dot plots reflected immunophenotypic aberrancy and degree of cell maturation. The patients were classified into AML subtypes M0- M2, M3, M4 and M5 according to the main morphological, cytochemical and immunophenotypical features. The variable combinations of MPO, SBB, CAE and ANBE were identified in relation to immunophenotype. The cytochemical profile of blasts was in concordance with immunophenotype, particularly in more differentiated AML subtypes, M3, M4 and M5. The findings of myeloid antigens expression and cytochemical features in poorly differentiated AML subtypes showed no practical relevance of cytochemical analysis. Notwithstanding that the cytochemical analysis of AML subtypes not sufficiently identifies the distinct aberrancies in heterogeneous leukemic blast cell populations, evaluation of the cytochemical profile in connection with immunophenotyping may help to classify the AML patients to relevant subtypes with more accuracy.

Acute Disease↗

PubMed1995

Immunophenotypic changes between diagnosis and relapse in childhood acute lymphoblastic leukemia.

To get more insight into the phenotypic changes of childhood acute lymphoblastic leukemia (ALL) at relapse, a detailed morphological and immunophenotypic study in 40 childhood ALL cases (32 precursor B-ALL and 8 T-ALL) was performed. Expression patterns of non-lineage specific markers (terminal deoxynucleotidyl transferase (TdT), CD34, and HLA-DR), B-lineage markers (CD10, CD19, CD20, and CD22), T-lineage markers (CD1, CD2, CD3, CD4, CD5, CD7, and CD8), and cross-lineage myeloid markers (CD14, CD15, and CD33) were compared at diagnosis and relapse. In case of low blast counts (< or = 70%) at relapse, double labeling for membrane markers and TdT was used in order to define the precise immunophenotype of the TdT+ leukemic cells. An immunological marker-shift was defined as either a conversion from positive to negative and vice versa or a difference in positivity of > or = 50%. Morphological differences between diagnosis and relapse were detected in 34% of precursor B-ALL and 14% of T-ALL. Differences in immunological marker expression were found in 72% of precursor B-ALL and in 75% of T-ALL, and generally concerned minor shifts with loss or acquisition of a few markers. The morphological shifts and immunophenotypic shifts were not correlated. Immunophenotypic shifts were found for all markers tested in precursor B-ALL, except for HLA-DR. Shifts in CD10 expression (16% of cases) were only observed in relapses occurring 30 months or more after diagnosis. In four precursor B-ALL an intra-lineage shift was found at relapse (one common ALL to null ALL and three pre-B-ALL to common ALL or null ALL) and two precursor B-ALL cases were diagnosed as acute non-lymphocytic leukemia at relapse based on morphology and immunophenotype. In T-ALL, neither intra-lineage nor inter-lineage shifts were observed, although shifts were detected in all T cell markers tested, except for the lineage specific CD3 and T cell receptor (TcR) markers. In conclusion, immunophenotypic shifts at relapse frequently occur in precursor B-ALL and T-ALL, in a small percentage leading to an intra-lineage shift (10%) or inter-lineage shift (5%). Therefore immunophenotypic monitoring of minimal residual disease in ALL patients should be based on multiple marker combinations, preferably together with polymerase chain reaction analysis of rearranged immunoglobulin and/or TcR genes or chromosome aberrations.

Adolescent↗

PubMed1997

Immunophenotype of Reed-Sternberg and Hodgkin's cells in sequential biopsy specimens of Hodgkin's disease: a paraffin-section immunohistochemical study using the heat-induced epitope retrieval method.

Other studies have shown that the immunophenotype of Reed-Sternberg and Hodgkin's (RS-H) cells in Hodgkin's disease commonly changes over time, as shown by examination of multiple biopsy specimens obtained from an individual patient. In this study we analyzed 96 sequential biopsy specimens (>1 month apart) obtained from 44 patients (nodular sclerosis, 34 specimens; mixed cellularity, 5; lymphocyte depletion, 1; unclassified, 4) using fixed, paraffin-embedded sections; heat-induced epitope retrieval (HIER); a panel of antibodies specific for the CD3, CD15, CD20, CD30, CD43, CD45/45RB, and CD79a antigens and Epstein-Barr virus latent-membrane protein; and a streptavidin-biotin method. In selected cases in which immunophenotypic changes occurred, studies were repeated using enzyme predigestion instead of HIER. There was no change in the immunophenotype of the RS-H cells in 36 (82%) of 44 patients. In 8 patients (18%), the immunophenotype of the RS-H cells varied in expression of one or two antigens. The antigens that varied were as follows: CD30, 3 patients; CD15, 3 patients; CD20, 1 patient; and CD15 and CD30, 1 patient. We conclude that the immunophenotype of RS-H cells in Hodgkin's disease is relatively stable over time and that CD15 and CD30 are the most common antigens that change. The frequency of immunophenotypic changes, 18%, is substantially lower than that reported previously. One likely explanation for this discrepancy is that we used HIER, a relatively recent innovation in diagnostic immunohistochemistry that has been shown to reduce artifacts attributable to inconsistent fixation and processing. The significance of immunophenotypic variation in eight cases (18%) is uncertain. This phenomenon may represent true biologic changes in RS-H cells. Alternatively, these changes may be attributable to artifacts secondary to inconsistent fixation or processing that HIER cannot overcome.

Adolescent↗

PubMed2001

SIHONSCORE: a scoring system for external quality control of leukaemia/lymphoma immunophenotyping measuring all analytical phases of laboratory performance.

For the diagnosis of leukaemia and leukaemic lymphoma, clinicians frequently have to rely on the results of immunophenotyping. To improve the quality of these results, the Dutch Foundation for Immunophenotyping of Haematological Malignancies (SIHON) initiated external quality rounds in 1986. Over a period of more than 10 years, this has led to improvements in the interpretation of immunophenotyping results. However, the evaluation of results focused mainly on the correctness of the interpretation of the immunophenotypical data, leaving the preceding analytical phases unevaluated. Therefore, in 1996 SIHON developed a more comprehensive scoring system, called SIHONSCORE, covering all three phases of immunophenotyping, namely the pre-analytical (i.e. choice of the staining panels), analytical (i.e. the technical part consisting of sample preparation, data acquisition and analysis) and the post-analytical phase (i.e. the interpretation) of the laboratory process. Here, we report how SIHONSCORE was successfully applied to three consecutive external quality rounds consisting of a total of nine different cases tested. For laboratory certification, participation in external quality control programmes is required. Evidently, criteria are needed to define the minimum acceptable performance of a certified laboratory. With SIHONSCORE, a useful instrument is obtained evaluating all phases of the performance of laboratories in leukaemia and lymphoma immunophenotyping.

Clinical Laboratory Techniques↗

PubMed1994

B-cell chronic lymphocytic leukaemia stage 0. An immunophenotypic study of 66 cases and comparison with B small cell lymphomas.

The cells of 66 B-CLL stage 0 patients were analyzed using a large panel of monoclonal antibodies in order to better define the immunophenotype of B-CLL. The data were compared with the immunophenotypes of lymph node cells from 51 patients with diffuse B small cell lymphoma or leukaemia with lymph node enlargement. The most frequent immunophenotype of B-CLL stage 0 was SIgM(D)+ EmR+ CD5+ CD9+ CD21+ CD23+ CD35- CD38-. Among the lymphomas, EmR-positive and EmR-negative cases were identified. The vast majority of the EmR-positive cases usually showed the leukaemic pattern, immunophenotype and lymph node histology of B-CLL. The EmR-negative cases usually had immunophenotypes quite different from those of B-CLL and the histology of indented cell lymphoma (centrocytic or intermediate) or features of lymphoplasmacytoid/cytic lymphoma. More than 20% of EmR-positive cells proved to be the most important marker to distinguish B-CLL from other lymphocytic lymphomas. Indeed this was a sign of better prognosis. Lymphoplasmacytoid/cytic lymphomas were EmR-positive with the immunophenotype of B-CLL or EmR-negative with a definitely different immunological profile. This suggests two morphologically similar but biologically different subgroups of these diseases.

Antigens, CD↗

PubMed2002

Immunophenotypic clustering of myelodysplastic syndromes.

Myelodysplastic syndromes (MDSs) are heterogeneous diseases of bone marrow (BM) cell precursors for which immunophenotypic characterization is still considered irrelevant despite the accuracy and sensitivity of flow cytometry techniques. The aim of this study was to determine whether immunophenotypic abnormalities could be defined in MDSs and could correlate with the French-American-British classification and cytogenetics. Analysis was performed on 275 BM samples (207 MDS patients, 68 controls) and 25 control blood samples. Immunophenotyping was based on a primary gating of blast cells, monocytes, and granulocytes according to CD45 antigen expression and side scatter light diffraction. Immunophenotypic hierarchical clustering was performed to analyze the results. The data obtained show that (1) immunophenotypic clustering partly discriminates patients with refractory anemia with excess blasts/refractory anemia with excess blasts in transformation (RAEB/RAEB-T), chronic myelomonocytic leukemia (CMML), and refractory anemia/refractory anemia with ring sideroblasts (RA/RARS) for CD45(lo) blast cells and patients with RA/CMML, RARS, and RAEB/RAEB-T for CD45(hi)/side scatter(hi) (SS(hi)) granulocytes; (2) the most discriminating markers were CD16, CD34, CD36, CD38, CD71, and HLA-DR for blast cells and CD11b, CD13, CD33, CD36, CD38, CD71, and HLA-DR for CD45(hi)/SS(hi) granulocytes; (3) clusters related to CD34 expression were associated with high levels of blast cells on BM smear; (4) clusters related to high levels of CD36 expression on CD45(lo) blast cells and CD45(hi)/SS(hi) granulocytes were associated with a poor International Prognosis Scoring System score; and (5) high levels of CD71 expression on CD45(hi)/SS(hi) granulocytes were associated with the RARS category. These results show a close relationship between immunophenotypic abnormalities and BM dysplasia and suggest that flow cytometry could be a future tool for the characterization of MDSs.

Antigens, CD↗

PubMed1998

Primary non-Hodgkin's lymphoma of the nose and nasopharynx: clinical features, tumor immunophenotype, and treatment outcome in 113 patients.

PURPOSE: To study the clinical features and outcome for primary non-Hodgkin's lymphomas of the nose/nasopharynx (NNP-NHLs) according to immunophenotype. PATIENTS AND METHODS: One hundred thirteen Chinese patients with primary NNP-NHLs that belonged to the categories E, F, G, or H according to the Working Formulation (WF), with full immunophenotypic data and complete clinical follow-up data, were analyzed in this retrospective study. RESULTS: Ninety (79.6%) patients had localized (stage I or II) disease, while 23 (20.4%) had stage III or IV disease. The lymphomas in 51 (45.1%), 24 (21.3%), and 38 (33.6%) patients showed natural killer (NK)/T- (CD56-positive), T-cell, and B-cell immunophenotype, respectively. Seventy-three patients (65.8%) achieved a complete remission, of whom 34 (46.6%) subsequently relapsed. The median follow-up time for those alive was 88 months. The 5-year actuarial disease-free and overall survival rates were 34.4% and 37.9%, respectively. Multivariate analysis showed that only stage and immunophenotype were significant for survival. NK/T lymphomas were distinctive among the three immunophenotypes in the following aspects: the highest male-to-female ratio, more frequent involvement of the nasal cavity alone, higher risk of dissemination to the skin, more frequent development of hemophagocytic syndrome, and the worst prognosis (overall median survival, 12.5 months). CONCLUSION: The three immunophenotypes studied are shown to exhibit different clinical patterns. Since the NK/T phenotype carries the worst prognosis, patients who present with NNP-NHL should have their tumors analyzed for CD56 expression.

Adolescent↗

PubMed1992

Acute leukemias with unusual immunophenotypes.

Over a two-year period, immunophenotypic patterns of 266 acute leukemia cases were analyzed using a panel of tests including TdT, SmIg and 9 surface antigens by the immunofluorescence stains for the assessment of the incidence and grade of phenotypic ambiguity (lineage infidelity) and the possible clinical significance of unusual immunophenotypes. Immunophenotypes were classified into four groups according to the degree of ectopic antigen expression. We classified as Group A (91.7%, 244 of 266 cases) those expressing conventional pattern without ectopic antigen. Group B (3.0%, 8 of 266 cases) was defined to have at least two lineage specific markers and single ectopic antigen. Such a "low grade deviation" did not prevent a definite immunodiagnosis. Group C (4.2%, 11 of 266 cases) revealed a promiscuous coexpression of markers related to different lineages, including two cases (0.8%, 2 cases) of biphenotypic leukemia. Group D (1.1%, 3 cases) included unclassifiable immunophenotypes with no antigen or HLA-DR only expression. Both patients with biphenotypic leukemia and one patient with unclassifiable immunophenotypes failed to respond to induction chemotherapy, suggesting a poor prognosis in these patients. The incidence of acute myelogenous leukemia (AML) cases with one or more ectopic surface antigens was 10 (8.1%) of the 124 AML cases. Ectopic antigen expression was seen in 5 (4%) of the 125 B-lineage acute lymphoblastic leukemia (ALL) cases and 3 (25%) of the 12 T-ALL cases. It is concluded that nearly 95% of cases of acute leukemia cases can be diagnosed accurately with immunophenotyping alone including patients with a mild degree of deviation from expected antigenic patterns.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗

PubMed1999

Clinically useful information provided by the flow cytometric immunophenotyping of hematological malignancies: current status and future directions.

BACKGROUND: At present, immunophenotyping of hematological malignancies represents one of the most relevant clinical applications of flow cytometry. In recent years, its use has extended from clinical research to diagnostic laboratories. The aim of this report is to critically review the type of information provided by the flow cytometric immunophenotyping of hematological malignancies and its clinical impact as well as to highlight its potential future applications. METHODS: The currently available information, including that provided by different international consensus groups on the phenotypic characterization of hematologic malignancies, was reviewed. Additionally, recent reports on the immunophenotypic analysis of hematological malignancies published in hematology, oncology, pathology, immunology, and cell biology journals were also analyzed. RESULTS: A careful review of the literature showed that in spite of the well-established utility of immunophenotyping for the diagnosis, classification, prognostic stratification, and monitoring of hematological malignancies, only a small part of the information on the immunophenotypic characteristics of pathological hemopoietic cells has been used routinely. Specific and sensitive identification of neoplastic cells and their accurate enumeration and phenotypic characterization represent the major aims of these procedures. Similarities between leukemic and healthy cells allow the establishment of the lineage and maturation stage of the pathologic cells, this information being of great utility for the diagnosis, classification, and prognostic evaluation of different subtypes of hematological malignancies. On the other hand, the phenotypic aberrations displayed by leukemic cells could allow the selection of cases carrying specific genetic abnormalities in which further confirmatory molecular studies will be performed. CONCLUSIONS: The information provided by the flow cytometric immunophenotyping of hematological malignancies is of great clinical utility, with a major challenge for the near future being the standardization of technical procedures, data interpretation, and reporting.

Flow Cytometry↗

PubMed2000

[Morphology and immunophenotyping in hematological malignancies].

We evaluated 443 outpatients and inpatients in Keio University Hospital between 1994 and 1999. Morphologic features from peripheral blood and bone marrow aspiration were evaluated in our hematology laboratory, using Wright-Giemsa, peroxidase staining films and other cytochemistry. Immunophenotype was determined by cell surface antigen analysis by laser flow cytometry, FACscan, using various monoclonal antibodies. Information on cytogenetic and molecular genetic characteristics can be also integrated for diagnosis. One hundred fifty patients were diagnosed with acute leukemia, in which 59 cases were ALL and 91 cases were AML. Seventy-four cases were MDS, 76 cases were myeloproliferative disorders, 21 cases were CLL related disorders, 104 patients were malignant lymphoma, and 18 cases were multiple myeloma. The ratio of male to female was 1.7. The probability of diagnostic rate by Immunophenotyping was estimated by Discriminant analysis in 189 patients, using multivariate analysis of immunophenotype compared to morphology. The average probability by immunophenotypic analysis for diagnostic rate was 91.7%, in which the probability for NHL was very high of 97.1%. Thus, morphologic and immunophenotypic analysis is most essential and basic approach in laboratory hematology, from the perspective of rapid and precise diagnostic methods. Recent advance appreciates the rapid contribution for diagnosis by immunophenotypic analysis. Furthermore, Tele-hematology would contribute the standardization for morphologic method in the near future.

Bone Marrow Cells↗

PubMed2001

New methodologic approaches for immunophenotyping acute leukemias.

BACKGROUND AND OBJECTIVES: Flow cytometry is nowadays the preferred method for immunophenotypic identification, enumeration and characterization of blast cells at diagnosis. Despite widespread application of standardized protocols, inter-laboratory reproducibility has still not been achieved. The complexity of diagnosis and evaluation of minimal residual disease, in immunophenotyping acute leukemia, demands the use of a test that provides all the necessary information. DATA SOURCES AND METHODS: The information given here is derived from the experience of the authors and from literature files. The most relevant studies with adequate conclusions were considered. We report on the current status of multiparametric immunophenotyping using simultaneous three and four-color staining and the applications of this technique. RESULTS: Multiparametric immunophenotyping is a powerful method for achieving a clear discrimination between normal and pathologic cells. The specific identification of leukemic cells by immunologic gating forms the basis for immunophenotypic diagnosis, classification as well as prognostic evaluation of patients with acute leukemias. The performance of the procedure with regards to the panels of reagents and the analytic processes, is necessarily different in lymphoblastic and myeloblastic leukemias, since the diagnostic questions are different. Phenotypic information should be specifically provided for the blast cells and antigen expression should preferably be reported in quantitative units and CV. This would allow a standardized cross evaluation of immunophenotypic results between different investigators and laboratories. INTERPRETATION AND CONCLUSIONS: Recent reports indicate that phenotypic aberrations reflect genetic abnormalities of leukemic cells and therefore their definition and identification is of clinical relevance not only for minimal residual disease monitoring but also for subclassifying acute myeloid and lymphocytic leukemias.

Acute Disease↗

PubMed2004

Contribution of flow cytometric immunophenotyping and bone marrow trephine biopsy in the detection of lymphoid bone marrow infiltration in non-Hodgkin's lymphomas.

The bone marrow (BM) is a frequent site of involvement in non-Hodgkińs lymphomas (NHL) and evidence of an infiltrated BM may implicate different therapeutical regimens. Flow cytometric immunophenotyping of bone marrow aspirates now is included in the assessment of patients with NHL and used as an adjunct to morphologic evaluation in the staging of lymphoma. The aim of the study was to compare flow cytometric immunophenotyping of BM and paraffin section staining of BM biopsies in the marrow involvement of NHL. Cytometric immunophenotyping of bone marrow and immunohistochemical paraffin section staining of bone marrow biopsies in 53 B- and T-cell lymphoma patients were performed. We used the following fluorochrom conjugated monoclonal antibodies specific for: CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD23, CD79B, FMC7 and Ig kappagamma light chain. Unilateral BM trephine biopsies were obtained in all cases, fixed, decalcified and paraffin-embedded. Morphologic marrow involvement by lymphoma was found in 24 cases; flow immunophenotyping identified 26 cases with NHL: morphology-positive/flow-positive (n=21), morphology positive/flow-negative (n=3), morphology-negative/flow-positive (n=4), and morphology-negative/flow-negative (n=23). The concurrence rate of BM trephine biopsy and flow cytometric immunophenotyping in evaluation of NHL bone marrow infiltration was 88.7%. Immunophenotyping of the bone marrow of NHL patients by flow cytometry is helpful for assessment of bone marrow infiltration, especially in B-cell disorders. Both trephine biopsies and flow cytometry are better than single investigation for detection of infiltration in NHL.

Adult↗

PubMed2004

[Immunophenotype analysis in 121 patients with lymphoproliferative diseases by flow cytometry].

BACKGROUND & OBJECTIVE: Multiple parameter immunophenotype analysis by flow cytometry (FCM) could improve the accuracy of diagnosis in lymphoproliferative diseases. This study was to analyze the immunophenotype of 135 samples, including lymph nodes, blood, bone marrow, and cerebrospinal fluid samples, from 121 patients with suspected lymphoid malignancies, to evaluate its role in diagnosis. METHODS: All samples were tested by routine morphological, pathological, and immunohistochemical methods, and analyzed by FCM in suspended single cells, to compare the accuracy of different diagnostic methods. RESULTS: (1) Three of 23 lymph nodes, which failed to be diagnosed by routine methods, were determined by flow cytometric immunophenotype analysis. (2) According to new WHO classification, 96 of 97 blood or bone marrow aspiration samples were diagnosed by flow cytometric immunophenotype analysis, while 88 of 97 samples were diagnosed by routine immunohistochemical method. (3) Multiple parameter immunophenotype analysis in cerebrospinal fluid samples by FCM improved the diagnostic accuracy of leukemia or lymphoma involvement of central nervous system. CONCLUSIONS: Multiple parameter immunophenotype analysis by FCM improves the accuracy of diagnosis in lymphoid malignancies, and can be used in diagnosis, differentiated diagnosis, and detection of minimal residue in lymphoproliferative diseases.

Adolescent↗