The human fetal thymus generates invariant effector γδ T cells.

In the mouse thymus, invariant γδ T cells are generated at well-defined times during development and acquire effector functions before exiting the thymus. However, whether such thymic programming and age-dependent generation of invariant γδ T cells occur in humans is not known. Here we found that, unlike postnatal γδ thymocytes, human fetal γδ thymocytes were functionally programmed (e.g., IFNγ, granzymes) and expressed low levels of terminal deoxynucleotidyl transferase (TdT). This low level of TdT resulted in a low number of N nucleotide insertions in the complementarity-determining region-3 (CDR3) of their TCR repertoire, allowing the usage of short homology repeats within the germline-encoded VDJ segments to generate invariant/public cytomegalovirus-reactive CDR3 sequences (TRGV8-TRJP1-CATWDTTGWFKIF, TRDV2-TRDD3-CACDTGGY, and TRDV1-TRDD3-CALGELGD). Furthermore, both the generation of invariant TCRs and the intrathymic acquisition of effector functions were due to an intrinsic property of fetal hematopoietic stem and precursor cells (HSPCs) caused by high expression of the RNA-binding protein Lin28b. In conclusion, our data indicate that the human fetal thymus generates, in an HSPC/Lin28b-dependent manner, invariant γδ T cells with programmed effector functions.

TdT expression in germ cell tumours: a possible immunohistochemical cross-reaction and diagnostic pitfall.

AIMS: Very recent papers proposed a possible role for the expression of terminal deoxynucleotidyl transferase (TdT) in the tumourigenesis of gonadal and extragonadal germ cell-derived tumours (GCTs). Our multicentric study evaluated the magnitude of the immunoreactivity for TdT in GCTs, encompassing seminoma, dysgerminoma, mature teratoma and mixed GCTs. METHODS AND RESULTS: The histological series was stained with both monoclonal and polyclonal antibodies, yielding a positivity of 80% of cases with well-defined nuclear reactivity. A significant difference in staining intensity between monoclonal and polyclonal antibodies was observed (p=0.005). However, exploiting western blot and more innovative proteomic approaches, no clear-cut evidence of the TdT protein was observed in the neoplastic tissues of the series. CONCLUSIONS: Alternatively to the pathogenetic link between TdT expression and GCTs tumourigenesis, we hypothesised the occurrence of a spurious immunohistochemical nuclear cross-reaction, a well-known phenomenon with important implications and a possible source of diagnostic pitfalls in routine practice for pathologists.

Regulation of cellular proliferation in acute lymphoblastic leukemia by Casein Kinase II (CK2) and Ikaros.

The IKZF1 gene encodes the Ikaros protein, a zinc finger transcriptional factor that acts as a master regulator of hematopoiesis and a tumor suppressor in leukemia. Impaired activity of Ikaros is associated with the development of high-risk acute lymphoblastic leukemia (ALL) with a poor prognosis. The molecular mechanisms that regulate Ikaros' function as a tumor suppressor and regulator of cellular proliferation are not well understood. We demonstrated that Ikaros is a substrate for Casein Kinase II (CK2), an oncogenic kinase that is overexpressed in ALL. Phosphorylation of Ikaros by CK2 impairs Ikaros' DNA-binding ability, as well as Ikaros' ability to regulate gene expression and function as a tumor suppressor in leukemia. Targeting CK2 with specific inhibitors restores Ikaros' function as a transcriptional regulator and tumor suppressor resulting in a therapeutic, anti-leukemia effect in a preclinical model of ALL. Here, we review the genes and pathways that are regulated by Ikaros and the molecular mechanisms through which Ikaros and CK2 regulate cellular proliferation in leukemia.

Minimal residual disease analysis by eight-color flow cytometry in relapsed childhood acute lymphoblastic leukemia.

Multiparametric flow cytometry is an alternative approach to the polymerase chain reaction method for evaluating minimal residual disease in treatment protocols for primary acute lymphoblastic leukemia. Given considerable differences between primary and relapsed acute lymphoblastic leukemia treatment regimens, flow cytometric assessment of minimal residual disease in relapsed leukemia requires an independent comprehensive investigation. In the present study we addressed evaluation of minimal residual disease by flow cytometry in the clinical trial for childhood relapsed acute lymphoblastic leukemia using eight-color flow cytometry. The major challenge of the study was to reliably identify low amounts of residual leukemic cells against the complex background of regeneration, characteristic of follow-up samples during relapse treatment. In a prospective study of 263 follow-up bone marrow samples from 122 patients with B-cell precursor acute lymphoblastic leukemia, we tested various B-cell markers, adapted the antibody panel to the treatment protocol, and evaluated its performance by a blinded parallel comparison with the polymerase chain reaction data. The resulting eight-color single-tube panel showed a consistently high overall concordance (P<0.001) and, under optimal conditions, sensitivity similar to that of the reference polymerase chain reaction method. Overall, evaluation of minimal residual disease by flow cytometry can be successfully integrated into the clinical management of relapsed childhood acute lymphoblastic leukemia either as complementary to the polymerase chain reaction or as an independent risk stratification tool. ALL-REZ BFM 2002 clinical trial information: NCT00114348.

The immunophenotype of adult T acute lymphoblastic leukemia in Morocco.

BACKGROUND: There is paucity of detailed studies of adult T cell acute lymphoblastic leukemia (T-ALL) in developing countries reflecting the condition of these patients including clinical and biological features. OBJECTIVE: This study was carried out to analyze the immunophenotypic characteristics of 40 Moroccan patients with T-ALL and its association with biological and clinical features. PATIENTS AND METHODS: Between 2006 and 2009, 130 adult patients diagnosed with acute lymphoblastic leukemia (ALL) were immunophenotyped by 3-color flow cytometry using a panel of monoclonal antibodies. Cases presenting features of a T-lineage phenotype were subjected to detailed analysis including immunophenotypic, clinical and biological parameters. RESULTS: Proportion of T-ALL among ALL Moroccan patients was 31.0%. Median age of patients was 28 years. Twenty-nine patients were females and 11 were males. 45.0% of patients (18/40) had features of immature T-ALL stages (pro-T and pre-T ALL), 30.0% (12/40) of CD1a+ cortical T-ALL stage and 25.0% (10/40) had a characteristic phenotype of medullary T-ALL. The frequencies of progenitor cell markers CD10, CD34 and TdT expression were 14.0; 57.5% and 50.0% respectively. The aberrant expression of B lineage associated antigen CD79a were positive in 20.5% of the cases and the aberrant expression of myeloid antigens CD13 and/or CD33 was found in 22 (55.0%) cases. No significant association was encountered between TdT, CD34 or myeloid antigens positivity and high risk features at presentation as age, sex, and white blood cells. However, myeloid antigens (CD13 and/or CD33) was significantly associated with T-cell maturation stages (p = 0.009). CONCLUSION: To the best of our knowledge, this is the first report from North Africa of immunophenotypic study on adult T-ALL. Our findings indicate that the proportion of T-ALL among ALL in Morocco is similar to that reported in others Mediterranean countries like France and Italy and that myeloid-associated antigens expression is frequently associated with immature immunophenotype.

Flow cytometry CD45-negative B-NHL: a case report of a diffuse large B-cell lymphoma without extranodal involvement.

BACKGROUND: The loss of CD45, the leukocyte-common antigen, has been described in rare cases of large B-cell lymphoma (LBCL) subtypes with extranodal involvement by immunohistochemical methods. Here we report a case of a patient with LBCL, with no extranodal lesions, which is CD45 negative by flow cytometry (FC) immunophenotyping. METHODS: Immunophenotyping and DNA content analysis was performed by multiparametric FC on lymph node and bone marrow aspirate obtained from a 65 year old male patient. RESULTS: Malignant B-lymphocytes were CD5-, CD10+/++, CD11c-, CD19+, CD20+/++, CD23-, CD34-, CD38-/+, CD45-, CD79b++/+++, BCL2 overexpressed, FMC7++, IgM++/+++, TdT- with Lambda light chain restriction. This pathological cellular population showed near-diploid DNA content, with a high proliferate rate. CONCLUSIONS: To our knowledge, we describe the first case of a CD19+ B-cell non-Hodgkin lymphoma without expression of CD45 detected by FC, and the first case without extranodal involvement presentation. This case is reported not only because it is a rare one but also to raise awareness of FC users of its correct diagnosis.

Absence of N addition facilitates B cell development, but impairs immune responses.

The programmed, stepwise acquisition of immunocompetence that marks the development of the fetal immune response proceeds during a period when both T cell receptor and immunoglobulin (Ig) repertoires exhibit reduced junctional diversity due to physiologic terminal deoxynucleotidyl transferase (TdT) insufficiency. To test the effect of N addition on humoral responses, we transplanted bone marrow from TdT-deficient (TdT(-/-)) and wild-type (TdT(+/+)) BALB/c mice into recombination activation gene 1-deficient BALB/c hosts. Mice transplanted with TdT(-/-) cells exhibited diminished humoral responses to the T-independent antigens α-1-dextran and (2,4,6-trinitrophenyl) hapten conjugated to AminoEthylCarboxymethyl-FICOLL, to the T-dependent antigens NP(19)CGG and hen egg lysozyme, and to Enterobacter cloacae, a commensal bacteria that can become an opportunistic pathogen in immature and immunocompromised hosts. An exception to this pattern of reduction was the T-independent anti-phosphorylcholine response to Streptococcus pneumoniae, which is normally dominated by the N-deficient T15 idiotype. Most of the humoral immune responses in the recipients of TdT(-/-) bone marrow were impaired, yet population of the blood with B and T cells occurred more rapidly. To further test the effect of N-deficiency on B cell and T cell population growth, transplanted TdT-sufficient and -deficient BALB/c IgM(a) and congenic TdT-sufficient CB17 IgM(b) bone marrow were placed in competition. TdT(-/-) cells demonstrated an advantage in populating the bone marrow, the spleen, and the peritoneal cavity. TdT deficiency, which characterizes fetal lymphocytes, thus appears to facilitate filling both central and peripheral lymphoid compartments, but at the cost of altered responses to a broad set of antigens.

Relevance of immunophenotypes to prognostic subgroups of age, WBC, platelet count, and cytogenetics in de novo acute myeloid leukemia.

Immunophenotyping is one of the independent prognostic factors in acute myeloid leukemia (AML). Relevance of immunophenotypes to prognostic subgroups of age, white blood cells (WBC), platelet count, and cytogenetics in de novo AML was comprehensively investigated in this study for the first time. Human leukocyte antigen (HLA)-DR and CD14 expression associated with the elderly, highest WBC count, and unfavorable-risk cytogenetics; CD4, CD7, and CD11b expression correlated with highest WBC count and unfavorable-risk cytogenetics; CD64 expression was associated with higher WBC count while that of CD13 was associated with lower platelet count; CD22, CD34, CD123, and terminal deoxynucleotidyl transferase (TdT) expression correlated with unfavorable-risk cytogenetics; CD5 expression was associated with normal platelet count while that of CD19 was associated with children and favorable-risk cytogenetics; CD117 expression was associated with low WBC and lower platelet counts; myeloperoxidase (MPO) expression correlated with lower platelet count; and MPO and glycophorin A (Gly-A) expression was associated with lower WBC count and favorable-risk cytogenetics. The results of the relevance analysis revealed the distribution characteristics of antigen expression in different AML prognostic subgroups. The majority of antigens associated with good or poor prognostic subgroups were in accordance with the previous reports of correlation of expression of these antigens with prognosis. Antigens associated with good (or poor) prognostic subgroups were defined as good (or poor)-risk antigens.

Resolution of unique Sca-1highc-Kit- lymphoid-biased progenitors in adult bone marrow.

We have identified a distinctive lymphoid-restricted progenitor population in adult mouse bone marrow based on a unique c-Kit(-)Sca-1(high)Flt3(+) AA4(+) surface phenotype. These cells are highly lymphoid biased and rapidly generate B and T cells after adoptive transfer. However, whereas previously described lymphoid progenitors such as common lymphoid progenitors express TdT and relatively high levels of RAG2, and are enriched for cells with an active V(D)J recombinase, Flt3(+) AA4(+) cells within the c-Kit(-)Sca-1(high) bone marrow fraction are TdT(-), are RAG2(low), and do not display evidence for ongoing or past recombinase activity. Furthermore, unlike common lymphoid progenitors that readily generate B cells upon stimulation with IL-7, c-Kit(-)Sca-1(high)Flt3(+) precursors do not express abundant levels of the IL-7R, and require costimulation with Flt3 ligand and IL-7 to generate B cells in vitro. Moreover, these findings suggest that hematopoietic stem cells in adults generate an array of lymphoid-biased progenitor populations characterized by distinct gene expression and cytokine response profiles.

[Diagnostic value of anti-terminal deoxynucleotidyl transferase antibody (TdT) in hematologic pathology]. / Intérêt diagnostique de l'anticorps anti-terminal désoxynucleotidyl transférase (TdT) en pathologie hématologique.

AIMS: The diagnosis of primitive hematologic malignancies depends on a panel of monoclonal antibodies which is growing over time. The distinction between immature (lymphoblastic lymphoma/acute lymphoblastic leukaemia) and mature lymphoma is sometimes difficult. In this study, we evaluated anti-TdT antibody in the diagnosis and classification of these proliferations. MATERIALS AND METHODS: 13 lesions were examined by immunohistochemistry: 4 B and T lymphoblastic lymphomas, 2 Burkitt's lymphomas, 5 B and T acute lymphoblastic leukemias and 2 acute monoblastic leukemias. RESULTS: TdT expression is specific of immature lymphoid proliferations (T or B lymphoblasts). TdT is not expressed by mature B or T cell lymphomas such as Burkitt's lymphomas. Significant numbers of cases of acute myeloblastic leukemias are TdT positive but could be easily distinguished from lymphoblastic proliferations. CONCLUSION: Anti-TdT antibody represents a useful marker for differentiating lymphoma/acute lymphoblastic leukemia from other lymphomas. This marker, available in routine diagnosis should be systematically included in the panel of antibodies used for immunophenotyping hematologic malignancies.

Rare detection of phenotypically immature lymphocytes in Hashimoto thyroiditis and rheumatoid arthritis.

It has been suggested that recombination activating gene (RAG)-dependent revision of the immunoglobulin genes in germinal centres may contribute to local production of autoantibodies in Hashimoto thyroiditis (HT) and rheumatoid arthritis (RA). To test this hypothesis we examined HT and RA tissues for expression of RAG and terminal deoxynucleotidyl transferase (TdT) in situ. Paraffin-embedded tissues from 19 HT patients and from 20 RA patients were subjected to immunohistochemistry using TdT-specific antibodies. Expression of the RAGs was studied by in situ hybridisation. Tonsil sections were used as a control. Expression of TdT and RAGs was detected in extrafollicular lymphocytes in control tonsil sections. By contrast, only rare TdT-expressing cells were identified in 11 of 19 HT and in 2 of 20 RA samples. Germinal centre B-cells were consistently TdT- and RAG-negative. These results suggest that local RAG-dependent receptor revision in germinal centres is unlikely to contribute to production of autoantibodies in HT and RA. The presence of TdT-positive extrafollicular cells may represent an influx of immature cells in the context of chronic immune stimulation.

Flow cytometric analysis of acute leukemias. Diagnostic utility and critical analysis of data.

CONTEXT: Acute leukemia displays characteristic patterns of surface antigen expression (CD antigens), which facilitate their identification and proper classification and hence play an important role in instituting proper treatment plans. In addition to enzyme cytochemical analysis, multiparameter flow cytometric analysis has become commonplace in most laboratories for that purpose. The essential role and caveats of flow cytometry in that regard, however, have received little scrutiny. OBJECTIVE: To evaluate the expression of commonly used immunomarkers and patterns in various acute leukemias to help define the best use and role of multiparameter flow cytometry in the diagnosis and proper classification of acute leukemias. DESIGN: We have retrospectively analyzed the immunophenotypic data from 508 de novo adult and pediatric acute leukemia patients, as studied using multiparameter flow cytometry in addition to routine morphologic and enzyme cytochemical analysis. Cytogenetic and/or molecular data were correlated in all 41 cases of acute promyelocytic leukemia (APL) and in 203 other cases of acute leukemia where those data were available. We have also determined the positive and negative predictive values of a combined CD34 and HLA-DR expression pattern for the differentiation of APL from other myeloid leukemias. RESULTS: In acute myeloid leukemia (AML) other than APL, expression of CD34 was seen in 62% and expression of HLA-DR in 86% of all cases. Twenty-six (10%) of 259 cases of non-APL AML were negative for both CD34 and HLA-DR as opposed to 33 (80%) of 41 cases of APL. None of the cases of APL were positive for both CD34 and HLA-DR in contrast to 149 (58%) of 259 cases of non-APL AML. Fifty-three cases were found to be examples of minimally differentiated AML (AML M0) based on the lack of expression of cytoplasmic CD3 and cytoplasmic CD79a and expression of one or more myelomonocytic-associated antigens and/or myeloperoxidase. Expression of CD20 was seen in 40 (24%) of 168 cases of precursor B-cell acute lymphoblastic leukemia (pB-ALL) and 52 (29%) lacked CD34 expression. Five of 180 cases of pB-ALL and 2 cases of precursor T-cell ALL (pT-ALL) were negative for terminal deoxynucleotidyl transferase (TdT). Aside from cytoplasmic CD3, CD5 and CD7 were the most sensitive antigens present in all 21 cases of pT-ALL. CD33 was more sensitive but less specific than CD13 for myeloid lineage. CONCLUSION: Aside from identification of blasts, flow cytometry was found to be especially useful in the correct identification of AML M0, differentiation of APL from AML M1/M2, and correct identification of TdT-negative ALL and unusual variants, such as transitional B-cell ALL and undifferentiated and biphenotypic acute leukemias.

MIC2, TdT, bcl-2, and CD34 expression in paraffin-embedded high-grade lymphoma/acute lymphoblastic leukemia distinguishes between distinct clinicopathologic entities.

We propose that 12E7 (CD99) expression, along with TdT, bcl-2, and CD34 reactivity in lymphoblastic lymphoma (LyL)/acute lymphoblastic leukemia (ALL), distinguishes this group of neoplasms from small noncleaved cell lymphomas (SNCLs) in both pediatric and adult patients, thereby narrowing the differential diagnosis of high-grade non-Hodgkin's lymphomas and acute lymphoblastic leukemias in paraffin sections. 12E7 (CD99) is one of a group of available antibodies that recognizes the product of the mic-2 gene, which was originally identified in ALL. Despite this, most clinicopathological research has focused on the reactivity of 12E7 in a subset of the small round cell tumors of childhood. Although TdT is widely used in the subtyping of blastic leukemias, its use in the distinction of high-grade lymphomas in paraffin sections has been limited. We collected 24 cases of LyL/ALL (13 B-cell and 11 T-cell) and 15 cases of SNCL from 1984 through 1993. We confirmed the diagnoses using morphology and analysis of immunologic data. We performed immunohistochemistry with the 12E7 antibody, TdT, bcl-2, and CD34 on formalin-fixed, paraffin-embedded material. The patients' ages ranged from 4 to 81 years; nine of the study patients were children. Sixteen of the 24 LyL/ALLs stained with 12E7. In contrast, none of the 15 cases of SNCL reacted with this antibody (chi-square P < .0001). A larger percentage of T-cell LyL/ALLs reacted with 12E7 than did B-cell LyL/ALLs (82% v 54%). Sixteen of 20 LyL/ALLs reacted with the anti-TdT antibody, as compared with none of 11 SNCLs (chi-square P < .0001). Six LyL/ALLs were CD34 positive (of 23), and none of the SNCLs were CD34 positive (0 of 12) (chi-square P = .0519). Bcl-2-positive cases were found among both LyL/ ALLs and SNCLs, although they were more prevalent among LyL/ ALLs (92% v 25%; chi-square P < .0001). When one considers the differential diagnosis of a high-grade lymphoma/acute lymphoblastic leukemia, positive reactions with 12E7, TdT, bcl-2, and CD34 support the diagnosis of LyL/ALL over SNCL. Moreover, we present data that suggests that evaluating for TdT in formalin-fixed paraffin-embedded tissue is a more sensitive test than using either 12E7, bcl-2 or CD34 alone.

Clinical, morphologic, cytogenetic and prognostic implications of CD34 expression in childhood and adult de novo AML.

Expression of CD34 by leukemic blasts was analyzed in 230 pediatric and 251 adult patients with de novo AML enrolled in two large multicenter trials (AML-BFM-87 and AMLCG respectively). The association between CD34 positivity and morphological classification according to FAB criteria, cytogenetic aberrations, immunophenotypic features and clinical characteristics was investigated. CD34 was expressed (> or = 20%) by leukemic cells from 45% of childhood and 43% of adult AML patients. CD34+ AML was often associated with M1/M2 morphology as well as the coexpression of CD7 and TdT. Translocation t(8;21), inv(16) and chromosome 5 and 7 aberrations were more frequently observed in CD34+ AML. There was a low frequency of CD34 expression in infant AML but no age dependency was evident in adult patients. CD34 expression exerted no influence on the rate of complete remissions (CR) after intensive multidrug induction therapy. In adults, 56% of the CD34-positive and 64% of CD34-negative cases achieved CR (P = 0.29), and the childhood trial even revealed a slight advantage for CD34+ AML with a CR rate of 80% vs. 71% for CD34-negative cases (P = 0.068). Long-term follow-up disclosed no significant differences in remission duration or event-free survival between the CD34-positive and CD34-negative groups. In conclusion, CD34+ AML patients comprise a heterogeneous group with good as well as poor risk factors. Though characterized by some distinct features, CD34 lacks prognostic significance in de novo AML patients submitted to intensive polychemotherapy.

Detection of terminal transferase in acute myeloid leukemia by flow cytometry.

The purpose of this study was twofold: (1) to develop an optimized, reliable method for the flow cytometric analysis of the intranuclear DNA polymerase, terminal deoxynucleotidyl transferase (TdT) in acute myeloid leukemia, and (2) to establish the usefulness of a novel, fluorescein-isothiocyanate conjugated monoclonal anti-TdT antibody (HT-6) in double-fluorescence staining for surface antigens in the characterization of leukemic cells. Inclusion of an aldehyde blocking buffer in the staining protocol reduced background fluorescence sufficiently to allow for the detection of the low-level fluorescent TdT+ myeloblasts. When admixed to normal peripheral blood mononuclear cells, 0.4-0.5% of HLA-DR+ or myeloid surface antigen+, TdT+ double-stained myeloblasts could be reliably detected above background levels. Flow cytometric TdT measurements using the HT-6 antibody in 55 patients with TdT+ acute lymphocytic or myelocytic leukemia or blast crisis of chronic myelogenous leukemia were equal or superior to the results obtained with a mixture of monoclonal anti-TdT antibodies (anti-HTDT-Mix) and comparable to those obtained by the conventional slide method employing polyclonal rabbit anti-human TdT antiserum. This flow cytometric TdT determination in combination with surface antigen staining using a novel anti-TdT monoclonal antibody (HT-6) allows for the recognition of minimal leukemic blast cells during clinical remission in acute myeloid leukemia.

Production of interleukin-1 alpha and interleukin-2 by separate, phenotypically different leukaemia and human T cell lymphotropic virus-1-transformed T cell clones.

The relationship between interleukin-1 (IL-1) and interleukin-2 (IL-2) production and immunophenotype marker profiles was studied in a panel of 29 leukaemia and human T cell lymphotropic virus-1 (HTLV-1)-transformed T cell lines. Culture supernatants from six of the 29 T cell lines tested increased IL-2 production by the MOLT-16 cell line in a manner similar to that of rIL-1 alpha or rIL-1 beta. The enhancing activity in the cell culture supernatants was inhibited by antibody against IL-1 alpha. Anti-IL-1 beta antibody had no inhibitory effect. All the cell lines producing IL-1 alpha had characteristics of activated mature T cells. They were terminal deoxyribonucleotidyl transferase (TdT)-, CD4+, CD8-, HLA-DR+ and all were strongly positive for IL-2R alpha (Tac antigen) expression. However, none of the IL-1 alpha producing cell lines secreted detectable IL-2. A significant quantity of IL-2 was found, after stimulation with phytohaemagglutinin, in supernatants from nine of the 29 cell lines tested. The majority of IL-2 producing cell lines originated from less mature, non-activated T cells, as they were characterized by the expression of TdT, lack of HLA-DR antigens and > 50% had no detectable IL-2R alpha. The results thus show that separate, phenotypically different leukaemia and HTLV-1-transformed T cell clones produce IL-1 alpha and IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric analysis of terminal deoxynucleotidyl transferase. A simplified method.

The authors evaluated a new cell membrane permeabilization method for the flow cytometric detection of terminal deoxynucleotidyl transferase (TdT). In this method, gradient-separated leukocytes or unseparated blood or bone marrow cells were incubated in a commercially available diethylene glycol-based red blood cell lysing solution, which not only lyses red blood cells, but also permeabilizes leukocyte cell membranes; the light scattering properties of the cells are retained. The validity of the current method was demonstrated by the good concordance of the findings with previously published data as follows: (1) practically identical results were obtained when an established method for cell permeabilization was used in parallel on the same samples; (2) the proportion of TdT-positive cells in normal peripheral blood was negligible; (3) the proportion of TdT-positive cells in normal bone marrow averaged 1%, and a significant portion of TdT-positive cells in normal bone marrow expressed CD10 and CD34; and (4) TdT-positive cell populations were seen with the expected frequencies in various types of leukemia. This method for TdT flow cytometry provides significant advantages over previously used methods and is especially suitable for TdT detection in routine laboratories.