Immunophenotyping Of Leukocytes Research Articles

Effect of Recombinant Heat Shock Protein 70 of Mycobacterial Origin on Cytotoxic Activity and Immunophenotype of Human Peripheral Blood Mononuclear Leukocytes

Recombinant heat shock protein of mycobacterial origin with a molecular weight 70 kDa in concentration 0.1 microg/ml in vitro activates cytotoxic activity of mononuclear lymphocytes from peripheral blood of healthy donors against K-562 human erythroblastic leukemia cells sensitive to natural killers. In other concentrations (1.0 microg/ml and 10 microg/ml) this heat shock protein did not significantly affect functional activity of mononuclear leukocytes. Expression of CD4, CD25, CD16 and CD56 on membrane of mononuclear leukocytes was also studied.

Malignant and Tuberculous Pleural Effusions: Immunophenotypic Cellular Characterization

INTRODUCTION AND OBJECTIVESTuberculosis and cancer are the main causes of pleural effusion. Pleural involvement is associated with migration of immune cells to the pleural cavity. We sought to characterize the immunophenotype of leukocytes in the pleural effusion and peripheral blood of patients with tuberculosis or malignancy.METHODSThirty patients with tuberculosis (14) or malignancy (16) were studied. A control group included 20 healthy blood donors.RESULTSMalignant phycoerythrin pleural effusions showed higher percentages of CD3, CD4, CD3CD45RO, and CD20CD25 lymphocytes and lower percentages of CD3CD25 and CD20HLA-DR when compared to PB lymphocytes. Compared to PB, tuberculous effusions had a higher percentage of lymphocytes that co-expressed CD3, CD4, CD3CD45RO, CD3TCRαβ, CD3CD28, and CD20 and a lower percentage of CD14, CD8 and CD3TCRγδ-positive lymphocytes. Malignant effusions presented higher expression of CD14 whereas tuberculous effusions had higher expression of CD3 and CD3CD95L. Peripheral blood cells from tuberculosis patients showed higher expression of CD14, CD20CD25 and CD3CD95L. Compared with the control cells, tuberculosis and cancer peripheral blood cells presented a lower percentage of CD3CD4 and CD3CD28-positive cells as well as a higher percentage of CD3CD8, CD3CD25 and CD3CD80-positive cells.CONCLUSIONSTuberculous and malignant peripheral blood is enriched with lymphocytes with a helper/inducer T cell phenotype, which are mainly of memory cells. CD14-positive cells were more frequently found in malignant effusions, while CD3-positive cells expressing Fas ligand were more frequently found in tuberculous effusions.

Long‐term stabilized blood samples as controls for flow cytometric HLA‐B27 screening: A feasibility study

Long-term stabilized blood samples are potentially useful as positive or negative procedure controls for flow cytometric HLA-B27 screening, and could serve as test samples in an external quality assessment (EQA) scheme. We evaluated long-term stabilized whole blood specimens as prepared for the UK NEQAS for Leucocyte Immunophenotyping EQA scheme (Sheffield, UK). Peripheral blood samples were obtained from nine blood bank donors with known HLA-B typing. Short-term stabilization with Trans-FIXtrade mark was performed before shipment to Sheffield. Thereafter, long-term stabilization was performed. Commercially available HLA-B27 mAb were tested periodically between 1 week and 12 months on (i) fresh, (ii) short-term stabilized, and (iii) long-term stabilized blood samples using a stain, lyse, and wash technique. We compared the forward scatter (FSC), sideward scatter (SSC), and fluorescence signals of lymphocytes as a function of time. Furthermore, a pilot send-out with stabilized blood samples of four blood bank donors was distributed among the participants to the Benelux EQA scheme for HLA-B27 screening, and results were compared with historical EQA data obtained using nonstabilized blood samples from the same donors. There were no major effects on FSC and SSC characteristics of lymphocytes. Background fluorescence of stabilized samples increased and specific fluorescence of stabilized HLA-B27 positive samples decreased as compared with fresh samples. However, discrimination between the investigated HLA-B27 positive and HLA-B27 negative samples remained feasible poststabilization. In the pilot send-out, the results obtained with stabilized samples were less concordant than with the corresponding fresh samples due to variable quality of the stabilized samples. Long-term stabilized whole blood samples are potentially useful as true HLA-B27 positive and true HLA-B27 negative control cells for daily and longitudinal quality control of flow cytometric HLA-B27 screening. In the same way, long-term stabilized samples may be used for EQA purposes. However, these samples are currently not feasible for reagent validation purposes. Extensive quality control of long-term stabilized samples is necessary before distribution in multicenter surveys.

Toxicity of High-Dose Chlorambucil in Wistar Rats

The aim of this study was to determine the maximum tolerated dose and the haematological and non-haematological toxicity of chlorambucil in male Wistar rats. Chlorambucil (pulverized pills or pure substance) was dissolved in ethanol and administered orally at a dose ranging from 0.15 to 50 mg/kg/day for 5 days. Evaluations of experimental rats included general clinical state of health, body weight, haematological indicators (red blood cell count, white blood cell count, platelet count, differential leukocyte count, packed cell volume and haemoglobin concentration), biochemical plasma indicators (urea, creatinine, ALP, ALT, AST, Na, K, Cl), immunophenotype of leukocytes (CD3, CD8 and CD4 markers), as well as histopathology. Doses of chlorambucil from 0.15 to 0.75 mg/kg/day had no effect on the general clinical state of health as well as on other laboratory variables. Doses of 2–12 mg/kg/day were well tolerated, but mild myelotoxicity (leukopaenia) and elevated activities of liver transaminases were found. Doses of 20 to 50 mg/kg/day were myelotoxic (p < 0.01) with prevailing lymphopaenia but only minimum effects on red blood cells and platelets. Histology revealed pneumotoxicity, nephrotoxicity and damage to the gastrointestinal tract. Doses of 40 and 50 mg/kg/day resulted in acute lethal effects in 50% and 75% of individuals, respectively. Results of this study are intended for use in the testing of chlorambucil in combination with other drugs as a conditioning for bone marrow transplantation.

Aflatoxin-related immune dysfunction in health and in human immunodeficiency virus disease.

Both aflatoxin and the human immunodeficiency virus (HIV) cause immune suppression and millions of HIV-infected people in developing countries are chronically exposed to aflatoxin in their diets. We investigated the possible interaction of aflatoxin and HIV on immune suppression by comparing immune parameters in 116 HIV positive and 80 aged-matched HIV negative Ghanaians with high (≥0.91 pmol/mg albumin) and low (<0.91 pmol/mg albumin) aflatoxin B1 albumin adduct (AF-ALB) levels. AF-ALB levels and HIV viral load were measured in plasma and the percentages of leukocyte immunophenotypes and cytokine expression were determined using flow cytometry. The cross-sectional comparisons found that (1) among both HIV positive and negative participants, high AF-ALB was associated with lower perforin expression on CD8+ T-cells (P = .012); (2) HIV positive participants with high AF-ALB had significantly lower percentages of CD4+ T regulatory cells (Tregs; P = .009) and naive CD4+ T cells (P = .029) compared to HIV positive participants with low AF-ALB; and (3) HIV positive participants with high AF-ALB had a significantly reduced percentage of B-cells (P = .03) compared to those with low AF-ALB. High AF-ALB appeared to accentuate some HIV associated changes in T-cell phenotypes and in B-cells in HIV positive participants.

P.2.e.006 Clinical features of early onset bipolar disorder

The mechanisms mediating the short- and long-term improvements in glucose homeostasis following bariatric/metabolic surgery remain incompletely understood.To investigate whether a reduction in adipose tissue inflammation plays a role in the metabolic improvements seen after bariatric/metabolic surgery, both in the short-term and longer-term.Fasting blood and subcutaneous abdominal adipose tissue were obtained before (n = 14), at one month (n = 9), and 6–12 months (n = 14) after bariatric/metabolic surgery from individuals with obesity who were not on insulin or anti-diabetes medication. Adipose tissue inflammation was assessed by a combination of whole-tissue gene expression and flow cytometry-based quantification of tissue leukocytes.One month after surgery, body weight was reduced by 13.5 ± 4.4 kg (p < 0.001), with improvements in glucose tolerance reflected by a decrease in area-under-the-curve (AUC) glucose in 3-h oral glucose tolerance tests (− 105 ± 98 mmol/L * min; p = 0.009) and enhanced pancreatic β-cell function (insulinogenic index: + 0.8 ± 0.9 pmol/mmol; p = 0.032), but no change in estimated insulin sensitivity (Matsuda insulin sensitivity index [ISI]; p = 0.720). Furthermore, although biomarkers of systemic inflammation and pro-inflammatory gene expression in adipose tissue remained unchanged, the number of neutrophils increased in adipose tissue 15–20 fold (p < 0.001), with less substantial increases in other leukocyte populations. By the 6–12 month follow-up visit, body weight was reduced by 34.8 ± 10.8 kg (p < 0.001) relative to baseline, and glucose tolerance was further improved (AUC glucose − 276 ± 229; p < 0.001) along with estimated insulin sensitivity (Matsuda ISI: + 4.6 ± 3.2; p < 0.001). In addition, improvements in systemic inflammation were reflected by reductions in circulating C-reactive protein (CRP; − 2.0 ± 5.3 mg/dL; p = 0.002), and increased serum adiponectin (+ 1358 ± 1406 pg/mL; p = 0.003). However, leukocyte infiltration of adipose tissue remained elevated relative to baseline, with pro-inflammatory cytokine mRNA expression unchanged, while adiponectin mRNA expression trended downward (p = 0.069).Both the short- and longer-term metabolic improvements following bariatric/metabolic surgery occur without significant reductions in measures of adipose tissue inflammation, as assessed by measuring the expression of genes encoding key mediators of inflammation and by flow cytometric immunophenotyping and quantification of adipose tissue leukocytes.

External quality control program: Experience in the department of Microbiology and Immunology, Muhimbili University College of Health Sciences, Dar es Salaam, Tanzania

Objective: To share experiences and lessons learned from 2002 National External Quality Assessment Schemes for Leukocyte Immunophenotyping (UK NEQAS) and College of American Pathologists (CAP) proficiency testing (PT) panels received and tested as part of External Quality Control Program Setting: Department of Microbiology and Immunology, Muhimbili University College of Health Sciences, Dar es Salaam. Methods: All PT panel records from January – December 2002 were reviewed and summarized to show the type of panel and date received, date results sent after analysis and our performances. Results: Six and fourteen panels were received from UKNEQAS and CAP, respectively. The mean time interval between receipt of panels and reporting back of the results was 9.4 days (range 2-17). UKNEQAS performance was good in 5/6 panels (83.3%) analyzed. Among CAP panels, satisfactory (100%) performances included two viral markers, two C. trachomatis antigen detection and two comprehensive hematology with automated differentials whereas unsatisfactory (79%, 66%, 71%) performances were all 3 general chemistry with an overall score of (6/9) 66.7%. Conclusion: To implement an effective and efficient quality assurance in an established laboratory requires committed long-term efforts that are potentially constrained with multiple obstacles. Continuing education and training of laboratory personnel is an integral part of quality control/quality assurance program. Keywords: Proficiency Testing, Quality Control, and Quality Assurance. > Tanzania Medical Journal Vol. 19 (1) 2004

Changes in fluorescence intensity of selected leukocyte surface markers following fixation

Immunophenotyping of blood leukocytes often involves fixation with paraformaldehyde prior to cytometry analysis. However, the influence of cell type and marker specificity on the stability of fluorescence intensity after fixation has not been well studied. Human whole blood was stained using a panel of fluorescein isothiocyanate-labeled antibodies to surface markers. Unfixed and fixed samples were analyzed by flow cytometry at 0, 2, 4, 6, 24, 48, and 96 h after staining. Fluorescence measurements were converted to molecules of equivalent soluble fluorochrome for comparison. Fixation caused a significant decrease in both forward and side scatter at 48 h which required gating adjustments to achieve resolution of cell populations. The autofluorescence increased progressively in fixed samples (ninefold at 96 h for monocytes). Variable decreases in marker-associated fluorescence became apparent after correction for autofluorescence. The magnitude of the decrease at 96 h varied with cell type and marker, from 5% for CD32 on monocytes to 39% for CD16 on neutrophils. The change in fluorescence intensity following staining and fixation of leukocytes varies with cell type and surface marker. Fluorescence stability should be determined for each cell type and marker used, and the confounding effects of fixation on cell autofluorescence should be considered.

Treponema pallidum elicits innate and adaptive cellular immune responses in skin and blood during secondary syphilis: a flow-cytometric analysis.

Syphilis is caused by the spirochetal pathogen Treponema pallidum. The local and systemic cellular immune responses elicited by the bacterium have not been well studied in humans. We used multiparameter flow cytometry to characterize leukocyte immunophenotypes in skin and peripheral blood from 23 patients with secondary syphilis and 5 healthy control subjects recruited in Cali, Colombia. Dermal leukocytes were obtained from fluid aspirated from epidermal suction blisters raised over secondary syphilis skin lesions. Compared with peripheral blood (PB), blister fluids (BFs) were enriched for CD4(+) and CD8(+) T cells, activated monocytes/macrophages, and CD11c(+) monocytoid and CD11c(-) plasmacytoid dendritic cells (mDCs and pDCs, respectively). Nearly all mDCs in BFs expressed the human immunodeficiency virus (HIV) coreceptors CCR5 and DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and high levels of human leukocyte antigen (HLA)-DR. Dermal pDCs expressed both HIV coreceptors without increases in HLA-DR intensity. Compared with normal blood, circulating mDCs in patients with syphilis expressed higher levels of both CCR5 and DC-SIGN, whereas circulating pDCs in patients expressed only higher levels of DC-SIGN. Most dermal T cells were CCR5(+) and displayed a memory (CD27(+)/CD45RO(+)) or memory/effector (CD27(-)/CD45RO(+)) immunophenotype. A corresponding shift toward memory and memory/effector immunophenotype was clearly discernible among circulating CD4(+) T cells. Compared with PB from control subjects, a larger percentage of CD4(+) T cells in PB from patients with syphilis expressed the activation markers CD69 and CD38. During secondary syphilis, T. pallidum simultaneously elicits local and systemic innate and adaptive immune responses that may set the stage for the bidirectional transmission of HIV.

Innate Immune Responses in Swine Resuscitated from Severe Traumatic Hemorrhagic Shock with Hemoglobin-Based Oxygen Carrier-201

Hemoglobin-based oxygen carrier-201 transports oxygen and improves survival in swine with hemorrhagic shock, but has potential to be immune activating. Herein, we evaluated HBOC-201's immune effects in swine with more severe hemorrhagic shock due to soft tissue injury and 55% blood volume catheter withdrawal over 15 minutes followed by fluid resuscitation at 20 minutes with HBOC-201, Hextend, or no treatment (NON) before hospital arrival. Survival rates were similar with HBOC-201 and Hextend (p > 0.05), but were higher than in (p = 0.007). There were no significant group differences in blood cell count, percentages of leukocyte sub-populations and immunophenotype (CD4:CD8 ratio), adhesion markers expression (neutrophil CD11b; monocyte or neutrophil CD49d) and apoptosis. There was a trend to higher plasma IL-10 in HBOC-201 and groups vs. Hextend. We conclude that in swine with severe controlled HS and soft tissue injury, immune responses are similar with resuscitation with HBOC-201 and Hextend.

Twenty years of external quality assurance in clinical cell analysis — A tribute to Jean‐Luc D'Hautcourt

External quality assurance (EQA) programs in clinical cell analysis are now a consolidated item of laboratory practice. All the flow cytometric testings with an impact on clinical decision making have been submitted to regular EQA programs during the last 20 years, and this has produced internationally homogeneous guidelines, with a remarkable improvement in result reproducibility.Jean-Luc D'Hautcourt was a pioneer in this field, and his valuable contributions to flow cytometric method standardization and to the dissemination of the educational aspects of EQA programs are recognized. The different methodological approaches undertaken in the United States and Europe are discussed. The educational role of SIHON in the Benelux Countries and of UKNEQAS for Leucocyte Immunophenotyping worldwide is emphasized. Accredited and accreditating EQA programs require an impressive degree of organization and technical knowledge, so that only major international providers can afford such a task nowadays. However, small local studies still provide the necessary stimulus to the continuous improvement of the scientifical aspects of EQA schemes.

A Pilot Phase II Trial of a Synthetic Tumor-Specific Breakpoint Peptide Vaccine in Patients with Chronic Myeloid Leukemia (CML) and Minimal Residual Disease.

Peptides derived from BCR-ABL have been used to trigger immune responses in CML pts. The native peptides may have low immunogenicity because they bind with low affinity to the major histocompatibility complex (MHC) molecules. Synthetic peptides containing mutations in the sequences of these peptides can bind with higher affinity than parental peptides and may generate a cross-reactive T cell response to the native sequence (known as a heteroclitic response). We used a mixture of heteroclitic and native peptides derived from both b3a2 and b2a2 sequences in a pilot study to vaccinate pts with CML in complete cytogenetic remission on imatinib (IM) therapy with stable bcr-abl transcript levels. Montanide ISA51 and GM-CSF were used as immunological adjuvants. Pts were required to have received IM for ≥12 mo with no change in dose for at least 6 mo and were not allowed any dose increases after registration. Pts were vaccinated with the peptides corresponding to their BCR-ABL breakpoint as follows: every 2 wks x4, then once 3 wks later, followed by 10 monthly vaccinations for a total of 15 vaccinations in 12 mo. After registration, pts had 3 additional measures of bcr-abl transcripts to better define baseline values. 8 pts have been vaccinated. At enrollment, pts had a median age of 45 yrs (range 29–63) and had been receiving IM for a median of 63 mo (range 35–68 mo) on a median dose of 600mg (range 300–800mg). Pts have been followed for a median of 15 wks (range 2–20) and have received a median of 7 vaccinations (range 1 to 8). To determine the effect of vaccination on immune leukocytes, we measured peripheral blood T-cell subsets, B, NK, and dendritic cells by FACS at baseline and at 3-month intervals thereafter. The percentage of myeloid DC (DC1) and plasmacytoid DC (DC2) cells were identified by the mutually exclusive expression of CD11c and CD123; Treg cells by the surface co-expression of CD3, CD4 and CD25 together with cytoplasmic expression of CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and FoxP3 (forkhead/winged helix transcription factor). Vaccinations have been well tolerated. The only adverse event attributable to the vaccine is local site reactions grade 1 or 2 in 5 pts. Among the 5 pts who have had an evaluation at 3 mo from the 1st vaccination, 1 has achieved a major molecular response (bcr-abl/abl <0.05%). The Wilcoxon signed ranks test was used to assess changes in the immunophenotype of peripheral leukocytes of 5 pts evaluated for leukocyte FACS analyses at baseline and after 3 mo on study. Compared with baseline, there were significant decreases in the percentages of B (p = 0.042) and NKT (p = 0.043) cells, and significant increases in the percentages of DC2 (p = 0.043) and Treg (p = 0.043) cells. All pts continue receiving vaccination as planned. We conclude that vaccination with b2a2 and b3a2 synthetic peptides is well tolerated. Further follow-up is required to determine the immunogenecity and efficacy of the vaccine in this setting.

Can Renal Leukemic Infiltration Cause Hypertension in Children?

Out of 334 children with acute lymphoblastic leukemia who were treated with St Jude Total XI and Total XIII chemotherapy protocols were investigated and 21 (6.3%) were hypertensive. The incidence of tumor lysis syndrome was higher in the hypertensive group than in the nonhypertensive group (28.6% vs. 11.5%) (P = 0.035). There were no differences between patients treated with high-dose methylprednisolone and prednisolone St Jude Total XI and Total XIII, St Jude Total XIII LR and St Jude Total XIII HR groups in respect of the above-mentioned parameters. Central nervous system involvement, skeletal system involvement, abdominal lymphadenopathy, elevated lactate dehydrogenase and leukocyte count, French-American-British types and immunophenotypes were not found to be statistically significant to the development of hypertension (P > 0.05). We found that renal leukemic infiltration is a risk factor in hypertension development (P = 0.04) and hypertension is a risk factor for renal parenchymal disorder in the follow-up period (P = 0.0001). Six patients presenting with hypertension in the first week of disease therapy were evaluated for renal parenchymal disorder and glomerular filtration rate abnormality in the follow-up period. Glomerular filtration rate abnormality was found in 1 and renal scintigraphic dimercaptosuccinic acid abnormalities (reduced uptake and dilated hypoactivity) were found in 4 patients. Hypertension was also found to be a risk factor for renal parenchymal disorder in the follow-up period.

Obligatory Role for Interleukin-13 in Obstructive Lesion Development in Airway Allografts

The pathogenesis of bronchiolitis obliterans (BO), a common and devastating obliterative disorder of small airways following lung transplantation, remains poorly understood. Lesions are characterized in their early stages by lymphocyte influx that evolves into dense fibrotic infiltrates. Airway specimens taken from patients with histological BO revealed infiltrating myofibroblasts, which strongly expressed the signaling chain of the high affinity interleukin-13 (IL-13) receptor IL-13Ralpha1. Because IL-13 has proinflammatory and profibrotic actions, a contributory role for IL-13 in BO development was examined using murine models of orthotopic and heterotopic tracheal transplantation. Compared with airway isografts, allografts exhibited a significant increase in relative IL-13 mRNA and protein levels. Allogeneic tracheas transplanted into IL-13-deficient mice were protected from BO in both transplant models. Flow cytometric analysis of orthotopic transplant tissue digests revealed markedly fewer infiltrating mononuclear phagocytes and CD3(+) T lymphocytes in IL-13-deficient recipients. Furthermore, protection from luminal obliteration, collagen deposition, and myofibroblast infiltration was observed in heterotopic airways transplanted into the IL-13(-/-) recipients. Transforming growth factor-beta1 expression was significantly decreased in tracheal allografts into IL-13(-/-) recipients, compared to wild-type counterparts. These human and murine data implicate IL-13 as a critical effector cytokine driving cellular recruitment and subsequent fibrosis in clinical and ex-perimental BO.

Panning of multiple subsets of leukocytes on antibody-decorated poly(ethylene) glycol-coated glass slides

The antibody (Ab) array format provides a unique opportunity to pan and characterize multiple leukocyte subsets in parallel. However, the questions of reproducibility and robustness of leukocyte panning on Ab arrays need to be answered for this technology to become an immunophenotyping tool. The present study sought to address several of these questions, including: (1) purity of leukocyte subsets captured on Ab regions, (2) dynamics of leukocyte binding, (3) elimination of non-specific cell adhesion, and (4) standardization of cell washing conditions. Abs for CD4 T-cells, CD8 T-cells, CD36 monocytes, and CD16b neutrophils were dispensed onto standard glass slides containing a thin film of poly(ethylene glycol) (PEG) hydrogel. PEG gel coating was highly effective in eliminated non-specific cell adhesion on the surface. Incubation of the Ab arrays with red blood cell (RBC) depleted whole blood resulting in antigen-specific panning of leukocyte subsets on the respective Ab domains. A flow through chamber was employed to determine optimal shear stress conditions for removal of non-specifically attached cells. The purity of the four subsets remaining on the surface after washing was determined by Wright staining and immunofluorescence, and was found to be as follows: CD4 T-cells (99.2 ± 0.3%), CD8 T-cells (98.7 ± 0.3%), CD36 monocytes (97.2 ± 0.9%), and CD16b neutrophils (99.1 ± 0.6%). In conclusion, the methods described in this study allow to separate whole blood into pure leukocyte subsets with minimal sample preparation and handling. These approaches will be valuable in the future development of Ab arrays as tools for quantitative immunophenotyping of leukocytes.

A combined HIV‐1 protein bead array for serology assay and T‐cell subset immunophenotyping with a hybrid flow cytometer: A step in the direction of a comprehensive multitasking instrument platform for infectious disease diagnosis and monitoring

A new generation of bench-top flow cytometers with digital signal processing to perform suspension array technology (SAT) based bead array assays as well as leukocyte immunophenotyping is now available. These hybrid instruments provide an opportunity for the development of a more cost effective multitasking platform to support infectious disease treatment in resource limited countries. We report the development and testing of two modules compatible with the hybrid flow cytometers. The first module is an eleven HIV-1 protein bead array (PBA) for the detection of circulating antibodies and the second is a cell based T-cell enumeration assay. The HIV-1 PBA was tested in parallel with two enzyme immunoassays (EIAs) for the detection of plasma antibodies from 4 HIV-1 seroconversion panels and a low antibody titer panel. The PBA as well as the two EIAs performed equally for the detection of antibody positive samples from all seroconversion panels. One antibody positive sample from the low antibody titer panel was missed by the PBA together with one of the two EIAs tested. A parallel analysis of the HIV-1 PBA with Western blot (a confirmatory test for HIV infection) using plasma from nine HIV-1(+) individuals showed that the HIV-1 PBA detected more of the gp41 and gp120 antibody positive samples. Preliminary CD4 T-cell immunophenotyping results from 14 HIV(+) and 10 HIV(-) whole blood specimens with the hybrid flow cytometer platform compared well to conventional flow cytometry data. The successful combination of bead and cell based assays on a single hybrid instrument demonstrated the potential utility of a multitasking platform. The results presented are providing groundwork for future development of more cost effective modular architecture for a flexible flow cytometry based platform.

Comparison of immunophenotyping by slide-based cytometry and by flow cytometry

Background Flow cytometry (FCM) is the gold standard for immunophenotyping of peripheral blood leukocytes (PBLs). Slide-based cytometry (SBC) systems, for example the laser scanning cytometer (LSC®, CompuCyte), can give additional information (repeated staining and scanning, morphology). In order to adequately judge the clinical usefulness of LSC for immunophenotyping it is obligatory to compare it with FCM. Aim The aim of this study was to systematically compare immunophenotyping by both FCM and LSC methods and to test the correlation of the results. Methods PBLs were stained with directly labeled monoclonal antibodies with the whole blood staining method. Aliquots of the same paraformaldehyde fixed specimens were analyzed in parallel by a FACScan (BD-Biosciences) using standard protocols and by LSC with different triggers (forward scatter, CD45 FITC, or 7-AAD). For 7-AAD measurements by LSC, slides were additionally fixed with acetone before 7-AAD staining. Results Calculating the percentage distribution of PBLs obtained by LSC and by FCM showed very good correlation with regression coefficients close to 1.0 for the major populations and the lymphocyte sub-populations (neutrophils, monocytes, and lymphocytes; T-helper-, T-cytotoxic-, B-, NK-cells). The best trigger for LSC was 7-AAD. Conclusion LSC can be recommended for immunophenotyping of PBLs especially in cases where only limited sample volumes are available or where additional analysis of the cells' morphology is important. The detection of rare leukocytes or weak antigens is limited; in these cases appropriate amplification steps for immunofluorescence should be engaged.

Iterative restaining as a pivotal tool for n‐color immunophenotyping by slide‐based cytometry

Slide-based cytometry (SBC) allows to "ask a cell a second time." We used this tool for detailed immunophenotyping of peripheral blood leukocytes (PBLs). PBLs primarily stained for CD-markers and DNA were immobilized on a glass slide and analyzed by laser scanning cytometry. Then, iterative restaining was applied for a second and a third analysis. Based on the cells' fixed location, analyses were merged on a single-cell level. We analyzed six virtual immunostainings by "recycling" the PE-channel for four CD-markers. Iterative restaining might prove to be a pivotal tool for n-color immunophenotyping exclusive to SBC concepts.

Aflatoxin B1 albumin adduct levels and cellular immune status in Ghanaians

Although aflatoxins (AFs) have been shown to be immune-suppressive agents in animals, the potential role of AFs in modifying the distribution and function of leukocyte subsets in humans has never been assessed. We examined the cellular immune status of 64 Ghanaians in relation to levels of aflatoxin B1 (AFB1)-albumin adducts in plasma. The percentages of leukocyte immunophenotypes in peripheral blood, CD4+ T cell proliferative response, CD4+ T(h) and CD8+ T cell cytokine profiles and monocyte phagocytic activity were measured using flow cytometry. NK cell cytotoxic function was determined by perforin and tumor necrosis factor-alpha expression in CD3-CD56+ NK cells. AFB1-albumin adducts levels ranged from 0.3325 to 2.2703 (mean = 0.9972 +/- 0.40, median = 0.9068) pmol mg(-1) albumin. Study participants with high AFB1 levels had significantly lower percentages of CD3+ and CD19+ cells that showed the CD69+ activation marker (CD3+CD69+ and CD19+CD69+) than participants with low AFB1 levels (P = 0.002 for both). Also, the percentages of CD8+ T cells that contained perforin or both perforin and granzyme A were significantly lower in participants with high AFB1 levels compared with those with low AFB1 (P = 0.012 for both). Low levels of CD3+CD69+ (r = -0.32, P = 0.016) and CD19+CD69+ (r = -0.334, P = 0.010) cells were significantly associated with high AFB1 levels using correlation analysis. By multivariate analysis, there were strong negative correlations between the percentages of these cells (CD3+CD69+: b = -0.574, P = 0.001, and CD19+CD69+: b = -0.330, P = 0.032) and AFB1 levels. These alterations in immunological parameters in participants with high AFB1 levels could result in impairments in cellular immunity that could decrease host resistance to infections.

Reduced gravity evaluation of potential spaceflight‐compatible flow cytometer technology

The presence of a spaceflight-compatible flow cytometer onboard the International Space Station would be extremely beneficial for in-flight medicine and reduced gravity research. In the past, commercially available flow cytometers were not suitable for use during spaceflight due to their size, weight, power, and sheath fluid requirements. Recent advances in cytometer technology have enabled features that now make a spaceflight-compatible cytometer possible. We evaluated a small, robust novel cytometer with design features that minimize or eliminate many incompatibilities with spaceflight. This cytometer is highly miniaturized and lightweight, does not require sheath fluid, and uses a low-energy diode laser. The ability to achieve laminar particle flow without sheath fluid is important because an instrument using large liquid volumes and producing an equivalent amount of hazardous liquid waste would not meet spaceflight constraints. For this study the cytometer was modified so that stained liquid cell samples could be delivered during reduced gravity. The cytometer was then evaluated onboard the NASA KC-135 reduced gravity research aircraft. The KC-135 uses a parabolic flight path to generate essentially zero gravity conditions for 30-s increments. Typically 40 parabolas are flown per mission, resulting in approximately 20 min of reduced gravity during which research may be performed. During this evaluation, bead-based cytometer precision, photomultiplier tube linearity, and leukocyte immunophenotype analysis were performed during reduced gravity. The flight data were then compared with ground-based control data and data generated using a reference cytometer (Beckman-Coulter XL). This novel cytometer functioned well during reduced gravity and produced data comparable to those of ground-based controls with only minor caveats. The reduced gravity cell immunophenotype data were indistinguishable from ground control data and reference cytometric data. Bead-based instrument precision (coefficient of variation) was slightly increased during reduced gravity operation, but not to a degree that would affect most common flow cytometric applications. The ability of the instrument to collect absolute cell counts was validated. This study represents the first generation of real-time flow cytometry data during zero gravity. With modifications, the evaluated cytometer technology could be the basis from which an operational spaceflight-compatible flow cytometer is developed.