Performance of a novel eight-color flow cytometry panel for measurable residual disease assessment of chronic lymphocytic leukemia.

BACKGROUND: Measurable residual disease (MRD) is an important prognostic indicator of chronic lymphocytic leukemia (CLL). Different flow cytometric panels have been developed for the MRD assessment of CLL in Western countries; however, the application of these panels in China remains largely unexplored. METHODS: Owing to the requirements for high accuracy, reproducibility, and comparability of MRD assessment in China, we investigated the performance of a flow cytometric approach (CD45-ROR1 panel) to assess MRD in patients with CLL. The European Research Initiative on CLL (ERIC) eight-color panel was used as the "gold standard." RESULTS: The sensitivity, specificity, and concordance rate of the CD45-ROR1 panel in the MRD assessment of CLL were 100% (87/87), 88.5% (23/26), and 97.3% (110/113), respectively. Two of the three inconsistent samples were further verified using next-generation sequencing. In addition, the MRD results obtained from the CD45-ROR1 panel were positively associated with the ERIC eight-color panel results for MRD assessment (R = 0.98, p < 0.0001). MRD detection at low levels (≤1.0%) demonstrated a smaller difference between the two methods (bias, -0.11; 95% CI, -0.90 to 0.68) than that at high levels (>1%). In the reproducibility assessment, the bias was smaller at three data points (within 24, 48, and 72 h) in the CD45-ROR1 panel than in the ERIC eight-color panel. Moreover, MRD levels detected using the CD45-ROR1 panel for the same samples from different laboratories showed a strong statistical correlation (R = 0.99, p < 0.0001) with trivial interlaboratory variation (bias, 0.135; 95% CI, -0.439 to 0.709). In addition, the positivity rate of MRD in the bone marrow samples was higher than that in the peripheral blood samples. CONCLUSIONS: Collectively, this study demonstrated that the CD45-ROR1 panel is a reliable method for MRD assessment of CLL with high sensitivity, reproducibility, and reliability.

Direct bone marrow HSC transplantation enhances local engraftment at the expense of systemic engraftment in NSG mice.

Direct bone marrow (BM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic stem cell (HSC) transplantation. Despite physical delivery into the BM cavity, many donor cells are rapidly redistributed by vascular perfusion, perhaps compromising efficacy. Anchoring donor cells to 3-dimensional (3D) multicellular spheroids, formed from mesenchymal stem/stromal cells (MSC) might improve direct BM transplantation. To test this hypothesis, relevant combinations of human umbilical cord blood-derived CD34(+) cells and BM-derived MSC were transplanted into NOD/SCID gamma (NSG) mice using either IV or intrafemoral (IF) routes. IF transplantation resulted in higher human CD45(+) and CD34(+) cell engraftment within injected femurs relative to distal femurs regardless of cell combination, but did not improve overall CD45(+) engraftment at 8 weeks. Analysis within individual mice revealed that despite engraftment reaching near saturation within the injected femur, engraftment at distal hematopoietic sites including peripheral blood, spleen and non-injected femur, could be poor. Our data suggest that the retention of human HSC within the BM following direct BM injection enhances local chimerism at the expense of systemic chimerism in this xenogeneic model.

Assessment of CD27 expression as a tool for active and latent tuberculosis diagnosis.

UNLABELLED: There are still no reliable tests to distinguish active tuberculosis (TB) from latent TB infection (LTBI). Assessment of CD27 modulation on CD4⁺ T-cells has been suggested as a tool to diagnose different TB stages. OBJECTIVES: To use several cytometric approaches to evaluate CD27 expression on Mycobacterium tuberculosis (Mtb)-specific CD4⁺ T-cells to differentiate TB stages. METHODS: 55 HIV-uninfected subjects were enrolled: 13 active TB; 12 cured TB; 30 LTBI. Whole blood was stimulated with RD1-proteins or Cytomegalovirus-lysate (CMV). Interferon (IFN)-γ response was evaluated by cytometry. The proportion of CD27(±) within the IFN-γ⁺ CD4⁺ T-cells or RATIO of the CD27-median fluorescence intensity (MFI) of CD4⁺ T-cells over the CD27 MFI of IFN-γ⁺ CD4⁺ T-cells was evaluated. RESULTS: The greatest diagnostic accuracy in discriminating active TB vs. LTBI or cured TB was reached by evaluating the CD27(+) CD45RA(-) cells within the IFN-γ⁺ CD4⁺ T-cell subset (76.92 sensitivity for both, and 90% and 91.67% specificity, respectively), although the use of the CD27 MFI RATIO allows for stricter data analysis, independent of the operator. CONCLUSIONS: the study of CD27 expression using different approaches, whether it involves evaluation of CD45RA expression or not, is a robust biomarker for discriminating TB stages.

Combined flow cytometric assessment of CD45, HLA-DR, CD34, and CD117 expression is a useful approach for reliable quantification of blast cells in myelodysplastic syndromes.

BACKGROUND: Quantification of bone marrow (BM) blasts by cytomorphology is essential for the diagnosis of myelodysplastic syndromes (MDS). Owing to its subjectivity and the potential impact of dysplastic features on accurate identification of blast cells, more objective approaches are required, multiparameter flow cytometry (MFC) being a particularly promising approach in this regard. However, no consensus exists about the optimal combination of markers and strategy to be used. METHODS: BM blast counts from 74 MDS patients were evaluated by morphology versus four different MFC phenotypic criteria: "CD34⁺", "CD34⁺ and/or CD117⁺", "CD34⁺, and/or CD117⁺ HLA-DR⁺", and "CD34⁺ and CD117⁺ HLA-DR⁺ plus CD64⁺ CD14(-/lo) " cells. For each criterium, the percentage of blasts was calculated using either all BM nucleated cells or non-erythroid CD45⁺ cells as denominator. RESULTS.: The number of "CD34⁺ and/or CD117⁺ HLA-DR⁺"cells showed the highest correlation and agreement with morphological counts, only a minor proportion of cases being misclassified by MFC vs. morphology for the >5% and >10% classification thresholds. In turn, a CD34⁺ phenotype was insufficient to correctly identify and quantify blasts. Conversely, usage of non-erythroid BM cells as denominator, or inclusion of "CD34⁺ and/or CD117⁺ HLA-DR⁺ plus CD64⁺ CD14(-lo") cells were both associated with overestimated blast counts. CONCLUSIONS: Quantification of "CD34⁺ and/or CD117⁺ HLA-DR⁺" cells (from all nucleated BM cells) by MFC is an efficient method for the enumeration of blasts in MDS. However, caution should be taken with replacing morphology by MFC blast counts; its combined use may rather provide complementary information increasing the accuracy and reproducibility of BM blast cell counts in these patients.

B- and T-cell memory elicited by a seasonal live attenuated reassortant influenza vaccine: assessment of local antibody avidity and virus-specific memory T-cells using trogocytosis-based method.

PURPOSE: The main purpose of vaccination is to generate immunological memory providing enhanced immune responses against infectious pathogens. The standard and most commonly used assay for influenza vaccine immunogenicity evaluation is a hemagglutination inhibition assay (HAI). It is clear now that HAI assay is unable to properly assess the proven protective immunity elicited by live attenuated influenza vaccines (LAIV). New methods need to be developed for more accurate LAIV immunogenicity assessment and prediction of vaccine efficacy among target populations. OBJECTIVE: Randomized placebo-controlled study of memory B- and T-cell responses to intranasal LAIV in young adults. METHODS: A total of 56 healthy young adults 18-20 years old received seasonal monovalent LAIV. Mucosal memory B-cell responses were measured by IgA avidity assessment in nasal swabs. CD4 memory T cells in peripheral blood were examined by the expression of CD45RO marker and in functional test by the ability of virus-specific T cells to maintain the trogocytosis with antigen-loaded target cells. RESULTS: Intranasal LAIV immunization enhances mucosal IgA avidity even without reliable increases in antibody titers. At the day 21 after vaccination, up to 40% of subjects demonstrated significant increases in both total and virus-specific CD4 memory T cells that were observed regardless of seroconversion rate measured by HAI assay. CONCLUSION: The data suggest that immunogenicity of LAIV vaccines should be evaluated on the mucosal and cellular immunity basis. The assays applied could be used to support influenza clinical trials through preliminary screening of volunteers and subsequent measurement of anti-influenza in immunity.

Accurate assessment of cell count and viability with a flow cytometer.

BACKGROUND: In this study we developed a method to measure cell concentration and viability in specimens received in flow cytometry and cytogenetics laboratories. METHODS: Specimens are stained with a vital fluorescent dye, SYTO13, the cell impermeant viability dye, 7-AAD, and a leukocyte marker, CD45. After the addition of an internal calibrator microsphere, FLOW-COUNT, the flow cytometer is capable of measuring the viability of nucleated cells, giving a general assessment of leukocyte populations and measuring their concentration. RESULTS: An accurate assessment of specimen quality is an important parameter when performing flow cytometric and cytogenetic leukemia/lymphoma assessment. High quality specimen is desired to avoid the pitfalls of non-specific staining and limited cellularity/viability. CONCLUSIONS: Use of a cell count and viability measurement prior to leukemia and lymphoma assessment by flow cytometry and cytogenetics helps to increase the rate of successful immunophenotypic and cytogenetic analysis.

Comparative analysis of methods for assessment of circulating endothelial progenitor cells.

The number and properties of endothelial progenitor cells (EPC) in disease states is of considerable interest due to the importance attributed to this distinct cell population. However, there has been no study comparing each of the methods employed in the same sampled individuals. Herein, we performed an analysis of several methods used for circulating EPC assessment and correlated them with humoral factors known to influence their numbers. Thirty-eight individuals (mean age of 34 +/- 9 years) were tested. Peripheral blood mononuclear cells were obtained and stained for FACS analysis with antibodies to CD34, CD45, CD133, and KDR and the remaining cells grown under endothelial cell conditions for assessment of colony-forming unit (CFU) numbers and adhesive properties. Levels of circulating vascular endothelial growth factor (VEGF), erythropoietin (EPO), and C-reactive protein (CRP) were determined and correlated with each of the EPC markers. CFU numbers did not correlate with CD34/KDR or CD34/CD133/KDR and negatively correlated with CD34/ CD133 numbers. CD34/KDR numbers correlated with CD34/CD133/KDR, but not with CD34/ CD133. Only CD34/KDR and CD34/CD133/KDR correlated with VEGF serum levels. The number of EPC adhering to fibronectin and endothelial cells correlated with CFU numbers and not with either of the EPC membrane markers. Current methods for quantitatively assessing numbers of circulating EPC are not correlated. VEGF serum levels are associated only with CD34/KDR and CD34/ CD133/KDR, whereas CFU numbers correlate with EPC functional properties. These findings may suggest that CD34/KDR is more appropriate for the definition of circulating EPC, whereas CFU numbers are more likely to reflect their ability to proliferate.

A standardized cytological and immunochemical method for the analysis of fine-needle spleen aspirates: assessment of leukocyte population changes in canine visceral leishmaniosis.

A method for the evaluation of splenic cellularity using samples collected by fine-needle aspirative biopsy was standardized in this work. The procedure includes erythrocyte lysing, preparation of cytospin films and staining by histochemical and immunocytochemical techniques. The cellular profiles of spleen preparations were compared with those observed in peripheral blood samples subjected to the same procedure. Two groups were compared, one consisting of 14 healthy uninfected and the other of 15 polysymptomatic Leishmania chagasi/infantum-infected dogs, from an endemic area for visceral leishmaniosis. Cell populations were identified by conventional hematoxilin-eosin and Wright' stainings, and by immunocytochemistry using monoclonal antibodies against canine CD45RA and CD45RB, phagocytes and a pan-leukocyte antigen. Larger neutrophil (P < 0.0001) and monocyte/macrophage (P = 0.0036) relative counts and lower lymphocyte relative counts (P < 0.0001) were found in the spleen, and not in the blood, of the animals with leishmaniosis than in those of the healthy animals. The proportions of CD45RB+ cells were higher, and of CD45RA+ cells were lower, both in the spleen and in the blood of animals with leishmaniosis than in those of healthy dogs (P < 0.05). Additionally, hematoxilin-eosin-stained cytospins of spleen aspirates from Leishmania-infected animals permitted the easy visualization of amastigote forms inside phagocytes, under light microscopy.

Flow cytometer in the infrared: inexpensive modifications to a commercial instrument.

BACKGROUND: The application of molecules that fluoresce in the infrared (IR) region to measure cell products would be enhanced by a flow cytometer capable of measuring them. To our knowledge, none exist at this time. Accordingly, we have developed such an instrument. METHODS: A Becton Dickinson LSR flow cytometer was modified to include a small 785-nm IR diode laser the size of a C cell battery with 44-mW output power. The instrument was modified further to accommodate this laser in addition to a 405-nm solid-state laser, a 488-nm air-cooled argon laser, and a 658-nm solid-state laser. Because the IR laser is dangerous to the eye, the laser beams were viewed for optical alignment using a CCD camera and video monitor. An avalanche photodiode was used in place of a photomultiplier tube because its detection sensitivity in the IR region is superior. RESULTS: To assess performance, scatter and fluorescence measurements were made using microspheres that fluoresce in the IR region, and human leukocytes were stained with CD45 biotin followed by a streptavidin conjugated with an IR dye. An avalanche photodiode was 2.3 to 2.8 times more sensitive than a photomultiplier tube for detecting IR fluorescence. Cells stained with CD45 biotin and avidin conjugated with an IR dye could easily be resolved and their fluorescence quantified; there was virtually no autofluorescence. In addition, a lipophilic membrane dye that emits in the IR region was studied. HL60 cells were stained with this dye and they exhibited bright fluorescence intensity. CONCLUSION: A commercial instrument could be modified to accommodate an IR laser for exciting dyes that fluoresce in the IR region. This new capability will extend the range of fluorescence that can be measured by flow cytometry.

Four-fold staining including CD45 gating improves the sensitivity of multiparameter flow cytometric assessment of minimal residual disease in patients with acute myeloid leukemia.

Multiparameter flow cytometry (MFC) is capable of quantifying minimal residual disease (MRD) in acute myeloid leukemia (AML). Its broad application, however, is limited by a lack of sensitivity in about 20% of patients. CD45 gating may improve sensitivity. A broad panel of four-fold combinations of monoclonal antibodies including CD45 in each was used to define leukemia-associated aberrant immunophenotypes (LAIP), to define their sensitivities in normal bone marrow samples, and to compare results to data obtained without CD45 gating using triple staining. In 45 patients, a LAIP was defined, 11 normal bone marrow samples were analyzed as controls. The median percentage of LAIP-positive AML cells with and without CD45 gating was 21.95% (range, 3.31-82.52%) and 20.52% (range, 3.22-81.94%). The median percentage of LAIP-positive normal bone marrow cells ranged from 0.01 to 0.42% (median, 0.02%) and 0.02 to 0.58% (median, 0.15%) with and without CD45 gating. The difference of LAIP-positive cells between AML and normal bone marrow samples amounted to a median of 3.08 log (range, 1.22-4.01) and 2.28 log (range, 1.12-3.34) with and without CD45 gating. CD45 gating improves the sensitivity of MFC-based MRD monitoring in AML by 1 log.

Ethylenediaminetetraacetic acid plus citrate-theophylline-adenosine-dipyridamole (EDTA-CTAD): a novel anticoagulant for the flow cytometric assessment of platelet and neutrophil activation ex vivo in whole blood.

Ethylenediaminetetraacetic acid (EDTA) is the anticoagulant recommended for full blood counts, citrate is recommended for coagulation and platelet studies, and citrate-theophylline-adenosine-dipyridamole (CTAD) inhibits platelet activation. Because the combination of EDTA and CTAD (E/C) is better than EDTA or CTAD alone for measuring platelet parameters on the ADVIA 120 Haematology System, we investigated whether it also offers advantages for the flow cytometric assessment of platelet and/or neutrophil activation and platelet-leucocyte aggregate formation ex vivo. Blood from healthy subjects was collected into E/C or citrate, kept at room temperature or at 4 degrees C, and analysed 0 to 360 min later in the ADVIA 120 and by immunofluorescent flow cytometry. Platelet count, mean platelet volume, number of platelet clumps, mean platelet component, numbers of CD62P(+) platelets and platelet-leucocyte aggregates, and expression of CD11b on neutrophils changed little over 360 min in blood with E/C kept at 4 degrees C. In contrast, one or more parameter changed when blood was kept with E/C at ambient temperature or with citrate at either temperature. The use of E/C in in vitro and in vivo studies is illustrated. Platelet and neutrophil activation status ex vivo can be reliably assessed if blood is collected into E/C, held at 4 degrees C, and analysed within 6 h.

CD4 cell monitoring for HIV/AIDS: old options; new insights.

Cost effective solutions are needed for laboratory monitoring that do not compromise on quality but address costs. Current recommended methods of CD4+ T cell enumeration are complex and costly. Monitoring typically utilises viral load assessment (PCR based) and CD4+ T cell counting to assess disease progression and response to therapy in HIV/AIDS. This paper reviews CD4 testing with the focus on different methods of CD4+ T cell enumeration including state-of-the-art flow cytometric testing, the advantages and disadvantages of these systems and quality control. Lastly, recent new work undertaken at the University of the Witwatersrand is discussed that addresses the problems of cost and precision and accuracy of CD4 T cell testing.

Analyses of quality assessment studies using CD45 for gating lymphocytes for CD3(+)4(+)%.

BACKGROUND: The purpose of this study was to assess whether laboratories which do not use CD45 for gating lymphocytes with three- (or four-) color flow cytometry (non-CD45 laboratories) for CD3(+)4(+)% and CD3(+)8(+)% do worse on quality assessment (QA) studies than laboratories which do use CD45 (CD45 laboratories). METHODS: Data came from blood specimens donated by 62 donors (50 HIV-positive) assayed over 2 years (November, 1996-October, 1998) by 35 laboratories in the NIAID DAIDS Flow Cytometry QA Program. RESULTS: Non-CD45 laboratories were significantly more likely to be classified as having unacceptable inter-laboratory results (far from the group median) than CD45 laboratories (5.6% vs 1.5%, P = 0.005 for CD3(+)4(+)%; 10.4% vs 5.0%, P = 0.007 for CD3(+)8(+)%). The intra-laboratory range of results on blinded replicates was significantly more likely to be deemed unacceptable (range >4%) in non-CD45 laboratories than in CD45 laboratories for CD3(+)8(+)% (14. 5% vs 3.5%, P = 0.002) but not for CD3(+)4(+)% (2.6% vs 1.5%, P = 0. 62). These differences in favor of CD45 gating were observed even though the non-CD45 laboratories had been doing three-color flow cytometry in the QA program significantly longer (P = 0.05) than the CD45 laboratories, and so would be expected to have fewer problems with the assay. CONCLUSIONS: Laboratories which choose to use a single CD3/CD4/CD8 tube for immunophenotyping may be sacrificing both accuracy and reproducibility.

How many gated lymphocytes are needed for accurate assessment of T-subset percentages by flow cytometry?

Current regulatory agencies specify the use of 2,500 gated lymphocytes for accurate lymphocyte immunophenotyping by flow cytometry. However, acquisition of 2,500 gated lymphocytes is often technically infeasible when testing whole blood from lymphopenic patients. Our laboratory thus compared CD3, CD4, and CD8 percentages obtained from a lymphocyte acquisition gate of 2,500 events with those obtained, respectively, from 1,000 and 500 event acquisition gates. The study group consisted of 59 specimens with CD4 values ranging from 1% to 66%; for data analysis purposes, the group was considered as a whole and was then subdivided according to CD4 percentage (> 25%, < 25%, < 5%). For all groupings analyzed, percentages of CD3+, CD4+, and CD8+ lymphocytes were not significantly different for either 1,000-event or 500-event gates when compared to the standard 2,500 gate (paired t-test). Replicate parallel analyses of some samples indicated that comparable precision is obtained by using the alternative gates. These findings indicate that the use of smaller numbers of acquired lymphocytes is a reasonable alternative in situations where 2,500 lymphocytes cannot be attained.

Flow cytometric assessment of peripheral blood contamination and proliferative activity of human bone marrow cell populations.

Bone marrow aspiration is superior to bone marrow biopsies due to less discomfort to the volunteer or patient, but it is inferior concerning the reproducibility of cytokinetic information. Therefore, a method that could select aspirates of quality and reproducibility equal to those of biopsies was sought. Low-density (mononucleated) bone marrow cells were labelled with T200 common leukocyte antigen, CD45, which differentiate cells into erythroid, myeloid, and lymphocyte + monocyte subpopulations based on their immunofluorescence intensity. A hypotonic propidium iodide solution was added, and DNA cell cycle characteristics of the total cells and the subpopulations were obtained. Twenty-two aspirations were performed on three healthy men. There was a strong negative correlation between the amount of CD45-gated lymphocytes + monocytes, indicative of peripheral blood cell contamination in the aspirate, and the percentage of total cells and subpopulations in DNA S phase. A marked reduction in the percentage of cells in S phase was observed when the lymphocyte + monocyte counts were higher than 30%; this level was used to exclude aspirates with an unacceptable degree of peripheral blood cell admixture. Twelve of the aspirates were found to be of acceptable quality due to their low lymphocyte + monocyte count. These aspirates were compared with 11 bone marrow biopsy expellates from hematologically normal patients undergoing open cardiac surgery. The 12 aspirates were found to have almost identical mean percent S-phase cells as the biopsy expellates, both for the total cell population (14% +/- 3.45% vs. 15% +/- 1.5%) and for the erythroid (24% +/- 6% vs. 24.4% +/- 3.3%) and myeloid (10% +/- 2.4% vs. 10.7% +/- 2.5%) subpopulations.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced assessment of DNA/proliferative index by depletion of tumor infiltrating leukocytes prior to monoclonal antibody gated analysis of tumor cell DNA.

Flow cytometry has become an important tool for the analysis of breast tumors, and assessment of S phase fraction and DNA ploidy are potential indicators of tumor aggression. Due to masking or dilution of infrequent tumor cell events, the presence of normal cell types, such as inflammatory cells and fibroblasts, can interfere with accurate DNA analysis of solid tumor samples. MDA-MB-175-VII human breast carcinoma cells, WI-38 human lung fibroblast cells, and peripheral blood leukocytes were mixed, in varying proportions, in order to represent human breast tumor samples. The cells were subsequently treated with CD45 conjugated magnetic microspheres to deplete tumor infiltrating leukocytes, thus enriching for tumor cells. The tumor cell mixtures were then stained with a pan-cytokeratin specific monoclonal antibody or with a monoclonal antibody that reacts with breast epithelial membrane antigen (EMA). When used in combination with monoclonal antibody gating, utilization of this bead-based technology resulted in enhanced precision of DNA analysis.

Assessment of the effects of instrumentation, monoclonal antibody, and fluorochrome on flow cytometric immunophenotyping: a report based on 2 years of the NIAID DAIDS flow cytometry quality assessment program.

This study of the effect on CD4%, CD8%, CD3+8+%, and CD3% of flow cytometer, monoclonal antibody, and fluorochrome was based on 71 whole-blood samples, each evaluated by 42 to 59 laboratories during 2 years of a flow cytometry quality assessment program. For the 24 HIV-positive specimens, FACScans produced significantly lower CD4% values than EPICS-Cs or EPICS Profiles, and for the 47 HIV-negative specimens, FITC was associated with significantly lower CD4% values than PE or RD1, but differences were never larger than 2% and regressions accounted for only 3-12% of the variability. The labs using the most common CD4 technique had significantly higher between-laboratory variability than all other labs grouped together. For both CD8 and CD3+8+, measurements on FACScans were significantly higher than measurements on EPICS, and measurements using Leu2 were significantly higher than measurements using T8, with regressions accounting for 12-31% of the variability. The machine differences in medians were 3-7% for labs using Leu2-FITC. It might be worthwhile to discourage the use of Leu2-FITC for measuring CD8% but no change in instrument, monoclonal antibody, or fluorochrome would greatly improve interlaboratory agreement on CD4%.