Detection Of Lymphocyte Subsets Research Articles

Circulating immune index predicting the prognosis of patients with hepatocellular carcinoma treated with lenvatinib and immunotherapy.

Immune checkpoint inhibitor (ICI)-based combination therapy has opened a new avenue for the treatment of multiple malignancies including hepatocellular carcinoma (HCC). However, considering the unsatisfactory efficacy, biomarkers are urgently needed to identify the patients most likely to benefit from ICI-based combination therapy. A total of 194 patients undergoing ICI-based combination therapy for unresectable HCC were retrospectively enrolled and divided into a training cohort (n = 129) and a validation cohort (n = 65) randomly. A novel circulating immune index (CII) defined as the ratio of white blood cell count (×109/L) to lymphocyte proportion (%) was constructed and its prognostic value was determined and validated. Patients with CII ≤ 43.1 reported prolonged overall survival (OS) compared to those with CII > 43.1 (median OS: 24.7 vs 15.1 months; 6-, 12-, 18-month OS: 94.2%, 76.7%, 66.1% vs 86.4%, 68.2%, 22.8%, P = 0.019), and CII was identified as an independent prognostic factor for OS (hazard ratio, 2.24; 95% confidence interval, 1.17-4.31; P = 0.015). These results were subsequently verified in the validation cohort. Additionally, patients with low CII levels had improved best radiological tumor response (complete response, partial response, stable disease, progressive disease: 3%, 36%, 50%, 11% vs 0%, 27%, 46%, 27%; P = 0.037) and disease control rate (89% vs 73%; P = 0.031) in the pooled cohort and better pathologic response (pathologic complete response, major pathologic response, partial pathologic response, no pathologic response: 20%, 44%, 28%, 8% vs 0%, 0%, 40%, 60%; P = 0.005) in the neoadjuvant cohort. Detection of lymphocyte subsets revealed that an elevated proportion of CD4+ T cells was related to better OS, while the proportion of CD8+ T cells was not. We constructed a novel circulating immune biomarker that was capable of predicting OS and therapeutic efficacy for HCC patients undergoing ICI and lenvatinib combination therapy.

Comparison of different combinations of antibodies and labeled fluorescein in the detection of lymphocyte subsets by flow cytometry.

Flow cytometry is a classical method for analyzing human peripheral blood lymphocyte subsets. This study aims to explore a new combination of antibody and labeled fluorescein for detecting lymphocyte subsets by comparing the effects of different combinations of antibody and labeled fluorescein in flow cytometry. We conducted a prospective study and enrolled 362 healthy patients undergoing physical examination in the medical examination center of the third hospital of hebei medical university. Venous blood was drawn from volunteers at the same time in the morning and divided into 3 tubes (Tube A, Tube B and Tube C). T lymphocytes were detected by 3-colors method (CD4-FITC/CD8-PE/CD3-PC5) in Tube A, B lymphocytes were detected by 2-colors method (CD19-FITC/CD3-PE) in Tube B, and T lymphocytes and B lymphocytes were detected by 4-colors method (CD4-FITC/CD8-PE/CD3-PC5/CD19-FITC) in Tube C. The repeatability and accuracy of the test scheme for Tube C shall not be inferior to that of Tube A and Tube B. There were no significant difference in the results of CD3 + and CD4+/CD8 + between Tube A and C, as well as in the results of CD3 + and CD19 + between Tube B and C. Pearson correlation analysis showed that the test results of a and C and B and C were highly correlated. The 4-colors method (CD4-FITC/CD8-PE/CD3-PC5/CD19-FITC) can detect T lymphocytes and B lymphocytes at the same time, reduce the use of fluorescence channels and save the detection cost, which is worthy of recommendation.

Clinical significance of circulating neutrophils and lymphocyte subsets in newly diagnosed patients with diffuse large B-cell lymphoma.

Lymphocytes play crucial roles in tumor surveillance in diffuse large B-cell lymphoma (DLBCL). Neutrophil-to-lymphocyte ratio (NLR), a biomarker for systematic inflammation, has been confirmed to be a prognostic factor for many malignant diseases. Herein, we conducted a systemic in-depth study of circulating neutrophils and lymphocyte subsets in DLBCL patients and their dynamics along with chemoimmunotherapy. A total of 61 patients with DLBCL were enrolled. Detection of lymphocyte subsets by flow cytometry was conducted at diagnosis and after 2/4/6/8 cycles' treatment of R-CHOP. Clinical significance, including incidence of infection, curative effect and disease-free survival (DFS), was analyzed based on the patients' clinical data and the quantity of lymphocyte subsets. The absolute numbers of neutrophils in stage III-IV DLBCL patients were obviously increased (p = 0.012), while the absolute numbers of lymphocytes were decreased (p = 0.025). Consequently, DLBCL patients had significantly higher NLR than healthy controls (p < 0.001). Further analysis of lymphocyte subsets showed a significantly reduced CD4 + T cells in DLBCL patients (p = 0.001). Patients with a lower lymphocyte counts (< 1.26*10E9/L) were more susceptible to infection (p < 0.001). NK cells were much higher in patients achieving complete remission than those of non-complete remission (p = 0.032). Higher neutrophils and NLR were closely associated with poorer DFS (p = 0.001 and p = 0.045, respectively). Circulating cells in DLBCL patients were dysregulated, featured with increased neutrophils and reduced lymphocytes. Higher NK cells before treatment predicted better therapeutic outcome. Higher neutrophils and NLR can be regarded as inferior prognostic predictors for DLBCL patients at diagnosis.

Role of Increased Syncytin-1 Expression in Pathogenesis of Anti-N-Methyl-d-Aspartate Receptor Encephalitis.

PurposeSyncytin-1 may play a role in several neuropsychiatric disorders, but its function in anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is unknown. The purpose of this study was to examine the possible mechanism of action of syncytin-1 in patients with anti-NMDAR encephalitis.Patients and MethodsTwenty patients with anti-NMDAR encephalitis and eight controls were recruited. The protein levels of syncytin-1 in serum were determined using an enzyme-linked immunosorbent assay, and the transcript levels of syncytin-1 were determined using real-time quantitative PCR. Flow cytometry was used for peripheral blood lymphocyte subset detection. Further, the relationship between syncytin-1 levels and clinical features of anti-NMDAR encephalitis and peripheral blood lymphocyte subsets was analyzed.ResultsCompared with those in controls, higher syncytin-1 levels and percentage of B cells (CD3-CD19+) were observed in patients with anti-NMDAR encephalitis. Among anti-NMDAR encephalitis patients, the level of syncytin-1 positively correlated with the proportion of B cells and modified Rankin scale score at onset and after immunotherapy and negatively correlated with the proportion of CD3+ T cells.ConclusionAn increased expression of Syncytin-1 is associated with the pathogenesis of anti-NMDAR encephalitis, providing evidence for elucidating the pathogenesis of the disease and suggesting novel therapeutic targets. Further, this study clarifies the role of syncytin-1 in neuroimmune disorders.

The development of autoverification system of lymphocyte subset assays on the flow cytometry platform.

Peripheral blood lymphocyte subsets are important parameters for monitoring immune status; however, lymphocyte subset detection is time-consuming and error-prone. This study aimed to explore a highly efficient and clinically useful autoverification system for lymphocyte subset assays performed on the flow cytometry platform. A total of 94,402 lymphocyte subset test results were collected. To establish the limited-range rules, 80,427 results were first used (69,135T lymphocyte subset tests and 11,292 NK, B, T lymphocyte tests), of which 15,000T lymphocyte subset tests from human immunodeficiency virus (HIV) infected patients were used to set customized limited-range rules for HIV infected patients. Subsequently, 13,975 results were used for historical data validation and online test validation. Three key autoverification rules were established, including limited-range, delta-check, and logical rules. Guidelines for addressing the issues that trigger these rules were summarized. The historical data during the validation phase showed that the total autoverification passing rate of lymphocyte subset assays was 69.65% (6,941/9,966), with a 67.93% (5,268/7,755) passing rate for T lymphocyte subset tests and 75.67% (1,673/2,211) for NK, B, T lymphocyte tests. For online test validation, the total autoverification passing rate was 75.26% (3,017/4,009), with 73.23% (2,191/2,992) for the T lymphocyte subset test and 81.22% (826/1,017) for the NK, B, T lymphocyte test. The turnaround time (TAT) was reduced from 228 to 167min using the autoverification system. The autoverification system based on the laboratory information system for lymphocyte subset assays reduced TAT and the number of error reports and helped in the identification of abnormal cell populations that may offer clues for clinical interventions.

The clinical, immunological and genetic features of 12 Chinese patients with STAT3 mutations

BackgroundLoss-of-function (LOF) mutations in signal transducer and activator of transcription 3 (STAT3) is one of the causes of STAT3 hyperimmunoglobulin E (IgE) syndrome (STAT3-HIES), while gain-of-function (GOF) mutations in STAT3 lead to immune dysregulation diseases. We retrospectively analyzed the age, common clinical symptoms, immunologic and molecular manifestations in 11 patients with LOF STAT3 mutations and 1 patient with a GOF STAT3 mutation.MethodsTwelve patients were enrolled in our study. Serum immunoglobulin measurements, lymphocyte subset detection and whole-exome sequencing were performed.ResultsThe median age at diagnosis of STAT3-HIES patients was 4.74 years. Eczema, recurrent respiratory infections, fevers, abscesses and Staphylococcus aureus infections were the classic manifestations. Elevated serum IgE levels are not always observed in conjunction with high eosinophil counts. A moderate viral DNA load was also measured in peripheral blood mononuclear cells. We noticed that c. 1144C>T was the most common mutation site, followed by c.1311C>A. Additionally, c.1311C>A and c. 1826G>C are two novel mutations. Eight patients achieved notable improvement after receiving intravenous immunoglobulin.ConclusionWe updated the current knowledge of this topic. We found an earlier median age at diagnosis, a higher survival rate, and a general lack of nonimmunological abnormalities; we also described the treatment details and novel mutations involve in STAT3-HIES and compared STAT3 LOF and GOF mutations.

Prognostic Significance of Circulating CD28 Negative Suppressor T Cells and Memory B Cells in Patients with Breast Cancer.

It has been suggested that routine assessment and quantification of different lymphocyte subsets can provide clinically meaningful prognostic information in breast cancer (BC). To determine the relationship between peripheral blood lymphocyte subsets and pathological parameters and response to therapy in patients with BC. Thirty patients with operable breast cancer treated surgically with either modified radical mastectomy or breast conservative surgery, and 20 healthy controls were included. For detection of lymphocyte subsets in peripheral blood; Fluorochrome-labeled monoclonal antibodies were used andcells were analyzed by flow cytometry. Patients were treated with chemotherapy, radiotherapy and hormonal treatment, and followed up to determine relapse and recurrence-free survival (RFS). Significant differences were found in the frequencies of B, T, NK, NKT, and CD28‒T cells between patients with BC and controls. Moreover, a significant difference was found in the percentage of CD8+CD28‒ T cells between patients with different pathologic subtypes of BC and negative correlations were observed between the frequency of CD8+CD28‒T cells and memory B cells, and RFS. Also, a significant difference in the frequency of naïve B cells was found in patients with different tumor grades and a negative correlation was found between the frequencies of B cells and NKT cells. NK, NKT, lymphocytes, and CD28‒ T cells significantly differed between healthy controls and BC patients. Also, memory B cells were associated with good response to treatment while CD28‒ T cells were associated with shorter RFS.

T-Cell Subset Counts in Peripheral Blood Can Be Used as Discriminatory Biomarkers for Diagnosis and Severity Prediction of Coronavirus Disease 2019.

This study evaluated the significance of lymphocyte subsets detection in peripheral blood in the diagnosis and prognosis of coronavirus disease 2019 (COVID-19). Our results revealed that CD3+T, CD4+T, CD8+T cells and NK cells were significantly decreased in COVID-19 patients. COVID-19 patients had a relatively slight decrease in CD4+T cells but a severe decrease of CD8+T cells. The significantly elevated CD4/CD8 ratio was observed in COVID-19 patients. T cell subset counts were related to the severity and prognosis of COVID-19. The counts of CD8+T and CD4+T cells can be used as diagnostic markers of COVID-19 and predictors of disease severity.

Effects of different sample preservation methods on the lymphocyte subset detection by flow cytometry

Objective To discuss the effects of sample storage time and temperature on lymphocyte subsets detected by flow cytometry. Methods Use flow cytometry to detect lymphocyte subsets of a total of 53 blood samples from hospitalized and out-patient patients in Qinghai Provincial People′s Hospital from October 26, 2018 to March 30, 2019. The test was completed within 4 hours after sample collection, which is the control group. The treatment groups are as follows: pretreatment was completed within 4 hours and detected after saving samples at room temperature (group A) for 24 and 36 hours; the tests were performed after keeping samples at room temperature (group B) for 24, 48, 72 hours; completed detection after preserving samples in 4 ℃ condition (group C) for 24, 48, 72 hours. Results In treatment group A, there was no significant difference in the results of lymphocyte subsets detected after 24 hours of storage compared with the control group(P>0.05); after 36 h of storage, CD3+and CD3+CD8+T lymphocytes percentages were significantly increased [(74.28±11.31)% vs (73.78±11.33)%, (32.15±14.82)% vs (31.00±14.79)%; all P 0.05); after 48 hours of storage, CD19+cells percentage were observably reduced [(15.60±12.09)% vs (16.11±12.38)%; P 0.05); after 72 hours of storage, CD3+and CD3+CD8+T cells percentages elevated significantly [(75.78±11.18)% vs (73.78±11.33)%, (32.57±14.90)% vs (31.00±14.79)%; all P 0.05); after 48 hours of storage, CD3+CD8+T cells percentage elevated obviously [(32.03±14.95)% vs (31.00±14.79)%; P<0.05] and CD19+cells percentage decreased markedly [(15.32±11.97)% vs (16.11±12.38)%; P<0.05]; after 72 hours of storage, CD3+and CD3+CD8+T cells percentages were significantly increased [(75.63±11.08)% vs (73.78±11.33)%, (32.62±14.98)% vs (31.00±14.79)%; all P<0.05], while CD56+and CD19+cells percentages were reduced observably [(8.21±6.52)% vs (9.02±6.80)%, (14.83±11.79)% vs (16.11±12.38)%; all P<0.05]. Conclusions The blood samples can be kept at room temperature and the testshould be completed within 48 h if the detection of lymphocyte subsets by flow cytometry cannot be performed in time; for the samples that have been pretreated, detection can be completed after saving at room temperature for 24 h, and the above treatments had no significant effect on the inspection results. Key words: Lymphocyte subsets; Flow cytometry; Sample preservation

Comparison of three different flow cytometers in the clinical assessment of lymphocyte subsets

Objective To evaluate the consistency and accuracy among 3 brands of flow cytometers(BriCyte E6, BD FACSCanto Ⅱ and Beckman Coulter FC 500) in the detection of lymphocyte subsets. Methods According to the methodology, the BriCyte E6 was compared with 2 flow cytometers commonly used in clinical detection. Seventy-three cases (40 male and 33 female) of anticoagulation peripheral blood specimens were collected in the clinical laborartory department of Xinhua Hospital in July 2015 and the percentage (%) and absolute number (#) of the lymphocyte subsets were detected by 3 different flow cytometers within samples collected 4 h. Results There were good consistency among the 3 flow cytometers (R2>0.95, R2 from 0.969 5 to 0.992 4) in the detection of lymphocyte subsets percentage, so did in the detection of absolute number (R2>0.95, R2 from 0.969 1 to 0.993 3). As to the precision evaluation, in the detectionof CD8%, T#, CD4+ T# and CD8+ T#, BriCyte E6 achieved a low CV% compared with FACSCanto Ⅱ and FC 500 (Friedman statistics are 16.720, 11.840, 15.760 and 15.430, P=0.000 2, 0.027, 0.000 4, 0.000 4, respectively). In the detection of T%, CD4%, NK%, B%, NK#, B#, there was no significant difference among the 3 flow cytometers (Friedman statistics are 4.242, 3.916, 0.852, 2.595, 1.835 and 0.578, P=0.119 9, 0.141 2, 0.653 2, 0.273 3, 0.399 6, 0.749 0, respectively). Conclusions The 3 flow cytometers have a good consistency in the detection of lymphocyte subsets. BriCyte E6 may be an alternative or complement of existing flow cytometers.(Chin J Lab Med, 2016, 39: 361-365) Key words: Flow cytometry; Comparative study; Lymphocyte subsets; Diagnostic equipment

The clinical study of dynamic detection of lymphocyte subsets in children with septic shock

Objective To investigate the dynamic changes of lymphocyte subsets (lymphocyte count, CD3+ T, CD4+ T, CD8+ T, CD19+ B, CD56+ NK)in children with septic shock. Methods Peripheral blood lymphocyte subsets were analysed in 25 cases with severe septic shock and 24 cases with mild septic shock on day 1, 3, 8, and compared with those of 25 healthy volunteers.Children with severe septic shock were divided into survival group and dying group according to the outcome.The detection data were compared. Results In the first day of admission, peripheral blood lymphocyte subsets in children with septic shock decreased significantly compared with the normal children, and there was significant difference in the three groups(P 0.05), but children with severe septic shock had statistical increase of CD4+ T, CD3+ T and lymphocyte count(P<0.05). There were 5 cases died who had severe septic shock in day 2 to day 5 after admission.The count of lymphocyte subsets were detected significantly lower in dying group than survival group in the third day of admission(P<0.05). Conclusion The continuous low level of lymphocyte subsets indicates bad outcome.The patient's condition improved with increase of CD4+ T, CD3+ T, lymphocyte count.CD4+ T is the most sensitive.Dynamic detection of lymphocyte subsets in the peripheral blood of children with septic shock is of great clinical significance in judging the severity of disease and curative effect. Key words: Septic shock; Lymphocyte subsets

Immune system disturbances in Clouston syndrome

Clouston syndrome belongs to the family of ectodermal dysplasias. So far, a defective immune response has not been reported in Clouston syndrome. We report, for the first time, immunological particularities of a large multigenerational Polish family with Clouston syndrome. Five members of the same family with Clouston syndrome, aged 6-76 years, and 20 healthy volunteers, aged 19-73 years, were enrolled in the study. In all participants, the ability of neutrophils to phagocytize opsonized Escherichia coli was assessed. Granulocyte oxidative burst was determined quantitatively, and an isolation of peripheral blood mononuclear cells and the detection of lymphocyte subsets were performed. All patients with Clouston syndrome underwent microscopic assessment of hair shafts, x-rays of the skull and hand bones, extra- and intraoral examination, and panoramic x-rays. Compared to the controls, all patients with Clouston syndrome presented with significantly reduced phagocytic activities of granulocytes and monocytes (P < 0.05). The percentages of granulocytes and monocytes being positive for oxidative burst were also significantly reduced in all patients with Clouston syndrome (P < 0.05). No disturbances in the percentages and absolute counts of T CD3+, T CD3+/CD4+, T CD3+/CD8+, natural killer, and B CD19+ cells were found. Although this study expands knowledge about Clouston syndrome, it also raises many questions. The results provide evidence of significantly reduced phagocytic activity and oxidative bursts of cells playing crucial roles in a nonspecific immune response. Further studies are required to understand the underlying mechanism of the hereby described abnormalities.

Diphencyprone and topical tacrolimus as two topical immunotherapeutic modalities. Are they effective in the treatment of alopecia areata among Egyptian patients? A study using CD4, CD8 and MHC II as markers

Objective: To evaluate the efficacy of two topically applied immunomodulative agents through the detection of lymphocyte subsets using monoclonal antibodies against CD4, CD8 and MHC II. Methods: Fifty patients from the Departments of Medical Biochemistry, Dermatology and Pathology at Cairo University with different degrees of alopecia areata (AA) were included in this study. They were classified into two groups each of 25 patients. Each patient was treated with the immunomodulative agent on one side of the scalp and the other side was left as a control. Biopsies were taken from all patients at the beginning of treatment and at the end of the study. Tissue specimens were prepared for histologic and immunophenotypic analysis. The main outcome measures were the uses of diphencyprone (DPCP) and topical tacrolimus as two topical immunotherapeutic modalities in the treatment of AA. Results: A clinical response of 68% was achieved in group A (treated with DPCP) while group B (treated with 0.1% tacrolimus) showed an insignificant clinical response. Decreased expression of CD4 and increased expression of CD8 and MHC II was detected in the post-treated areas compared with pretreated areas in cases treated with DCPC. In tacrolimus-treated cases, there was a decrease in CD4 and MHC II, with no change in CD8 between the pre- and post-treated areas. Conclusion: DCPC is one of the most accepted therapeutic modalities in the treatment of AA, with a favourable prognosis among patchy hair loss. MHC II expression was the one correlating with clinical response. Tacrolimus, though beneficial in other dermatoses, could not be considered effective in the treatment of AA.

Allergology on a chip

Allergology on a chip

Rapid quantification of lymphocyte subsets in heterogeneous cell populations by flow cytometry

Determination of the number of viable cells or quantification of lymphocyte subsets in heterogeneous cell populations is critically important for cytotoxicity assays, apoptosis assays, or the analysis of differential activation of T-cell subsets by distinct stimuli. In this report, we describe a rapid flow cytometry method termed Standard Cell Dilution Analysis (SCDA) specifically to quantify any subset of phenotypically definable, viable cells in heterogeneous populations using a FACScan flow cytometer. This method combines: (1) specific detection of lymphocyte subsets by phycoerythrin-conjugated monoclonal antibodies, (2) electronic exclusion of dead cells or cell debris by propidium-iodide staining and gating on forward vs. sidescatter, respectively, and (3) admixture of a known amount of fixed, fluorescein isothiocyanate stained cells immediately before analysis as a constant parameter to allow for calculation of cell quantity. We have used SCDA to analyze the in vitro growth characteristics of various human T-lymphocyte subpopulations in response to different activation stimuli.

Identification of Lymphocyte Subsets in the Human Fallopian Tube

Immunohistochemical investigations for the detection of lymphocyte subsets in the human oviduct have been performed. Knowledge about local immunity especially cell-mediated immunity, in the fallopian tube has been, up to now, limited. As an essential structure for the human reproduction process, the tubal mucous membrane is exposed to a variety of antigens. A total number of 20 tubal biopsies obtained from fertile women during gynecological operations like tubal ligations or hysterectomy were examined by the immunoperoxidase technique. Seven specimens were obtained during the proliferative phase, ten during the secretory phase and three during a caesarean section with tubal ligations. It could be established that the presence of lymphocytes in the oviductal mucous membrane is physiological. These cells can be identified by their typical immunohistochemical patterns. There were no significant differences of the type and number of lymphocytes in the mucosa within the phases of menstrual cycle. The dominant cell types in the tubal mucosa were the CD3+ and CD8+ lymphocytes. It can be suggested that the lymphocytes in the tubal mucosa may involved in the process of immune tolerance, which could realize the transport of sperms and blastocysts through the oviduct under normal conditions without activation of local immune mechanisms. The lymphoid tissue of the oviduct is a specialized form of mucosal-associated lymphoid tissue (MALT).

Detection of lymphocyte subsets using three-color/single-laser flow cytometry and the fluorescent dye Peridinin chlorophyll-a protein

The fluorescent dye, Peridinin chlorophyll A protein (PerCP) derived from dinoflagellate organisms (Glenodinium sp.) can be excited by a 488 nm laser and emits light with a large Stokes shift and no major spectral overlap with commonly used chromophores such as fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE). PerCP was conjugated directly to various mouse monoclonal antibodies (mAb) specific for human leukocyte markers or to avidin for use with biotinylated-mAb, and used to perform three color single-laser flow cytometry. The efficacy of this method was demonstrated by analyzing the heterogeneity of thymus T lineage subsets and B lymphocyte subsets in blood. CD4-CD8-, CD4+CD8+ and CD4+CD8- or CD4-CD8+ subsets differ in their expression of cell-cell interaction markers including CD18, CD28, CD44 and Leu 8, and activation/subset markers CD45RO, CD45RA and CD26. Some CD5+ peripheral blood B cells, unlike CD5-B cells, expressed CD45RO or high levels of CD54 (ICAM-1) suggesting the CD5+ B cell population contains activated lymphocytes. The availability of such an accessible method for three color analysis will make it possible to do routine three color monitoring of immunologic diseases such as AIDS, and autoimmune or periodontal diseases.