Whole Blood Staining Research Articles

A 47-marker immune profiling flow cytometry assay to enable comprehensive antigen-specific immune analysis

Abstract The complex nature of the immune system requires deep interrogation at the single-cell level. Flow cytometry using CyTOF® technology employs antibodies tagged with isotopically enriched metals instead of fluorophores, resulting in discrete signals that facilitate highly multiparametric characterization on a single-cell level. CyTOF technology enables analysis of over 50 parameters simultaneously without the impediments of spectral overlap or autofluorescence, allowing for a reliable evaluation of immune responses. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated, dried-down, and validated 30-marker antibody cocktail for single-tube staining of human whole blood or PBMC and acquisition on CyTOF systems. Using the assay and Maxpar Pathsetter™ software, stained samples are automatically resolved into 37 immune populations. In this study, we highlight the customizability of the Maxpar® Direct panel by adding an array of 17 surface and intracellular parameters with the Maxpar Direct™ T Cell Activation Expansion Panel and live-cell barcoding. The simultaneous detection of key surface and intracellular markers in this expanded panel enabled comprehensive analysis of immune cell activation and antigen-specific recall responses to PBMC stimulated with a pool of immunodominant microbial peptides. Taken together, this 47-marker panel and automated analysis unlocked deeper immunophenotyping and functional profiling of activated immune cells and provided insight into the cell-mediated adaptive immune response to foreign targets. Such phenomena are hallmarks for research on infection, vaccine development, and immunotherapy. For Research Use Only. Not for use in diagnostic procedures.

Changes in mononuclear cell subsets in capillary and venous blood of patients with psoriasis depending on the treatment

Topical glucocorticoids are conventionally used to treat psoriasis, but such treatment provides a short-term effect, and may cause various complications during long-term usage. A detailed study of the immunopathogenesis of psoriasis has made it possible to use bioengineered drugs that block the main cytokines. It has been shown that IL-36 plays an important regulatory role in pathogenesis of psoriasis. The aim of the study was to study therapeutic effect of patients with psoriasis using topical glucocorticoid hormone versus IL- 36 receptor antagonist (RAIL-36), with respect to clinical course of psoriasis and the subsets of mononuclear cells in venous and capillary blood taken close to the focus of inflammation. 16 patients with psoriasis (group 1a) received 0.1% mometasone cream for 14 days; 20 patients of group 1b received a gel containing 0.4% recombinant RAIL-36 for 14 days. Control group included 20 healthy adults. Treatment efficacy was assessed by PASI, DISHS and DLQI indices. 19 lymphocyte subsets and 3 monocyte subsets were assessed by four-color staining of whole capillary and venous blood with erythrocyte lysis using BD Biosciences (USA) technologies and reagents. It was shown that both drugs led to a decrease in the severity of the disease at the end of treatment. However, 2 weeks after the end of treatment in group 1a, the disease indexes nearly returned to the initial values. Meanwhile, the reduced index levels persisted 2 weeks later in group 1b. Significant deviations (more pronounced in capillary blood) were revealed for the levels of several leukocyte subsets in the psoriasis patients compared with healthy persons. As a result of treatment, we have revealed some changes in the levels of leukocyte subsets common to the two groups, and special differences for the two treatment options, that were more pronounced in capillary blood samples. Both medical preparations used are suitable for treatment of psoriasis.

Mass Cytometry Analysis of Whole Blood Response to an Allergen.

Mass cytometry allows for the use of highly multiplexed antibody panels due to the lack of spill-over between channels detected by mass spectrometry. An advantage over fluorescence cytometry is the relative lack of background, which provides excellent resolution for detection of phosphoproteins and quantification of cell signaling. We have applied mass cytometry to the analysis of whole blood staining after ex vivo stimulation with peanut allergen (Tordesillas et al., J Allergy Clin Immunol 138:1741-4.e9, 2016). This allows for high-dimensional analysis of basophil activation, and analysis of the entire composition of the blood compartment in response to allergen exposure. Here, we describe our optimized protocol for activation and staining of whole blood for mass cytometry analysis that is currently in use in multicenter clinical trials. The protocol can be easily adopted to analyze blood leukocytes in other diseases, including asthma.

Mononuclear subsets and cytokine profile of venous and capillary blood in patients with psoriasis and healthy people

Psoriasis is considered an autoimmune disease with a predominantly cellular mechanism for the development of disorder. Studies in immune pathogenesis of psoriasis were performed either in animal model, which is not just similar to humans, or the data were obtained in patients by means of skin window method, which is traumatic, or by examining venous blood. However, it is difficult to discern parameters of the local immune response in venous blood samples. We have attempted to find an adequate method which would be convenient both for the patient and for the researcher, in order to assess local immune processes occurring in the skin affected by psoriasis. We examined 20 patients with a verified diagnosis of psoriasis, the average age was 44.3 years. The control group included 15 healthy adults, with average age of 46.6 years. Capillary blood was taken by fingerprick, whereas, in psoriatic patients, the samples were taken near the psoriatic lesion at a final volume of 400 μL in two microvettes. Venous blood (3 mL) was taken from the cubital vein into a vacuum tube with EDTA. Clinical analysis of venous and capillary blood was performed in automated hematological analyzer. Immunophenotyping was performed by four-color staining of whole capillary and venous blood followed by lysis of erythrocytes. Cytofluorometry was performed using techniques and reagents from BD Biosciences (USA). Plasma cytokines were determined by multiplex approach (MagPix, BioRad, USA). Upon clinical analysis of blood, the difference between capillary and venous blood was not found, either in healthy group, or among patients with psoriasis. In healthy people, the subsets of mononuclear cells, did not differ between venous and capillary blood. The samples of capillary and venous blood in the patients with psoriasis showed significantly increased levels of double-positive lymphocytes (CD45RA+/CD45R0+), B lymphocytes and NKT lymphocytes (both for relative and and absolute values). A significant increase in the percentage of naive T lymphocytes, activated helpers (Thact) and Treg, as well as B1 cells and Breg, and a significant decrease in B2 lymphocytes was registered in capillary blood of the patients with psoriasis. In venous blood samples from psoriatic patients, only a significant increase in Thact, Treg, and Breg was revealed. In the capillary blood of patients with psoriasis, we found a significant increase in the levels of non-classical M2 monocytes and inflammatory Minfl monocytes, and a decrease in classical M1 monocyte levels; in venous blood of psoriatic patients, only an increase in inflammatory Minfl monocytes was revealed. In capillary blood, all the studied cytokines in psoriasis patients significantly exceeded the levels of corresponding cytokines in healthy controls, except of IL-10. The levels of this cytokine did not differ from healthy group. In venous blood, the levels of most studied cytokines in the group of patients with psoriasis did not differ from the group of healthy ones. Approximately two-fold increase was revealed for IL-4, IL-21, IL-23 and TNF. First, the subsets of mononuclear cells and the cytokine profile of capillary and venous blood of healthy people did not differ significantly. Secondly, our proposed method for determining the subsets of mononuclear cells and capillary blood cytokines profile from the area of psoriatic lesions may be used to monitor local immunity in the patients with psoriasis. This approach is significantly less traumatic than the skin window method and more informative than the studies of venous blood.

BIOLOGICAL AGING IS ASSOCIATED WITH INCREASED MONOCYTE INFLAMMATORY ACTIVITY IN OLDER ADULTS

Chronic inflammation is thought to play a central role in biological aging. However, the causes of chronic inflammation are not fully elucidated. We hypothesized that a dysregulation in monocyte inflammatory activity may contribute to chronic inflammation and biological aging. There are no validated methods for Biological Age (BA) estimation. Therefore, we also hypothesized that older adults with a recent ED (Emergency Department) admission had a higher BA compared to age-matched older adults without a recent ED admission. Two groups of older adults were enrolled: a “high BA”-group who were discharged from the ED four weeks preceding data collection (n=52), and a “low BA”-group consisting of age and sex matched participants without ED admission within the two years preceding data collection (n=52). We assessed NF-κB phosphorylation (Ser529) and NLRP3 inflammasome levels in monocytes using flow cytometry staining of whole blood. Preliminary analyses showed that participants had a median age of 74.8 (IQR: 70.7–82.0) years, 48% were women. Participants in the high-BA group had reduced lower body strength (30 seconds chair stand test p=0.02 and 4 meters gait speed p=0.001) and cognitive function (Digit Symbol Substitution Test p=0.001 and Trail Making Test p=0.002) compared to the low-BA group. Monocytes of participants in the low BA group had lower constitutive p-NF-κB (p< 0.0001) and NLRP3 (p=0.0001) median fluorescence intensity compared to the high BA group. Increased monocyte inflammatory activity assessed by p-NF-κB and NLRP3 was associated with a higher BA. We will investigate associations between monocyte inflammatory activity and markers of chronic inflammation.

New laboratory parameters in the diagnosis of acute mesenteric ischemia.

Lack of clear clinical and laboratory picture of subjective evaluation of bowel viability, the progression of bowel necrosis in the postoperative period with acute mesenteric ischemia (АМI) contributes to the high mortality rate. Earlier experiments we proved that gut necrosis leads to changes in the subpopulation structure of blood lymphocytes. This prompted us to determine the clinical significance of the subpopulation structure of venous blood lymphocytes in patients with acute mesenteric ischemia. The paper is based on a retrospective analysis of the results of a controlled clinical and immunological examination of 18 patients aged 62 to 78 years (control group and a group of АМI). Evaluation lymphocyte subpopulation structure was performed by the standard method of direct immunofluorescence staining of whole blood. The obtained data were processed with nonparametric statistical methods. Study of lymphocyte subpopulation structure in patients with АМI patients showed a decrease in the absolute and relative number of CD8, CD4, B, NK cells on the indicators in the control group. Ischemia and necrosis of the intestinal mucosa accompanied by a massive translocation of intestinal microflora through the impaired intestinal barrier along with the migration of lymphocytes into the lesion and death, which is manifested in a decrease in the number of lymphocytes of the peripheral blood. Comprehensive assessment of venous blood lymphocyte subpopulation structure can be used as an additional diagnostic criterion necrotic step АМI, serve as criteria for selection of patients for immunotherapy.

Flow Cytometry Psychomotor Exercise in Medical Laboratory Science Curriculum Facilitates Correct Interpretation of Lymphocyte Subset Analysis

Abstract Objectives To effectively include flow cytometry psychomotor objectives in medical laboratory science curriculum as recommended in the entry-level curriculum for MLS published by the American Society for Clinical Laboratory Science. Until fall 2018, our university-based MLS program’s coursework included one to two lectures by the FC operator from the local hospital, tour of their facility, interpretation of cases using histograms, and occasional observations of analyses during clinical practicum. Hands-on performance by 18 to 20 students annually was not possible. We secured an ASCP Foundation Laboratory Science Program Educational Grant for faculty training in flow cytometry and reached out to flow cytometry core facility on the university campus for permission to use the facility with supervision and at a discounted rate. Methods Each group of three to four students spent 120 to 160 minutes at the facility, including instruction by the manager, hands-on contact running the instrument by each student, and observation of other classmates. The activity included whole-blood antibody staining prior entry to the facility and CD3/CD4/CD8 lymphocyte subset analysis using MACSQuant Analyzer 10 (Miltenyi Biotec) to distinguish normal versus abnormal control and to infer the status of an assigned deidentified patient with prior HIV-1 infection (University Biosafety IBC# 18–021). Following the experience, students completed online homework, including multiple-choice and open-ended questions, which required interpretation of images recorded during the exercise. Results Blackboard test item analysis revealed that median percent of students correctly answering each question was 88 (n = 17). Students received individual feedback on each missed question. On the final Immunology exam, 100% of students provided correct interpretation (active infection or infection under control/normal result). Conclusion The results indicated good comprehension of lymphocyte subset analysis concepts. Additional exercise in immunophenotyping is under way in the Hematology class. We believe that inclusion of the flow cytometry psychomotor objectives in the curriculum will improve the students’ preparation for practice.

AGE-DEPENDENT CHANGES OF T-REGULATORY AND Th17 SUBSET LEVELS IN PERIPHERAL BLOOD FROM HEALTHY HUMANS

Age-dependent development of Th17 and Treg lymphocyte subsets in healthy humans is studied insufficiently. The present study aimed to investigate quantitative characteristics of Th17 and Treg subsets in peripheral blood of healthy subjects for various age groups. 352 healthy humans (168 female and 184 male), one month to 85 years old, were subject to examination, including 79 infants in their first year of life; 34 children at aged 1 year to 2 years 11 months; 24, at 3 to 4 years 11 months; 28, at the age of 5 to 6 years 11 months; 25 children aged 7-8 years 11 month; 36 children aged 9 to 11 years 11 months; 39 adolescents aged 12 to 14 years 11 months; 26 adolescents aged 15 to 18 years; 25 young adults aged 20 to 35 years; 11 adults at 36 to 49 years old; 16 adults aged 59-70 years, and 9 elderly people over 70 years old. The study was performed with capillary blood in children under 2 years, and venous blood taken in elder persons. The basic and ‘minor’ subsets of peripheral blood lymphocytes were evaluated by flow cytometry using four-color staining of whole blood and following erythrocyte lysis. We used the following surface markers: CD3, CD4, CD8, CD25, CD127, CD161, CD45R0 for lymphocyte subsets detection. It has been shown that Treg percentage (a ratio of CD4 + CD25 hi CD127 low/neg in the CD3 + CD4 + gate) did not depend on age of the people under study, and can be approximated by a linear function. The absolute number of Tregs in childhood is progressively decreased and, after 10 years old, it reaches plateau values. This age-dependent relationship may be approximated by a logarithmic function. Evaluation of Th17 subset levels demonstrated a strong relative and absolute age-dependent growth of this cell subpopulation. Percentage and absolute numbers of Th17 lymphocyte (share of CD4 + CD161 + CD45R0 + in the CD3 + CD4 + gate), can be approximated by a square function. The age of 10-12 years seems to be critical to the immune system formation. We suppose the process of the immune system development to be completed at this age, and maturation of the immune cell populations is then observed. A decrease in both relative and absolute numbers of Treg and Th17 lymphocyte subsets was found in elderly persons (> 70 years old). Our data on peripheral blood Tregs and Th17 subsets, with respect to their percentage and absolute numbers in healthy humans, may be used as age-related reference values.

The effects of hydrogen sulfide on platelet–leukocyte aggregation and microvascular thrombolysis

The volatile transmitter hydrogen sulfide (H2S) is known for its various functions in vascular biology. This study evaluates the effect of the H2S-donor GYY4137 (GYY) on thrombus stability and microvascular thrombolysis. Human whole blood served for all in vitro studies and was analyzed in a resting state, after stimulation with thrombin-receptor activating peptide (TRAP) and after incubation with 10 or 30 mM GYY or its vehicle DMSO following TRAP-activation, respectively. As a marker for thrombus stability, platelet–leukocyte aggregation was assessed using flow cytometry after staining of human whole blood against CD62P and CD45, respectively. Furthermore, morphology and quantity of platelet–leukocyte aggregation were studied by means of scanning electron microscopy (scanning EM). Therefore, platelets were stained for CD62P followed by immuno gold labeling. In vivo, the dorsal skinfold chamber preparation was performed for light/dye induction of thrombi in arterioles and venules using intravital fluorescence microscopy. Thrombolysis was assessed 10 and 22 h after thrombus induction and treatment with the vehicle, GYY, or recombinant tissue plasminogen activator (rtPA).Flow cytometry revealed an increase of CD62P/CD45 positive aggregates after TRAP stimulation of human whole blood, which was significantly reduced by preincubation with 30 mM GYY. Scanning EM additionally showed a reduced platelet–leukocyte aggregation and a decreased leukocyte count within the aggregates after preincubation with GYY compared to TRAP stimulation alone. Further on, morphological signs of platelet activation were found markedly reduced upon treatment with GYY. In mice, both GYY and rtPA significantly accelerated arteriolar and venular thrombolysis compared to the vehicle control. In conclusion, GYY impairs thrombus stability by reducing platelet–leukocyte aggregation and thereby facilitates endogenous thrombolysis.

Chronic mucocutaneous candidiasis: characterization of a family with STAT-1 gain-of-function and development of an ex-vivo assay for Th17 deficiency of diagnostic utility.

SummaryChronic mucocutaneous candidiasis (CMC) is characterized by recurrent and persistent superficial infections, with Candida albicans affecting the mucous membranes, skin and nails. It can be acquired or caused by primary immune deficiencies, particularly those that impair interleukin (IL)−17 and IL‐22 immunity. We describe a single kindred with CMC and the identification of a STAT1 GOF mutation by whole exome sequencing (WES). We show how detailed clinical and immunological phenotyping of this family in the context of WES has enabled revision of disease status and clinical management. Together with analysis of other CMC cases within our cohort of patients, we used knowledge arising from the characterization of this family to develop a rapid ex‐vivo screening assay for the detection of T helper type 17 (Th17) deficiency better suited to the routine diagnostic setting than established in‐vitro techniques, such as intracellular cytokine staining and enzyme‐linked immunosorbent assay (ELISA) using cell culture supernatants. We demonstrate that cell surface staining of unstimulated whole blood for CCR6+CXCR3–CCR4+CD161+ T helper cells generates results that correlate with intracellular cytokine staining for IL‐17A, and is able to discriminate between patients with molecularly defined CMC and healthy controls with 100% sensitivity and specificity within the cohort tested. Furthermore, removal of CCR4 and CD161 from the antibody staining panel did not affect assay performance, suggesting that the enumeration of CCR6+CXCR3–CD4+ T cells is sufficient for screening for Th17 deficiency in patients with CMC and could be used to guide further investigation aimed at identifying the underlying molecular cause.

Characterizing the hematopoietic stem/progenitor cells in the placenta and umbilical cord blood from normal and preeclamptic subjects

Homocysteine lowering with folic acid has been shown to be ineffective in reducing vascular events in patients with established cardiovascular disease. However, it may be beneficial in the early stages of atherosclerosis particularly for the population at risk. We have investigated the effect of folic acid administration on homocysteine levels, endothelial function, and leukocyte adhesion molecules in atorvastatin-treated patients. Thirty-nine patients were randomized to folic acid 5 mg daily or no additional treatment. Plasma total homocysteine levels were determined by high-performance liquid chromatography. Biochemical and hematologic tests were performed using routine automated analyzer methods. We examined the expression of l-selectin (CD62L); α-subunits of integrins LFA-1 (CD11a), Mac-1 (CD11b), and VLA-4 (CD49d); and β-subunit of β-2 integrins (CD18) by an immunofluorescence method using single-step staining of whole blood with monoclonal antibodies. The fluorescence was quantified by flow cytometry. Reactivity of the forearm skin microvascular bed to thermal and ischemic stimuli was measured using laser Doppler flowmetry. Postocclusive and thermal reactive hyperemia tests were performed. Statistical analysis used 2-sample t test and paired t test. Homocysteine levels decreased significantly in the folic acid–treated group (11.78 ± 2.7 vs 9.75 ± 1.89 μmol/L, P < .0001) but did not change in the control group (11.17 ± 2.09 vs 11.88 ± 2.88 μmol/L). There was no effect of folic acid treatment on leukocyte expression of cell adhesion molecules or microvascular reactivity. The lack of effect of folic acid on microvascular reactivity and leukocyte adhesion molecules supports the evidence against the beneficial effects of folic acid administration in the early stages of atherogenesis.

Vaccination with Irradiated Autologous Tumor Cells Mixed with Irradiated GM-K562 Cells Provokes Lymphocyte Activation and Specific Antitumor Immunity in Patients with Recurrent Glioblastoma

We have previously reported the safety and feasibility of vaccinating recurrent malignant glioma patients with irradiated autologous cells mixed with irradiated GM-K562 cells. Here, we report the impact of vaccination on the expression of T-lymphocyte co-stimulatory and co-inbhibitory molecules as well as on regulatory T-lymphocytes. Furthermore, we explored the impact of vaccination on patient humoral immune responses with particular attention to antibody titers against molecules associated with tumor angiogenesis. Blood was collected from 9 of 10 treated patients at regular intervals and submitted for immune analysis. 5-color flow cytometry was employed after staining of whole blood by an immunophenotyping antibody panel. Antibody titers to tumor-associated antigens were measured by ELISA of patient plasma - all assays were performed in duplicate. Treatment-associated change in percent expression of various markers was analyzed via the non-parametric sign-rank test. p < 0.05 was considered statistically significant. In patients undergoing craniotomy for recurrent malignant glioma, vaccination with irradiated autologous tumor cells and irradiated GM-K562 cells was associated with statistically significant increases in CD4+ T-lymphocyte expression of costimulatory molecules OX40 and 4-1BB (CD137). CD8+ T-lymphocytes saw increased expression of 4-1BB as well. Likewise, there were increases in CD4+ T-lymphocyte expression of CTLA-4, PD1, and FOXP3 - each of which is associated with negative immune regulation and are targetable with existing monoclonal antibodies. 7 of 9 patients had marked treatment-driven increases in antibody titers to Angiopoietin 2, which may represent a novel glioma vasculature-associated antigen. Vaccination with irradiated autologous tumor cells mixed with irradiated GM-K562 cells in patients with recurrent malignant glioma is biologically active and activates both T-lymphoctes and humoral antitumor immunity. Vaccine-induced expression of costimulatory molecules may provide the rationale for combination therapies with either co-stimulatory receptor agonist or co-inhibitory receptor antagonist molecules.

Increased Plasma Levels of Soluble CD27 among HIV/HCV Co-infected and HIV/HCV/GBV-C Triply Infected Subjects.

CD27 is a biomarker associated with both T-cells and B-cells activation .Plasma soluble CD27 (sCD27) was identified as a marker of disease outcome in Human Immunodeficiency Virus (HIV) infection .Testing of plasma sCD27 represents a good tool to monitor the change of immune activation during HIV infection.We sought to analyses role of Hepatitis C Virus (HCV) and also GB Virus type C (GBV-C) co-infections on HIV-related immune activation, through measuring sCD27 plasma levels.Blood samples from a total of 86 patients with HIV infection were taken. Plasmas were analyzed for HCV using serologic test and GBV-C by reverse transcriptase polymerase chain reaction (RT-PCR). CD4+ and CD8+T-cell counts were evaluated by CD3/CD4+ and CD3/CD8+ double staining of whole blood followed by flow cytometric analysis .Then Cross-sectional comparison of sCD27 plasma levels was carried out among patients : HIV (n=20), HIV/ GBV-C (n=14), HIV/ (HCV) (n=26) and HIV/HCV/GBV-C (n=26).Plasma level of sCD27 was higher in HIV/HCV/GBV-C patients as compared to HIV mono-infected patients (p= 0.006) and based on results there was significant differences in the plasma levels of sCD27 between HIV-infected individuals with and without HCV coinfection (P=0.017) and also correlation between sCD27 and percent of CD4+T-cells was in highest level among HIV/HCV co-infected patients group [r= -0.59 (p=0.001)]. High levels of sCD27 among HIV/HCV patients argues in favor of sCD27 plasma level determination for monitoring of clinical features among HIV/HCV coinfected patients.

A Statistical Interaction between Circumsporozoite Protein-Specific T Cell and Antibody Responses and Risk of Clinical Malaria Episodes following Vaccination with RTS,S/AS01E

The candidate malaria vaccine RTS,S/AS01E provides significant but partial protection from clinical malaria. On in vitro circumsporozoite protein (CSP) peptide stimulation and intra-cellular cytokine staining of whole blood taken from 407 5–17 month-old children in a phase IIb trial of RTS,S/AS01E, we identified significantly increased frequencies of two CSP-specific CD4+ T cells phenotypes among RTS,S/AS01E vaccinees (IFNγ-IL2+TNF− and IFNγ-IL2+TNF+ CD4+ T cells), and increased frequency of IFNγ-IL2-TNF+ CD4+ T cells after natural exposure. All these T cells phenotypes were individually associated with reductions in the risk of clinical malaria, but IFNγ-IL2-TNF+ CD4+ T cells independently predicted reduced risk of clinical malaria on multi-variable analysis (HR = 0.29, 95% confidence intervals 0.15–0.54, p<0.0005). Furthermore, there was a strongly significant synergistic interaction between CSP-specific IFNγ-IL2-TNF+ CD4+ T cells and anti-CSP antibodies in determining protection against clinical malaria (p = 0.002). Vaccination strategies that combine potent cellular and antibody responses may enhance protection against malaria.

Association of an increased frequency of CD14+HLA‐DRlo/neg monocytes with decreased time to progression in chronic lymphocytic leukaemia (CLL)

Association of an increased frequency of CD14⁺HLA‐DR^lo/neg monocytes with decreased time to progression in chronic lymphocytic leukaemia (CLL)

Use of Whole-Blood Samples in In-House Bulk and Single-Cell Antigen-Specific Gamma Interferon Assays for Surveillance of Mycobacterium tuberculosis Infections

Tests based on the gamma interferon (IFN-gamma) assay (IGA) are used as adjunctive tools for the diagnosis of Mycobacterium tuberculosis infection. Here we compared in-house and commercial whole-blood IGAs to identify a suitable assay for the surveillance of tuberculosis in population studies. The IGAs were selected on the basis of the ease with which they are performed and because they require a small amount of a biological sample and do not require cell purification. Since a "gold standard" for latently M. tuberculosis-infected individuals is not available, the sensitivities and the specificities of the IGAs were determined with samples from patients with clinically diagnosed active tuberculosis and in Mycobacterium bovis BCG-unvaccinated healthy controls. The in-house tests consisted of a bulk assay based on diluted whole blood and a single-cell assay based on IFN-gamma intracellular staining. The commercial assays used were the QuantiFERON-TB-Gold (Q-TB) and the Q-TB in-tube tests. When the purified protein derivative was used as the antigen, in-house whole-blood intracellular staining was found to be highly discriminatory between active tuberculosis patients and BCG-vaccinated healthy controls, whereas the other IGAs did not discriminate between the two categories of patients. When M. tuberculosis-specific antigens were used, a very strong agreement between the results of the Q-TB in-tube assay and the clinical diagnosis was observed, while the Q-TB assay, performed according to the manufacturer's instructions, showed a significantly lower performance. Intriguingly, when the test was performed with RD1 proteins instead of peptides, its sensitivity was significantly increased. The in-house assay with diluted whole blood showed an elevated sensitivity and an elevated specificity, and the results agreed with the clinical diagnosis. Considering that the in-house assay uses 1/20 of the sample compared with the amount of sample used in the commercial IGA, it appears to be particularly promising for use in pediatric studies. Overall, the different assays showed different performance characteristics that need to be considered for surveillance of tuberculosis in population studies.

Decreased Numbers of Tregs in Aplastic Anemia Is Detected by Immunohistochemistry and Flow Cytometry.

Idiopathic aplastic anemia (AA) is characterized by immune-mediated destruction of hematopoietic stem cells, leading to peripheral pancytopenia. Immune pathogenesis in AA is supported by experimental data, as well as clinical observations and may be related to the breach of peripheral or central tolerance. Regulatory T cells (Treg) constitute one of the most important mechanisms of central tolerance engaged in the down-modulation of autoreactive T cells. Tregs have been found to be reduced in several autoimmune diseases and decreased frequencies of Tregs were also reported in AA and MDS. Overexpression of the high affinity IL-2 receptor alpha chain (CD25) and the forkhead family transcription factor P3 (FoxP3), required for the development and function of Tregs, serve as phenotypic markers for Tregs. We investigated Treg levels in a cohort of AA patients (N=21) and healthy individuals (N=15); flow cytometric quantification of Treg was carried out after surface/intracellular staining of whole blood for Treg markers (CD3, CD4, CD25, FoxP3). After proper gating (light scatter properties, CD3, CD4, CD25), CD4+ T cells were subdivided into CD25−, CD25int and CD25hi populations, and the co-expression of CD25hi and Foxp3 was analyzed. In comparison to controls, AA patients (N=12) show not only lower frequencies of CD4+CD25hi+ T cells within the total lymphocyte population (median 0.07% vs. 0.21%; p=.03), but also absolute lower absolute numbers (1.31/uL vs. 5.78/uL, p=.0002). Similarly, CD4+CD25hi+FoxP3+ T cells were found to be depressed in untreated AA patients in comparison to controls (median 0.07% vs. 0.21% and 1.06/uL vs. 4.76/uL; p=.03 and p=.003). While Tregs were lower in patients with active disease unresponsive to immunosuppressive treatment (responder 0.1% vs non responder 0.07%, CD4+CD25hi Tcells, p=.02), serial testing performed in 6 patients treated with ATG/CsA did not reveal correlation between hematologic improvement and recovery of Treg numbers. When double immunohistochemical staining for CD3 and Foxp3 was performed in pre-treatment bone marrow core biopsies of AA patients (N=3) and controls (N=2) a mean of 3 CD3+Foxp3+ cells/10 high power fields (hpf) were counted (vs. mean 28/10 hpf, p<.05 in controls), suggesting that lower numbers of Tregs were also present in the bone marrow of AA patients. In conclusion, our results suggest that Tregs are decreased in blood and marrow of patients with idiopathic AA, consistent with the breach of peripheral tolerance in AA. In addition to flow cytometry, immunohistochemical staining of histologic specimens can be used for the quantitative analysis of Tregs in bone marrow failure syndromes and other immune-mediated conditions such as GvHD.

Folic acid does not improve surrogate markers of early atherosclerosis in atorvastatin-treated patients

Abstract Homocysteine lowering with folic acid has been shown to be ineffective in reducing vascular events in patients with established cardiovascular disease. However, it may be beneficial in the early stages of atherosclerosis particularly for the population at risk. We have investigated the effect of folic acid administration on homocysteine levels, endothelial function, and leukocyte adhesion molecules in atorvastatin-treated patients. Thirty-nine patients were randomized to folic acid 5 mg daily or no additional treatment. Plasma total homocysteine levels were determined by high-performance liquid chromatography. Biochemical and hematologic tests were performed using routine automated analyzer methods. We examined the expression of l-selectin (CD62L); α -subunits of integrins LFA-1 (CD11a), Mac-1 (CD11b), and VLA-4 (CD49d); and β -subunit of β -2 integrins (CD18) by an immunofluorescence method using single-step staining of whole blood with monoclonal antibodies. The fluorescence was quantified by flow cytometry. Reactivity of the forearm skin microvascular bed to thermal and ischemic stimuli was measured using laser Doppler flowmetry. Postocclusive and thermal reactive hyperemia tests were performed. Statistical analysis used 2-sample t test and paired t test. Homocysteine levels decreased significantly in the folic acid–treated group (11.78 ± 2.7 vs 9.75 ± 1.89 μ mol/L, P μ mol/L). There was no effect of folic acid treatment on leukocyte expression of cell adhesion molecules or microvascular reactivity. The lack of effect of folic acid on microvascular reactivity and leukocyte adhesion molecules supports the evidence against the beneficial effects of folic acid administration in the early stages of atherogenesis.

B‐cell antigen D8/17 is a marker of rheumatic fever susceptibility in Aboriginal Australians and can be tested in remote settings

To test the B-cell antigen D8/17 as a marker of past rheumatic fever (RF) in a predominantly Aboriginal Australian population, and to evaluate technical modifications to allow its use in remote settings. Cross-sectional survey in a remote Aboriginal community, a regional tertiary referral hospital and a tertiary paediatric centre in Melbourne. 106 people, including three with acute RF, 38 with a history of past RF, 20 relatives of these people, and 45 healthy controls. D8/17 expression in B cells. Blood was collected from each participant and the expression of D8/17 and CD19 in each sample was analysed by flow cytometry. The mean proportion of D8/17-positive B cells was 39.3% (SD, 11.8) in patients with previous RF, 22.5% (SD, 5.2) in first-degree relatives, 11.6% (SD, 7.2) in controls, and 83.7% (SD, 10.1) in patients with acute RF (analysis of variance test between means, P = 0.001). A cut-off of 22.1% of D8/17-positive B cells to indicate past RF yielded the highest percentage of correct results (95.4%). Delayed staining of whole blood (mean, 0.55 days; SD, 0.2) gave equivalent results to immediate staining, but the D8/17 assay on peripheral blood mononuclear cells was unreliable. The B-cell antigen D8/17 accurately identifies Australians with a past history of RF, and the assay is feasible in remote settings with access to facilities capable of performing D8/17 staining within half a day of sample collection.

Multiparameter Analysis of Intracellular Phosphoepitopes in Immunophenotyped Cell Populations by Flow Cytometry

This unit presents protocols for measuring intracellular phosphoepitopes by flow cytometry for biochemical investigations in both human and murine primary cells as well as in cell lines. Conventional methods that require cellular lysis cannot discriminate between proteins from different cellular subsets. Intracellular detection of phosphorylated and nonphosphorylated signaling molecules, on the other hand, has recently exposed the heterogeneity that can be observed upon signal transduction. Although staining methodologies for cytokines and cell cycle antigens have been appreciated for years, detection of phosphorylated molecules presents several new challenges, including generation of reagents and details of the staining technique. As these techniques are adapted to new applications, the protocols continue to be refined. This unit describes signal amplification of intracellular signals upon detergent-based permeabilizations, staining protocol for adherent cells, methodology for whole-blood staining, and multiparameter staining procedures for surface and intracellular antigens.