Cluster Of Differentiation Markers Research Articles

Cluster of Differentiation Markers and Human Leukocyte Antigen Expression in Chronic Lymphocytic Leukemia Patients: Correlations and Clinical Relevance.

Chronic lymphocytic leukemia (CLL) is a distinct category of lymphoproliferative disorder characterized by the clonal expansion of mature B cells, followed by their accumulation in primary and secondary lymphoid organs. Cluster of differentiation (CD) markers such as CD79b, CD45, CD23, CD22 and CD81 serve as reliable prognostic indicators in CLL as well as the human leukocyte antigen (HLA) with its well-documented associations with various cancers. This study aims to investigate, for the first time, potential connections between HLA typing and CD marker expression in CLL. Although it is one of the most prevalent neoplasms, there is a need for biomarkers that can improve survival. This study included 66 CLL patients and 100 controls, with all samples analyzed using biochemical methods, flow cytometry, and cytomorphology. Next-generation sequencing was performed for HLA typing. The results indicate that several CD markers are statistically associated with different HLA alleles, specifically CD45 with HLA-C*07:01:01; CD79b with HLA-DPA1*02:01:02; CD23 with HLA-B*39:01:01; CD22 with HLA-B*49:01:01, HLA-C*07:01:01, HLA-DPB1*02:01:02, and HLA-DRB1*07:01:01; and CD81 with HLA-DPB1*04:02:01, HLA-DQA1*01:04:01, and HLA-DQB1*05:03:01. In conclusion, this research demonstrates significant statistical links between HLA genes and immunophenotypic markers in CLL patients, shedding new light on the immunological context of CLL.

The impact of COVID-19 on microRNA and CD marker expression in AML patients

Acute myeloid leukaemia (AML) is an aggressive leukaemia characterised by uncontrolled blast cell proliferation. miRNAs and Clusters of Differentiation (CD) molecules play essential roles in AML progression. This study aims to investigate the effect of COVID-19 on the expression of circulating miRNA and CD molecules in AML. This cross-sectional study recruited 32 AML patients and 20 controls. Blood samples were collected and analysed using molecular cytogenetic, miRNA/mRNA expression, and flow cytometry techniques. The expression of miRNAs varied significantly between patients with AML and control individuals. The co-expression of these miRNAs was higher (P < 0.05), indicating that the presence of one miRNA led to increased expression of other miRNAs. A differential correlation was observed between miRNAs and CD markers. Additionally, miRNA 16, miRNA 21, and miRNA 221 showed significant downregulation (P < 0.05 and P < 0.01, respectively) in AML patients with COVID-19 infection compared to those without a disease. Interestingly, this study identified a higher expression level (P < 0.01) of miRNA 137 as a novel biomarker for AML patients. Moreover, the expression of miRNA 137 showed a high correlation (P < 0.05) with most of the CD markers examined in this study and FISH features data. Furthermore, a strong correlation (P < 0.01) was observed between CD markers and miRNA among AML patients with positive and negative COVID-19 infection. These data demonstrated that COVID-19 contributed to increased expression of microRNAs in AML patients. MicroRNA 137 was identified as a novel microRNA that exhibited significant differences between patients and healthy individuals, highlighting its role in AML pathogenesis.

Frequency and Aberrant CD Marker Profile of Acute Leukaemias.

To determine the frequency of different types of acute leukaemia and their subtypes along with associated aberrant CD markers. Descriptive study. Place and Duration of the Study: Department of Immunology Armed Forces Institute of Pathology,National University of Medical Sciences, Rawalpindi, Pakistan, from November 2021 to October 2023. All samples received for flow cytometric immunophenotyping with suspicion of acute leukaemia were included in the study. Cells were stained with fluorochrome labelled monoclonal antibodies against lineage-specific cluster of differentiation (CD) markers through a lyse-wash procedure. Acquisition and analysis were done using multi-parameter BD FACS Canto II Flow cytometer and BD FACS Diva software, respectively. Data were entered and analysed using SPSS v 23.0. Over a period of 2 years, a total of 1,115 suspected patients were tested for acute leukaemia. Among them, 728 (65.3%) were males and 387 (34.7%) were females, with mean age 28 ± 21 years, ranging from 1 week to 87 years. Among a total of 875/1115 (78.5%) diagnosed cases of acute leukaemia, AML was the most common leukaemia present in 408/875 (46.6%) patients followed by B-ALL and T-ALL in 384/875 (43.8%) and 70/87 (8%) patients, respectively (p = 0.5712). Aberrant CD markers were detected in 109/875 (12.5%) leukaemias (p = 0.0628). The most common aberrant CD markers in B-ALL were CD13 and CD33 present in 30/384 (7.8%) cases separately. Among AML and T-ALL most common aberrant CD markers were CD7 and CD33 present in 25/408 (6.13%) and 7/70 (10%) cases, respectively. Special consideration should be given to the presence of aberrant CD markers when assigning lineages to acute leukaemias. They may be important diagnostic, prognostic, and management tools for institution of immunotherapy. Aberrant CD markers, Acute leukaemia, CD Markers, Flow cytometry, Immunophenotyping.

Assessing Pancytopenia in Leukemia Patients through flow Cytometry and ELISA to Evaluate the Complete Blood Counts and Cluster of Differentiation Markers

To diagnose cases involving pancytopenia or leukopenia, a comprehensive assessment of various factors is necessary, including blood count, peripheral blood, and bone marrow analysis, immunophenotyping, and cytogenetics. This study aims to examine the complete blood count parameters and CD markers in Sudanese patients with leukemia and pancytopenia, utilizing flow cytometry and ELISA techniques. This study is a laboratory-based addressing the assessment of the target population (acute Leukaemia with pancytopenia) by complete blood count, flow cytometry, and ELISA techniques. The research group was comprised of patients who were diagnosed with acute leukemia and had pancytopenia before undergoing treatment. Another group of patients with acute leukemia but without pancytopenia was also included. In addition, there was a control group consisting of healthy individuals who volunteered for the study. Essentially, the control group was made up of healthy individuals who were not affected by acute leukemia or pancytopenia. In our study, we enrolled a total of 150 participants, comprising three groups: 50 cases of acute leukemia, 50 patients with acute leukemia who subsequently developed pancytopenia, and 50 healthy volunteers. The majority of participants were female, constituting 56% of the sample (84 individuals), while the most prevalent age group represented was individuals aged 65, accounting for 43.3% of the participants. Our analysis revealed a statistically significant correlation between age and both leukemia and leukemia with pancytopenia, with a p-value of 0.00. Furthermore, the presence of either AML or ALL also exhibited a substantial association with the disease, indicated by a p-value of 0.00. Specifically, the use of a flow cytometer allowed us to identify the presence of CD3 with a p-value of 0.00 and CD4 with a slightly higher p-value of 0.04. Improvement of patient management by introducing effective tools for predicting prognosis is the key to success in managing diseases. We recommend that flowcytometry be used routinely to diagnose leukemia and leukemia with cytopenia in patients at all stages of the disease.

The Prognostic Value of Aberrant Expression of Cluster Differentiation Markers in Patients with Acute Leukemia

The Prognostic Value of Aberrant Expression of Cluster Differentiation Markers in Patients with Acute Leukemia

Immunohistochemical study of the expressed cluster differentiation markers proteins type 20 and 56 in breast tissues from a group of Iraqi patients with breast cancers.

Tumor-infiltrating lymphocytes (TIL) are important immunological components in response to cancers. Patients with higher numbers of TIL in breast cancerous tissues, comprising T- cytotoxic and T - helper cells along with B- and rare natural killer (NK) cells, have more favorable clinical outcomes. To analyze the rate of the expressed surface biomarker proteins of CD20-B cells and CD56- NK cells on the infiltrative lymphocytic subpopulations in a group of breast tumorous tissues (invasive and benign) from female patients in Iraq and explore the relations to the grade of the invasive breast cancerous tissues. One hundred and 75 archived breast tissues were enrolled in this retrospective research: 100 archived breast from female patients with invasive breast cancers (BC) [20 well differentiated BC tissues; 48 moderately differentiated BC and 32 poorly differentiated BC tissues]; 50 tissue biopsies from female patients with benign breast tumors and 25 apparently normal individuals with healthy breast tissues (included as the control group for this study). Immunohistochemistry was achieved for the detection of the expressed surface biomarker proteins related to B cell CD20 and NK cell CD56 present on the infiltrative lymphocytic subpopulations in breast tissues by using specific primary antibodies for these proteins via utilizing an immune-enzymatic antigen detection system. The detection of IHC reactions for the expressed B cell CD20 - cell surface ( CD) biomarker proteins were observed in 53 out of 100 (53.0%) BC tissues, and in 24 out of 50 (48.0%) benign breast tumorous tissues, while CD20- positive cell surface markers was detected in apparently healthy breast tissues of the control group in a percentage of 32.0% (8 out of 25 tissues). Statistical significant differences (P<0.05) between both groups of malignant and benign breast tumors and the control group were found. However, between breast malignant and benign tumor groups, no significant difference was found ( p >0.05). Detection of CD56- IHC reactions revealed in 14% (14 out of 100 BC tissues), in 16% (8 out of 50 benign breast tissues) and none of control breast tissues revealed CD56- IHC reactions. Among all the enrolled groups, no significant differences (P>0.05) were detected. The observed significant rates that showed highly significant differences between both studied groups of breast malignant and benign tumor in comparison to the control group indicate that the CD20- positive infiltrative B cell- lymphocytic subpopulations might contributed in the defense against these subsets of benign and malignant breast tumors. However, the observed rates of NK cell CD56 present on the lymphocytic subpopulations infiltrating the examined malignant and benign breast tumorous tissues seeming to play irrelevant roles in the defense against these studied breast tumor groups.

67 Infant Health Status Impacts the Neutrophil Phenotypes and Polyunsaturated Fatty Acids Composition in Human Milk

Abstract Background The abundance of human milk leukocytes, including neutrophils, changes in response to the health status of mothers and their infants. Elevated leukocyte counts occur during infant infection and return to baseline levels upon recovery. However, it is unclear how infant health status can affect the neutrophil phenotypes and the concentration of fatty acids in their mothers’ human milk. Objectives This study investigates the association between infant health status and human milk neutrophil counts and activation state, and fatty acid levels, in human milk. Design/Methods This is a prospective cohort study of 50 healthy breastfeeding mothers recruited from St. Michael’s hospital, in Toronto, Ontario, who were followed up from 2-4 weeks until 4 months postpartum. Human milk samples were collected from participants and data regarding infant health status were collected from self-reported questionnaires completed by participants at both timepoints, then imputed based on whether infants had taken antibiotics, had been hospitalized, or had been diagnosed with a medical condition, at least 2 weeks prior to samples collection. Neutrophils were quantified using flow cytometry and labelled with a panel of antibodies to detect specific cluster of differentiation (CD) biomarkers. Fatty acids were identified and quantified using a gas chromatography-flame ionization detector (GC-FID). Linear mixed-effects models were used to evaluate the correlation between infant health status and changes in neutrophil counts, along with their expressed CD markers, and fatty acids composition in human milk during lactation. Results The CD markers were classified into 4 categories based on their function: degranulation/activation markers (CD63, CD64, and CD66a), an immunoregulation marker (CD16), adhesion markers (CD11b, CD18), and a lipopolysaccharide receptor (CD14). Human milk from mothers whose infants had a health condition had neutrophils which significantly expressed elevated levels of the CD64 biomarker (β: 85.65, p=0.009 in adjusted models), compared to human milk from mothers of healthy infants. However, there were no significant associations between infant health status, absolute hmPMN counts, andother CD biomarkers (p&amp;gt;0.05). There were also elevated levels of arachidonic acid (β: 0.12, p=0.013) and C22:5n-6 (β: 0.06, p=0.015) fatty acids in the human milk of mothers whose infants had a health condition, compared with human milk from mothers of healthy infants, after adjusting for maternal age, post-pregnancy body mass index, infant sex, and infant feeding pattern. Conclusion This study demonstrates that infant health status is associated with changes in human milk immunological components, suggesting an inflammatory protective response mechanism in the mammary glands triggered by infants infection. How these alterations can affect infant health outcomes in the short and longer terms need critical consideration.

Evaluation of aberrant expression of CD markers in acute leukemia cells

Background and objective: Worldwide immunophenotyping by flow cytometry (FCM) in acute leukemia (AL) is the golden step in the diagnosis. It’s very common for acute leukemias to aberrantly express antigens or cluster of differentiation (CD) markers which are usually expressed in other lineages of the disease hence this study aimed at determining the prevalence of aberrancy in AL and to find out the frequency of each aberrant CD marker and their association with the clinic-hematological profile of the cases. Methods: Following history and clinical examination of enrolled patients, blood and/or bone marrow aspirate was drawn for morphological examination and immunophenotyping by FCM from 86 newly diagnosed acute leukemia cases then multiple steps procedure was applied followed by interpretation of the results. Results: The prevalence of aberrant phenotype was 46.5%. The proportional frequency of aberrant phenotype in acute myeloid leukemia (AML) was 41%, in B-acute lymphoblastic leukemia (B-ALL) was 48.8% and in T-acute lymphoblastic leukemia (T-ALL) was 66.6%. The commonest aberrant CD markers in AML were CD22 and CD2, in B-ALL were CD66c and CD13 while in T-ALL were CD13 and CD33. The aberrant phenotype harbored lower white blood cell (WBC) count and blast percentage in PB, also splenomegaly was more frequent in lymphoid positive (Ly+) AML and myeloid positive (My+) T-ALL while in B-ALL, splenomegaly was more frequent in myeloid negative (My-) B-ALL. Conclusion: Aberrant phenotype prevalence in our study sample was comparable to other studies, considerable frequency of aberrant markers is present in cases of AL and some variations exist regarding the clinical and hematological profile of the aberrant group.

Hematological, clinical, immunophenotypic characterization, and treatment outcomes of prognostically significant genetic subtypes of B-lineage acute lymphoblastic leukemia: A report of 1021 patients from India.

The published literature on hematological, clinical, flowcytometric-immunophenotyping, and minimal residual disease outcomes of the prognostically important genetic subtypes of acute lymphoblastic leukemia (ALL) is scarce from low-income countries. For newer classifications such as BCR::ABL1-like ALLs, the scarcity of patient-level data is even more pronounced. The authors performed comprehensive detection of recurrent gene fusions and BCR::ABL1-like ALL cases followed by immunophenotypic profiling and obtained clinical outcome parameters for a large cohort (n=1021) of patients from India. This cohort included a significant number of patients with BCR::ABL1-like ALL subtype and other genetic subtypes of ALL. Patients with BCR::ABL1-positive and BCR::ABL1-like ALL were significantly older, had male preponderance, and expressed a higher white blood cell count than BCR::ABL1-negative cases (p<.05). Logistic regression modeling of B-lineage-ALL (B-ALL) subtypes revealed that cluster of differentiation (CD)36 is a strong statistically significant predictive marker of BCR::ABL1-like ALL (p<.05). Furthermore, patients with BCR::ABL1-like ALLs show a significantly higher frequency of CD36 expression compared to BCR::ABL1-negative ALLs (p<.05). In terms of clinical symptoms, lymphadenopathy is a strong statistically significant predictive marker in BCR::ABL1-like ALLs compared to BCR::ABL1-negative ALL cases (p<.05). In terms of treatment outcomes, minimal residual disease (MRD) positivity in BCR::ABL1-positive ALL cases were statistically significant (p<.05), and BCR::ABL1-like ALL cases had high MRD-positivity as compared to BCR::ABL1-negative ALL cases but did not show statistical significance. The findings evince the use of novel therapies and personalized treatment regimens to improve the overall survival of the newer incorporated entities in B-ALLs. This is the first report characterizing the hematological, clinical, flowcytometric-immunophenotyping, and minimal residual disease outcomes of the prognostically significant subtypes of ALLs in patients from India. Characterizing the hematological, clinical, flowcytometric-immunophenotyping, and minimal residual disease outcomes of the prognostically significant subtypes (n=1021) of acute lymphoblastic leukemia (ALLs) in patients from India. We have made two independent logistic regression models of cluster of differentiation (CD) markers and clinical symptoms to differentiate prognostically significant subtypes of ALLs. Logistic regression analysis of CD markers revealed CD36 as a strong predictor in BCR::ABL1-like ALL cases compared to BCR::ABL1-negative ALL cases. Logistic regression analysis of clinical symptoms revealed lymphadenopathy significantly predicts BCR::ABL1-like ALLs (p<.05). In terms of treatment outcomes, BCR::ABL1-positive ALL had statistically significant minimal residual disease (MRD) (p<.05), and BCR::ABL1-like ALL cases had high MRD-positivity but did not show statistical significance as compared to BCR::ABL1-negative ALLs.

Targeted surface marker screening on neuronal structures in the human choroid

While choroidal neuronal control is known to be essential for retinal and ocular health, its mechanisms are not understood. Especially, the local choroidal innervation mediated by intrinsic choroidal neurons (ICN) remains enigmatic. Neuronal functionality depends on the synaptic neurotransmitters and neuroregulatory peptides involved as well as from membrane components presented on the cell surface. Since the neuronal surface molecular expression patterns in the choroid are currently unknown, we sought to determine the presence of various cluster-of-differentiation (CD) antigens in choroidal neuronal structures with a particular focus on ICN. Human choroids were prepared for immunohistochemistry and the pan-neuronal marker PGP9.5 was combined with CD15, CD24, CD29, CD34, CD46, CD49b, CD49e, CD56, CD58, CD59, CD71, CD81, CD90, CD146, CD147, CD151, CD165, CD171, CD184, CD200, CD271 and fluorescence- and confocal laser scanning-microscopy was used for documentation. The following antigens were found to be co-localized in PGP.9.5+ nerve fibers and ICN perikarya: CD29, CD34, CD56, CD81, CD90, CD146, CD147, CD151, CD171, CD200 and CD271, while all other CD markers where not detectable. Whereas CD24− and CD59− immunoreactivity was clearly absent in ICN perikarya, some neural processes of the choroidal stroma displayed CD24 and CD59 immunopositivity. While a multitude of the aforementioned CD-markers were indeed detected in nervous structures of the choroid, the CD24+ and CD59+ nerve fibers most likely have extrinsic origin from cranial ganglia since ICN cell bodies were found to lack both markers. These findings illustrate how the detailed analysis of CD molecules described here opens novel avenues for future functional studies on choroidal innervation and its control.

A Systematic Review Comparing Bone Graft Harvest From the Iliac Crest, Tibia, and Calcaneus Donor Sites in Foot and Ankle Surgery

Category: Basic Sciences/Biologics; Other Introduction/Purpose: Bone autografts are frequently harvested for use in foot and ankle surgery. The gold standard harvest site is the iliac crest; however there is known morbidity with this harvest site. Alternative harvest sites include the tibia and calcaneus which are easily accessible for lower extremity surgery. Controversy exists regarding the osteogenic potential of autografts from each location. We performed a systematic review of the known data on cellular autografts from the iliac crest, tibia, and calcaneus focusing on the total cells harvested from each site as well as the presence of the critically important osteogenic osteoprogenitor cells. We also reviewed autograft volume, autograft consistency, surgical techniques, and the morbidities and complications associated with each bone autograft harvest location. Methods: In accordance with the PRISMA 2020 guidelines, a literature search of the PubMed, Cochrane Library, Web of Science, and Google databases from inception through January 2022 was performed. The search terms included: 'autograft,' 'bone graft,' 'foot and ankle surgery,' 'iliac crest,' 'tibia,' 'calcaneus,' reamer irrigator aspirator,' 'RIA,' 'mesenchymal cells,' 'osteoprogenitor cells,' 'quantity,' 'number,' 'amount,' 'yield,' 'concentration,' 'surface antigens,' 'surface markers,' 'CD markers.' Studies were included if autologous bone graft was harvested from the iliac crest, femur, tibia, or calcaneus and the cells in the harvest underwent histologic evaluation, quantification, or CD surface antigen identification. We included studies with bone graft as well as bone marrow aspirates used as samples. The nucleated cell and osteoprogenitor cell yield was recorded from each location; and MSC CD markers present at each harvest site were extracted from the eligible studies. Results: 13 studies met our inclusion criteria. Eight studies performed cell quantification from the harvest sites. Notably these studies only provide counting of total nucleated cells without specific identification of the osteoprogenitors. Colony-forming units were also reported and their findings are reviewed. Total cellular material was on average greatest in the iliac crest, with intermediate counts in the proximal tibia, and lowest in the calcaneus autograft samples. Five studies identified mesenchymal stromal cell (MSC) cluster of differentiation (CD) antigens at the harvest sites of interest. CD markers confirm osteoprogenitor cells are present in each sample location. Total number of osteoprogenitor cells remains unknown, however authors have attempted to extrapolate this number based on number of total nucleated cells and other data. Conclusion: Osteoprogenitor cells can be harvested from the iliac crest, tibia, and calcaneus. Greater total nucleated cell yields were identified in harvests from the iliac crest with descending amounts moving location distally in the limb. Nucleated cells were counted to provident estimates of osteoprogenitor cells yields in most studies, with flow cytometry confirmation that osteoprogenitors are present. Strict quantification of osteoprogenitor cells remains unknown. Multiple studies compared cellular material from bone marrow aspirates versus bone graft harvests themselves. Studies with modern techniques are needed to identify the true number of osteoprogenitors in each location.

A glycan-based approach to cell characterization and isolation: Hematopoiesis as a paradigm.

Cell surfaces display a wide array of molecules that confer identity. While flow cytometry and cluster of differentiation (CD) markers have revolutionized cell characterization and purification, functionally heterogeneous cellular subtypes remain unresolvable by the CD marker system alone. Using hematopoietic lineages as a paradigm, we leverage the extraordinary molecular diversity of heparan sulfate (HS) glycans to establish cellular "glycotypes" by utilizing a panel of anti-HS single-chain variable fragment antibodies (scFvs). Prospective sorting with anti-HS scFvs identifies functionally distinct glycotypes within heterogeneous pools of mouse and human hematopoietic progenitor cells and enables further stratification of immunophenotypically pure megakaryocyte-erythrocyte progenitors. This stratification correlates with expression of a heptad of HS-related genes that is reflective of the HS epitope recognized by specific anti-HS scFvs. While we show that HS glycotyping provides an orthogonal set of tools for resolution of hematopoietic lineages, we anticipate broad utility of this approach in defining and isolating novel, viable cell types across diverse tissues and species.

An NF-κB- and Therapy-Related Regulatory Network in Glioma: A Potential Mechanism of Action for Natural Antiglioma Agents.

High-grade gliomas are among the most aggressive malignancies, with significantly low median survival. Recent experimental research in the field has highlighted the importance of natural substances as possible antiglioma agents, also known for their antioxidant and anti-inflammatory action. We have previously shown that natural substances target several surface cluster of differentiation (CD) markers in glioma cells, as part of their mechanism of action. We analyzed the genome-wide NF-κB binding sites residing in consensus regulatory elements, based on ENCODE data. We found that NF-κB binding sites reside adjacent to the promoter regions of genes encoding CD markers targeted by antiglioma agents (namely, CD15/FUT4, CD28, CD44, CD58, CD61/SELL, CD71/TFRC, and CD122/IL2RB). Network and pathway analysis revealed that the markers are associated with a core network of genes that, altogether, participate in processes that associate tumorigenesis with inflammation and immune evasion. Our results reveal a core regulatory network that can be targeted in glioblastoma, with apparent implications in individuals that suffer from this devastating malignancy.

Elevated CD9 expression as a potential biomarker for diagnosis of Bernard-Soulier syndrome.

Diagnosis of inherited platelet glycoprotein disorders is based on specific laboratory techniques such as aggregometry and flow cytometry. Flowcytometry is a powerful method, but equivocal results are produced in some cases. New cluster of differentiation markers could resolve the diagnostic dilemmas. Abnormal expression of CD9 in Bernard-Soulier syndrome (BSS) is recently reported. We aimed to determine the diagnostic significance of CD9 expression in a cohort of Iranian patients with inherited platelet glycoprotein defects. Twelve BSS, 21 Glanzmann thrombasthenia and 16 healthy controls were included in the present study. Flowcytometric diagnosis of BSS and Glanzmann thrombasthenia was made by analysis of CD41/61 and CD42a/42b CD markers. Moreover, phycoerythrin-labelled anti CD9 was examined in patients and healthy controls. The mean fluorescence intensity (MFI) of CD9 among the three groups was compared using suitable statistical methods and a P value of less than 0.05 considered statistically significant. Mean MFI of CD9 was 990.0 in BSS patients versus 421.2 and 317.3 in individuals with Glanzmann thrombasthenia and healthy controls, respectively (P < 0.05). Between the two-group comparison of means by the Mann-Whitney test revealed a P value of less than 0.001 for BSS group versus GT (2.4-fold) and BSS versus healthy controls (2.9-fold). CD9 molecule also expressed differently in patients with Glanzmann thrombasthenia in comparison with healthy controls (P < 0.001), although with a less magnitude (1.3-fold). According to our findings, CD9 is a potential biomarker for laboratory diagnosis of inherited glycoprotein defects, especially to elucidate the ambiguous results in BSS cases.

Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative.

BackgroundThe Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking.ObjectiveTo develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories.MethodsA high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ+, regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation.ResultsUsing automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen.ConclusionWe optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.

Effect of autologous fat transfer in acute burn wound management: A randomized controlled study

ObjectiveThe use of fat grafting is being widely used for different indications one of which is wound healing. In this study we compare the use of autologous fat grafting (AFG) as a novel indication in acute burn wounds healing and burn scarring to the conventional methods of burn wound management both clinically and histologically. Several small observational studies demonstrated the effect of the AFG in healing of chronic wounds, different vascular ulcers or effect on scars yet no randomized controlled trial is available to compare its role with conventional methods. MethodsThe study was a prospective, open-label single center, randomized control clinical trial included 100 patients with superficial and deep dermal burns from March 2019 to March 2020 randomized to AFG protocol consisted of a single injection of autologous fat grafting then dressed with nano fat (Group A) or conventional methods of serial dressings with 1% silver sulphadiazine or other topical agents (Group B). Inclusion criteria included newly admitted burn patients with affected total body surface area (TBSA) (10%−25%) while exclusion criteria included burns patients with affected TBSA of< 10% or> 25%, or loss of subcutaneous fat, fascia, muscles and bones, inhalational burn, and burns in genitalia, perineum and peri-anal areas and co-morbidity(ies) that might affect wound healing or eligibility for anaesthesia and surgery. Also, results were confirmed by histological analysis for samples from both groups by light microscopic examination, and the nano-fat was subjected to flow cytometric analysis of the cluster of differentiation (CD) markers of mesenchymal stem cells markers CD 90, CD44, CD45, CD 73, and CD 34. (ClinicalTrials.gov Identifier: NCT03791710) ResultsWe found a significant reduction in total hospital stay days (p = <0.001), less further skin grafting (p = 0.003), less contracture formation (p = <0.002) while scar texture improved (p = <0.001) in group A compared to group B. Flow cytometric analysis documented that the nano-fat was positive to CD 90, 73, 44, 45 and 34. ConclusionIn a comparison between AFG protocol to the conventional methods in the treatment of acute burn wounds, AFG protocol was associated with significant clinical improvement in the form of lower hospital stay time, lower incidence of scaring or contracture and lower skin grafting use which was confirmed by serial photographic and histological assessment.

Engineered Chitosan-based Nanoparticles Modulate Macrophage–Periodontal Ligament Fibroblast Interactions in Biofilm-mediated Inflammation

IntroductionCrosstalk between immune cells and tissue-resident cells regulates the pathophysiology and posttreatment healing of apical periodontitis. This investigation aimed to understand the influence of residual root canal biofilm on macrophage (MQ)–periodontal ligament fibroblast (PdLF) interaction and evaluate the effect of engineered chitosan-based nanoparticles (CSnp) on MQ-PdLF interactions in residual biofilm-mediated inflammation. MethodsSix-week-old Enterococcus faecalis biofilms in root canal models were disinfected conventionally using sodium hypochlorite alone or followed by calcium hydroxide medication or CSnp dispersed in carboxymethylated chitosan (CMCS). The effect of the treated biofilms (n = 25/group) on the inflammatory response of THP-1–differentiated MQ monoculture versus coculture with PdLF was evaluated for cell viability, MQ morphometric characterization, inflammatory mediators (nitric oxide, tumor necrosis factor alpha, interleukin [IL]-1 beta, IL-1RA, IL-6, transforming growth factor beta 1 [TGF-β1], and IL-10), and the expression of transcription factors (pSTAT1/pSTAT6)/cluster of differentiation markers (CD80/206) after 24, 48, and 72 hours of interaction. PdLF transwell migration was evaluated after 8 and 24 hours. Unstimulated cells served as the negative control, whereas untreated biofilm was the positive control. ResultsBiofilm increased nitric oxide and IL-1β but suppressed IL-10, IL-1RA, and PdLF migration with significant cytotoxic effects. CSnp/CMCS reduced nitric oxide and IL-1β (P < .01) while maintaining ≥90% cell survival up to 72 hours with evident M2-like MQ phenotypic changes in coculture. CSnp/CMCS also increased the IL-1RA/IL-1β ratio and enhanced TGF-β1 production over time (P < .05, 72 hours). In coculture, CSnp/CMCS showed the highest IL-10 level at 72 hours (P < .01), reduced the pSTAT1/pSTAT6 ratio, and enhanced PdLF migration (P < .01, 24 hours). ConclusionsCSnp/CMCS medication facilitated MQ switch toward M2 (regulatory/anti-inflammatory) phenotype and PdLF migration via paracrine signaling.

Amnion and Umbilical Cord–Derived Products in Sports Medicine: From Basic Science to Clinical Application

Background: Birth tissue products from amnion, chorion, umbilical cord, amniotic fluid, or cord blood are frequently marketed as viable sources of stem cells and growth factors. It can be difficult for health care professionals to differentiate implied from explicit conclusions in reported product analyses. Purpose: To provide an educational platform for health care professionals to interpret data presented in the promotion of birth tissue products. Study Design: Descriptive laboratory study and expert opinion; Level of evidence, 5. Methods: A cord blood product was analyzed by 3 methods for cell viability, 2 methods for assessment of cell morphology and cell type, multicolor flow cytometry to identify stem cells, and enzyme-linked immunosorbent assay (ELISA) plus Western blot for analysis of interleukin 1 receptor antagonist protein (IL-1ra). These data were compared with analyses reported by the manufacturer. Results: Cell viability in the cord blood product was less than reported by the manufacturer, the cells were primarily leukocytes, no stem cells were present, and the concentration of IL-1ra was falsely increased due to nonspecific antibody binding in the sample. Conclusion: To assess birth tissue products, health care professionals should consider the following: (1) Understanding fluorescent dyes is important for assessing cell viability data—green does not always mean alive. (2) The report of “cells” in the product does not necessarily mean “stem cells”; microscopic images of at least ×20 or a hemogram should be evaluated to determine cell type (leukocyte, red blood cells, etc). (3) There is no single cluster of differentiation (CD) marker on flow cytometry to identify stem cells. (4) Biological tissues are complex substances, and inaccurately increased measurements of growth factors could be present in ELISA results because most ELISAs are not designed or validated for use in biologics. Furthermore, the reported measurement of growth factors should be considered relative to concentrations in native biological tissues and plasma. Clinical Relevance: Health care professionals should be able to interpret cell viability, cell morphology, stem cell analysis using CD markers, and growth factor analysis when considering use of a birth tissue product in patients.

Cluster of differentiation (CD) 9-positive mouse pituitary cells are adult stem/progenitor cells.

SOX2-positive cells are stem/progenitor cells that supply hormone-producing cells; they are found in the anterior lobe of the rodent pituitary gland. However, they are likely composed of several subpopulations. In rats, a SOX2-positive cell populations can be distinguished by the presence of S100β. We identified the novel markers cluster of differentiation (CD) CD9 and CD81, members of the tetraspanin superfamily, for the identification of S100β/SOX2-positive cells. Recently, CD9/CD81 double-knockout mice were generated. Although they grew normally until 3weeks after birth, they exhibited atrophy of the pituitary gland. These findings suggested that CD9/CD81/S100β/SOX2-positive cells in the mouse pituitary are adult stem/progenitor cells. To substantiate this hypothesis, we examined CD9 and CD81 expression in the adult and developing anterior lobe. Immunohistochemistry showed that CD9/CD81-positive cells began appearing from postnatal day 0 and settled in the stem cell niches (marginal cell layer and parenchyma) of the adult anterior lobe while expressing S100β. We next isolated CD9 -positive cells from the adult anterior lobe, using the anti-CD9 antibody for cell characterisation. The cells in culture formed free-floating three-dimensional clusters (pituispheres); moreover, induction into all types of hormone-producing cells was successful. Furthermore, reduction of CD9 and CD81 mRNAs by siRNAs inhibited cell proliferation. These findings indicate that CD9/CD81/S100β/SOX2-positive cells may play a role as adult stem/progenitor cells in SOX2-positive subpopulations, thus supplying hormone-producing cells in the postnatal anterior lobe. Furthermore, CD9 and CD81 are implicated in cell proliferation. The current findings provide novel insights into adult pituitary stem/progenitor cells.

Tissue-specific murine neutrophil activation states in health and inflammation.

Neutrophils are quickly recruited to tissues in response to proinflammatory cues; however, little is known about tissue neutrophil phenotypes in health. We employ a multicolor flow cytometric approach to assess surface markers of activation on neutrophils from the bone marrow, blood, peritoneum, spleen, liver, fat, colon, and oral cavity of healthy mice. Cell preparations were promptly fixed to preserve native surface marker expression levels. Peritoneal, colonic, and oral neutrophils were also assessed in the setting of pHrodo-induced peritonitis, dextran sodium sulfate-induced colitis, and ligature-induced periodontal disease, respectively. Our results demonstrate consistent detectable neutrophil populations in various sterile and nonsterile tissues of healthy mice, and these cells had tissue-specific neutrophil immunophenotypes. Neutrophils derived from biofilm-associated mucosal tissues had 2- to 3-fold higher expression of surface markers of activation, including CD66a, CD11b, and CD62L, compared to neutrophils derived from both sterile healthy tissues as well as tissues in animals treated with broad-spectrum antibiotics. Furthermore, the unique cluster of differentiation (CD) marker activation signatures of tissue-specific neutrophils from the peritoneum, colon, and oral cavity were altered to a proinflammatory immunophenotype with the presence of an inflammatory stimulus. Based on our results, we propose a model whereby a hierarchy of tissue neutrophil immunophenotypes, based on the differential expression of CD markers of activation, correlates with sterile, healthy commensal biofilm-associated and inflamed tissue states.