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The flow cytometric detection of minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemias (precursor-B-ALL) mainly relies on the identification of minor leukemic cell populations that can be discriminated from their normal counterparts on the basis of phenotypic aberrancies observed at diagnosis. This technique is not very complex, but discordancies are frequently observed between laboratories, due to the lack of standardized methodological procedures and technical conditions. To develop standardized flow cytometric techniques for MRD detection, a European BIOMED-1 Concerted Action was initiated with the participation of laboratories from six different countries. The goal of this concerted action was to define aberrant phenotypic profiles in a series of 264 consecutive de novo precursor-B-ALL cases, systematically studied with one to five triple-labelings (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) using common flow cytometric protocols in all participating laboratories. The use of four or five triple-stainings allowed the identification of aberrant phenotypes in virtually all cases tested (127 out of 130, 98%). These phenotypic aberrancies could be identified in at least two and often three triple-labelings per case. When the analysis was based on two or three triple-stainings, lower incidences of aberrancies were identified (75% and 81% of cases, respectively) that could be detected in one and sometimes two triple-stainings per case. The most informative triple staining was the TdT/CD10/CD19 combination, which enabled the identification of aberrancies in 78% of cases. The frequencies of phenotypic aberrations detected with the other four triple-stainings were 64% for CD10/CD20/CD19, 56% for CD34/CD38/CD19, 46% for CD34/CD22/CD19, and 22% for CD19/CD34/CD45. In addition, cross-lineage antigen expression was detected in 45% of cases, mainly coexpression of the myeloid antigens CD13 and/or CD33 (40%). Parallel flow cytometric studies in different laboratories finally resulted in highly concordant results (>90%) for all five antibody combinations, indicating the high reproducibility of our approach. In conclusion, the technique presented here with triple-labelings forms an excellent basis for standardized flow cytometric MRD studies in multicenter international treatment protocols for precursor-B-ALL patients. [ABSTRACT FROM AUTHOR]

The flow cytometric detection of minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemias (precursor-B-ALL) mainly relies on the identification of minor leukemic cell populations that can be discriminated from their normal counterparts on the basis of phenotypic aberrancies observed at diagnosis. This technique is not very complex, but discordancies are frequently observed between laboratories, due to the lack of standardized methodological procedures and technical conditions. To develop standardized flow cytometric techniques for MRD detection, a European BIOMED-1 Concerted Action was initiated with the participation of laboratories from six different countries. The goal of this concerted action was to define aberrant phenotypic profiles in a series of 264 consecutive de novo precursor-B-ALL cases, systematically studied with one to five triple-labelings (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) using common flow cytometric protocols in all participating laboratories. The use of four or five triple-stainings allowed the identification of aberrant phenotypes in virtually all cases tested (127 out of 130, 98%). These phenotypic aberrancies could be identified in at least two and often three triple-labelings per case. When the analysis was based on two or three triple-stainings, lower incidences of aberrancies were identified (75% and 81% of cases, respectively) that could be detected in one and sometimes two triple-stainings per case. The most informative triple staining was the TdT/CD10/CD19 combination, which enabled the identification of aberrancies in 78% of cases. The frequencies of phenotypic aberrations detected with the other four triple-stainings were 64% for CD10/CD20/CD19, 56% for CD34/CD38/CD19, 46% for CD34/CD22/CD19, and 22% for CD19/CD34/CD45. In addition, cross-lineage antigen expression was detected in 45% of cases, mainly coexpression of the myeloid antigens CD13 and/or CD33 (40%).... [ABSTRACT FROM AUTHOR]

The European BIOMED-1 Concerted Action was initiated in 1994 to improve and standardize the flow cytometric detection of minimal residual disease (MRD) in acute leukemia (AL). Three different protocols were defined to identify the normal subsets of B, T and myeloid cells in bone marrow (BM), and were applied to the different types of AL in order to study aberrant immunophenotypes. Using sensitive acquisition methods ('live gate') T cell subsets in normal BM could be identified with five triple-stains: CD7/CD5/CD3, CD7/CD4/CD8, CD7/CD2/CD3, CD7/CD38/CD34 and TdT/CD7/surface or cytoplasmic (cy)CD3 (antibodies conjugated with FITC/PE/PECy5 or PerCP, respectively). The identification of T cell subsets in BM allowed definition of 'empty spaces' (ie areas of flow cytometric plots where normally no cells are found). All studied T-ALL cases (n = 65) were located in 'empty spaces' and could be discriminated from normal T cells. The most informative triple staining was TdT/CD7/cyCD3, which was aberrant in 91% of T-ALL cases. In most cases, two or more aberrant patterns were found. Apparently the immunophenotypes of T-ALL differ significantly from normal BM T cells. This is mostly caused by their thymocytic origin, but also the neoplastic transformation might have affected antigen expression patterns. Application of the five proposed marker combinations in T-ALL contributes to standardized detection of MRD, since cells persistent or reappearing in the 'empty spaces' can be easily identified in follow-up BM samples during and after treatment. [ABSTRACT FROM AUTHOR]

Immunofluorescence stainings for the CD10 antigen and terminal deoxynucleotidyl transferase (TdT) can be used for the detection of leukemic blasts in CD10+ precursor-B-acute lymphoblastic leukemia (precursor-B-ALL) patients, but can also provide insight into the regeneration of normal precursor-B-cells in bone marrow (BM). Over a period of 15 years, we studied the regeneration of CD10+, TdT+, and CD10+/TdT+ cells in BM of children with (CD10+) precursor-B-ALL during and after treatment according to three different treatment protocols of the Dutch Childhood Leukemia Study Group (DCLSG) which differed both in medication and time schedule. This study included a total of 634 BM samples from 46 patients who remained in continuous complete remission (CCR) after treatment according to DCLSG protocols VI (1984-1988; n = 8), VII (1988-1991; n = 10) and VIII (1991-1997; n = 28). After the cytomorphologically defined state of complete remission with CD10+ and CD10+/TdT+ frequencies generally below 1% of total BM cells, a 10-fold increase in precursor-B-cells was observed in protocol VII and protocol VIII, but not in protocol VI. At first sight this precursor-B-cell regeneration during treatment resembled the massive regeneration of the precursor-B-cell compartment after maintenance treatment, and appeared to be related to the post-induction or post-central nervous system (CNS) therapy stops in protocols VII and VIII. However, careful evaluation of the distribution between the 'more mature' (CD10+/TdT-) and the 'immature' (CD10+/TdT+) precursor-B-cells revealed major differences between the post-induction/post-re-induction precursor-B-cell regeneration (low 'mature/immature' ratio: generally <1.0), the post-CNS treatment regeneration (moderate 'mature/immature' ratio: 1.2-2.8), and the post-maintenance regeneration (high 'mature/ immature' ratio: 5.7-7.6). We conclude that a therapy stop of approximately 2 weeks is already sufficient to induce significant precursor-B-cell regeneration even from aplastic BM after induction treatment. Moreover, differences in precursor-B-cell regeneration patterns are related to the intensity of the preceding treatment block, with lower 'mature/immature' ratios after the highly intensive treatment blocks. This information is essential for a correct interpretation of flow cytometric immunophenotyping results of BM samples during follow-up of leukemia patients. Particularly in precursor-B-ALL patients, regeneration of normal precursor-B-cells should not be mistaken for a relapse. [ABSTRACT FROM AUTHOR]

During the last two decades, major progress has been made in the technology of flow cytometry and in the availability of a large series of monoclonal antibodies against surface membrane and intracellular antigens. Flow cytometric immunophenotyping has become a diagnostic tool for the analysis of normal and malignant leukocytes and it has proven to be a reliable approach for the investigation of minimal residual disease (MRD) in leukemia patients during and after treatment. In order to standardize the flow cytometric detection of MRD in acute leukemia, a BIOMED-1 Concerted Action was initiated with the participation of six laboratories in five different European countries. This European co-operative study included the immunophenotypic characterization and enumeration of different precursor and mature B cell subpopulations in normal bone marrow (BM). The phenotypic profiles in normal B cell differentiation may form a frame of reference for the identification of aberrant phenotypes of precursor-B cell acute lymphoblastic leukemias (precursor-B-ALL) and may therefore be helpful in MRD detection. Thirty-eight normal BM samples were analyzed with five different pre-selected monoclonal antibody combinations: CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19, CD19/CD34/CD45 and TdT/CD10/CD19. Two CD19- immature subpopulations which coexpressed B cell-associated antigens were identified: CD34+/CD22+/CD19- and TdT+/CD10+/CD19-, which represented 0.11 +/- 0.09% and 0.04 +/- 0.05% of the total BM nucleated cells, respectively. These immunophenotypes may correspond to the earliest stages of B cell differentiation. In addition to these minor subpopulations, three major CD19+ B cell subpopulations were identified, representing three consecutive maturation stages; CD19dim/CD34+/TdT+/CD10bright/CD22dim/CD45dim /CD38bright/CD20- (subpopulation 1), CD19+/CD34-/TdT-/CD10+/CD22dim/CD45+/CD38bright/ CD20dim (subpopulation 2) and CD19+/CD34-/TdT-/CD10-/CD22bright/CD45bright/ CD38dim/CD20bright (subpopulation 3). The relative sizes of subpopulations 1 and 2 were found to be age related: at the age of 15 years, the phenotypic precursor-B cell profile in BM changed from the childhood 'immature' profile (large subpopulations 1 and 2/small subpopulation 3) to the adult 'mature' profile (small subpopulation 1 and 2/large subpopulation 3). When the immunophenotypically defined precursor-B cell subpopulations from normal BM samples are projected in fluorescence dot-plots, templates for the normal B cell differentiation pathways can be defined and so-called 'empty spaces' where no cell populations are located become evident. This allows discrimination between normal and malignant precursor-B cells and can therefore be used for MRD detection. [ABSTRACT FROM AUTHOR]

Analyzes commercially available fixation and permeabilization solutions for the flow cytometric detection of intracellular antigens. Methods to detect flow cytometry of intracellular antigens; Flow cytometric analysis of terminal deoxynucleotidyl transferase; Importance of flow cytometric detection of intracellular antigens for immunophenotyping of leukemia and lymphomas.

IgG4-related disease (IgG4-RD) is an increasingly recognised entity characterised by tumefied lesions that can affect multiple organs. Awareness of IgG4-RD is important, as it has been shown to mimic other diseases and may result in irreversible organ damage if not treated. If suspected, immunostaining for IgG4-positive plasma cells is essential for diagnosis and revision of old biopsies may be necessary.

Multiparameter flowcytometry offers an insight into differentiation pathways, maturation stages and abnormal features of cell (sub)populations thus helping to establish and classify hematological malignancies. The Dutch Foundation for Immunophenotyping of Hematological Malignancies (SIHON) has formulated a guideline for a rapid screening followed by confirmation and classification in a standardized way. For this aim seven carefully composed monoclonal antibody combinations are elucidated for screening the test sample in a first phase. In this phase a relative frequency distribution of the cells will be established and a decision will be made about abnormal cells present, as well as their mature or immature state and the cell lineage they belong to. In a second phase, panels with cell lineage dependent monoclonal antibody combinations may be used to confirm and classify the abnormal cell population indicated in phase 1, as well as to establish the presence or absence of an abberant immunophenotype.

Background: The abundance of monoclonal antibodies (mAb) and the routine use of quadruple stainings in flow cytometry allow stepwise analysis of bone marrow (BM) samples that are suspected for abnormal hematopoiesis. A screening phase that precedes lineage-specific classification phases should be sufficient to assess whether the BM has a normal or abnormal composition, as well as to identify the abnormal differentiation lineage., Methods: For a quick and easy flow cytometric screening of BM samples, we selected six quadruple immunostainings that cover multiple differentiation stages of the B-cell, monocytic, granulocytic, and erythroid lineages: TdT/CD20/CD19/CD10 and CD45/CD34/CD19/CD22 for B cells, CD34/CD117/CD45/CD13.33 for precursor granulocytic and precursor monocytic cells (myelo/monoblasts), CD14/CD33/CD45/CD34 for monocytic cells, CD16/CD13/CD45/CD11b for granulocytic cells, and CD71/CD235a/CD45/CD117 for erythroid cells., Results: The six quadruple immunostainings reveal specific staining patterns in normal BM, which allow the recognition of various subpopulations of the respective lineages. These staining patterns can be used as a frame of reference for recognition of normal and abnormal BM development. Examples of normal (age-related) variations in these otherwise stable staining patterns are presented together with several abnormal differentiation patterns., Conclusions: Although alternative immunostainings can be used (e.g., including NK- and T-cell markers), we feel that the selected six stainings represent a comprehensive and easy screening phase for quick identification of shifts in the composition of the studied differentiation lineages, reflecting age-related changes or disease-induced BM abnormalities., (Copyright 2004 Wiley-Liss, Inc.)

Objective: To evaluate long-term immune reconstitution of children treated with highly active antiretroviral therapy (HAART)., Methods: The long-term immunological response to HAART was studied in 71 HIV-1-infected children (aged 1 month to 18 years) in two prospective, open, uncontrolled national multicentre studies. Blood samples were taken before and after HAART was initiated, with a follow-up of 96 weeks, and peripheral CD4 and CD8 T cells plus naive and memory subsets were identified in whole blood samples. Relative cell counts were calculated in relation to the median of the age-specific reference., Results: The absolute CD4 cell count and percentage and the CD4 cell count as a percentage of normal increased significantly (P < 0.001) to medians of 939 x 106 cells/l (range, 10-3520), 32% (range, 1-50) and 84% (range, 1-161), respectively, after 48 weeks. This increase was predominantly owing to naive CD4 T cells. There was a correlation between the increase of absolute naive CD4 T cell counts and age. However, when CD4 T cell restoration was studied as percentage of normal values, the inverse correlation between the increase of naive CD4 T cell count and age was not observed. In addition, no difference in immunological reconstitution was observed at any time point between virological responders and non-responders., Conclusions: Normalization of the CD4 cell counts in children treated with HAART is independent of age, indicating that children of all age groups can meet their CD4 T cell production demands. In general, it appears that children restore their CD4 T cell counts better and more rapidly than adults, even in a late stage of HIV-1 infection.

Clonality assessment through Southern blot (SB) analysis of TCRB genes or polymerase chain reaction (PCR) analysis of TCRG genes is important for diagnosing suspect mature T-cell proliferations. Clonality assessment through reverse transcription (RT)-PCR analysis of Vbeta-Cbeta transcripts and flow cytometry with a Vbeta antibody panel covering more than 65% of Vbeta domains was validated using 28 SB-defined clonal T-cell receptor (TCR)alphabeta(+) T-ALL samples and T-cell lines. Next, the diagnostic applicability of the V(beta) RT-PCR and flow cytometric clonality assays was studied in 47 mature T-cell proliferations. Clonal Vbeta-Cbeta RT-PCR products were detected in all 47 samples, whereas single Vbeta domain usage was found in 31 (66%) of 47 patients. The suspect leukemic cell populations in the other 16 patients showed a complete lack of Vbeta monoclonal antibody reactivity that was confirmed by molecular data showing the usage of Vbeta gene segments not covered by the applied Vbeta monoclonal antibodies. Nevertheless, this could be considered indirect evidence for the "clonal" character of these cells. Remarkably, RT-PCR revealed an oligoclonal pattern in addition to dominant Vbeta-Cbeta products and single Vbeta domain expression in many T-LGL proliferations, providing further evidence for the hypothesis raised earlier that T-LGL derive from polyclonal and oligoclonal proliferations of antigen-activated cytotoxic T cells. It is concluded that molecular Vbeta analysis serves to assess clonality in suspect T-cell proliferations. However, the faster and cheaper Vbeta antibody studies can be used as a powerful screening method for the detection of single Vbeta domain expression, followed by molecular studies in patients with more than 20% single Vbeta domain expression or large suspect T-cell populations (more than 50%-60%) without Vbeta reactivity.

Background: Analysis of the T-cell receptor (TCR)-Vbeta repertoire has been used for studying selective T-cell responses in autoimmune disease, alloreactivity in transplantation, and protective immunity against microbial and tumor antigens. For the interpretation of these studies, we need information about the Vbeta repertoire usage in healthy individuals., Methods: We analyzed blood T-lymphocyte (sub)populations of 36 healthy controls (age range: from neonates to 86 years) with a carefully selected most complete panel of 22 Vbeta monoclonal antibodies, which together recognized 70-75% of all blood TCRalphabeta(+) T lymphocytes. Subsequently, we developed a six-tube test kit with selected Vbeta antibody combinations for easy and rapid detection of single ("clonal") Vbeta domain usage in large T-cell expansions., Results: The mean values of the Vbeta repertoire usage were stable during aging in blood TCRalphabeta(+) T lymphocytes as well as in the CD4(+) and CD8(+) T-cell subsets, although the standard deviations increased in the elderly. The increased standard deviations were caused by the occurrence of oligoclonal T-cell expansions in the elderly, mainly consisting of CD8(+) T lymphocytes. The 15 detected T-cell expansions did not reach 40% of total TCRalphabeta(+) T lymphocytes and represented less than 0.4 x 10(9) cells per liter in our study. Vbeta usage of the CD4(+) and CD8(+) subsets was comparable for most tested Vbeta domains, but significant differences (P < 0.01) between the two subsets were found for Vbeta2, Vbeta5.1, Vbeta6.7, Vbeta9.1, and Vbeta22 (higher in CD4(+)), as well as for Vbeta1, Vbeta7.1, Vbeta14, and Vbeta23 (higher in CD8(+)). Finally, single Vbeta domain expression in large T-cell expansions can indeed be detected by the six-tube test kit., Conclusions: The results of our study can now be used as reference values in studies on distortions of the Vbeta repertoire in disease states. The six-tube test kit can be used for detection of single Vbeta domain expression in large T-cell expansions (>2.0 x 10(9)/l), which are clinically suspicious of T-cell leukemia., (Copyright 2000 Wiley-Liss, Inc.)

DCs are known for their superior antigen-processing and antigen-presenting capacities. They are capable of processing intact protein: either endocytosed exogenous proteins or newly synthesized endogenous viral and bacterial proteins. They are potent inducers of primary T-cell immune responses such as in allogeneic MLRs. It is also known that DCs can provide a strong stimulus for autologous T-cell proliferation. So far no information exists on the capacity of DCs to induce primary mH antigen-specific T-cell responses. Therefore, we investigated whether human DCs, isolated from peripheral blood, were able to generate specific T-cell responses between MLR-negative HLA genotypically identical individuals in vitro. To this end, unfractionated cells, monocytes, and B cells were assayed in parallel with DCs to compare their capacity to activate unprimed T cells in a primary MLR. DCs indeed induced significant proliferation between HLA genotypically identical siblings, whereas the other APCs were unable to evoke any T-cell response at all. As expected, besides these allogeneic T-cell responses, autologous T-cell responses were initiated by the DCs as well. Nonetheless, despite further detailed analyses of the responding T cells, neither proliferative nor cytotoxic mH antigen-specific reactivities could yet be detected using the stimulation protocols described herein.

After BMT, donor T cells are activated which can display GvHD as well as GvL activities. In order to study this GvL-specific T-cell response in vitro, proliferative T-cell clones from post-BMT PBMCs were generated by stimulation with a patient's leukemic cells. One CD4+ T-cell clone (designated M-33) displayed strong proliferative activity against the patient's leukemic cells but not against the patient's EBV-LCLs. The induction of proliferation, however, appeared not to be leukemia specific. Detailed analysis of the reactivity patterns revealed that T-cell clone M-33 recognizes an as yet unknown nonpolymorphic determinant in the context of self HLA-DRw52, presented by all but one type of APC. T-cell clone M-33 proliferated upon stimulation by PB-MCs, freshly isolated B cells, monocytes, dendritic cells, leukemic B cells, and nonleukemic B-cell blasts; solely in vitro EBV-transformed B cells and in vivo EBV-infected B cells failed to induce proliferation of T-cell clone M-33. Neither surface expression of MHC or accessory molecules on the EBV cells nor suppression caused by the EBV-infected cells could explain their failure to stimulate T-cell clone M-33. We therefore hypothesize that the absence of the stimulatory capacity once the B cells are virally infected could be the result of competition for MHC class II binding of the Epstein-Barr viral peptides, thus affecting the postulated DRw52-restricted peptide for recognition by T-cell clone M-33.