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PubMed2007

Influence of 4- and 6-color flow cytometers and acquisition/analysis softwares on the determination of lymphocyte subsets in HIV infection.

BACKGROUND AND OBJECTIVES: Lymphocyte immunophenotyping provides valuable information for the diagnosis and monitoring of patients with cellular immunodeficiencies, such as HIV/AIDS. In this study, we have assessed the influence of 4-color and 6-color flow cytometers, and respective analytical softwares on the enumeration of lymphocytes in HIV infected individuals. METHODS: The expression of various cell surface markers on lymphocytes was measured from the EDTA blood of 66 HIV infected patients on the FACSCalibur (4-color) and FACSCanto (6-color) flow cytometers. Percentage of lymphocytes expressing a particular cell surface marker was analyzed on FACSCalibur using the Cell Quest Pro software (v 5.2), while the analysis on FACSCanto was done using FACSCanto (v 1.0.3) and FACSDiva (v 4.1) softwares respectively. RESULTS: The data shows significantly higher mean CD3 T-cell counts on FACSCalibur, Cell Quest Pro (1,864 +/- 1,044 cells/microl) as compared to FACSCanto (1,840 +/- 1,040 cells/microl) (P < 0.05). The CD4 T-cell counts were also higher on FACSCalibur, Cell Quest Pro (885 +/- 770 cells/microl), and FACSDiva (892 +/- 773 cells/microl) versus FACSCanto (867 +/- 767 cells/microl) (P < 0.05). FACSCalibur, Cell Quest Pro, and FACSDiva showed similar values except for CD8 T-lymphocytes where FACSDiva had significantly lower values (P < 0.05). The B-cell counts were unaffected when either of the instruments or softwares were used, while the natural killer (NK) cells (CD16 + 56 positive cells) showed similar trend like CD3 and CD4 counts with significant differences in the mean cell counts between FACSCalibur, Cell Quest Pro (240 +/- 165 cells/microl), and FACSDiva (238 +/- 163 cells/microl) versus FACSCanto with higher NK cell counts (260 +/- 176 cells/microl). CONCLUSIONS: The enumeration of lymphocyte subsets was comparable between FACSCalibur, Cell Quest Pro, and FACSDiva, based analysis and it was significantly different than FACSCanto software based analysis. Our observations suggest that FACSDiva software should be preferred over the FACSCanto software for immunophenotyping on FACSCanto flow cytometer and the laboratories should report the instrument and software used for the specimen analysis while reporting immunophenotyping results.

Antigens, Surface↗
PubMed2002

T-lymphocyte subsets in patients with AJCC stage III gastric cancer during postoperative adjuvant chemotherapy. American Joint Committee on Cancer.

BACKGROUND AND AIMS: Advanced neoplastic diseases alter the immune response in cancer patients. The aim of this study was to evaluate the changes of T-lymphocyte subsets during postoperative adjuvant chemotherapy, and the relationship between T-lymphocyte subsets and tumor recurrence in AJCC stage III gastric cancers. MATERIAL AND METHODS: Analysis of T-lymphocyte subsets was performed in 39 patients with stage III gastric adenocarcinoma who had undergone a curative gastric resection and postoperative chemotherapy. CirculatingT-lymphocyte subsets were measured on venous blood by using flow cytometry and monoclonal antibodies on preoperative day 1, and postoperative months 1, 3, and 6. RESULTS: The 5-year disease-free survival rates of patients with stage 3a and 3b gastric cancer were 57.1% and 33.3%, respectively (p = 0.06). Values of CD3+ and CD4+ T-cells, and CD4+/CD8+ ratios were consistently lower in the recurrence group throughout the observation period. CD4+ T-cell counts were significantly lower in the recurrence group on preoperative day 1, and postoperative months 1 and 6. However, most values of the T-lymphocyte subsets showed no statistically significant difference when comparing the stage 3a and 3b disease patient groups. CONCLUSIONS: The results of this study suggest that immunosuppression associated with CD3+ and CD4+ T-cell depression is a risk factor for postoperative recurrence in patients with stage III gastric cancer.

Adenocarcinoma↗
PubMed1992

Changes in lymphocyte subsets in myasthenia gravis: correlation with level of antibodies to acetylcholine receptor and age of patient.

We report a detailed analysis of the subsets of lymphocytes in patients with myasthenia gravis (MG). There was a slight, nonsignificant increase in the level of CD5+ B lymphocytes among MG patients as compared with normal controls. The proportion of CD5+ T cells in MG was similar to that in controls. However, whereas age had no effect on the level of these cells in normal individuals, a significant age-related decrease of these cells was present in MG patients. The proportion of double-positive CD4+CD8+ T cells was significantly increased in MG. The level of the CD29+CD4+ (helper-inducer) subset was significantly higher in MG patients than in controls. There was no correlation between the titer of autoantibodies to acetylcholine receptor and the level of either CD29+CD4+ T cells or CD5+ B cells among MG patients. The only T-cell subset that correlated with the autoantibody titer was the CD45RA+CD4+ (suppressor-inducer) subset of CD4+ T cells.

Adolescent↗
PubMed1988

Lymphocyte subsets in human thymoma studied with monoclonal antibodies.

Lymphocyte subsets were investigated using OKT series monoclonal antibodies and flowcytometry in 16 cases of thymoma. From the viewpoint of lymphocyte subsets, thymoma could be divided into three types: thymus lymphocyte type, peripheral lymphocyte type, and intermediate type. In thymus lymphocyte type, the number of OKT-6+ cells exceed that of OKT-3+ cells, and are more than 50%. In peripheral lymphocyte type, the number of OKT-6+ cells are less than that of OKT-3+ cells and less than 10%. In intermediate type, OKT-6+ cells are between 10% and 30%. These three types correlate well with the histologic features with respect to the number and distribution of lymphocytes in thymoma tissue. Lymphocytes were infiltrating abundantly and intermingled in the tumor cell nests in thymus lymphocyte type, and were infiltrating rather scantly and outside the nests in peripheral lymphocyte type.

Adolescent↗
PubMed2001

Lymphocyte subsets in peripheral blood of dogs--a flow cytometric study.

Slight differences in the results of papers describing lymphocyte subsets distribution in the peripheral blood of healthy dogs may be explained by differences in monoclonal antibody clones and sources, breed and age of animals examined, methods of sample treatment, or methods of result analysis. In this paper, we described the effect of sample processing and of sample storage as well as the effect of age, breed, and gender of dogs on lymphocyte subset distribution. No significant differences were found between samples processed following a whole-blood lysis method and samples processed after density gradient separation. Furthermore, no significant differences were found between samples processed within 2h after collection and those stored at 4 degrees C for 12-16 h before processing. Age-related changes were evident in lymphocyte subset distribution in the peripheral blood of 38 Beagles divided according to their age into the six groups: (1) 5-6 days; (2) 2 months; (3) 6 months; (4) 1-2 years; (5) 3-5 years; and (6) >5 years. The percentage of B-lymphocytes (CD21-like positive cells) in the peripheral blood of newborn pups was 39.5+/-5.7 and decreased with advancing age. The percentage of CD8+ lymphocytes was 7.7+/-3.4 after birth and increased with advancing age. No age-related changes were observed in the percentages of CD4+ lymphocytes. The CD4+:CD8+ ratio decreased with advancing age. No significant age-related change was observed for lymphocytes bearing the gammadelta-TCR. Some breed differences were evident. Adult (1-5-year-old) Beagles, German Shepherds, Dalmatians, and Dachshunds were examined. The percentages of lymphocytes were higher in Beagles and Dachshunds than in Dalmatians and German Shepherds. The highest and the lowest absolute lymphocyte counts were found in Beagles and German Shepherds, respectively. As a consequence, German Shepherds showed the lowest absolute counts of the individual lymphocyte subpopulations and the widest neutrophil:lymphocyte ratio. Dalmatians showed the lowest percentage of CD3+ cells, the highest percentage of CD21+ cells, and the lowest CD4+:CD8+ ratio. German Shepherds showed the lowest percentage of CD21+ cells and the highest CD4+:CD8+ ratio. Females in Beagles and Dachshuns had nonsignificantly higher percentages of total lymphocytes, CD3+, CD4+, and nonsignificantly lower percentages of CD21+ lymphocytes. We concluded that there are age-, breed-, and perhaps also gender-related differences in lymphocyte subset distribution in the peripheral blood of dogs. Therefore, there is need to use appropriate control group in the experimental protocols. Among-breed differences could explain, at least partly, breed predisposition for some diseases.

Age Factors↗
PubMed1993

Effect of lentinan on lymphocyte subsets of peripheral blood, lymph nodes, and tumor tissues in patients with gastric cancer.

The effect of lentinan on lymphocyte subsets of peripheral blood, lymph nodes, and tumor tissues in gastric cancer patients was investigated. A 2-mg dose of lentinan was administered to 12 patients intravenously twice, the first at 3-9 days before and the second the day before surgery. The results were then compared with a control group without lentinan administration comprising 12 patients. Regarding peripheral blood and lymph nodes without metastasis, lymphocyte subsets defined with anti-CD3, anti-CD4, anti-CD8, anti-Leu7, and anti-Leu11 were analyzed by flow-cytometry. As for tumor tissues, lymphocyte subsets defined with anti-CD3, anti-CD4, anti-CD8, anti-Leu11, and anti-M3 were analyzed after immunohistochemical staining. There were no significant changes in the lymphocyte subsets of peripheral blood between the two groups. In the lymph nodes, the CD4 cell ratios increased; otherwise in regard to tumor-infiltrating lymphocytes (TILs), the number of CD4, Leu11 and LeuM3 cells showed a prominent increase. Therefore, lentinan was observed to produce different effects in accordance with the subjective organs.

Aged↗
PubMed1992

Effect of labor on lymphocyte subsets in full-term neonates.

PROBLEM: To determine the effect of labor on lymphocyte subsets in full-term neonates. METHOD OF STUDY: Cord blood obtained at delivery from full-term neonates, six born vaginally and six born by elective Cesarean section, was analyzed for lymphocyte subsets. Monoclonal antibodies, immunofluorescence, and flow cytometry were utilized to determine the lymphocyte phenotype frequencies in these neonates. These frequencies were compared by mode of delivery and to adult peripheral blood reference ranges using a two-tailed Student's t-test, P < 0.05. RESULTS: A profile of significantly elevated T (CD2, CD3) and helper cells (CD4) and depressed Natural Killer cells (CD16, CD56) is characteristic of term Cesarean section neonates. Significantly depressed frequency of T cells (CD2, CD3) and helper T cells (CD4) and elevated Natural Killer cells (CD16, CD56) is characteristic of vaginally delivered neonates. CONCLUSIONS: The mode of delivery affects the lymphocyte subset frequencies in full-term neonates.

Antigens, CD↗
PubMed1999

Effects of a short-term strength training programme on lymphocyte subsets at rest in elderly humans.

The effects of a short-term strength training programme on resting lymphocyte subsets and stress hormone concentrations were analysed in 32 elderly sedentary subjects. Out of these 32 subjects, 8 women and 8 men [mean age 70.1 (SEM 1.0) years] were randomly assigned to a 8-week strength training programme which consisted of three sets of eight repetitions at 80% of one repetition maximum, for leg press, bilateral leg extension and seated chest press, 3 days a week. The remaining 8 women and 8 men [mean age 70.5 (SEM 0.9) years] served as controls. Absolute counts of lymphocyte subsets (CD20+, CD3+, CD3+CD4+, CD3+CD8+, CD3-CD56+CD16+) were measured with a new technique combining fluorescent microspheres and flow cytometry. In the trained subjects, substantial increases in strength took place in one repetition maximum during the 8-week training period for leg press [from means of 20.7 (SEM 1.0) to 23.6 (SEM 1.0) N x kg(-1) LBM (lean body mass)], chest press [from means of 5.4 (SEM 0.3) to 6.2 (SEM 0.3) N x kg(-1) LBM] and bilateral leg extension [from means of 6.3 (SEM 0.2) to 7.4 (SEM 0.3) N x kg(-1) LBM] movements. Baseline cortisol concentration (P < 0.01), CD20+ cell count (P < 0.05), CD3+ cell count (P < 0.05), and CD4+ cell count (P < 0.01) decreased in both groups secondary to circannual variations between winter and summer. No significant effect of strength training on resting adrenaline, noradrenaline and cortisol concentrations or distributions of lymphocyte subsets at rest was observed. The main finding of this study was to demonstrate that 8-week is too short a duration for a strength training programme to modify counts of lymphocyte subsets at rest in elderly sedentary adults.

Aged↗
PubMed1986

An automated method for the detection of lymphocyte subset specific antibodies in multiple sclerosis sera.

Cytotoxic antibodies specific for lymphocyte subsets have been described in patients with several autoimmune diseases and a role for these antibodies in the pathogenesis of these diseases has been suggested. We describe a quantitative and rapid technique for the detection of cytotoxic antibodies and for the delineation of their lymphocyte subset specificity. The technique is based on the automated technique for tissue typing and makes use of reference monoclonal antibodies which are mixed with the sera to be tested. Percentage lysis obtained with this technique correlated well with percentage staining by FACS analysis. The general applicability of the technique is illustrated by the demonstration of lymphocyte subset specific cytotoxic antibodies in sera from multiple sclerosis patients.

Antibodies, Monoclonal↗
PubMed1989

Heterogeneous distribution and transmembrane signaling properties of lymphocyte function-associated antigen (LFA-1) in human lymphocyte subsets.

The role of LFA-1, a member of the integrin supergene family, in intercellular adhesion, including lymphocyte-endothelial cell (EC) binding, has been established. We now demonstrate that differences in LFA-1 cell surface density are responsible for the variable adhesion efficiency of lymphocyte subsets to EC. Electrophoretic analysis revealed multiple glycosylated isoforms of both alpha and beta subunits, largely as a result of different degrees of sialylation, with variable expression among different lymphocyte subsets. Neuraminidase digestion before EC adhesion increased the binding efficiency of all lymphocyte subsets, although the relative increase in each subset was proportional to the initial LFA-1 sialic acid content. LFA-1 cross-linking resulted in phosphoinositide hydrolysis and a rise in [Ca2+]i when using anti-alpha but not anti-beta subunit antibodies. These findings indicate that the density of LFA-1 on lymphocyte subsets controls their adhesive properties, and that the LFA-1 alpha subunit has transmembrane signaling properties that may result in activation events after interaction with its natural ligands.

Adult↗
PubMed1995

Exercise-induced muscle damage: effect on circulating leukocyte and lymphocyte subsets.

The purpose of this study was to determine the effect of downhill and level running on circulating leukocyte and lymphocyte subsets and T lymphocyte activation. Using a random cross-over design, 10 runners completed two trials of 60 min of level running (0% grade; LR) and downhill running (-10% grade; DHR) at 70% of level VO2max. Blood samples were obtained preexercise and immediately postexercise (POST) and at 1.5, 12, 24, and 48 h of recovery. Creatine kinase activity peaked at 12 h of recovery from DHR and was not significantly altered following LR. The number of total T, CD16+, CD3+CD56+ cells were significantly higher POST DHR compared with LR. Leukocyte and neutrophil counts were significantly higher at 1.5 and 12 h of recovery from DHR compared with LR. The number of activated CD8+ cells (CD25+ CD8+) was significantly higher at 12 h of DHR compared to LR. Total T cells were significantly reduced at various time points during the 48 h of recovery from LR and DHR. In summary, DHR relative to LR resulted in a greater mobilization of lymphocytes (post), neutrophils (1.5-12 h of recovery) and activation of CD8+ cells at 12 h of recovery. In addition, reductions in circulating T lymphocyte subsets occurred following both conditions.

Adult↗
PubMed2000

Differences in lymphocyte subsets in the wall of high endothelial venules and the lymphatics of human palatine tonsils.

Lymphocyte trafficking plays a critical role in disseminating specifically primed lymphocytes all over the body. Most concepts on the interaction of adhesion molecules on lymphocyte subsets and specialized endothelia such as those in high endothelial venules (HEV) are based on animal experiments as kinetic studies cannot be performed in humans. We therefore characterized lymphocyte subsets in the wall of HEV and in the lumen of lymphatics of 18 human palatine tonsils by immunohistology. All subsets studied were found in the wall of HEV (% of lymphocytes): 32% CD20+, 50% CD3+, 14% CD4+, 32% CD8+ and also 21% CD45RA+ and 39% CD45RO+. In the lymphatics, used to indicate lymphocytes emigrating from the tonsils, a different composition was found; e.g. many more T cells and three times more CD45RA+ than RO+ lymphocytes. Thus, HEV are not a selective entry site nor lymphatics an exit for specific lymphocyte subsets only, at least in these tonsils with chronic stimulation.

Adult↗
PubMed1998

Characterization of lymphocytic subsets and cytokine production in gastric biopsy samples from Helicobacter pylori patients.

BACKGROUND: This study characterized the phenotypic subsets of isolated gastric lymphocytes and the cellular immune response in cultured gastric biopsy specimens. METHODS: Endoscopy specimens from 40 Helicobacter pylori-positive and 40 H. pylori-negative patients were studied. a) Isolated gastric lymphocytes were analysed for CD4+, CD8+ T-lymphocyte subsets, activated T cells, and natural killer cells on a fluorescence-activated cell sorter, using monoclonal antibodies. b) The supernatant of cultured gastric biopsy specimens were assayed for interleukin (IL)-2, IL-4, and IL-6 levels. RESULTS: In H. pylori-positive patients there was (a) a decrease in CD4+/CD8+ T cells, no change in activated T cells, and an increase in natural killer cells, and (b) no change in IL-2 levels and a significant increase in IL-4 and IL-6 levels. CONCLUSIONS: There is an increase in CD8+ lymphocytes and natural killer cells, and the observed increase in IL-4 and IL-6 might be important in H. pylori-associated gastritis.

Adult↗
PubMed2005

Detection of T lymphocyte subsets of children with Helicobacter pylori infection.

AIM: To study the transformation of T lymphocyte subsets in children with Heliobacter pylori (H pylori) infection. METHODS: The H pylori infection status were determined by a combination of ELISA and Western blot (immunoblot) technique in 98 children and T lymphocyte subsets in peripheral blood were determined by flow cytometrical analysis. RESULTS: There were 75 children positive with H pylori infection and 23 negative in 98 children. Comparing the proportion of peripheral blood T lymphocytic subsets in children with H pylori infection and without H pylori infection, it was found that a higher proportion of CD4 T-cells in infected children (39.02+/-7.71 vs 34.25+/-10.73, t = 2.246,P<0.05) and higher value of CD4 to CD8 T-cells ratio (1.51+/-0.52 vs 1.25, t = 2.104, P<0.05) were present, but there were not significant differences in CD3 T-cells and CD8 T-cells (73.11+/-10.02 vs 69.49+/-17.08, 27.22+/-6.07 vs 28.27+/-8.67, P>0.05). CONCLUSION: Th1 cell-mediated immune responses may be induced by H pylori infection in children.

Adolescent↗
PubMed1985

Lymphocyte subsets and EB virus antibodies in nasopharyngeal carcinoma.

Lymphocyte subsets and Epstein-Barr virus (EBV)-associated antibodies were studied in 108 patients with nasopharyngeal carcinoma (NPC) and in 34 normal controls. Lymphocyte subsets were identified with monoclonal antibodies (Ortho Co.) by indirect immunofluorescent antibody (IFA) method. The helper T lymphocytes (OKT4+) in NPC patients comprised 38.2 +/- 10.5% which is significantly different from 45.2 +/- 8.0% in controls. The helper/suppressor ratio in NPC patients was 1.33 +/- 0.65 which is significantly different from 1.64 +/- 0.48 in controls, but the ratio was not correlated with disease extent, sex, age, total lymphocyte counts, WBC counts and EBV-associated antibodies of NPC patients. There were no remarkable differences between NPC patients and controls in B lymphocytes (OKIa+), total T lymphocytes (OKT3+), and suppressor lymphocyte (OKT8+) percentages, total lymphocyte counts and WBC counts. The EBV-associated antibodies were titrated by the IFA method using P3HR-1 cells and Raji cells induced by IUdR as target. Mean antibody titers and seropositive rates showed significant increase in NPC patients (1:12-1:502 and 45.4%-68.5%, respectively) compared with controls (1:1-1:87 and 1.0%-5.9%, respectively). The increase in antibodies was positively correlated with NPC disease extent, but was not correlated with the sex, age, total lymphocyte counts, helper/suppressor ratio, and WBC counts of NPC patients.

Adult↗
PubMed2002

Evaluation of a universal template for single-platform absolute T-lymphocyte subset enumeration.

The single-platform absolute T-lymphocyte subset analysis was evaluated utilizing a universal protocol in a Canadian multicenter study with the collaboration of the members of the Canadian HIV Trials Network (CTN). Participants used flow cytometers and reagents of their choice for labeling and lysing whole blood. Over a 2-year period, CTN laboratories performed single-platform absolute T-lymphocyte subset enumerations on fresh and commercial stabilized blood products using commercially available microfluorospheres TruCount and Flow-Count. This multicenter evaluation demonstrated that the application of a universal template for single-platform analysis provides a generic approach that embraces a wide array of immunophenotyping settings available in clinical laboratory.

CD3 Complex↗
PubMed1994

Analysis of lymphocyte subsets in a renal allograft recipient infected with human immunodeficiency virus.

OBJECTIVE: To study the change in lymphocyte subsets of a renal allograft recipient who acquired human immunodeficiency virus (HIV) infection. PATIENT AND METHODS: A 53-year-old Japanese man who acquired HIV infection at the time of renal transplantation and died from opportunistic infection 3 years later was studied. Lymphocyte subsets were examined serially throughout his clinical course. RESULTS: The numbers of CD4 positive cells were severely decreased but those of CD8 positive cells were stable; the CD 4/8 ratio was extremely low. The numbers of B cells were also decreased but production of immunoglobulin was increased. In spite of this immunosuppressive state, graft rejection was noted when the patient's immunosuppressive medication was reduced. CONCLUSION: The lymphocyte subsets of renal transplant patients with HIV infection should be closely observed, as should the nature and timing of opportunistic infection in relation to transplantation, and the administration of immunosuppressive medication.

AIDS-Related Opportunistic Infections↗
PubMed2002

T and B lymphocyte subsets in patients with unexplained recurrent spontaneous abortion: IVIG versus placebo treatment.

PROBLEM: To investigate circulating lymphocyte subsets in women with recurrent spontaneous abortion (RSA) in relation to pregnancy outcome and to treatment with intravenous immunoglobulin (IVIG). METHOD OF STUDY: Forty-one women with a history of unexplained RSA were examined during first trimester of pregnancy before IVIG or placebo treatment and after pregnancy. The results were compared with five healthy, non-pregnant women and five women in the first trimester of normal pregnancy. Circulating lymphocyte subsets with focus on T-cell subpopulations were determined by flow cytometry. RESULTS: The proportions of human leukocyte antigen (HLA)-DR positive T cells (CD3+ HLA-DR+), T-killer/effector cells (CD8+ S6F1+) and B cells (CD19+) were increased, whereas the proportion of T-suppressor/inducer cells (CD4+ CD45RA+) was decreased during first trimester pregnancy of RSA women compared with pregnant normal controls. T and B lymphocyte subsets did not correlate with pregnancy outcome on either IVIG or placebo group. CONCLUSIONS: In RSA patients, the immune system seems to be activated in contrast to the suppression noted in normal pregnancy.

Abortion, Habitual↗