Showing papers in "Cytometry Part B-clinical Cytometry in 2011"

Central memory CD4+ T cells dominate the normal cerebrospinal fluid.

Abstract: Background: To use cerebrospinal fluid (CSF) immune phenotyping as a diagnostic and research tool, we have set out to establish reference values of white blood cell (WBC) subsets in CSF. Methods: We assessed the absolute numbers and percentages of WBC subsets by 6-color flow cytometry in paired CSF and blood samples of 84 individuals without neurological disease who underwent spinal anaesthesia for surgery. Leukocyte (i.e., lymphocytes, granulocytes, and monocytes), lymphocyte (i.e., T [CD4+ and CD8+], NK, NKT and B cells), T cell (i.e., naive, central memory, effector memory, and regulatory) and dendritic cell subsets (i.e., myeloid and plasmacytoid) were studied. Results: CSF showed a predominance of T cells, while granulocytes, B and NK cells were relatively rare compared to blood. The majority of T cells in CSF consisted of CD4+ T cells (∼70%), most of them (∼90%) with a central memory phenotype, while B cells were almost absent (<1%). Among the small population of dendritic cells in CSF, those of the myeloid subtype were more frequent than plasmacytoid dendritic cells (medians: 1.7% and 0.4% of leukocytes, respectively), whilst both subsets made up 0.2% of leukocytes in blood. Conclusions: This study reports reference values of absolute numbers and percentages of WBC subsets in CSF, which are essential for further investigation of the immunopathogenesis of neuro-inflammatory diseases. Furthermore, the relative abundance of CD4+ T cells, mainly with a central memory phenotype, and the presence of dendritic cells in CSF suggests an active adaptive immune response under normal conditions in the central nervous system (CNS). © 2010 International Clinical Cytometry Society

Flow cytometric characterization of cerebrospinal fluid cells

Abstract: Flow cytometry facilitates the detection of a large spectrum of cellular characteristics on a per cell basis, determination of absolute cell numbers and detection of rare events with high sensitivity and specificity. White blood cell (WBC) counts in cerebrospinal fluid (CSF) are important for the diagnosis of many neurological disorders. WBC counting and differential can be performed by microscopy, hematology analyzers, or flow cytometry. Flow cytometry of CSF is increasingly being considered as the method of choice in patients suspected of leptomeningeal localization of hematological malignancies. Additionally, in several neuroinflammatory diseases such as multiple sclerosis and paraneoplastic neurological syndromes, flow cytometry is commonly performed to obtain insight into the immunopathogenesis of these diseases. Technically, the low cellularity of CSF samples, combined with the rapidly declining WBC viability, makes CSF flow cytometry challenging. Comparison of flow cytometry with microscopic and molecular techniques shows that each technique has its own advantages and is ideally combined. We expect that increasing the number of flow cytometric parameters that can be simultaneously studied within one sample, will further refine the information on CSF cell subsets in low-cellular CSF samples and enable to define cell populations more accurately.

External quality assurance of circulating tumor cell enumeration using the CellSearch(®) system: a feasibility study.

Abstract: Background: Circulating tumor cells (CTCs) are cells that have detached from solid tumors and entered the blood. CTCs can be detected, among others, by semi-automated immunomagnetic enrichment and image cytometry using CellSearch® (Veridex, Raritan, NJ). We studied the feasibility of external quality assurance (EQA) of the entire CellSearch procedure from blood draw to interpretation of results in multiple laboratories. Methods: Blood samples from six cancer patients and controls were distributed to 14 independent laboratories to test between-laboratory, between-assay, and between-instrument variation. Additionally, between-operator variability was assessed through the interpretation of blinded images of all blood samples on a website. Results: Shipment and storage of samples had no influence on CTC values. Between-instrument (coefficient of variation (CV) < 12%) and between-assay variation was low (CV ≤ 20%), indicating high reproducibility. However, between-laboratory CV ranged from 45 to 64%. Although inter-operator agreement on image interpretation (Fleiss' κ statistics) ranged from “substantial” to “almost perfect,” image interpretation, particularly of samples containing high numbers of apoptotic cells, was the main contributor to between-laboratory variation. Conclusions: This multicenter study shows the feasibility of an EQA program for CTC detection in patient samples, and the importance of continuation of such a program for the harmonization of CTC enumeration. © 2010 International International Clinical Cytometry Society

Enrichment of circulating tumor cells from a large blood volume using leukapheresis and elutriation: Proof of concept

Abstract: Background: The aim of this study was to determine the applicability of a sequential process using leukapheresis, elutriation, and fluorescence-activated cell sorting (FACS) to enrich and isolate circulating tumor cells from a large blood volume to allow further molecular analysis. Methods: Mononuclear cells were collected from 10 L of blood by leukapheresis, to which carboxyfluorescein succinimidyl ester prelabeled CaOV-3 tumor cells were spiked at a ratio of 26 to 106 leukocytes. Elutriation separated the spiked leukapheresates primarily by cell size into distinct fractions, and leukocytes and tumor cells, characterized as carboxyfluorescein succinimidyl ester positive, EpCAM positive and CD45 negative events, were quantified by flow cytometry. Tumor cells were isolated from the last fraction using FACS or anti-EpCAM coupled immunomagnetic beads, and their recovery and purity determined by fluorescent microscopy and real-time PCR. Results: Leukapheresis collected 13.5 × 109 mononuclear cells with 87% efficiency. In total, 53 to 78% of spiked tumor cells were pre-enriched in the last elutriation fraction among 1.6 × 109 monocytes. Flow cytometry predicted a circulating tumor cell purity of ∼90% giving an enrichment of 100,000-fold following leukapheresis, elutriation, and FACS, where CaOV-3 cells were identified as EpCAM positive and CD45 negative events. FACS confirmed this purity. Alternatively, immunomagnetic bead adsorption recovered 10% of tumor cells with a median purity of 3.5%. Conclusions: This proof of concept study demonstrated that elutriation and FACS following leukapheresis are able to enrich and isolate tumor cells from a large blood volume for molecular characterization. © 2010 International Clinical Cytometry Society

Variables affecting the quantitation of CD22 in neoplastic B cells.

Abstract: Background: Quantitative flow cytometry (QFCM) is being applied in the clinical flow cytometry laboratory for diagnosis, prognosis, and assessment of patients receiving antibody-based therapy. ABC values and the effect of technical variables on CD22 quantitation in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FCL), hairy cell leukemia (HCL) and normal B cells were studied. Methods: The QuantiBrite System V R was used to determine the level of CD22 expression (mean antibody bound per cell, ABC) by malignant and normal B cells. The intra-assay variability, number of cells required for precision, effect of delayed processing as well as shipment of peripheral blood specimens (delayed processing and exposure to noncontrolled environments), and the effect of paraformaldehyde fixation on assay results were studied. Results: The QuantiBRITE method of measuring CD22 ABC is precise (median CV 1.6%, 95% confidence interval, 1.2–2.3%) but a threshold of 250 malignant cells is required for reliable CD22 ABC values. Delayed processing and overnight shipment of specimens resulted in significantly different ABC values whereas fixation for up to 12 h had no significant effect. ABC measurements determined that CD22 expression is lower than normal in ALL, CLL, FCL, and MCL but higher than normal in HCL. Conclusions: CD22 expression was atypical in the hematolymphoid malignancies studied and may have diagnostic utility. Technical variables such as cell number analyzed and delayed processing or overnight shipment of specimens impact significantly on the measurement of antigen expression by QFCM in the clinical laboratory. Published 2010 Wiley-Liss, Inc. † Key terms: CD22; quantitative flow cytometry; QuantiBRITE; ABC

Cellular features of psoriatic skin: Imaging and quantification using in vivo reflectance confocal microscopy

Abstract: Background: In vivo reflectance confocal microscopy (RCM) is a novel, exciting imaging technique. It provides images of cell-and tissue structures and dynamics in situ, in real time, without the need for ex vivo tissue samples. RCM visualizes the superficial part of human skin up to a depth of 250 μm. In psoriasis, an erythematosquamous skin disease, we evaluated well known histological features of stable psoriasis vulgaris (PP) with RCM. RCM images were correlated to morphological and cell biological findings in routine HE and immunohistochemical stained histology with CD3 and antifilaggrin antibodies. Methods: Lesional and nonlesional skin of eight patients with PP were evaluated with RCM, after which 4-mm punch biopsies were taken and cut vertically in two equal parts. One part was processed in the conventional vertical way, the other horizontally (en face) for optimal correlation to RCM images. We evaluated and quantificated nine histopathological features of psoriasis: parakeratosis, epidermal and dermal inflammatory infiltrate, diminished or absent stratum granulosum, epidermal thickening, thinning of the suprapapillary epidermal plate, increased height of the papillary dermis, increase in number of dermal papillae and increase in number and volume of papillary capillaries. Results: Quantification and evaluation of cell biological and histological features of PP with RCM correlated highly to evaluation in HE, CD3 and filaggrin-stained histology. Conclusions: RCM is a novel technique which can be used for real time, cytometric evaluation and quantification of PP features. RCM might be suited equally for cytometric evaluation of other superficial tissues. © 2010 International Clinical Cytometry Society

Flow cytometric analysis of microparticle phenotype and their role in thrombin generation.

Abstract: Background: Microparticles may be generated from a number of cell types and are known to play a role in haemostasis by a variety of mechanisms. We investigated the role of platelet, red cell, and leucocyte-derived microparticles in the measurement of thrombin generation. Methods: Four parameters of thrombin generation (the endogenous thrombin potential (ETP), lag time, time to peak, peak height) and microparticle content was determined in 35 plasma samples from normal individuals pre and post filtration to remove microparticles. Immunofluorescent flow cytometry was used to identify and enumerate platelet, leucocyte, monocyte and red cell derived microparticles in plasma samples based on the expression of CD42b, CD45, CD15, and Glycophorin A respectively. Expression of phosphatidylserine and tissue factor by microparticles was determined by Annexin V and anti CD142 binding. The pre and post filtration results were compared. Results: There was a significant decrease in ETP and Peak Height, and an increase in the time to peak post filtration (P < 0.001). A significant decrease in the number of CD42+, CD45+, CD15+, CD142+, and Annexin V+ microparticles was also observed. The change in CD42b+ microparticles correlated highly with the change in Annexin V+ microparticles (r = 0.68). Whilst the change in ETP correlated best with the change in CD15+ microparticles (r = 0.45) and the change in time to peak correlated with the change in Annexin V binding (r = 0.52) (P < 0.01). Conclusion: The presence of micropartcles in plasma significantly affects thrombin generation. © 2010 International Clinical Cytometry Society

Flow cytometry immunophenotyping for the evaluation of bone marrow dysplasia.

Abstract: The pathological hallmark of myelodysplastic syndromes (MDS) is marrow dysplasia, which represents the basis of the WHO classification of these disorders. This classification provides clinicians with a useful tool for defining the different subtypes of MDS and determining individual prognosis. The WHO proposal has raised some concern regarding minimal diagnostic criteria particularly in patients with normal karyotype without robust morphological markers of dysplasia (such as ring sideroblasts or excess of blasts). Therefore, there is clearly a need to refine the accuracy to detect marrow dysplasia. Flow cytometry (FCM) immunophenotyping has been proposed as a tool to improve the evaluation of marrow dysplasia. Rationale for the application of FCM in the diagnostic work up of MDS is that immunophenotyping is an accurate method for quantitative and qualitative evaluation of hematopoietic cells and that MDS have been found to have abnormal expression of several cellular antigens. To become clinically applicable, FCM analysis should be based on parameters with sufficient specificity and sensitivity, data should be reproducible between different operators and the results should be easily understood by clinicians. In this report, we reviewed the most relevant progresses in detection of marrow dysplasia by FCM in MDS as defined by WHO criteria.

Flow cytometric osmotic fragility—An effective screening approach for red cell membranopathies

Abstract: Background: Among the red cell membrane disorders, hereditary spherocytosis (HS) is one of the most common causes of inherited hemolytic anemia. The aim of this study was to compare the flow-cytometric approach for screening of red cell membrane disorders based on osmotic fragility with the eosin-5-maleimide (E5 0 M) dye test. A group of b-thalassemia heterozygotes were also studied. Methods: A red cell suspension was spiked with deionized water during acquisition and the count of residual red cells measured sequentially in real-time using flow cytometry. Fluorescence intensity of red cells stained with eosin-5-maleimide was also measured. Results: The hereditary spherocytosis (HS) group showed significantly decreased percentage residual red cells (9.31% 6 3.75%) (P 5 0.0091) whereas the b-thalassemia group showed a significant increase (93.56% 6 12.98%) (P 5 0.0008) compared to the normal control group (46.26% 6 11.33%). The cut off value of the flow cytometric osmotic fragility (FCM OF) test for red cell membrane disorders was 23.59% giving a sensitivity of 100% and specificity of 98%. Conclusions: The advantages of the FCM OF test are that it is quantitative, time effective and requires only deionized water for the measurement of osmotic fragility. It could be an effective first line screening approach for red cell membrane disorders in hematology laboratories where a flow cytometer is available. V C 2011 International Clinical Cytometry Society Key terms: red cell membranopathies; b-thalassemia; screening by flow-cytometry; osmotic fragility; eosin-5-maleimide fluorescence test

Variation of MDR Proteins Expression and Activity Levels According to Clinical Status and Evolution of CML Patients

Abstract: The involvement of the multidrug resistance (MDR) mediated by ABC transporter proteins P-glycoprotein (Pgp) and multidrug resistance-associated protein-1 (MRP1) overexpressions in patients with chronic myeloid leukemia (CML) are not completely understood. Pgp and MRP1 expressions and activity were analyzed in samples from 158 patients with chronic myeloid leukemia (CML). Using flow cytometry, Pgp expression was more frequently observed in early chronic (P = 0.00) and in advanced (P = 0.02) CML phases when it was compared to MRP1 expression. Variation of MDR expression and activity were observed during the CML evolution in patients previously treated with interferon and imatinib. In the K562-Lucena cell line, Pgp positive, imatinib caused an enhancing in Pgp expression at protein and mRNA levels, whereas in the Pgp negative cell line, this drug was capable of decreasing MDR1/Pgp mRNA levels. Our result emphasizes the importance of understanding the different aspects of MDR status in patients with CML when they are under investigation in determining imatinib resistance. © 2010 International Clinical Cytometry Society

Flow cytometric analysis of CD123 is useful for immunophenotyping classical Hodgkin lymphoma.

Abstract: Although the diagnosis of classical Hodgkin lymphoma (CHL) is traditionally made morphologically in tissue sections, it is now possible to diagnose CHL by flow cytometry (FC). In an effort to identify additional antigens on Hodgkin and Reed-Sternberg (HRS) cells that might be useful for immunophenotyping this lymphoma by FC, we examined the expression of CD123 (α chain of the IL-3R) on HRS cells and compared this with the expression of CD123 in other lymph node samples (372 tissue specimens, including 98 reactive and 274 neoplastic cases), using a nine-color FC reagent combination including anti-CD123. CD123 was found to be expressed on the majority of HRS cell populations (59% of 59 CHL lymph nodes examined), rarely on B-cell non-Hodgkin lymphomas (NHLs) (3 of 3 hairy cell leukemia cases, 3 of 29 chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) cases, 1 of 55 large B-cell lymphoma cases, but not on 95 other B-cell NHL examined), and in none of the 16 T-cell NHL cases examined. In reactive lymph nodes, CD123 expression was largely limited to plasmacytoid dendritic cells and rare histiocyte populations that can readily be distinguished from HRS populations. As the expression of this antigen is shown to be relatively limited in B- and T-NHL and in reactive lymph node populations, assessment of CD123 expression is useful for supporting the FC diagnosis of CHL.

Evaluation of basophil activation in mastocytosis with Hymenoptera venom anaphylaxis

Abstract: Background: Basophil activation tests (BATs) have been demonstrated to be useful in detecting IgE-mediated sensitization by measuring basophil activation surface markers (CD63 and CD203c). Hymenoptera venom is one of the best known mediators-release trigger in patients with systemic mastocytosis (SM). The aim of this study was to investigate the use of BATs as an additional diagnostic tool in patients with mastocytosis suffering from hymenoptera venom anaphylaxis (HVA). Methods: A total of 22 patients with history of HVA and SM, together with a group of 11 patients with HVA in whom SM was ruled out after a complete bone marrow study, were analyzed. Results: Among 11 SM patients who had specific serum IgE (sIgE) against hymenoptera venom and an evaluable BAT, a positive BAT was found in nine. Additionally, a positive BAT was detected in three of seven patients who had no sIgE. These three patients had low levels of total IgE compared with control population (mean of 20 vs. 78 IU/mL); one had discontinued immunotherapy after 5 years, when sIgE levels had turned negative, and, in the other two patients, BAT identified the culprit insect. Conclusions: BAT is a useful complementary diagnostic tool to sIgE in mastocytosis patients with HVA, and it may contribute to predict or confirm these nearly fatal reactions, especially before discontinuing venom immunotherapy in patients who are negative for skin tests or sIgE or display low total IgE levels; in such cases, it also provides evidence on the culprit insect prompting HVA. © 2010 International Clinical Cytometry Society

Flow cytometric analysis of peripheral blood dendritic cells in patients with severe sepsis.

Abstract: Background: Dendritic cells (DC) play a key role in cell-mediated immunity. We aimed to analyse the number and function of peripheral blood (PB) myeloid and plasmacytoid DC (mDC/pDC) in patients with severe sepsis. Methods: Twenty-six septic patients, 20 surgical patients (abdominal aortic aneurysm) and 20 healthy controls were enrolled in this prospective study. Results: At day 1 (enrollment in the study), septic patients showed in comparison with healthy controls, decreased mDC (P < 0.001) and increased pDC (P = 0.03), resulting in a reduction of the mDC/pDC ratio (P < 0.001). Surgery induced a decrease in both mDCs and pDCs level, without modification of mDC/pDC ratio. Septic patients included 15 survivors and 11 nonsurvivors. At day 1 no significant difference was found in mDC between the two groups, while pDCs were significantly higher in nonsurvivors (P = 0.03). At the outcome, mDC were selectively increased with respect to day 1 in survivors (P = 0.001), while no significant differences were observed as far as pDC count is concerned in both groups. Sorted DC from septic patients showed in comparison with healthy controls, reduced levels of HLA DR (P < 0.001), CD11c (P < 0.001), CD83 (P = 0.006) and of costimulatory molecule CD86 (P = 0.003); an up-regulation of chemokine receptor CXCR4 (P = 0.031) and increased apoptosis (P < 0.001). Conclusions: Sepsis has a profound effect on PB DC compartment. These alterations appear to be sepsis-specific. Increased apoptosis and alteration of migration and trafficking mechanism may be involved in DC compartment modification. DC alterations could contribute to sepsis-mediated immunoparalysis. © 2010 International Clinical Cytometry Society

Six color flow cytometry detects plasma cells expressing aberrant immunophenotype in bone marrow of healthy donors

Abstract: Background: Both normal and malignant plasma cell (PC) populations can be identified using modern flow cytometry (FC) technique in multiple myeloma (MM) patients Expression of CD19 and CD56 markers is heterogenous on bone marrow PCs of healthy individuals Little is known about immunophenotypically aberrant (CD19−/CD56+) PCs subpopulation of healthy people Methods: Using six color FC, we analyzed PCs in BM samples of 11 healthy donors (HD) and compared their immunophenotypic properties with clonal PC populations from MM patients Results: Both immunophenotypically normal (CD19+/CD56−) and aberrant (CD19−/CD56+) PC populations could be detected in 10 of 11 HDs' BM samples and constituted the median of 603% (373–723) and 96% (0–357) of BM PCs, respectively CD19, CD56, CD38, CD45, and CD20 marker expression characteristics were of little value discriminating clonal PCs of MM patients from immunophenotypically aberrant PCs of healthy donors Conclusions: Our findings suggest that aberrant immunophenotype is common in BM PCs of healthy people Improvements in FC methodology to separate normal and malignant PCs remain an open area for future investigations © 2011 International Clinical Cytometry Society

A cytometric study of the red blood cells in Gaucher disease reveals their abnormal shape that may be involved in increased erythrophagocytosis

Abstract: Background: Gaucher disease is a sphingolipidosis caused by a deficiency of the enzyme glucocerebrosidase. Macrophages transform into pathogenic Gaucher cells following the phagocytosis of red blood cells (RBCs) and subsequent accumulation of glucosylceramide. Enhanced erythrophagocytosis is one feature of the disease indicating abnormal macrophage-RBC interactions. We hypothesized that the erythrophagocytosis observed in Gaucher disease may be at least partly due to abnormalities in the RBCs themselves. Methods: To investigate this hypothesis, we used flow cytometry FSC/SSC to study RBCs sampled from seven patients with Gaucher disease in terms of their shape and the expression of markers of senescence and phagocytosis. Cells from two of the seven patients were evaluated before and 9 months after the start of enzyme-replacement therapy. Results: Untreated patients were found to have abnormal flow-cytometry profiles suggesting an alteration of Gaucher RBC morphology. Scanning electron microscopy confirmed this finding by revealing many abnormally shaped RBCs. Whereas there was no evidence of desialylation of membrane glycoconjugates or phosphatidylserine exposure, RBC viability (calcein-AM test) and CD47 expression were reduced. These anomalies found in RBCs sampled from two patients before treatment, were no longer present after a 9 month-long enzyme-replacement therapy. Conclusions: We report on previously overlooked alterations of Gaucher RBCs that may facilitate erythrophagocytosis in untreated patients. Their potential role in the anemia, the excess of aggregation and rheological anomalies associated with Gaucher disease must now be addressed. RBC anomalies may

An improved flow cytometric assay for detection and discrimination between malignant cells and atypical mesothelial cells, in serous cavity effusions.

Abstract: Background: The aim of this study was to evaluate a flow cytometric assay for the detection of malignant effusions. Methods: During the last 4-year period, 125 effusions suspicious for malignancy were prospectively analyzed by flow cytometry and conventional cytology. A three-step flow cytometric assay was performed, beginning with an initial informative panel of two protocols, containing SYTO-16, 7-AAD, CD71-PE, CD45-ECD, and CD66abce-FITC, CD64-PE, CD45-ECD, CD16-PECy5, CD14-PECy7, respectively. This was followed by a basic immunophenotypic panel of seven three-color combinations, containing in the first position, EMA, Ber-EP4, CD66abce, CD56, and intracellular desmin-33, combined with CD71-PE and CD45-PeCy5 in each tube. Finally, a cytokeratin-FITC/propidium iodide DNA panel was conducted, for the detection of aneuploidy in cytokeratin positive cells. Results: The sensitivity and specificity of flow cytometry were 85.1 and 97.8%, and of cytology 93.2 and 95.6%, respectively. A significant association was observed between the results of the two techniques (P < 0.001). Among eight atypical cases detected by cytology, five had been precisely characterized as malignant by flow cytometry. EMA and Ber-EP4 proved the most sensitive markers for malignancy diagnosis, while the detection of desmin-33 negative/cytokeratin positive cells had the simultaneous highest positive and negative predictive values. CD66abce was very specific, although nonsensitive, while DNA ploidy analysis was nonspecific, as hyperploidy was observed in reactive mesothelial cells. Conclusions: A flow cytometric assay of high sensitivity and specificity is proposed for the routine identification of carcinoma cells in effusions and their distinction from atypical mesothelial cells, as an ancillary to conventional cytology. © 2011 International Clinical Cytometry Society

Polychromatic flow cytometry identifies novel subsets of circulating cells with angiogenic potential in pediatric solid tumors.

Abstract: Background: Pediatric solid tumors depend upon angiogenesis for their growth and metastases. A new polychromatic flow cytometry (PFC) protocol has revealed circulating cells of hematopoietic and endothelial lineages from the peripheral blood (PB) of healthy individuals, and has defined the different cell types involved in the growth of tumor vasculature that are critical in angiogenesis. Methods: PB was collected from both healthy children and children with different malignant solid tumors and the mononuclear cells (MNCs) were subsequently isolated. PFC was applied and the MNCs were evaluated for proangiogenic and nonangiogenic circulating progenitor cells (CPCs), endothelial colony forming cells (ECFCs), and mature endothelial cells using the markers CD45, CD31, AC133, CD34, CD14, CD235a, CD41a, and a viability marker. Results: ECFCs and CPCs were significantly elevated in patients at day 21 compared to controls. The ratio of proangiogenic to nonangiogenic CPCs was significantly elevated compared to controls at baseline and returned to healthy baseline levels following treatment. Conclusions: We describe the successful identification of these hematopoietic and endothelial progenitor cells in both healthy children and children with solid tumors. In addition, this is a potential discovery of novel predictive biomarkers for future clinical trials. © 2011 International Clinical Cytometry Society

A GEIL flow cytometry consensus proposal for quantification of plasma cells: application to differential diagnosis between MGUS and myeloma.

Abstract: This standardized multiparameter flow cytometric approach allows for the detection and quantification of bone marrow tumor plasma-cell infiltration in nearly all cases of MGUS and myeloma, independently of debris and hemodilution This approach may also prove useful for the detection of minimal residual disease

Evaluation of degranulation and cytokine production in natural killer cells from spondyloarthritis patients at single-cell level

Abstract: Background: A pathogenetic role of natural killer (NK) cells has been hypothesized in spondyloarthritides (SpA), but still conflicting data exist. Aim of this study was to investigate, in a cohort of SpA patients, the peripheral blood NK cell number, the cytokine production (IFNc and TNFa), and the cytotoxic activity on target cell stimulation. Moreover, we aimed to evaluate the expression of KIR3DL1, an inhibitory receptor binding HLA class I alleles, including HLA-B27 strongly associated with SpA, between different NK cell subsets. Methods: We enrolled 21 SpA patients and 21 healthy controls. Multicolor flow cytometry was used to analyze the cytokine levels triggered by activating receptors, the degranulation as CD107a surface expression on target cell stimulation, the number and KIR3DL1 expression on peripheral blood NK cells. Results: When stimulated with P815 cells preincubated with antibodies against activating receptors, NK cells produced IFNc to a lesser extent respect to healthy controls (P 5 0.0012). No differences were found in TNFa production and NK cell degranulation in patients and controls. We observed a higher frequency of KIR3DL1-positive NK cells from SpA patients than in controls (P 5 0.02). Similar results were obtained in NK cell subsets. Conclusions: In our SpA patients, we observed a reduced IFNc production, which may be explained by the elevated frequency of KIR3DL1-expressing NK cells, while the other parameters were similar to those of healthy controls. These results may support the role of NK cells in the pathogenesis of SpA, although more data are needed to substantiate these observations. V C 2010 International Clinical Cytometry Society

Optimizing Antibody Panels for Efficient and Cost-Effective Flow Cytometric Diagnosis of Acute Leukemia

Abstract: Background: Differentiating acute myeloid leukemia (AML) from acute lymphoblastic leukemia (ALL) determines effective patient management and often depends on flow cytometry. Antibodies used in flow cytometry are costly, and the expenses are not always reimbursed. Having observed that AML and ALL have distinct patterns in the CD45/SSC panel, we set to analyze more leukemia cases and establish an algorithm for the efficient diagnosis of acute leukemia. Methods: We retrospectively analyzed 127 consecutive cases of acute leukemia within the last 2 years and correlated the blast distribution patterns in the CD45/SSC panel, with the morphology and the detailed immunophenotype. Results: Our results show that all the acute leukemias can be initially triaged into AML, ALL, and Indeterminate provisional groups based on the blast distribution patterns in the CD45/SSC panel and morphology. Each group was then further analyzed with tailored AML, ALL, and Indeterminate flow panels. Using this approach, we have efficiently and correctly diagnosed almost all the acute leukemias. Our analysis also determined the minimal numbers of immunological markers needed for the lineage assignment of acute leukemia. Conclusion: The algorithmic approach with tailored subsequent antibody selection could maintain diagnostic accuracy while significantly reducing reagent use, labor, and time. With a shrinking reimbursement for flow cytometric studies, an increase in laboratory efficiency without compromising diagnostic accuracy or turnaround time will contribute to preserving revenue and optimizing clinical service. © 2011 International Clinical Cytometry Society

Phenotypic abnormalities strongly reflect genotype in patients with unexplained cytopenias

Abstract: Background: In patients with unexplained cytopenias, abnormal karyotyping studies can be found with inconclusive light microscopic findings. Multidimensional flow cytometry (FCM) can identify myelomonocytic cells with aberrant phenotypes often not seen by standard morphology. Methods: In 431 patients presenting with unexplained cytopenia(s) FCM results were compared to abnormal karyotyping and FISH results recognized as associated with myelodysplastic syndrome (MDS) in the 2008 WHO classification, to assess the degree of and types of phenotypic abnormalities observed using a previously reported flow cytometric scoring system (FCSS). Fluorescence activated cell sorting was also used to identify subpopulations of abnormal maturing myelomonocytic cells that carry the genotypic abnormality. Results: For marrows with complex (three or more karyotypic abnormalities), two abnormalities, isolated chromosome seven anomalies, del(5q) or del(13q), 100% of cases were positive when using a FCSS cutoff of ≥2. Trisomy 8, del(20q), and minus Y had flow scores ≥2 in 72, 60, and 18%, respectively, but in some cases the flow score was high, indicating myeloid dysplasia. Most patients (16/22) with high myeloid progenitor cells (MyPC) (>20%) also exhibited maturing myeloid cell abnormalities by FCM. Morphology was negative in the maturing myeloid cells in many cases with phenotypically abnormal myeloid cells. Conclusions: The high correlation between genotypic and phenotypic abnormalities suggests a possible increased utility of flow cytometry in the diagnosis of patients with unexplained cytopenias and may be useful in future clinical studies and in the classification by the WHO, using the FCSS rather than simple counting of flow cytometric abnormalities. © 2010 International Clinical Cytometry Society

Early and rapid detection of X-linked lymphoproliferative syndrome with SH2D1A mutations by flow cytometry†

Abstract: Background: X-linked lymphoproliferative syndrome (XLP) is a rare immunodeficiency with extreme vulnerability to Epstein-Barr virus (EBV) infection. It presents with fatal infectious mononucleosis, lymphoproliferative disorder, or dysgammaglobulinemia. The majority of affected males have mutations in the SH2D1A/SLAM-associated protein (SAP) gene. We previously generated an antihuman SAP monoclonal antibody (KST-3) for a flow cytometric assay and described the activation of T cells to be necessary for the flow cytometric assessment of the SAP expression using an FITC-conjugated secondary antibody. Methods: Between 2005 and 2008, we recruited 23 male patients with suspected XLP, including mainly EBV-associated hemophagocytic lymphohistiocytosis (HLH), and attempted to evaluate SAP expression in fresh lymphoid cells using Alexa Fluor 488-conjugated secondary antibody instead of an FITC-conjugated one. Results: The method demonstrated that SAP was intensely expressed in CD8+ T cells and NK cells in normal fresh blood samples, thus suggesting the possible rapid identification of individuals with SAP deficiency. SH2D1A mutations were identified in six patients with SAP deficiency, but not in patients with normal SAP expression. Conclusion: The outcomes from this trial were verified by a flow cytometric assay using KST-3 and Alexa Fluor 488 secondary antibody. Based on the demonstration SAP deficiency in patients with suspected XLP, including mainly EBV-associated HLH, this approach could serve as a method for the early and rapid detection of patients with XLP-1. © 2010 International Clinical Cytometry Society

Cell-cycle distribution of different cell compartments in normal versus reactive bone marrow: a frame of reference for the study of dysplastic hematopoiesis.

Abstract: Limited information is currently available about the proliferation activity and cell-cycle distribution of different bone marrow (BM) cell subsets defined according to their lineage and maturation stage in normal versus cytopenia-associated reactive BM samples. Here, we report a three-color flow cytometry approach to investigate the cell-cycle distribution of different BM cell compartments—CD34+ hematopoietic progenitor and precursor cells (HPC), maturing neutrophils and monocytic cells, mature lymphocytes, eosinophils, and nucleated red blood cell precursors (NRBC)—from normal (n = 47) versus cytopenia-associated reactive (n = 47) BM samples. Highly similar proliferation profiles were detected in normal versus reactive BM, with a higher proliferation index (PI) for the more immature CD34+ HPC, CD11b− maturing neutrophils and NRBC versus other BM cell compartments. The only differences observed between normal and reactive BM were restricted to the more mature (CD13hi/CD11b+) bands/neutrophils and to monocytic cells, which showed an increased PI (0.9% ± 0.8% vs. 0.6% ± 0.5% and 6 ± 3.6 vs. 4.6 ± 4.5, respectively) at the expense of a lower PI of CD34+ HPC in reactive conditions. Of note, bands/mature neutrophils and mature lymphocytes showed either residual numbers or absence of S + G2/M-phase cells in both normal and reactive BM. Our results suggest that a slight shift of proliferation from the early precursors to the more mature granulomonocytic compartment occurs in reactive BM, which could reflect an attempt of the hematopoietic system to rapidly produce functional neutrophils and monocytes, at the expense of a lower expansion of the minor compartments of CD34+ HPC. © 2011 International Clinical Cytometry Society

The use of IL‐3 in basophil activation tests is the real pitfall

Abstract: I have recently read the article by Sturm et al. in the latest issue of Cytometry B Clinical Cytometry (1), to which I agree with many of the Author’s conclusions, though I have some consideration to address. The article tries to assess the use of basophil activation tests (BATs) based on CD203c upregulation as a reliable tool for allergy diagnosis. BATs, based either on the increased expression of CD63 or CD203c by activated basophils, have been proposed in many issues as a promising diagnostic tool for drug hypersensitivity, hymenoptera venom allergy, or other allergic pathologies (2); criticism has raised the question whether baso-tests are potential good analytical approaches or pitfalls (3). CD203c is rapidly upregulated as other less known activation markers (CD164 and CD13) and maximal ectoenzyme updisplacement on basophil membrane are reached after 5–15 min following stimulation (4,5). Both the granule-associated tetraspan CD63 and the ectoenzyme CD203c are upregulated to the plasma membrane of basophilic cells in response to FceRI crosslinking, which can be triggered both by allergens and anti-IgE immunoglobulins. Upregulation time-course of these markers seems to be rather tricky to evaluate the quality and the amount of basophil activation response to an IgEmediated triggering agent. In the work of Sturm and colleagues, CD63 and CD203c upregulation are affected both by activation time and by prestoring blood samples at room temperature at different times (1). This observation raises the question whether it is possible to standardize a basophil resting (nonactivated) condition and to evaluate the amount of expression of membrane markers with a reference standardized evaluation, which could be used in any circumstance: in this context, critical factors are represented by basophil spontaneously induced activation, by intracellular calcium and the presence of calcium chelators in the medium, and by time and temperature. However, the proposal is hampered by the wide variability of basophil response to allergens, due to different levels of non-releaser and releaser subjects, by seasonal factors and by experimental conditions (3), and we are possibly in a bottleneck quite similar to that of coagulation international normalized ratio values. Blood microenvironment can preactivate leukocytes by releasing several priming factors but also basophils can produce IL-3 within 4 h in response to IgE-dependent activation (6). Within 4 h, IL-3 production reaches its peak but its release begins in the first hour following peripheral blood withdrawal (6). Hence, autocrine IL-3 release by basophils in blood stressed by venipuncture and vial microenvironment is reported to be the most critical point of preanalytical factors able to influence BAT diagnostic performance. A low level of anti-IgE/FceRI crosslinking triggers IL-3 autocrine production to prime basophils to respond, so even anti-IgE concentration might be a critical point (6). Basophils rapidly bind and use the IL-3 they produce but this effect is inhibited in the presence of neutralizing anti-IL-3-a receptor, namely CD123 (6): this could raise the question whether incubation of anti-CD123-fluorochrome labeled antibodies and exogenous IL-3 in the same cellular environment could be optimized. One way to carry out experiments with resting basophils keeping a baseline level until their stimulation is to use ice bath. I wonder whether basophils could actually be primed by autocrine IL-3 in the 4 h following venous blood withdrawal, especially if receptor–ligand function is blocked by treating basophils in ice bath: a spontaneously induced priming by autocrine IL-3 should justify the conclusion with which Sturm EM et al. suggest how to use BATs that evaluate CD203c upregulation. Hence, one issue is either to start experimental condition very early or by using ice bath for treating basophils, even during staining with fluorochrome-labeled antibodies (5). Factors other than IgE-mediated agonists, such as anaphylotoxins or bacterial products, are not able to induce autocrine IL-3 by nonstimulated basophils; hence, preanalytical errors due to environmental contamination are not relevant for basophil evaluation, whereas what seems to be more critical is the role of intracellular calcium (6). The latter condition along with a relatively high temperature could be the main factor affecting basophil spontaneously induced autocrine priming; on the other hand, calcium chelators such as ethylene-diamine-tetraacetic-acid (EDTA) or ethylene-glycol-tetraacetic-acid (EGTA) in blood microenvironment might affect activation testing (3). Another issue is that basophils may very well regulate their own priming, namely that the priming mechanism involves a fine regulation of IL-3 receptor recycling and desensitization/downregulation to further IL-3 providing. This makes crucial the use IL-3 in the long-standing idea that in vitro basophil releasability is clinically relevant (7). Basophils from allergic patients can be primed compared with those of non-allergic subjects; in the latter case, priming effect induced by exogenous IL-3 of the former might lead to underestimate CD203c upregulation following anti-IgE stimulation, mainly when it is compared with exogenous IL-3 primed nonallergic samples. The same reasoning could be forwarded by considering IL-3 spontaneously induced priming by preanalytical condition. We are unable to know if by treating basophils with exogenous IL-3, IgE-mediated response of CD203c is downregulated or not. Therefore, besides to the preanalytical and experimental factors here Cytometry Part B (Clinical Cytometry) 80B:137–138 (2011)

Primary role of multiparametric flow cytometry in the diagnostic work-up of indolent clonal mast cell disorders.

Abstract: Background: According to the World Health Organization (WHO) classification the diagnosis of systemic mastocytosis (SM) relies on bone marrow (BM) examination and is based on one major and four minor criteria. Herein, we used WHO criteria to compare flow cytometry (FC) with other available techniques in the diagnosis of SM after BM examination. Methods: We analyzed a cohort of 95 patients with suspect SM. All patients underwent comprehensive BM examination by using cytology, immunohistochemistry, FC and molecular study for mutation of c-Kit and serum tryptase dosage. FC evaluation was based on a combination of monoclonal antibodies, specifically CD25/CD2/CD45/CD34/CD117. Results: Seventy-four out of ninety-five patients were diagnosed with indolent SM (n = 59) or monoclonal mast cell activation syndrome (n = 15) because satisfying less than 3 minor criteria. Thirty-nine out of these seventy-four patients fulfilled the major histological criterion, whereas the presence of a minor criterion was assessed by FC, molecular study, cytology, and tryptase level in 70/74, 52/67, 56/74, and 42/74 patients, respectively. FC showed higher sensitivity than IHC in detection of CD25+ mast cells (MC) (92.9% vs. 73.8%; P = 0.019), especially in the absence of the major histological criterion (90.5% vs. 47.6%; P = 0.003). Moreover, CD2 expression was documented by FC and IHC in 97.1% and 35.3% of cases, respectively (P < 0.001). Conclusions: FC showed the best sensitivity for identifying abnormal MC compared to other techniques, especially in cases with low MC burden. Therefore, we hope for a major role of FC in the diagnostic work-up of clonal MC disorders. © 2011 International Clinical Cytometry Society

Lymphocyte phenotypic subsets in umbilical cord blood compared to peripheral blood from related mothers.

Abstract: Lymphocyte Phenotypic Subsets in Umbilical Cord Blood Compared to Peripheral Blood from Related Mothers Salvatore Chirumbolo,* Riccardo Ortolani, Dino Veneri, Ricciarda Raffaelli, Diego Peroni, Roberta Pigozzi, Marco Colombatti, and Antonio Vella Department of Pathology and Diagnostics, Section of General Pathology, University of Verona, strada Le Grazie 8 37134 Verona, Italy Department of Pathology and Diagnostics, Section of Immunology, University Hospital-Azienda Ospedaliera Universitaria Integrata-Policlinico GB Rossi, piazzale AL Scuro, 10 37135 Verona, Italy Department of Clinical and Experimental Medicine, Section of Haematology, University Hospital-Azienda Ospedaliera Universitaria Integrata-Policlinico GB Rossi, piazzale AL Scuro, 10 37135 Verona, Italy Department of Mothers and Children Biology-Genetics, Section of Pediatrics, University Hospital-Azienda Ospedaliera Universitaria Integrata-Policlinico GB Rossi, piazzale AL Scuro, 10 37135 Verona, Italy Department of Mothers and Children Biology-Genetic, Section of Obstetrics and Gynecology, University Hospital-Azienda Ospedaliera Universitaria Integrata-Policlinico GB Rossi, piazzale AL Scuro, 10 37135 Verona, Italy

Aberrant expression of c-met and HGF/c-met pathway provides survival advantage in B-chronic lymphocytic leukemia.

Abstract: Background: B-chronic lymphocytic leukemia (B-CLL) is characterized by accumulation of CD5+ B lymphocytes. Decreased VLA-4 (Cd49d/CD29) and CD11a expression and defective adhesion in B-CLL have been previously shown, although there was no substantial data about its importance in immunobiology of B-CLL. The hepatocyte growth factor (HGF) receptor, c-met, plays a role in adhesion by acting on VLA-4. c-met and VLA-4 share crucial signaling molecules in cell survival. In this study, relationship between expressions of c-met and CD49d, CD11a, and additional common signaling molecules in B-CLL was investigated. Methods: White blood cells from 24 patients with CLL were studied by flow cytometry and/or western blotting prior to and after culturing with recombinant HGF. HGF level from sera was measured with a bead-based flow cytometric assay. Results: c-metα and c-metβ were expressed on B-CLL cells, while no expression was observed on normal donor CD19+ cells. This increase was inversely correlated with decreased expression of adhesion molecules. Serum level of HGF in B-CLL was found to be increased. In vitro experiments showed that HGF supported survival in B-CLL cells supporting the possible function of HGF/c-met pathway in B-CLL. Furthermore, expressions of critical signaling molecules shared by both VLA-4 and HGF/c-met systems including Bcl-XL, Akt, PI3K, and phospho-bad136 following HGF stimulations of B-CLL cells have been found to be increased. Conclusion: Increased expression of c-met and HGF may bypass the importance of expression of critical adhesion molecules and support survival of B-CLL cells. c-met, being one of the surface tyrosine kinases, may serve as a target for future therapies in B-CLL meriting more attention. © 2010 International Clinical Cytometry Society

Significance of the volume of fetomaternal hemorrhage after performing prenatal invasive tests.

Abstract: Background: Fetal erythrocytes cross the placenta during gestation, but invasive prenatal procedures might develop into fetomaternal hemorrhage (FMH). We examine whether flow cytometry immunophenotyping might be useful for measuring the volume of FMH after such procedures. Methods: Fetal erythrocytes (%) were determined in 153 pregnant women after amniocentesis (129) and chorionic villous sampling (24) using a monoclonal antibody against fetal hemoglobin. Fetal erythrocytes were identified for their high expression of fetal hemoglobin (HbF 11 ). Blood samples from two control groups, 53 healthy males and 21 pregnant women not submitted to invasive tests, were used to establish normal values of circulating HbF 11 erythrocytes in adults. Results: The highest percentage of HbF 11 erythrocytes in the control groups was 0.015%. The rate of HbF 11 erythrocytes in samples after invasive tests ranged between 1 ml of FMH (volume of packed cells corresponding to 0.054–0.15% HbF 11 erythrocytes), but only two had sonographic evidence of bleeding. Conclusions: Most women in our series had a very low volume of FMH after the invasive tests. Acute bleeding should be thoroughly investigated in women with either more than 1 ml of packed cells or more than 0.05% of HbF 11 erythrocytes. Intermediate values between >0.015% and <0.05%, should be carefully considered depending on the week of gestation. Data obtained before 15 weeks might reflect previous cell trafficking between fetus and mother instead of acute hemorrhage. V C 2010 International Clinical Cytometry Society Key terms: fetomaternal hemorrhage; flow cytometry; fetal hemoglobin; amniocentesis; prenatal invasive tests

Modulation of the cellular and humoral immune response to pediatric open heart surgery by methylprednisolone

Abstract: Background: With the intention to reduce overshooting immune response, glucocorticoids are frequently administered perioperatively in children undergoing open heart surgery. In a retrospective study we investigated extensively the modulation of the humoral and cellular immune response by methylprednisolone (MP). Methods: This study was carried out on blood samples from two groups of children who had undergone surgical correction of atrial or ventricular septal defects, either without (MP−, n = 10), or with MP administration (MP+, n = 23, dose median 11 (IQR 10–16) mg kg−1 body weight) before cardiopulmonary bypass (CPB, duration median 42 (IQR 36–65) min). EDTA blood was obtained 24 h preoperatively, after anesthesia, at CPB begin and end, 4, 24, and 48 h after surgery, at discharge and at out-patient follow-up (median 8.2 (IQR 3.3–12.2) months after surgery). Complex blood analysis including clinical chemistry and flow cytometry were performed to monitor humoral immune response, differential blood count, lymphocyte subsets, and the degree of activation of various leukocyte subpopulations. Results: The patients' postoperative courses and follow-up were uneventful. Release of IL-6 and IL8 was reduced and that of the anti-inflammatory cytokine IL-10 upregulated by MP. Significant increase of circulating neutrophils and monocytes as inflammatory reaction to surgery and CPB contact was detected in both groups. However, invasion of monocytes to the periphery was delayed with MP. CD4+ and CD8+ T-lymphocyte counts were lower with MP treatment. B-lymphocyte count increased significantly after surgery in MP+ but remained constant in MP− group. Conclusions: MP treatment partially decreased the pro-inflammatory effect of CPB surgery and induced anti-inflammatory effect on the cellular and humoral level. © 2011 International Clinical Cytometry Society

Persistence of immunological alterations after thymectomy in Good's syndrome: A clue to its pathogenesis

Abstract: Analyzing the phenotypic characterization of the immune system cells involved in the pathogenesis of immunodeficiency with thymoma (Good's syndrome) is difficult due to the low number of studies on that subject. We describe the immunological alterations observed in a case of Good's syndrome, and we summarize the pathogenic explanations found in the literature.