Two nomograms constructed for predicting the efficacy and prognosis of advanced non­small cell lung cancer patients treated with anti­PD­1 inhibitors based on the absolute counts of lymphocyte subsets.

BACKGROUND: Patients treated with immune checkpoint inhibitors (ICIs) are at risk of considerable adverse events, and the ongoing struggle is to accurately identify the subset of patients who will benefit. Lymphocyte subsets play a pivotal role in the antitumor response, this study attempted to combine the absolute counts of lymphocyte subsets (ACLS) with the clinicopathological parameters to construct nomograms to accurately predict the prognosis of advanced non-small cell lung cancer (aNSCLC) patients treated with anti-PD-1 inhibitors. METHODS: This retrospective study included a training cohort (n = 200) and validation cohort (n = 100) with aNSCLC patients treated with anti-PD-1 inhibitors. Logistic and Cox regression were conducted to identify factors associated with efficacy and progression-free survival (PFS) respectively. Nomograms were built based on independent influencing factors, and assessed by the concordance index (C-index), calibration curve and receiver operating characteristic (ROC) curve. RESULT: In training cohort, lower baseline absolute counts of CD3+ (P < 0.001) and CD4+ (P < 0.001) were associated with for poorer efficacy. Hepatic metastases (P = 0.019) and lower baseline absolute counts of CD3+ (P < 0.001), CD4+ (P < 0.001), CD8+ (P < 0.001), and B cells (P = 0.042) were associated with shorter PFS. Two nomograms to predict efficacy at 6-week after treatment and PFS at 4-, 8- and 12-months were constructed, and validated in validation cohort. The area under the ROC curve (AUC-ROC) of nomogram to predict response was 0.908 in training cohort and 0.984 in validation cohort. The C-index of nomogram to predict PFS was 0.825 in training cohort and 0.832 in validation cohort. AUC-ROC illustrated the nomograms had excellent discriminative ability. Calibration curves showed a superior consistence between the nomogram predicted probability and actual observation. CONCLUSION: We constructed two nomogram based on ACLS to help clinicians screen of patients with possible benefit and make individualized treatment decisions by accurately predicting efficacy and PFS for advanced NSCLC patient treated with anti-PD-1 inhibitors.

Reduced Counts of Various Subsets of Peripheral Blood T Lymphocytes in Patients with Severe Course of COVID-19.

This study was intended to define T lymphocyte subsets in different clinical groups of COVID-19-infected patients to explore the interaction between T cell-mediated immune response and the severity of COVID-19 course. Lymphopenia in patients with severe COVID-19 was found. In patients with severe COVID-19 course, the absolute counts of CD3+, CD4+, and CD8+ T lymphocytes at admission were lower than on day 14 after discharge. Further analysis showed that the older were the patients with COVID-19, the more likely they developed severe infection. The results confirmed the significance of T lymphocytes in the clearance of the COVID-19.

Reference values for T and B lymphocyte subpopulations in Turkish children and adults

Background/aim: Established reference values are critical for the interpretation of immunologic assessments. In particular, the proportion and absolute counts of T- and B- cell subpopulations are subject to change with age and ethnicity. We aimed to establish age- specific reference values for lymphocyte subsets using updated immunophenotyping panels. Materials and methods: We studied a total of 297 healthy Turkish subjects aged 0 to 50 years, stratified into major age brackets in a cluster factor of 10 per age-group. The predetermined age intervals contained randomly allocated participants enrolled over a period of 6 months, who were homogenously distributed by sex. We analyzed a complete blood count test and simultaneously with detailed immunophenotyping enumerated the percent and absolute cell counts of lymphocyte subsets. Results: The percentage and absolute counts of lymphocyte subsets show a marked surge across the age-span. T helper, T cytotoxic, and the natural killer cell numbers were increasing from birth until 6 months, followed by a gradual decrease thereafter. B cell numbers were rising until 2 years, followed by a gradual decrease for the upcoming years, accompanied by a steady expansion of unclass-switched- and class-switched- B cells. Conclusion: We provide updated extensive reference intervals for lymphocyte subpopulations in Turkish people.

Absolute counting of neutrophils in whole blood using flow cytometry.

Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens.

A clinical nomogram based on absolute count of lymphocyte subsets for predicting overall survival in patients with non-small cell lung cancer.

BACKGROUND: The absolute count of lymphocyte subsets (ACLS) is correlated to the prognosis of multiple malignancies. This study aimed to combine the ACLS with the clinicopathological parameters to develop a nomogram to accurately predict the prognosis of non-small cell lung cancer (NSCLC) patients. METHODS: This retrospective study included a training cohort (n = 1685) and validation cohort (n = 337) with NSCLC patients treated in First Teaching Hospital of Tianjin University of Traditional Chinese Medicine between January 2018 and January 2021. Cox regression were conducted to identify factors associated with overall survival. The nomogram was built based on 10 significant factors, and evaluated by the concordance index (C-index), calibration curve and receiver operating characteristic (ROC) curve. RESULTS: In the training cohort, the multivariate cox proportional hazard regression analysis showed that the independent factors for overall survival (OS) included age, brain metastases, hepatic metastases, respiratory system diseases, clinical stages, surgery, absolute count (AC) of CD3+, CD4+, CD8+, and NK cells, which were all applied in the nomogram. The C-index of the nomogram to predict OS was 0.777 (95% CI, 0.751-0.802) in training cohort and 0.822 (95% CI, 0.798-0.846) in validation cohort. The area under the ROC showed a good discriminative ability in both cohorts. Calibration curves presented an excellent consistence between the nomogram predicted probability and actual observation. CONCLUSIONS: We established a prognostic nomogram to predict OS of the NSCLC patient. This nomogram provided a more quantitative, scientific and objective basis for accurate diagnosis and individual management of NSCLC patients.

Flow Cytometric Identification and Enumeration of Monocyte Subsets in Bovine and Water Buffalo Peripheral Blood.

Monocytes are innate immune system key players with pivotal roles during infection and inflammation. They migrate into tissues and differentiate into myeloid effect cells (macrophages, dendritic cells) which orchestrate inflammatory processes and are interfaces between the innate and adaptive immune responses. Their clinical relevance to health and disease of cattle (Bos taurus) and water buffalo (Bubalus bubalis), two of the most important livestock species, has been highlighted in physiologic (pregnancy) and pathologic (mastitis, metritis, and viral infections) conditions. The existence of three different monocyte subsets in cattle was established by flow cytometry (FC), as follows: classical (cM; CD14++ CD16-/low ), intermediate (intM; CD14++/+ CD16+ ), and non-classical (ncM; CD14-/low CD16++ ) monocytes. FC applications for studying the immune system of cattle and water buffalo still have significant limitations. In this article, we describe some practical approaches to overcome these limitations and, in particular, allow the identification and enumeration of cM, intM, and ncM subpopulations in cattle and buffalo peripheral blood. Indeed, we propose the new procedure lyse/wash/no-centrifugation (L/W/NC) that can be combined with the FC absolute counting procedures and can overcome specific issues of the lyse/no-wash protocols (L/NW). Finally, for the first time, we demonstrated the existence of cM, intM, and ncM monocyte subsets also in the water buffalo, showing some interesting differences with cattle, such as the bubaline cM are mainly CD14+/++ /CD16+ . These subtle differences may influence inflammatory disease regulation in, for example, mastitis and metritis. The upregulation of CD16 expression on cM may reveal different monocyte priming, leading to different functional features of macrophages/dendritic cells in tissues after infection. © 2023 Wiley Periodicals LLC. Basic Protocol: Absolute count of cM, intM, and ncM without compensation Alternate Protocol: Absolute count of cM, intM, and ncM for single laser platform Support Protocol 1: In-house monoclonal antibody labeling using a Pacific Blue™ kit Support Protocol 2: In-house monoclonal antibody labeling using an Alexa Fluor® 647 kit Support Protocol 3: Titration of fluorochrome-conjugated antibodies.

Current Applications of Absolute Bacterial Quantification in Microbiome Studies and Decision-Making Regarding Different Biological Questions.

High throughput sequencing has emerged as one of the most important techniques for characterizing microbial dynamics and revealing bacteria and host interactions. However, data interpretation using this technique is mainly based on relative abundance and ignores total bacteria load. In certain cases, absolute abundance is more important than compositional relative data, and interpretation of microbiota data based solely on relative abundance can be misleading. The available approaches for absolute quantification are highly diverse and challenging, especially for quantification in differing biological situations, such as distinguishing between live and dead cells, quantification of specific taxa, enumeration of low biomass samples, large sample size feasibility, and the detection of various other cellular features. In this review, we first illustrate the importance of integrating absolute abundance into microbiome data interpretation. Second, we briefly discuss the most widely used cell-based and molecular-based bacterial load quantification methods, including fluorescence spectroscopy, flow cytometry, 16S qPCR, 16S qRT-PCR, ddPCR, and reference spike-in. Last, we present a specific decision-making scheme for absolute quantification methods based on different biological questions and some of the latest quantitative methods and procedure modifications.

A flow cytometric assay for the quantification of MSC lysis by peripheral blood mononucleated cells.

Mesenchymal stromal cells (MSC) are attractive candidates for the treatment of acute graft versus host disease (aGvHD) or autoimmune disorders. However, mechanisms of MSC recognition remain unclear and there are evidences that MSC are not totally immunoprivileged. Data suggest that MSC undergo apoptosis after infusion in presence of cytotoxic cells and their death could drive immunosuppression. In GvHD patients, that activity was associated with clinical response. It is mandatory to develop an in vitro potency testing predictor of the "in vivo" response to the therapy. We describe a flow cytometric assay based on differential immunostaining of target and effector cells where BM MSC are enumerated with fluorospheres to determine the loss of target cells after co-culture with PB MNC. 6/13 (46%) of BM MSC lots were lysed by PB MNC and the lysis was proportional to the E/T cell ratio. The method overcomes the problems linked to the use of dyes or radioactive, evidencing the limitations linked to the use of a single vital dye and proposing a precise gating strategy based on absolute cell counts where cells are left untouched. The assay is easy and could be used to predict the response of the patients to the therapy.

Absolute quantitation of microbes using 16S rRNA gene metabarcoding: A rapid normalization of relative abundances by quantitative PCR targeting a 16S rRNA gene spike-in standard.

Metabarcoding of the 16S rRNA gene is commonly used to characterize microbial communities, by estimating the relative abundance of microbes. Here, we present a method to retrieve the concentrations of the 16S rRNA gene per gram of any environmental sample using a synthetic standard in minuscule amounts (100 ppm to 1% of the 16S rRNA sequences) that is added to the sample before DNA extraction and quantified by two quantitative polymerase chain reaction (qPCR) reactions. This allows normalizing by the initial microbial density, taking into account the DNA recovery yield. We quantified the internal standard and the total load of 16S rRNA genes by qPCR. The qPCR for the latter uses the exact same primers as those used for Illumina sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene to increase accuracy. We are able to calculate the absolute concentration of the species per gram of sample, taking into account the DNA recovery yield. This is crucial for an accurate estimate as the yield varied between 40% and 84%. This method avoids sacrificing a high proportion of the sequencing effort to quantify the internal standard. If sacrificing a part of the sequencing effort to the internal standard is acceptable, we however recommend that the internal standard accounts for 30% of the environmental 16S rRNA genes to avoid the PCR bias associated with rare phylotypes. The method proposed here was tested on a feces sample but can be applied more broadly on any environmental sample. This method offers a real improvement of metabarcoding of microbial communities since it makes the method quantitative with limited efforts.

[Effects of "Tiaoyi Sanjiao" acupuncture and moxibustion on cancer-induced fatigue and immune function in patients with advanced non-small cell lung cancer].

OBJECTIVE: To observe the therapeutic effect of "Tiaoyi Sanjiao" （regulating and tonifying the triple energizer）acupuncture and moxibustion in the treatment of cancer-related fatigue of advanced non-small cell lung cancer and observe its influence on lymphocyte count. METHODS: The cancer-related fatigue patients with advanced non-small cell lung cancer who met the inclusive criteria were randomly divided into 2 groups, with 77 cases in each group. Patients in the control group were treated with conventional Chinese and Western medicine. In the treatment group, on the base of the treatment as the control group, "Tiaoyi Sanjiao" acupuncture and moxibustion was applied（mild moxibustion at Danzhong ［CV17］, Zhongwan［CV12］, Qihai［CV6］, bilateral Zusanli［ST36］, and acupuncture at bilateral Xuehai［SP10］, Waiguan［SJ5］, Taichong［LR3］）. KPS score, Piper scale, percentage and absolute count of lymphocyte subsets of the two groups before and after treatment were observed. RESULTS: Compared with pre-treatment, the KPS scores of both groups were significantly increased after treatment (P<0.05); the behavioral dimension score of the Piper scale, and the percentage of B cells of the control group decreased significantly(P<0.05); the behavioral dimension, emotional dimension, sensory dimension scores, and total score of the Piper scale in the treatment group were significantly reduced(P<0.05), while the percentage of CD3+T cells and absolute counts of CD3+T cells, CD4+T cells and CD8+T cells of the treatment group were significantly increased (P<0.05). After treatment, in comparison with the control group, behavioral dimension score of the Piper scale in the treatment group decreased significantly(P<0.05); the percentage and absolute counts of B cells and CD4+T cells, the absolute count of CD3+T cells in the treatment group were up-regulated (P<0.05). CONCLUSION: "Tiaoyi Sanjiao" acupuncture and moxibustion can significantly alleviate the fatigue state and improve the quality of life in patients with advanced non-small cell lung cancer, which may be achieved by regulating the number of lymphocytes and improving immune function.

Comparison of methods and TAT assessment: Volumetric AQUIOS CL and bead-based FACS CANTO II cytometers.

BACKGROUND: Measurement of lymphocyte subpopulations is a crucial parameter in the diagnosis and monitoring of therapy in a wide variety of clinical conditions. We compared different flow cytometry-based methods to determine lymphocyte subsets counts of routine samples by volumetric AQUIOS CL (Beckman Coulter) and bead-based FACS CANTO II (BD Biosciences) cytometers. We evaluated the possible decrease of the labor intensive technical work using the fully automated AQUIOS CL system in the pre and post analytical steps in comparison with our routine flow cytometer FACS CANTO II, toward the reduction of laboratory analytical turnaround time (TAT). METHODS: The analytical performance of AQUIOS CL flow cytometer compared with FACS CANTO II was evaluated testing 224 routine samples, attending the Immunology and Allergology Laboratory Unit, S. Giovanni di Dio Hospital, (Florence, Italy) between September and October 2015. RESULTS: Bland-Altman plot of the differences between the two methods showed an excellent agreement for absolute and percentage counts. Furthermore, the study showed that automated AQUIOS CL system is simple to be used during all analytical steps with a reduction of TAT. CONCLUSION: In routine conditions, AQUIOS CL flow cytometer could be a suitable tool for subpopulations subset analysis. © 2017 International Clinical Cytometry Society.

Evaluation of a novel automated volumetric flow cytometer for absolute CD4+ T lymphocyte quantitation.

BACKGROUND: Bead-based single platform cytometry technology (SPT) is the gold standard when performing CD4 absolute counting. However, it presents drawbacks as precision depends on various critical steps (for example, pipetting methodology, overtime stability of beads, stability of fluidics, regular recalibration…) and thus requires skilled operators. The fully automated volumetric SPT AQUIOS CL (Beckman Coulter) has recently emerged as an alternative with no need of beads. It may help improving results standardisation and fulfilling requirements for certification (ISO 15189). In this study, we assessed SPT AQUIOS CL performances in accordance to requirements for ISO 15189 accreditation. METHODS: We evaluated repeatability and reproducibility (precision), bias (trueness), uncertainty (total error), range limits (linearity, quantification, detection limits), and inter-reagent/inter-sample contaminations in enumerating CD4+ T-cells. Concomitantly, we compared AQUIOS CL CD4+ T-cell values with the results obtained with our routine bead-based SPT (that is, FC500 Beckman Coulter, bead-based SPT), on blood samples from 148 patients representative of clinical laboratory routine workload. RESULTS: Every result (repeatability, reproducibility, trueness, total error) was below the acceptable thresholds proposed in international recommendations. Contamination results and range limits (linearity, quantification, and detection limits) were all found perfectly suitable to routine analysis. The comparison between AQUIOS CL and FC500 exhibited excellent correlation and agreement (Pearson R = 0.99, P < 0.001; Lin's concordance correlation coefficient: Lin ρc = 0.991, Cb = 0.999), and Bland-Altman analysis did not reveal any systematic error. CONCLUSIONS: Our results demonstrate that, upon subsequent validation in more routine conditions, the AQUIOS CL could be a suitable tool for clinical flow cytometry laboratories facing accreditation process. © 2016 International Clinical Cytometry Society.

Major depressive disorder patients on antidepressant treatments display higher number of regulatory T cells

@#Introduction: Regulatory T cell (Treg) is a subtype of T lymphocyte that plays a crucial role in establishing immunologic self-tolerance and maintaining immune homeostasis. In this study, we set out to investigate the percentage and absolute count of Tregs in major depressive disorder (MDD) patients and their correlation with disease severity. Materials & Methods: This is a case-control study consisting of 47 MDD patients and 47 healthy controls. MDD patients were treated with antidepressant drugs according to their physician’s choice. The severity of MDD was assessed using Beck Depression Inventory (BDI) and Montgomery-Asberg Depression Rating Scale (MADRS) at the time of recruitment. Healthy controls completed the Depression Anxiety Scoring System (DASS21) questionnaire to ensure they were in good mental health without history of MDD. The percentage and absolute count of CD4+ CD25+ Tregs and CD4+ CD25+ FOXP3+ Tregs were identified by multiparameter flow cytometry. Results: The percentage and absolute count of CD4+ CD25+ Treg cells were significantly higher in MDD patients than in healthy controls (P<0.001, in both cases). Likewise, the percentage and absolute count of CD4+ CD25+ FOXP3+ Treg cells were also significantly higher in MDD patients compared to healthy controls (P=0.003 and P=0.002, respectively). However, there was no significant correlation between the percentage and absolute count of CD4+ CD25+ Treg and CD4+ CD25+ FOXP3+ Treg cells with BDI or MADRS score. Conclusions: Our results suggest that antidepressant treatments contributed to an upregulation of Tregs in MDD patients.