Four-color flow cytometric analysis of myeloma plasma cells

We monitored the behavior of residual myeloma plasma cells in patients with multiple myeloma after high-dose therapy and autologous or allogeneic transplantation using 3 methods of a flow cytometric technique using 4-color staining, immunofixation, and polymerase chain reaction approaches. We analyzed 17 cases by a relatively simple flow cytometric technique using CD38/CD45/CD19/CD56. Detectable myeloma plasma cells were found in 5 patients at diagnosis and 9 patients after treatment. Of 14 cases, 9 (64%) had CD19-CD56+ myeloma plasma cells, and 5 (36%) of 14 had CD19-CD56- myeloma plasma cells. When 37 bone marrow samples that had less than 5% myeloma plasma cells were assessed, myeloma plasma cells were detected in all 20 immunofixation-positive cases and 3 of 17 immunofixation-negative cases (P = .002). All 4 polymerase chain reaction-negative samples characterized as immunofixation-negative contained no detectable myeloma plasma cells. Flow cytometry can provide effective information to detect low levels of myeloma plasma cells.     

Optimized flow cytometric analysis of endothelial progenitor cells in peripheral blood

Although flow cytometry is a rapid and convenient way to measure the number of circulating endothelial progenitor cells (EPC), there is no standard technique for preparation and measurement. The aim of this study was to present an optimized preparation method for EPC measurement which should serve as a standard to facilitate the comparison of the results in stem cell investigations by different research groups. We have looked for the preparation method which delivered the best immunostaining with the directly conjugated antibodies against VEGF R2, CD133, CD34, and CD45. In order to test the sensitivity of the method, we determined the number of EPC in the peripheral blood of volunteers by flow cytometry and by cell culture assay. Furthermore, we have evaluated the influence of different durations of conservation on the EPC cell count. The pre-treatment of blood samples with 0.2% formaldehyde for 30 minutes delivers the best immunostaining, and blood samples can be stored overnight at 4 degrees C without loss of counting rate for EPC. We found an excellent correlation (r = 0.98) between the flow cytometric measurement and the cell count of the cell culture method. The presented protocol for the flow cytometric measurement of EPC in the peripheral blood can be used as a diagnostic or prognostic tool; we propose this protocol as the standard for EPC quantification.     

Flow cytometric "rare event analysis": a standardized approach to the analysis of donor cell chimerism

Lack of standardization of methods and detection limits contributes to the controversial results regarding microchimerism after organ transplantation and has prompted the development of a standardized, reproducible, "easy-to-use" flow cytometric method for this type of "rare event analysis".EDTA-blood of healthy, HLA-typed donors was stained simultaneously with FITC- and biotin-labeled HLA-class I antibodies (One Lambda) as well as Cy5-PE-labeled CD45 (Medac, Hamburg) according to a standard protocol and analysed on a Coulter EPICS XL Flow cytometer (FCM).An absolute range of positivity (mean MESF+/- 1 STD) was determined for 22 HLA-specific antibodies. The range of positivity ranged between 5000 and 20,000 MESF (anti-A23, 24(9) FITC) and 40,000-140,000 (anti-Bw6 FITC). The frequency of nonspecific positive signals using nonstained cells, isotype-controls and irrelevant HLA antibodies was between 0.01% and about 0.5%, in some samples up to 1.4%, with an MESF between 8000 and 150,000, thus interfering clearly with the defined positive range of most antibodies tested. Using an "HLA antibody cocktail", combining FITC- and PE-labeled antibodies for different HLA specificities and thereby creating an internal control, the identification of donor cells was improved but was only rarely applicable.Due to the lack of highly reactive antibodies, FCM analysis was not suitable for the reliable identification of very low numbers of donor hematopoetic cells despite the theoretical advantages of flow cytometric detection of rare events. The single parameter approach was hampered by a significant frequency of nonspecific positive signals, which were easily mistaken as specific (true) positive signals, whereas the multiparameter approach could only be used in selected cases.     

Identification of unsuspected PNH-type cells in flow cytometric immunophenotypic analysis of peripheral blood and bone marrow

In this report, the flow cytometric expression patterns for CD14 on monocytic cells and CD16 on granulocytic cells in peripheral blood or bone marrow specimens are illustratedfor 15 patients proven to have a paroxysmal nocturnal hemoglobinuria (PNH) phenotype by flow cytometric analysis for CD55 and CD59. The varied clinical manifestations of PNH and its rarity may make it difficult to recognize clinically. As a result, blood or bone marrow samples may be submitted for flow cytometric analysis to exclude bone marrow neoplasia or dysplasia in patients with cytopenias rather than to exclude PNH. This was true in 5 of 15 study cases. Unlike CD55 and CD59, CD14 and/or CD16 are assessed routinely in the flow cytometric analysis of blood and bone marrow samples. Recognition of abnormal patterns of CD14 and CD16 expression might permit the identification of clinically unsuspected PNH by routine flow cytometric analysis.     

Simultaneous identification of eight leucocyte subsets of human peripheral blood using three-colour immunofluorescence flow cytometric analysis

The full analytical potential of flow cytometry has been exploited in order to identify a maximum of human peripheral blood leucocyte subsets in a single tube. For this purpose a mixture composed of six different monoclonal antibodies directly coupled to FITC or phycoerythrin or indirectly linked to the DuoChrome reagent via the biotin/streptavidin system was used. This combination of monoclonal antibodies and fluorochromes offered the possibility of simultaneously determining eight leucocyte subsets of human peripheral blood using a single laser beam. These subsets were T4 cells, bright and dim T8 cells, T null (CD3+ CD4- CD8-) cells, NK cells and B cells, polymorphonuclear cells and monocytes. This method is very easy to perform, can be applied to samples from other lymphoid tissues such as tonsils and is particularly useful when starting with a low number of cells.     

A flow cytometric immune function assay for human peripheral blood dendritic cells

CD11c+ and CD11c- (CD123+) dendritic cells (DCs) have been described in blood. Both cell types express high levels of HLA-DR and lack the lineage markers CD3, CD14, CD19, CD20, CD16, and CD56. These immunophenotypic properties were used along with analysis of activation-related surface antigens and intracellular staining of cytokines to characterize functional responses of these DC subsets to stimuli in whole human blood (WB). Samples from healthy donors were activated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate plus ionomycin (PMA+I). The only distinct response in CD11c- DCs was the expression of CD25 upon PMA+I activation. CD11c+ cells responded to LPS stimulation by producing high levels of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), and lower levels of IL-6, IL-1Ra, and IL-8 and an increased expression of accessory molecules (CD25, CD40, CD80, CD86, HLA-DR, and HLA-DQ). PMA+I activation of CD11c+ cells resulted in high levels of IL-1beta and lower levels of IL-8, IL-1Ra, and TNF-alpha and up-regulation of CD80, CD86, HLA-DR, and HLA-DQ. Our data support prior observations of functional differences between peripheral blood DC subsets and demonstrate the power of multiparameter flow cytometry to characterize the pleiotropic responses of these cells to various stimuli.     

Immunophenotypic analysis of peripheral T-cell neoplasms. A multiparameter flow cytometric approach

We retrospectively reviewed multiparameter flow cytometric analyses in 50 peripheral T-cell neoplasms (PTCNs). Results were interpreted within the context of a large cohort of nonneoplastic T-cell populations. All PTCN diagnoses were confirmed with morphologic and/or molecular analysis. Aberrant populations were defined as discrete immunophenotypic clusters exhibiting loss of or increased or diminished expression of T-cell antigens relative to internal immunophenotypically normal T-cell populations. An antigenic pattern was considered abnormal if it exceeded ranges for T-cell subsets in specific anatomic sites or was not normally encountered. Forty-six of 50 and 41 of 50 demonstrated 1 or more and 2 or more aberrations, respectively. The most common abnormally expressed antigen was CD3, followed by CD7, CD5, and CD2. Except for CD7, abnormally dim or bright antigen expression was more common than deletion. Only 3 cases were abnormal solely based on expansion of an otherwise immunophenotypically normal population; the remainder had patterns of antigen expression not seen in nonneoplastic populations. These data indicate that most PTCNs are aberrant by multiparameter flow analysis. However, results must be interpreted within the context of thorough knowledge of the immunophenotypic spectrum of nonneoplastic T cells.     

Cytogenetic and flow cytometric analysis of a clear cell chondrosarcoma

Cytogenetic analysis of a rare tumor, a clear cell chondrosarcoma of the spine, showed unusual karyotypic findings. The tumor had a predominant clone with a near-haploid chromosome complement of 30 chromosomes with loss of one homologue of each chromosome pair except chromosomes 5, 7, 12, and 19-22. A second clone with 58-60 chromosomes appeared to have originated by a doubling of the near-haploid clone. No structural changes were present. Comparison with other solid tumors and leukemias with near-haploid chromosome complements showed an interesting difference in the chromosomes which were preferentially disomic or monosomic in the two groups. Quantitative DNA analysis also showed aneuploid clones of cells corresponding to the near-haploid and hyperdiploid chromosome counts obtained cytogenetically.     

Flow cytometric analysis of porcine peripheral blood leukocytes infected with pseudorabies virus

The susceptibility of fractionated porcine peripheral blood leukocytes (PBL) to pseudorabies virus (PRV) was studied by flow cytometry and defined by viral antigen expression. Viral antigens on the surface of infected cells and cell viability were evaluated by forward angle light scatter (FALS), 90-degree light scatter (90LS), green fluorescence (FITC-anti-PRV), and red fluorescence (propidium iodide). Approximately 10% of infected mononuclear cells from healthy pigs expressed cell-surface PRV antigen. Cell-surface fluorescence and cell type were confirmed by sorting live positive cells for microscopy. In sorted positive samples, the lymphocyte versus monocyte ratio was approximately 50%:50%, defined by morphology. Positive lymphocytes represent 5.75% of total mononuclear cells. When cells were stimulated with phytohemagglutinin (PHA) and lipopolysaccharide (LPS) before infection, mitogen-stimulated T-lymphoblasts showed increased susceptibility to PRV (40.7% positive) and died of infection. Monocytes, particularly adherent monocytes, were highly susceptible (40% to 71.4% positive). Granulocytes appeared to be refractory. The relative susceptibility of various PBL populations was compared by normalizing lymphocyte susceptibility to 1 as follows: resting total lymphocytes (1); B-lymphocytes (0.67); T-lymphoblasts (7.08); total monocytes (4.27); adherent cells (4.03 to 10.88); adherent monocytes (6.95 to 12.42); granulocytes (0.24). These findings suggest a possible mechanism by which PRV could have an immunosuppressive effect as well as a pathway for dissemination of PRV.     

Assessing donor chimerism using flow cytometry in paroxysmal nocturnal haemoglobinuria after stem cell transplantation--a case report

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemopoietic stem cell disorder arising from somatic mutation of the X-linked PIG-A gene which leads to deficiency of the glycosylphosphatidylinositol (GP1) membrane anchor proteins such as CD 59 (MIRL: membrane inhibitor of reactive lysis) and CD 55 (DAF: decay accelerating factor). Allogeneic peripheral blood stem cell transplant (PBSCT) is a curative mode of treatment in symptomatic PNH patients. Assessment of donor chimerism for PBSCT can be performed by various methods including short tandem repeat loci (STR) and variable number of tandem repeats (VNTR). Flow cytometry, which is much cheaper and faster, also can be used to assess engraftment in patients with PNH. Engrafted patients will show the presence of CD 55 and CD 59 on their red cells and white cells. We describe here the usefulness of flow cytometry in the assessment of donor chimerism following allogeneic PBSCT, in a case of PNH.     

X射线工作者外周血细胞DNA含量检测和染色体畸变分析

本文用流式细胞术对５６例Ｘ射线工作者的外周血细胞做了ＤＮＡ含量分析，并与染色体畸变结果进行了对比研究。结果表明，在５０例Ｘ射线工作者中有５例出现了ＤＮＡ倍体异常。流式细胞术分析的ＤＮＡ超二倍体者，在其染色体数目分析中，＞２ｎ细胞百分比与其呈一致性的变化。Ｘ射线工作者高二倍体细胞百分比（＞２Ｃ％）和Ｓ期细胞比例（ＳＰＦ）均明显高于正常对照组（Ｐ＜０．０５）。Ｘ射线组染色体断裂率与＞２Ｃ％呈明显的正相关（ｒ＝０．８０）。     

Subtyping lymphocytes in peripheral blood by direct immunoalkaline phosphatase labeling and light scatter/absorption flow cytometric analysis.

Evaluation of blood cells to determine immunologic status is becoming an important clinical application of flow cytometric analysis. For a wider use of immunophenotyping technology in clinical laboratories, the authors developed a rapid method to detect monoclonal antibody-labeled cells using forward light scatter/absorption clinical flow cytometers such as the Technicon H*1 and Technicon H*2 differential complete blood count analyzers. Calf-intestinal alkaline phosphatase was conjugated to mouse monoclonal antibodies (anti-CD2, CD3, CD4, CD8, CD19) for direct immunoenzymatic labeling. The combination of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue-tetrazolium salt in diethanolamine buffer at pH 9.6 was selected as buffer/substrate to yield stable, insoluble, and very intense purplish-blue precipitates on the surface of the cells labeled with monoclonal antibody-alkaline phosphatase conjugates. Endogenous alkaline phosphatase in granulocytes was inhibited with levamisole. Early mild fixation of the white cells permitted incubation at 38 +/- 1 degrees C, which accelerated each step of the reaction without disrupting the cells throughout the procedure. The method is competitive with the direct immunofluorescence whole-blood method used on fluorescence flow cytometers in speed, sensitivity, and accuracy, as demonstrated with alkaline phosphatase-conjugated anti-CD2, CD3, CD4, CD8, CD19 monoclonal antibodies.     

流式细胞术在周围神经肿瘤诊断上的应用

Flow cytometric DNA analysis was performed on formalin fixed paraffin-embedded samples taken from 43 cases of peripheral nerve tumors. Fourteen samples were taken from benign tumors, 11 from actively proliferative tumors and 12 from malignant tumors. Ploidy pattern and proliferative index of tumors were estimated. All samples of benign tumors showed a diploid model of DNA content whereas DNA aneuploidy was encountered in 75% (12/16) of the malignant tumors and 25% (4/16) of the actively proliferative tumors. The proliferative index (PI) of benign tumors (medium: 24.8%) was lower than that of proliferative tumors (medium: 42.7%). The proliferative index of malignant tumors was the highest in the experimental group (medium: 51.2%). These results were conformable to the malignant degree of histologic grading and prognosis.     

Diagnosis of infective and inflammatory disorders by flow cytometric analysis of blood neutrophils

The utility of neutrophil parameters provided by two flow cytometric hematologic analyzers (the H-1 and H6000, Technicon Instruments Corporation, Tarrytown, NY) was investigated for the diagnosis of infective and/or inflammatory disorders. The test population of 156 hospital patients was selected on the basis of a blood culture request. Positivity or negativity for infective and/or inflammatory disease was inferred from chart review. The parameters evaluated included the absolute neutrophil count, the lobularity index, and the left shift flag from the H-1, the percentage of high peroxidase cells from the H6000, the routine laboratory band count, and a reference band count. Significant intercorrelations were observed between these parameters. The diagnostic performance of the routine laboratory band count was significantly inferior to that of all other parameters. At equivalent points on their receiver operating characteristic curves, the diagnostic efficiencies of the remaining tests ranged from 61.5% for the lobularity index to 67% for the left shift flag and the percentage of high peroxidase cells. These differences were not significant statistically.     

Rapid flow cytometric measurement of cytokine-induced phosphorylation pathways [CIPP] in human peripheral blood leukocytes

Current strategies designed to assess cells in the peripheral blood are limited to evaluation of phenotype or delayed measurement [>6 h] of function, usually quantifying cytokine production, cytolytic activity, or response to antigens. We reasoned that measurable abnormalities in signaling pathways could reflect pathological environs that cells experience in the setting of inflammatory states/cancer and could be represented in the peripheral blood. Two major pathways regulating the immune response are the JAK/STAT and MAPK/ERK pathways. These pathways are initiated by ligand-receptor binding and are rapidly propagated by subsequent protein phosphorylation cascades. We evaluated the brief application of cytokines in vitro to interrogate the early phosphorylation events of these signaling pathways in normal peripheral blood mononuclear cells (PBMC). Individual cytokine doses and time intervals of treatment were assessed to identify conditions useful in a clinical laboratory and as an initial goal to induce maximal phosphorylation. Surprisingly, all of the STAT proteins assessed and ERK1/2 are maximally phosphorylated within 15 min in human PBMC simply following addition of cytokines without preactivation of the cells. At 2 h, cells typically return to their basal phosphorylation states. For most of the cytokines tested, increased phosphorylation directly correlated with increased concentrations of the individual cytokines. These strategies will enable robust development of simple blood analyses to identify normal levels as well as impairments in STAT and MAPK/ERK signaling pathways associated with various human disease states including acute and chronic inflammatory conditions throughout clinical immunology.     

Automated flow cytometric analysis of blood cells in cerebrospinal fluid: analytic performance

We compared the performance of an automated method for obtaining RBC and WBC counts and WBC differential counts in cerebrospinal fluid (CSF) samples with the reference manual method. Results from 325 samples from 10 worldwide clinical sites were used to demonstrate the accuracy, precision, and linearity of the method. Accuracy statistics for absolute cell counts showed a high correlation between methods, with correlation coefficients for all reportable absolute counts greater than 0.9. Linearity results demonstrated that the method provides accurate results throughout the reportable ranges, including clinical decision points for WBCs of 0 to 10/microL. Interassay precision and intra-assay precision for the ADVIA 120 (Bayer HealthCare, Tarrytown, NY) method were acceptable at all levels. The ADVIA 120 CSF Assay enumerates and differentiates cells via flow cytometry in a minimally diluted sample, improving the analysis of typically hypocellular CSF samples. Study results demonstrate that the automated ADVIA 120 CSF Assay is an acceptable alternative to the labor-intensive manual method.     

Anaplastic large cell lymphoma: a flow cytometric analysis of 29 cases

We report our experience with flow cytometric (FC) analysis of 29 cases of anaplastic large cell lymphoma (ALCL). Morphologic analysis of processed cytocentrifuged preparations demonstrated neoplastic cells in 28 cases. In 25 of these, an aberrant lymphoid population was detected by FC analysis. The majority showed high orthogonal light scatter, similar to monocytes or granulocytes. Of the antigens CD2, CD3, CD4, CD5, and CD7, 5 cases expressed 1, 8 expressed 2, 6 expressed 3, 3 expressed 4, and 3 expressed all 5. CD4 was expressed most commonly (20/25 [80%]), followed by CD2 (18/25 [72%]), CD3 (10/25 [40%]), and CD5 and CD7 (8/25 [32%] each). CD45 was expressed in 23 of 25 cases and CD13 in 7 of 9. Of 21 cases, 13 were anaplastic lymphoma kinase (ALK)+, all of which were CD4+, vs 5 of 8 ALK - cases (P = .042). Most ALCLs can be detected and characterized by multiparameter FC analysis. However, light scatter gating on typical lymphoid regions may yield false-negative results in a substantial number of cases.     

Immuno-gold labeling for flow cytometric analysis

Colloidal gold particles coated with goat anti-mouse immunoglobulin antibodies were used to analyse surface antigens on various cell types by flow cytometry. The gold-labeled cells showed an increasing signal amplification in the 90 degree light scatter with increasing wavelength of the incident laser light, reaching a more than 10-fold amplification at 632.8 nm. This wavelength was provided by a 0.5 mW helium-neon laser. The magnitude of the signal amplification due to the gold label as well as the specificity of the label was sufficient for quantitative discrimination between positive and negative cells. Cell viability was not affected by the gold label. Mouse spleen cells were labeled with various combinations of FITC- and gold-conjugated antibodies. It was found that the gold and fluorescent labels did not interfere with each other. Colloidal gold may thus be used as an additional label for multiparameter cell analysis and sorting. Biparametric cell analysis/sorting of surface antigen-labeled cells (label versus low-angle scatter) becomes possible even with a low energy helium-neon laser.