BACKGROUND: We developed a single-color multitarget flow cytometry (SM-FC) assay, a single-tube assay with graded mean fluorescence intensities (MFIs). We evaluated the repeatability of SM-FC, and its correlation with multicolor flow cytometry (MFC), to assess its application as a routine FC assay. METHODS: We selected CD19, CD3, CD4, and CD8 as antigen targets to analyze a lymphocyte subset. MFIs were graded by adjusting monoclonal antibody (mAb) volumes to detect several cell populations. Dimly labeled mAb was prepared by decreasing mAb volume and the optimum diluted volume was determined by serial dilution. SM-FC repeatability was analyzed 10 times in 2 normal controls. The correlation between SM-FC and MFC was evaluated in 20 normal and 23 patient samples. RESULTS: CV values (0.8-5.0% and 1.3-4.1% in samples 1 and 2, respectively) acquired by SM-FC with CD3-fluorescein alpha-isothyocyanate (FITC)dim+CD4-FITCbright and with CD19-FITCdim+CD3-FITCbright showed good repeatability, comparable to that acquired by MFC (1.6-3.7% and 1.0-4.8% in samples 1 and 2, respectively). Excellent correlation was observed between the 2 methods in the 20 normal samples (B cells, T cells, non-Thelper cells, and Thelper cells; r2=0.87, 0.97, 0.97, and 0.98, respectively; P<0.05). There were also linear relationships between SM-FC with CD19-FITCdim+CD3-FITCbright and CD8-PEdim+CD4-PEbright, and MFC, in the 23 patient samples (B cells, T cells, Tcytotoxic cells, and Thelper cells; r2> or =0.98, 0.99, 0.99, and 0.99, respectively; P<0.05). CONCLUSIONS: The multicolor, single-tube SM-FC technique is a potential alternative tool for identifying a lymphocyte subset.
Antibodies, Monoclonal/chemistry/*immunology ; Antigens, CD19/chemistry/metabolism ; Antigens, CD3/chemistry/metabolism ; Antigens, CD4/chemistry/metabolism ; Antigens, CD8/chemistry/metabolism ; B-Lymphocyte Subsets/immunology/metabolism ; Color ; Flow Cytometry/*methods ; Fluorescein-5-isothiocyanate/*chemistry ; Humans ; T-Lymphocyte Subsets/immunology/metabolism

Objective: To explore the application value of T lymphocyte subsets combined with procalcitonin (PCT), C-reactive protein (CRP), neutrophil to lymphocyte ratio (NLR) and white blood cell count (WBC) in the auxiliary diagnosis and prognosis evaluation of sepsis. Methods: In a retrospective study, seventy-two patients with sepsis diagnosed and treated in Tianjin First Central Hospital from June 2018 to April 2021 were selected as the research objects, and included in the sepsis group were 46 males and 26 females, aged 68 (57.3, 80.3) years. In addition, 111 patients with local infection admitted to hospital during the same period were included in the local infection group, including 62 males and 49 females, aged 68 (51, 77) years. Sepsis patients were divided into survival group (43 cases) and death group (29 cases) according to the 28-day outcome. CD3⁺, CD4⁺, CD8⁺, CD4⁺/CD8⁺ ratio were detected by flow cytometry within 24 h after admission, PCT was detected by ELISA, CRP was detected by immunoturbidimetry, blood routine examination, blood lactic acid (Lac) and oxygen partial pressure (PO₂) were detected by instrumental method. Multivariate Logistic regression analysis was used to evaluate the correlation between each indicator and sepsis, and receiver operating characteristic curve (ROC) was drawn to evaluate the diagnostic value of each indicator for sepsis. Multivariate Logistic regression analysis and Kaplan Meier survival analysis were used to evaluate the prognostic value of each index for patients with sepsis. Results: Peripheral blood CD3⁺, CD4⁺, CD8⁺, CD4⁺/CD8⁺ ratio and PLT in sepsis group were significantly lower than those in local infection group(Z=-8.184,P<0.001;Z=-7.210,P<0.001;Z=-5.936,P<0.001;Z=-2.700,P=0.007;Z=-6.381,P<0.001); PCT, CRP, NLR and Lac levels were significantly higher than those in local infection group(Z=-8.262,P<0.001;Z=-3.094,P=0.002;Z=-9.004,P<0.001;Z=-4.770,P<0.001). Multivariate Logistic regression model showed that PCT, NLR, CD3⁺, CD8⁺, CD4⁺/CD8⁺ were independent risk factors for sepsis. According to ROC curve analysis, AUC of sepsis patients diagnosed by each indicator were 0.862, 0.894, 0.858, 0.760 and 0.618, respectively. The cut-off values were 3.075 ng/ml, 10.715, 44.935×10⁹/L, 27.463×10⁹/L and 0.750, respectively. The NLR sensitivity was 80.6%, and the CD3⁺ specificity was 94.6%. The AUC of combined detection of PCT and NLR was 0.947, sensitivity was 87.5% and specificity was 91.9%. The combined detection AUC of PCT, NLR, CD3⁺, CD4⁺/CD8⁺ was 0.958, the sensitivity and specificity were 90.3% and 91.0% respectively(P<0.001). PCT and Lac in death group were significantly higher than those in survival group(Z=-2.302,P=0.021;Z=-3.095,P=0.002);Peripheral blood CD4⁺/CD8⁺ levels were significantly lower than those in survival group(Z=-3.691,P<0.001),Multivariate Logistic regression model showed that CD4⁺/CD8⁺ ratio was an independent risk factor for 28 d mortality in patients with sepsis (P<0.001). The ROC curve showed that the AUC was 0.758, and the Youden index reached the maximum when the cut-off value was 1.27, the sensitivity and specificity were 79.3% and 60.5%, respectively. Compared with patients with CD4⁺/CD8⁺ ≥1.27, 28-day mortality was significantly increased in patients with CD4⁺/CD8⁺<1.27 (P=0.032). Conclusion: The combined detection of PCT, NLR, CD3⁺ and CD4⁺/CD8⁺ can improve the auxiliary diagnostic efficiency of sepsis, and the ratio of CD4⁺/CD8⁺ in peripheral blood may have certain predictive value for the prognosis of sepsis.
Aged ; C-Reactive Protein/analysis* ; Female ; Humans ; Male ; Middle Aged ; Procalcitonin ; Retrospective Studies ; Sepsis/diagnosis* ; T-Lymphocyte Subsets/chemistry*

The pathogenesis of some autoimmune diseases has been considered to be related to abnormal differentiation of T cell subsets. This study was aimed at investigating the change of Th1-like and Th2-like cells balance in ITP children, and analyzing the role of T cell subsets disequilibrium in the pathogenesis of ITP. Peripheral blood T cells were collected from 30 ITP patients, the T-cells were isolated and purified. The ratios of Th/Tc, Th1/Th2 and Tc1/Tc2 in peripheral blood T cells were analyzed by immunofluorescence staining and bicolor flow cytometry (FCM) in vitro. The results showed that as compared with the ratios of Th1/Th2 (48.76% +/- 6.17%) and Tc1/Tc2 (18.90% +/- 4.12%) in healthy children, the ratios of Th1/Th2 (56.21% +/- 5.95%) and Tc1/Tc2 (23.09% +/- 3.31%) in ITP children increased obviously. FCM analysis revealed that average percentages of Th, Th1, Th2, Tc, Tc1 and Tc2 were 22.31% +/- 6.51%, 21.92% +/- 6.42%, 0.39% +/- 0.14%, 31.12% +/- 6.15%, 30.95% +/- 5.45% and 1.34% +/- 0.84% in ITP children versus 39.24% +/- 5.82%, 39.01% +/- 5.47%, 0.80% +/- 0.16%, 30.25% +/- 5.63%, 28.72% +/- 5.20% and 1.52% +/- 0.68% in healthy children. The average percentages of Th, Th1 and Th2 decreased obviously, while the average percentages of Tc, Tc1 and Tc2 did not change. It is concluded that the ratios of Th1/Th2 and Tc1/Tc2 in peripheral blood T cells increase obviously in ITP children and the cellular immunity in ITP children shifts to Th1 type immunity superiority, which suggest that the abnormal differentiation of T cell subsets may play an important role in the pathologic process of ITP.
Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; T-Lymphocyte Subsets ; immunology ; metabolism ; pathology ; T-Lymphocytes, Cytotoxic ; chemistry ; Th1 Cells ; immunology ; Th2 Cells ; immunology

OBJECTIVETo observe effect of oral scorpio and scolopendra powder on T-cell subsets in peripheral blood and intestine from rats with collagen induced arthritis (CIA).

METHOD60 rats were randomly divided into 6 groups: normal control group, model control group, low-dose scorpio and scolopendra group, middle-dose scorpio and scolopendra group, high-dose scorpio and scolopendra group, and type II collagen group. Rat's rheumatoid arthritis was induced by collagen II (C II). Level of T-cell subsets from peripheral blood and intestine was measured by flow cytometry.

RESULTCD4+ T cellular level was obviously increased (P < 0.05 or P < 0.01) or kept increased tendency in peripheral blood and intestine from the model group compared with that of the normal group, while the ratio of CD4+/CD8+ in intestine was obviously descent but the contrary in peripheral blood (P < 0.05 or P < 0.01). CD4+, CD8+ T cellular level in intestine were obviously descent and the ratio of CD4+ /CD8+ increased in all treated groups when compared with in the model group (P < 0.05 or P < 0.01). However, CD4+ T cellular level and the ratio of CD4+/CD8+ in peripheral blood were remarkablely decreased.

CONCLUSIONThe mechanism that scorpio and scolopendra could treat rat's rheumatoid arthritis may be regulating balance of T-lymphocyte subsets in peripheral blood and intestine.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Arthritis, Experimental ; immunology ; Arthritis, Rheumatoid ; immunology ; CD4 Lymphocyte Count ; CD4-Positive T-Lymphocytes ; CD8-Positive T-Lymphocytes ; Female ; Intestinal Mucosa ; immunology ; Medicine, Chinese Traditional ; Rats ; Rats, Wistar ; Scorpions ; chemistry ; T-Lymphocyte Subsets ; drug effects ; immunology

OBJECTIVETo analyze the effect of Chinese herbs used by Prof. LI Tao on peripheral blood T subsets in treating multiple sclerosis (MS) by using association rules and statistical methods, thereby providing evidence for optimizing prescriptions.

METHODSData of MS inpatients and outpatients recorded by data collecting system, Xiyuan Hospital, China Academy of Chinese Medical Sciences were resorted. The relationship between Chinese herbs and T cell subsets were analyzed using SPSS17.0 and Aprior module in SPSS Clementine 12.0.

RESULTSRadix bupleuri, Radix Paeoniae alba, Fructus Aurantii, Atractylodes, and Radix Glycyrrhizae were most commonly used herbal combinations.Radix Aconiti lateralis preparata and Rhizoma Smilacis glabrae were often added. Radix Aconiti lateralis preparata was associated with decreased Th1 cells (confidence level 83.78%, supportive level 36.26%). Decreased Th1 cell was associated with Radix Aconiti lateralis preparata (confidence level 71.26%, supportive level 36.26%).Radix Aconiti lateralis preparata was obviously associated with decreased Th1 cells. Radix Bupleuri, Radix Paeoniae alba, bitter orange, Atractylodes , Radix glycyrrhizae, and Radix Aconiti lateralis preparata could reduce peripheral blood Th1 subsets of MS patients and elevate Th2 subsets (all P < 0.01).

CONCLUSIONSThe herbal combination of Radix Bupleuri, Radix Paeoniae alba, Fructus Aurantii, Atractylodes, Radix Glycyrrhizae, Rhizoma Smilacis glabrae, and Radix Aconiti lateralis preparata could lower peripheral blood Th1 cells and elevate Th2 cells, and prevent the relapse of MS possibly by reducing Th1 cells and elevating Th2 cells. Especially Radix Aconiti lateralis preparata played important roles in aforesaid changes of Th1 and Th2.

Aconitum ; chemistry ; Atractylodes ; chemistry ; Bupleurum ; chemistry ; China ; Drugs, Chinese Herbal ; therapeutic use ; Fruit ; chemistry ; Glycyrrhiza ; chemistry ; Humans ; Multiple Sclerosis ; therapy ; Paeonia ; chemistry ; Plant Roots ; chemistry ; Rhizome ; chemistry ; Smilacaceae ; chemistry ; T-Lymphocyte Subsets ; drug effects

To compare the effects of polysaccharide nucleic acid fraction of bacillus calmette guerin (BCG-PSN) and thymopeptides on T-lymphocytes of normal and immunosuppressed mice, CD4+ and CD8+ T-lymphocyte subsets of single nucleic cell in thymus, spleen and peripheral blood were detected successively by flow cytometry after application of BCG-PSN and thymopeptides. Meanwhile, CD4+/CD8+ ratio was also calculated. The results showed that both BCG-PSN and thymopeptides could decrease the proportion of CD4+ CD8+ T-lymphocyte subsets in the thymus, at the same time increase CD4+ T-lymphocyte, CD8+ T-lymphocyte proportion in the three tissues. The fluctuation in amplitude was greater in thymopeptides group than that in BCG-PSN group. It is concluded that acting location of thymopeptides is in thymus, its stimulating action is stronger than that of BCG-PSN, while BCG-PSN not only accelerates the differentiation in thymus, but also has some direct stimulation to peripheral CD4+ T-lymphocytes, and can maintain CD4+/CD8+ ratio within normal range. So, BCG-PSN is safer.
Adjuvants, Immunologic/*pharmacology ; Immunocompromised Host ; Mycobacterium bovis/*chemistry ; Nucleic Acids/pharmacology ; Peptide Fragments/*pharmacology ; Polysaccharides, Bacterial/*pharmacology ; T-Lymphocyte Subsets/*drug effects ; Thymus Gland/chemistry

The present study was conducted in order to investigate the immunologic alterations alongside the numerical changes in peripheral blood lymphocytes(PBL) and their subsets in stomach cancer patients. Lymphocyte surface markers were determined in 85 stomach cancer patients and 49 controls by indirect immunofluorescence technique using monoclonal antibodies. Monoclonal antibodies used were Leu 2a(CD8, suppressor/cytotoxic T cells), Leu 3a(CD4, inducer/helper T cells), Leu 4(CD3, pan T reagent), Leu 11(CD16, natural killer cells) and Leu 12(CD19, B cells). The numbers of PBL, CD3+, CD4+, CD8+, CD16+ and CD19+ cells significantly decreased and the CD4: CD8 value increased in 85 patients with stomach cancer compared to those in controls(p < 0.01). In stage I(n = 17), neither PBL, their subsets nor the CD4: CD8 value were significantly different from those of the controls. In stage II(n = 17), the numbers of PBL, CD3+, CD4+ and CD8+ cells decreased(p < 0.01). In stage III(n = 24) and IV(n = 27), PBL and all subsets measured decreased(p < 0.01). The CD4: CD8 value showed significant increases in stages III and IV(p < 0.01), because the CD8+ cells decreased to a greater extent than did the CD4+ cells. The results demonstrating that the lymphocyte subsets are depressed differentially with the stage suggest that host immunity is impaired with the progression of stomach cancer.
Adult ; Age Distribution ; Aged ; Antigens, CD19/analysis ; Antigens, CD3/analysis ; CD4-Positive T-Lymphocytes/cytology ; CD8-Positive T-Lymphocytes/cytology ; Female ; Human ; Lymphocyte Count ; Lymphocyte Subsets/chemistry/*immunology ; Male ; Middle Age ; Receptors, IgG/analysis ; Sex Distribution ; Stomach Neoplasms/blood/*immunologym

OBJECTIVE: To explore the immunoregulatory effect of Huangqi Fuzhengtang on immunosuppressive mice. METHODS: The immunosuppressive mouse model was established by using hydrocortisone. Huangqi Fuzhengtang and Yupingfeng San apozema were given to the mice intragastrically. The contents of hemolysin, IL-2, and INF-gamma in the mouse serum and the expression of CD3(+), CD4(+), and CD8(+) in the peripheral blood lymphocytes were measured. RESULTS: Huangqi Fuzhengtang could obviously elevate the contents of hemolysin, IL-2, INF-gamma and the ratio of CD4(+) and CD8+, which was more effective than Yupingfeng San. CONCLUSION Huangqi Fuzhengtang could improve the immune function in the immunosuppressive mice.
Adjuvants, Immunologic ; pharmacology ; Animals ; Astragalus propinquus ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Hydrocortisone ; Immunocompromised Host ; drug effects ; immunology ; Male ; Mice ; Random Allocation ; T-Lymphocyte Subsets ; drug effects

BACKGROUND: In the severe community-acquired pneumonia, it has been known that the immune status is occasionally suppressed. This study was performed to identify the immunologic markers related with the prognostic factors in severe community-acquired pneumonia. METHODS: 23 patients with severe community-acquired pneumonia were involved in this study, and divided into survivor (16) and nonsurvivor (7) groups. In this study, the medical history, laboratory tests(complete blood counts, routine chemistry profile, immunoglobulins, complements, lymphocyte subsets, cytokines, sputum and blood culture, urine analysis), and chest radiographs were scrutinized. RESULTS: 1) Both groups had lymphopenia(total lymphocyte count 995.6±505.7/mm2 in the survivor and 624.0±287.6/mm2 in the nonsurvivor group). 2) The T-lymphocyte count of the nonsurvivor group(295.9±203.0/mm2) was lower than the survivor group(723.6±406.5/mm2) (p<0.05). 3) The total serum protein(albumin) was 6.0±1.0(2.7±0.7) g/dl in the survivor and 5.2±1.5(2.3±0.8)g/dl in the nonsurvivor group. The BUN of the noncurvivor group(41.7±30.0mg/dl) was higher than that of the survivor group(18.9±9.8mg/dl)(p<0.05). The creatinine concentration was higher in the nonsurvivor group(1.8±1.0mg/dl) than that in the survivor group(1.0±0.3mg/dl)(p<0.05). 4) The immunoglobulin G level was higher in the survivor group (1433.0±729.5mg/dl) than in the nonsurvivor group(849.1±373.1mg/dl)(p<0.05). 5) The complement C3 level was 108.0±37.9mg/dl in the survivor group and 88.0±32.1mg/dl in the nonsurvivor group. 6) A cytokine study showed an insignificant differenne in both groups. 7) Chronic liver disease, DM, and COPD were major underlying diseases in both groups. CONCLUSION: These results suggest that decreased a T-lymphocyte count and immunoglobulin G level, and an increased BUN and creatinine level may be associated with the poor prognosis of severe community-acquired pneumonia.
Biomarkers ; Chemistry ; Complement C3 ; Complement System Proteins ; Creatinine ; Cytokines ; Humans ; Immunoglobulin G ; Immunoglobulins ; Immunologic Factors* ; Liver Diseases ; Lymphocyte Count ; Lymphocyte Subsets ; Pneumonia* ; Prognosis* ; Prospective Studies* ; Pulmonary Disease, Chronic Obstructive ; Radiography, Thoracic ; Sputum ; Survivors ; T-Lymphocytes

OBJECTIVETo study the protective effect of Sarcandra glabra extract (SGE) on immune system in restrained mice.

METHODThe male C57BL/6 mice were randomly divided into normal control group, stress control group, 125, 500 mg x kg(-1) SGE group. The spleen lymphocyte suspensions of each group were prepared. The parameters of spleen T cells subsets, NK cell and NKT cell proportion and number was detected by Flow cytometry.

RESULTSGE regulated the balance of T cell subsets, increased the percent of NK cells and NKT cell proportion and number in restrained mice.

CONCLUSIONSGE has immunologic protective effect in restrained mice probably via the amelioration of immune cells proportion and number.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Killer Cells, Natural ; drug effects ; immunology ; Magnoliopsida ; chemistry ; Male ; Mice ; Mice, Inbred C57BL ; Natural Killer T-Cells ; drug effects ; immunology ; Restraint, Physical ; Stress, Psychological ; immunology ; T-Lymphocyte Subsets ; drug effects ; immunology