Detection of intracytoplasmic immunoglobulin by flow cytometry in B-cell malignancies

The aim of this study was to compare the results of flow cytometric (FCM) determination of heavy and light chain cytoplasmic immunoglobulin (cIg) with those obtained by the peroxidase-antiperoxidase (PAP) method. Fifty-one patients, including five non-T-acute lymphoblastic leukemias, 16 B-chronic lymphocytic leukemias (CLL), 13 non-Hodgkin's lymphomas, seven hairy cell leukemias, four multiple myeloma/plasma cell leukemias, and six T-cell leukemia/lymphomas, as well as 12 normal controls, were studied. Saponin-permeabilized cell suspensions were indirectly stained with monoclonal antibodies and analyzed by flow cytometry. Acetone-fixed cytocentrifuge smears were stained for cIg by the PAP method. The results obtained indicate that: (a) detection of cIg by FCM is a feasible and useful technique to confirm the B-cell lineage of leukemias and lymphomas, particularly those characterized by low-density surface immunoglobulin, such as CLL; and (b) cIg detection by FCM and PAP staining are complementary methods to recognize with certainty the monoclonality of B-cell malignancies.     

Terminal deoxynucleotidyl transferase in diagnosis of lymphomas

TdT activities were determined on 29 specimens of mononuclear blood, bone marrow or lymph node cells from 18 patients with non Hodgkin's Lymphomas, 2 Hodgkin's patients and 3 patients with non neoplastic lymph nodes. The neoplastic cells were typed using tests detecting membrane markers (E, Em, SIg), and monoclonal antibodies (MoAb). In a group of 15 patients with Low Grade Malignant Lymphoma (L. lymphocytic, centrocytic and lymphoplasmocytic) 14 cases belonged to B cell phenotype lymphoma, with 3 cases among them with a moderate TdT activity. In one case of lymphocytic lymphoma the cells had the non T, non B, TdT+ characteristics. High TdT activity was observed in both examined patients with lymphoblastic lymphoma and in cells obtained from the lymph node of one Hodgkin's lymphoma case. Although our group was of heterogenic character, our investigations confirm the value of TdT as biochemical marker of immature lymphocytes and its usefulness for differential diagnosis of malignant lymphomas.     

185例儿童急性白血病流式细胞术免疫分型研究

目的:探讨儿童急性白血病流式细胞术免疫分型的意义。方法:采用流式细胞术三色荧光标记技术和CD45/SSC双参数散点图设门,检测185例儿童急性白血病的免疫表型,对抗原表达情况进行分析。结果:流式细胞术免疫分型和FAB分型的符合率为89.19%。185例儿童急性白血病中,ALL为121例,占AL的65.41%,B-ALL为113例,主要表达B系的CD19(99.12%)、CD22(98.13%)、CD79a(96.19%)、CD10(86.73%)。T-ALL占8例;主要表达CD5(100%)、CD7(100%)、cCD3(100%)、CD8(87.5%)。AML为47例,占25.41%,主要表达CD33(93.62%)、CD15(78.72%)、CD64(76.6%)、MPO(76.6%)、CD13(74.47%)。在B-ALL,AML,T-ALL中,敏感性最高的抗体分别是CD19,CD33,CD5和CD7,特异性最强的抗体分别是CD79a,MPO,cCD3。AMLL为17例,占9.19%,其中B/M为9例,T/B为5例,T/M为3例。My十-ALL为54例,占ALL的44.63%,表达的髓系抗原为CD13、CD15、CD33、CD64。Ly+-AML为18例,占AML的38.30%,表达的淋系抗原为CD19、CD4、CD7。系列非相关抗原CD34的表达率为67.57%,HLA-DR的表达率为85.41%,CD38的表达率为80.59%,TdT的表达率为62.59%。结论:流式细胞术免疫分型在白血病分型中起重要作用,是FAB分型的补充和修正,提高了儿童急性白血病诊断的准确率有必要进一步加强流式细胞术免疫分型的标准化工作。     

Immunophenotyping of acute lymphoblastic leukemia using immunohistochemistry in bone marrow biopsy specimens

Flow cytometry is the preferred method of diagnosing and immunophenotyping acute lymphoblastic leukemia (ALL). However, there are situations in which immunohistochemical staining (IH) of bone marrow trephine biopsy specimens can be used to provide immunophenotypic information. To evaluate the use of IH and to confirm its value in diagnosing and typing of ALL, we studied 50 cases of denovo ALL that were previously classified into pre B, T and B by morphologic, cytochemical and FC methods. Paraffin embedded bone marrow trephine biopsies sections were stained using a panel of antibodies,namely, myeloperoxidase (MPO), terminal deoxynucleotidyl transferase (TdT), CD10, CD20, CD79a, CD3. The cases included 37 pre BALL, 10 T ALL and 3 mature BALL. TdT was the most commonly expressed antibody and was positive in 41 of 50 cases of ALL (82%) and in 95% of pre B ALL cases. CD79a and CD10 were positive in 68% and 65% of pre B ALL cases, respectively. CD79a showed similar positivity in B ALL cases (66%). CD 20 was positive in 66% of mature B ALL cases but less positive in pre B ALL (22%). CD3 was positive in 70% of T ALL cases and negative in other ALL subtypes. All of the cases were negative for MPO. Diagnosis and immunophenotyping of acute lymphoblastic leukemia is possible using immunohistochemical staining of bone marrow trephine biopsies.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line kappa light chain genes and germ-line T-cell receptor beta- and gamma-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Construction of human-mouse T cell hybrids that express human T cell-associated surface antigens and allow the chromosomal localization of these antigens

We have constructed somatic cell hybrids between the murine T cell line BW5147 and cells from patients suffering from T cell acute lymphoblastic leukemia. The obtained hybrid clones were analyzed for expression of human T cell antigens and presence of human chromosomes. T cell hybrids derived from fusion between the BW5147 cell line and bone marrow cells from a patient with pre-T acute lymphoblastic leukemia (TdT+/HLA-DR+/Tp41+/T11+/T1-/T6-/T4-/T8-/T3-) appeared to express the human T cell antigen Tp41, which can be recognized by the monoclonal antibodies 3A1 and WT1. Although this panel of hybrid cells contained all human chromosomes, no other T cell antigens were expressed. Fusion of the BW5147 cell line with peripheral blood cells from a patient with a more mature T cell acute lymphoblastic leukemia (TdT+/HLA-DR+/Tp41+/T11+/T1+/T6-/T4+/T8+/T3-) resulted in a panel of hybrid clones that expressed not only the Tp41 antigen, but also the human T cell antigens T1 and T4; two hybrids even expressed the T3 antigen. This panel of hybrids also contained the whole human genome. The two panels of human-mouse T cell hybrids allowed us to assign the genes coding for the human T cell antigens Tp41, T1, and T4 to human chromosomes 17, 11, and 12, respectively. Furthermore, these data support our previous suggestion that the expression of human lymphoid differentiation antigens in human-mouse lymphoid hybrids is influenced by the differentiation stage of the fusion partners.     

Flow cytometric DNA index and karyotype in childhood lymphoblastic leukemia.

Flow cytometric DNA-index (DI(FCM)) and karyotype were analysed in 82 consecutive children with acute lymphoblastic leukemia (ALL) during a 10 year period. A statistically significant correlation existed between modal chromosome number and DI(FCM) (p = 0.009). DI(FCM) could reliably identify leukemias with >51 chromosomes, whereas only three out of 12 cases with modal chromosome numbers between 47-51 were classified as aneuploid by DI(FCM). In the pseudodiploid group only one out of 20 leukemias had a DI(FCM) > 1.0. Five leukemias with a diploid karyotype showed an aneuploid DI(FCM) and in three patients the flow cytometric measurement revealed biclonality undetected by karyotyping. During treatment aneuploid clones could be detected by DI(FCM) in a substantial number of cases where the cytogenetic analysis was normal, and the opposite was also demonstrated in one case. DI(FCM) gave prognostic information, showing that cases with a DI > 1.12 (corresponding to 51 chromosomes) had a superior outcome with treatment protocols today in use.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Automated fluorescence image cytometry. DNA quantification and detection of chlamydial infections

Digitized fluorescence microscopy in conjunction with automated image segmentation is a promising approach for screening clinical specimens quickly and reliably. This paper describes the hardware and software of a prototype image-based cytometer that can identify fluorescent objects, discriminate true objects from artifacts and divide overlapping pairs of objects. The use of this image cytometer is discussed for: (1) the measurement of the DNA ploidy distribution of isolated mature rat liver nuclei labeled with 4',6-diamidine-2-phenylindole; (2) the comparison of the DNA ploidy distributions of the same samples measured by image cytometry (ICM) and flow cytometry (FCM); and (3) the quantification of chlamydial infection by double labeling cells with antichlamydiae antibody and Hoechst 33258 for nuclear DNA analysis. Ploidy distributions measured by the automated image cytometer compared favorably to those obtained by FCM. All pairs of overlapping nuclei were automatically detected by an additional computer algorithm, and those pairs that were clearly more than one nucleus by visual inspection were correctly divided. The irregular morphology of the chlamydiae-infected cells meant that 26% of them were not correctly identified in the fluorescein-stained images (as judged by manual inspection), but all cells were nevertheless detected correctly from the images of the Hoechst-stained samples. Automated fluorescence ICM yielded results similar to those obtained with FCM and had the additional benefit of maintaining cell and tissue architecture while preserving the opportunity for subsequent manual inspection of the specimen.     

Antileukemic Efficacy of Continuous vs Discontinuous Dexamethasone in Murine Models of Acute Lymphoblastic Leukemia

Osteonecrosis is one of the most common, serious, toxicities resulting from the treatment of acute lymphoblastic leukemia. In recent years, pediatric acute lymphoblastic leukemia clinical trials have used discontinuous rather than continuous dosing of dexamethasone in an effort to reduce the incidence of osteonecrosis. However, it is not known whether discontinuous dosing would compromise antileukemic efficacy of glucocorticoids. Therefore, we tested the efficacy of discontinuous dexamethasone against continuous dexamethasone in murine models bearing human acute lymphoblastic leukemia xenografts (n = 8 patient samples) or murine BCR-ABL+ acute lymphoblastic leukemia. Plasma dexamethasone concentrations (7.9 to 212 nM) were similar to those achieved in children with acute lymphoblastic leukemia using conventional dosages. The median leukemia-free survival ranged from 16 to 59 days; dexamethasone prolonged survival from a median of 4 to 129 days in all seven dexamethasone-sensitive acute lymphoblastic leukemias. In the majority of cases (7 of 8 xenografts and the murine BCR-ABL model) we demonstrated equal efficacy of the two dexamethasone dosing regimens; whereas for one acute lymphoblastic leukemia sample, the discontinuous regimen yielded inferior antileukemic efficacy (log-rank p = 0.002). Our results support the clinical practice of using discontinuous rather than continuous dexamethasone dosing in patients with acute lymphoblastic leukemia.     

Diagnostic and prognostic value of the terminal deoxynucleotidyl transferase (TdT) activity in treating the blastic phase (bp) of chronic granulocytic leukaemia (CGL)

The blastic cell phenotypes of 26 cases of CGL in blastic phase were estimated and the patients were treated with different schemes. The following methods of typisation of the blast cells were used: cytochemical stainings (POX, Sudan B, PAS, nonspecific esterase), estimation of TdT activity, and in 11 patients the testing with monoclonal antibodies of VI series. Using these methods 10 patients (38%) with lymphoid form of the blastic phase, 11 (43%) with the myeloid type and 5 patients (19%) with undifferentiated type were diagnosed. In the group of lymphoblastic type a longer survival time and complete remissions were observed. High TdT activity in blastic cells did correspond with favourable response to Vincristin and Prednison. The introduction of TdT assessment into the diagnosis of CGL allows the cells to be classified more precisely, thus helping in defining the prognosis and in the choice of treatment programme.     

Effect of neuraminidase treatment on serum reactivity to autologous leukemic blast cells

Summary The sera of 35 patients with acute lymphoblastic leukemia (ALL) and acute non-lymphoblastic leukemia (ANLL) were tested for reactivity against cell surface antigens of autologous leukemic blast cells by protein A assay (PA), immune adherence assay (IA), and anti-C3 mixed hemadsorption assay (C3-MHA). Autologous serum reactivity was detectable by PA in four cases and by LA and C3-MHA in about half the patients. Autologous serum reactivity occurred more often in ALL than in ANLL. Absorption studies revealed that in one patient only the autologous reactivity was directed against a restricted antigen, which could be detected only on the individual T-ALL blast cells. All other autologous antibodies detected unspecific antigens. Neuraminidase treatment had two effects: first, it increased antibody attachment to antigens which are also present on untreated cells; secondly, after neuraminidase treatment an antigen was detectable on the cell surface which could also be demonstrated on neuraminidase-treated non-leukemic cells (e.g., erythrocytes). Neither of these two effects of neuraminidase treatment seems to be tumor-specific. Possible therapeutic effects of neuraminidase are probably caused by unspecific adjuvant effects of the enzyme.     

Determination of the rate of DNA synthesis of human tumor cells obtained by fine needle aspiration. Comparison of flow cytometry and an immunoperoxidase method for the detection of thymidine analogue incorporation.

Two methods of detecting thymidine analogue incorporation by lymphoma, leukemia and myeloma cells obtained by fine needle aspiration (FNA) are described. In one method, cells which have been incubated with the thymidine analogue iododeoxyuridine (IDURD) were exposed to primary monoclonal anti-IDURD antibody and a fluorescein-labeled linking antibody. The fluorescence of the antibody-labeled cells, which had synthesized DNA and incorporated the analogue, was detected by flow cytometry (FCM). In a second method, the cells that incorporated the analogue were detected on glass slide Cytospin preparations by an immunoperoxidase (IP) technique. The IDURD labeling index (LI), as determined by both FCM and IP staining, was compared to the percentage of cycling (S + G2/M) cells as determined by acridine-orange FCM. The data indicate that the IP method is reliable and correlated strongly with FCM determination of LI, percentage S-phase and lymphoma grade. Given the low cost and wide availability of IP technology, the IP method may be desirable for laboratories wishing to supplement cytology reports with cell cycle data.     

185例儿童急性白血病流式细胞术免疫分型研究

目的：探讨儿童急性白血病流式细胞术免疫分型的意义。方法：采用流式细胞术三色荧光标记技术和CD45／SSC双参数散点图设门,检测185例儿童急性白血病的免疫表型,对抗原表达情况进行分析。结果：流式细胞术免疫分型和FAB分型的符合率为89．19％。185例儿童急性白血病中,ALL为121例,占AL的65．41％,B—ALL为113例,主要表达B系的CD19（99．12％）、CD22（98．13％）、CD79a（96．19％）、CD10（86．73％）。T—ALL占8例;主要表达CD5（100％）、CD7（100％）、cCD3（100％）、CD8（87．5％）。AML为47例,占25．41％,主要表达CD33（93．62％）、CD15（78．72％）、CD64（76．6％）、MPO（76．6％）、CD13（74．47％）。在B—ALL,AML,T—ALL中,敏感性最高的抗体分别是CD19,CD33,CD5和CD7,特异性最强的抗体分别是CD79a,MPO,cCD3。AMLL为17例,占9．19％,其中B／M为9例,T／B为5例,T／M为3例。My＋-ALL为54例,占ALL的44．63％,表达的髓系抗原为CD13、CD15、CD33、CD64。Ly＋-AML为18例,占AML的38．30％,表达的淋系抗原为CD19、CD4、CD7。系列非相关抗原CD34的表达率为67．57％,HLA—DR的表达率为85．41％,CD38的表达率为80．59％,TdT的表达率为62．59％。结论：流式细胞术免疫分型在白血病分型中起重要作用,是FAB分型的补充和修正,提高了儿童急性白血病诊断的准确率。有必要进一步加强流式细胞术免疫分型的标准化工作。     

Accurate quantitation of Leishmania infection in cultured cells by flow cytometry

BACKGROUND: Leishmaniases are major parasitic diseases caused by protozoans that are obligate intracellular parasites during the mammalian phase of their life cycle. Quantitation of experimental mammalian cell infections is usually performed by time-consuming microscopic examination. In this report a flow cytometry (FCM)-based assay suitable for studying in vitro infections by L.amazonensis is presented. METHODS: Intense fluorescence staining of the amastigote forms with a stage- and species-specific monoclonal antibody was obtained after permeabilization of both the host-cell cytoplasmic membrane and the parasitophorous vacuole membrane by saponin treatment. RESULTS: Upon flow cytometry (FCM) analysis, parasitized cells separated sharply from the auto-fluorescence of the mammalian host cells, giving the assay a high degree of sensitivity and specificity. Ninety to 98% of cells in the more fluorescent population harbored parasites visible by phase-contrast and UV-light microscopy, while no parasites were observed in more than 95% of the cells in the population with background fluorescence. Comparisons of the FCM results with those from microscope counting and analysis of various dilutions of parasitized cells confirmed the reliability of the method. CONCLUSIONS: The FCM assay provided rapid quantitation of Leishmania infection either in mouse macrophages, the natural host cell in murine leishmaniasis, or in Chinese hamster ovary (CHO) cells, a non-macrophage cell line proposed as an in vitro model for studying host-parasite interactions. The protocol described here should be adaptable to studies involving other parasites residing in nucleated cells.     

Terminal deoxynucleotidyl transferase, TdT, as a marker for leukemia and lymphoma cells

Terminal deoxynucleotidyl transferase, TdT, was assayed in the mononucleate cells of blood and bone marrow from 121 patients with leukemias at the onset of disease and from 95 subjects with malignant lymphomas at diagnosis. This intracellular marker was also investigated by cytoimmunofluorescent tests in 17 other cases of initial leukemias and in 3 diagnosed lymphoblastic lymphomas. Generally, the TdT levels were significantly enhanced in the blasts of the following: acute undifferentiated leukemias; the more immature types of acute lymphoblastic leukemias i.e., the null, non-T non-B, common, early T and pre-B subgroups; a fraction of blastic crises in chronic myelogenous leukemias; and many lymphoblastic lymphomas. TdT might also be slightly increased in the mononucleate blood cells obtained from the most immature forms of acute myelogenous leukemias. Relapses with changes in cell phenotypes were occasionally observed in previously TdT-positive leukemias as a result of clonal evolution of the disease. The leukemias with blasts containing high levels of TdT were usually responsive to treatment with corticosteroids and vincristine. TdT is an oligoclonal marker characterizing several populations of undifferentiated or poorly differentiated blasts that tend to develop towards or along the lymphoid pathway. Together with specific immunological markers, this enzyme is useful to define the particular type of leukemic cells. It also serves to identify the quasi-lymphoblastic nature of the malignant clone, a helpful indication for the choice of therapy.     

血清GPAA1、SF、OPN与儿童急性淋巴细胞白血病危险度的关系及对血栓发生风险的评估效能研究

摘要 目的：分析血清糖基磷脂酰肌醇锚附着蛋白1（GPAA1）、铁蛋白（SF）、骨桥蛋白（OPN）与儿童急性淋巴细胞白血病危险度的关系及对血栓发生风险的评估效能。方法：选择我院自2017年1月至2022年12月接诊的112例急性淋巴细胞白血病患儿作为观察组，另选112例性别、年龄与观察组相匹配的健康体检儿童作为对照组。检测两组血清GPAA1、SF、OPN表达水平，分析不同危险度的急性淋巴细胞白血病患儿血清GPAA1、SF、OPN表达水平的差异性，观察急性淋巴细胞白血病患儿的血栓发生情况，通过受试者工作特征曲线（ROC）下面积（AUC）评价血清GPAA1、SF、OPN预测急性淋巴细胞白血病患儿发生血栓的效能。结果：观察组血清GPAA1、SF、OPN表达水平均高于对照组（P＜0.05）；在低危、中危和高危的急性淋巴细胞白血病患儿中，血清GPAA1、SF、OPN表达水平有差异（P＜0.05）；经Spearman相关性分析，血清GPAA1、SF、OPN表达水平与儿童急性淋巴细胞白血病危险度呈正相关（P＜0.05）；在112例急性淋巴细胞白血病患儿中，发生血栓12例，占10.71%；经多因素Logistic回归分析，血清GPAA1、SF、OPN均是急性淋巴细胞白血病患儿发生血栓的独立预测因素（P＜0.05）；经ROC曲线分析，血清GPAA1、SF联合OPN预测急性淋巴细胞白血病患儿发生血栓的AUC为0.901。结论：血清GPAA1、SF、OPN与儿童急性淋巴细胞白血病危险度密切相关，联合预测患儿发生血栓的效能较好，对此病的诊治具有重要指导意义。     

Electron microscopy: a contribution to further classification of acute unclassifiable childhood leukemia

The ultrastructural, light microscopical and immunological features of twelve cases of acute childhood leukemia are described. Nine cases were unclassifiable by light microscopy, morphology and cytochemistry, and three were difficult to classify because of a low percentage of Sudan-Black B positive blasts. By means of electron microscopy (including peroxidase cytochemistry), two main groups were seen: 1. Acute myeloid leukemia, in which could be distinguished a) a more differentiated myeloid leukemia, b) a leukemia with megakaryoblastic involvement and c) a minimally differentiated acute myeloid leukemia with granules present and 2. lymphoblastic leukemia. One case could not be classified. The first group included two possible cases of a hybrid leukemia with CD19 or CD10 positivity as well as ultrastructural peroxidase activity. We conclude that electron microscopy aids to further classification of minimally differentiated and hybrid acute leukemias.     

Human bone marrow cells positive for terminal deoxynucleotidyl transferase (TdT), HLA-DR, and a T cell marker may represent prothymocytes

Recent evidence suggests that prothymocytes, which occur in a low frequency in murine bone marrow (BM), are already committed to thymocyte differentiation and discrete from precursor B cells as well as pluripotent hematopoietic stem cells. Furthermore, it was suggested that, in rodents, prothymocytes are positive for the nuclear enzyme terminal deoxynucleotidyl transferase (TdT) and a T cell surface antigen. The human prothymocyte has not been identified as yet. We analyzed human BM cells by double immunofluorescence staining for TdT and the T cell surface markers Tp41 (recognized by the monoclonal antibodies WT1 and 3A1), T11, T1, and T6. In the BM samples tested, neither T1+/TdT+ nor T6+/TdT+ cells were detected, but Tp41+/TdT+ and T11+/TdT+ cells were present in low frequencies. In childhood BM, the frequency was about two to five in 10,000, whereas in adult BM and regenerating BM, these cells were not always detectable, but if detected, their frequency was five- to 10-fold lower. In a triple staining, using fluorescein, rhodamine, and colloidal gold particles as labels, it appeared that all Tp41+/TdT+ cells were also positive for HLA-DR. These Tp41+/HLA-DR+/TdT+ cells were also detectable in low frequencies in the thymus, and occasionally Tp41+/TdT+ and T11+/TdT+ cells were detected in the peripheral blood (PB), suggesting a migration from the BM to the thymus via the PB. The malignant counterpart of the Tp41+/HLA-DR+/TdT+ cell was detected in a patient with acute lymphoblastic leukemia with the Tp41+/T11+/HLA-DR+/TdT+/T1-/T6- phenotype and germ-line immunoglobulin heavy chain genes. We postulate that the Tp41+/T11+/HLA-DR+/TdT+/T1-/T6- cell represents a human prothymocyte.     

Discrepancy between flow cytometric analyses of CALLA using two monoclonal antibodies

Both the J5 and BA-3 monoclonal antibodies are considered to be specific for epitopes on the common acute lymphoblastic leukemia antigen (CALLA). Flow-cytometric analyses of three cell lines and one normal bone marrow sample using these antibodies as CALLA markers demonstrated that J5-labeled cells were always brighter than those labeled with BA-3, and that the ratio of their fluorescence intensities varied widely in the different systems. Furthermore, one of the lines, RPMI 8226, while positive for J5, appeared to be negative when labeled with BA-3, except for a slight displacement of the fluorescence distribution relative to the control. A possible explanation for the observed results is that the BA-3 binding epitope or epitopes on CALLA may vary in their number and/or accessibility to the antibody. These observations suggest that the use of a single monoclonal antibody to detect a cell surface antigen may be misleading, particularly when a negative result is obtained.