Immunophenotyping of Lymphocyte T and B in the Peripheral Blood of Systemic Lupus Erythematosus

The immunophenotyping expression levels of lymphocyte in the peripheral blood from 21 patients with active systemic lupus erythematosus were analyzed by using the immunofluorescence labeling flow cytometry technique to investigate the immunophenotyping expression of lymphocytes T and B in the peripheral blood of active SLE patients and its clinical value. It was showed that, compared with normal controls, the expression of CD + 3, CD + 4 and the ratio of CD + 4/CD + 8 in the peripheral blood of these patients were decreased , while the expression of CD + 8, CD + 20 was significantly increased . It was suggested that both T and B cells in patients with active SLE involved in immunoregulation, were activated. The abnormal expression of lymphocyte immunophenotyping could influence the immune reaction in SLE patients, which might be one of the important pathogenesis factors in SLE.

Immunophenotyping of hematopoietic progenitor cells: Comparison between cord blood and adult mobilized blood grafts

AIM:To study the immunophenotype of hematopoietic progenitor cells from cord blood (CB) grafts (n = 39) in comparison with adult apheresis grafts (AG, n = 229) and pre-apheresis peripheral blood (PAPB) samples (n = 908) using flow cytometry analysis.METHODS: First, we performed a qualitative analysis of CD34+ cell sub-populations in both CB and PAPB grafts using the standardized ISHAGE protocol and a wide panel of 20 monoclonal antibodies. Next, we stud-ied some parameters, such as the age of mothers and the weight of newborns, which can influence the qual-ity and the quantity of CD34+ cells from CB. RESULTS: We found that the percentage of apoptotic cells was high in CB in comparison to PAPB (PAPB: 4.6% ± 2.6% vs CB: 53.4% ± 5.2%, P < 0.001). In CB, the weight of newborn and the age of the mother have the influence on CD34+ cells. The follow-up of Ag CD133in the ISHAGE double platform protocol in association with CD45, CD34 and the 7’AAD shows an equal rate between the two cell populations CD133+CD45+CD34+ high and CD34+CD45+ high with a higher percentage. So, is the inclusion of Ac CD133 necessary in the pres-ent panel included in the ISHAGE methodflLast part, we showed a signif icant presence of interferon γ in CB in comparison to PAPB, the annexin showing the high number of apoptotic cells in CB. CONCLUSION: This study demonstrates that many different obstetric factors must be taken into account when processing and cryo-banking umbilical CB units for transplantation.

Immunophenotypings of Malignant Epithelial Mesothelioma and Their Roles in the Differential Diagnosis

To investigate the immunophenotypings of malignant epithelial mesothelioma (MEM), and to seek the valuable markers in distinguishing peritoneal MEM from peritoneal metastatic ovarian adenocarcinoma (OA) and colorectal adenocarcinoma (CA), immunohistochemical SP method was used to detect expressions of HBME-1, E-cadherin, CA19-9, MOC-31 and CK7 in paraffin-embedded tissues of 18 cases of MEM, 20 OA and 20 CA. The results showed that there was a significant difference in the expressions of E-cadherin, CA19-9 and MOC-31 between MEM and OA group (P<0.05). Similarly, the difference in the expression of HBME-1, E-cadherin, CA19-9, MOC-31 and CK7 between MEM and CA groups is significant (P<0.05). These results indicate that HBME-1 could be used as a positive marker in distinguishing MEM from CA. E-cadherin, CA19-9 and MOC-31 are considered to be useful negative markers in diagnostic distinction between MEM and metastatic adenocarcinomas, including OA and CA. CK7 is the best positive marker in distinguishing MEM from CA, but this marker appears to be valueless in discriminating MEM from OA.

IMMUNOPHENOTYPING OF CUTANEOUS GERMINAL CENTER CELL-DERIVED LYMPHOMAS

Monoclonal antibodies were used to label cutaneous germinal center cell-derived lymphomas <CGCCL) obtained from 10 patients. According to the Kiel classification, they were classified into 2 types. Eight patients had centroblastic/centrocytic <CB/CC) lymphomas while 2 patients and centrocytic (CC) lymphomas. After monoclonal antibody labelling, the results were consistent with those of the clinical and morphologic analyses. Of the 10 cases, 9 were B1 positive, 6 were K positive, and 4 were λ positive. In 8 cases labeled with immunoglobulin, 6 were IgGFab positive, 2 were IgM positive and 8 were IgA negative. Five cases (CB/CC 3, CC 2) were both Bl, K and IgG positive (γ K). Four cases CB/CC were both Bl and A positive. Only one case (CB/CC) was both K and IgM positive (μ K). Two cases (CB CC) were both A and IgG positive (γ λ). The results indicate that Bl, K and A are the most important markers to phenotype cutaneous B-cell lymphomas. Our findings also show a higher percentage of y K types in CGCCL as compared with Western countries.

Deep Immunophenotyping of Human Whole Blood by Standardized Multi‑parametric Flow Cytometry Analyses

Immunophenotyping is proving crucial to understanding the role of the immune system in health and disease.High-through-put flow cytometry has been used extensively to reveal changes in immune cell composition and function at the single-cell level.Here,we describe six optimized 11-color flow cytometry panels for deep immunophenotyping of human whole blood.A total of 51 surface antibodies,which are readily available and validated,were selected to identify the key immune cell populations and evaluate their functional state in a single assay.The gating strategies for effective flow cytometry data analysis are included in the protocol.To ensure data reproducibility,we provide detailed procedures in three parts,including(1)instrument characterization and detector gain optimization,(2)antibody titration and sample staining,and(3)data acquisition and quality checks.This standardized approach has been applied to a variety of donors for a better understanding of the complexity of the human immune system.

Alternative Phenotypic Profile for B-Cell Abnormality in a Case at Zinder National Hospital

Introduction: Since it is impossible to establish a diagnosis in the presence of hyperlymphocytosis not secondary to lymphocytic hyperactivation, we considered a B-lymphoid hematopathy with a non-specific phenotypic profile. We report one case of this. Observation: This is a forty-eight (48) year old patient with hyperlymphocytosis at 139,000 elements per cubic millimeter, heterogeneous splenomegaly at 25.6 cm in diameter on abdominal ultrasound without detectable deep or peripheral lymphadenopathy. Peripheral blood cytology shows lymphocyte cell proliferation suggestive of the circulating phase of chronic lymphoproliferative B syndrome. The expression profile of cell membrane markers did not allow for the definition of a specific phenotypic profile. Faced with this immunophenotyping result, we considered a B-lymphoid hemopathy with a non-specific phenotypic profile. After three courses, the MINICHOP treatment was used to achieve partial remission with wasting of more than 80% of the evaluable masses. Conclusion: Despite the contribution of immunophenotyping in the diagnosis of lymphoproliferative syndromes, it is possible to consider the diagnosis of a B-lymphoid hemopathy with a phenotypic non-specific profile of CD45+, monotypic kappa, CD19+, FMC7+, CD22+, CD5−, CD79b−, CD23−, CD43−, CD38−, CD200−.

Blastic plasmacytoid dendritic cell neoplasm in Jinhua,China:Two case reports

BACKGROUND Blastic plasmacytoid dendritic cell neoplasm(BPDCN)is a rare and clinically aggressive hematologic malignancy originating from the precursors of plasmacytoid dendritic cells.BPDCN often involves the skin,lymph nodes,and bone marrow,with rapid clinical progression and a poor prognosis.The BPDCN diagnosis is mainly based on the immunophenotype.CASE SUMMARY In this paper,we retrospectively analyzed 2 cases of BPDCN.Both patients were elderly males.The lesions manifested as skin masses.Morphological manifestations included diffuse and dense tumor cell infiltration of the dermis and subcutaneous tissues.Immunohistochemistry staining showed that cluster of differentiation CD4,CD56,CD43,and CD123 were positive.CONCLUSION In this paper,we retrospectively analyzed 2 cases of BPDCN.Both patients were elderly males.The lesions manifested as skin masses.Morphological manifestations included diffuse and dense tumor cell infiltration of the dermis and subcutaneous tissues.Immunohistochemistry staining showed that cluster of differentiation CD4,CD56,CD43,and CD123 were positive.

IMMUNOPHENOTYPING OF 515 CASES OF ACUTE LYMPHOBLASTIC LEUKEMIA IN CHINA

Using cell surface markers and a panel of monoclonal antibodies, 515 cases of acute lymphoblastic leukemia (ALL) were immunophenotyped. T cell type ALL (T-ALL), non-T cell type ALL (Non-T-ALL) including common ALL (C-ALL), Null-ALL and B cell type ALL (B-ALL) were found. These major subtypes of ALL were further divided according to their phenotypes in detail. It was noticed that the phenotypes of these subtypes of ALL reflected basically the phenotypes of normal T or B cells at various differentiation stages or certain population of lymphocytes. The diagnosis of cell lineage was more precise when based on immunophenotyping than morphological description. The combination of morphological and immunological classification can improve the diagnosis of acute leukemias. In addition, it was observed that the immunophenotyping was relevant to clinicopathologic features, responses to therapy and prognosis of ALL patients. The incidences of major subtypes of ALL, the age distribution of ALL subsets and male sex

Intestinal natural killer/T-cell lymphoma presenting as a pancreatic head space-occupying lesion:A case report

BACKGROUND Intestinal natural killer/T-cell lymphoma(NKTCL)is a rare and aggressive non-Hodgkin’s lymphoma,and its occurrence is closely related to Epstein-Barr virus infection.In addition,the clinical symptoms of NKTCL are not obvious,and the specific pathogenesis is still uncertain.While NKTCL may occur in any segment of the intestinal tract,its distinct location in the periampullary region,which leads clinicians to consider mimics of a pancreatic head mass,should also be addressed.Therefore,there remain huge challenges in the diagnosis and treatment of intestinal NKTCL.CASE SUMMARY In this case,we introduce a male who presented to the clinic with edema of both lower limbs,accompanied by diarrhea,and abdominal pain.Endoscopic ultrasound(EUS)showed well-defined homogeneous hypoechoic lesions with abundant blood flow signals and compression signs in the head of the pancreas.Under the guidance of EUS-fine needle biopsy(FNB)with 19 gauge or 22 gauge needles,combined with multicolor flow cytometry immunophenotyping(MFCI)helped us diagnose NKTCL.During treatments,the patient was prescribed the steroid(dexamethasone),methotrexate,ifosfamide,L-asparaginase,and etoposide chemotherapy regimen.Unfortunately,he died of leukopenia and severe septic shock in a local hospital.CONCLUSION Clinicians should enhance their understanding of NKTCL.Some key factors,including EUS characteristics,the right choice of FNB needle,and combination with MFCI,are crucial for improving the diagnostic rate and reducing the misdiagnosis rate.

Endometriosis of the lung: A case report and review of literature

BACKGROUND Lung endometriosis is an extremely rare gynecological disease.Current literature reports suggest that the majority of patients will present with only generic symptoms,such as hemoptysis,pneumothorax,and hemopneumothorax,which often leads to misdiagnosis.To date,there are 18 case reports of lung endometriosis that describe the clinical manifestation,imaging changes,treatment,and prognosis of the disease.To provide further insights into this rare disease,we present a new case report and a brief review of pulmonary endometriosis.CASE SUMMARY We report here about a 19-year-old woman who was admitted to the hospital for repeated catamenial hemoptysis over a 3-mo period.computed tomography(CT)imaging during menstruation revealed patchy high-density shadows,approximately 0.5 cm3 in size,in the right middle lobe of the lung.The patient’s hemoptysis and changes in the CT scans resolved after menstruation.Thoracoscopic right middle lobectomy,right lower lung repair,and closed thoracic drainage were performed.Postoperative histopathology confirmed lung endometriosis.There was no recurrence of symptoms at the 6 mo follow-up.CONCLUSION We propose diagnosing lung endometriosis by thoroughly taking reproductive history,clinical details,imaging,and histopathology followed by treatment with surgical resection.

间充质的干细胞的隔离和描述源于人的胎盘组织

AIM: To explore the feasibility of placenta tissue as a reliable and efficient source for generating mesenchymal stem cells (MSC). METHODS: MSC were generated from human placenta tissue by enzymatic digestion and mechanical dissociation. The placenta MSC (PLC-MSC) were characterized for expression of cell surface markers, embryonic stem cell (ECS) gene expression and their differentiation ability into adipocytes and osteocytes. The immunosuppressive properties of PLC-MSC on resting and phytohemagglutinin (PHA) stimulated allogenic T cells were assessed by means of cell proliferation via incorporation of tritium thymidine (3H-TdR). RESULTS: The generated PLC-MSC appeared as spindle-shaped cells, expressed common MSC surface markers and ESC transcriptional factors. They also differen-tiated into adipogenic and osteogenic lineages when induced. However, continuous cultivation up to passage 15 caused changes in morphological appearance and cellular senescence, although the stem cell nature of their protein expression was unchanged. In terms of their immunosuppressive properties, PLC-MSC were unable to stimulate resting T cell proliferation; they inhibited the PHA stimulated T cells in a dose dependent manner through cell to cell contact. In our study, MSC generated from human placenta exhibited similar mesenchymal cell surface markers; MSC-like gene expression pattern and MSC-like differentiation potential were comparable to other sources of MSC. CONCLUSION: We suggest that placenta tissues can serve as an alternative source of MSC for future experimental and clinical studies.

骨头的 Immunophenotype 和区别能力从 CBA/Ca， ICR 和 Balb/c 的导出髓的间充质的干细胞老鼠

AIM:To assess the capacity to isolate and expand mesenchymal stem cells(MSC)from bone marrow of CBA/Ca,ICR and Balb/c mice. METHODS:Bone marrow of tibia and femur were flushed,cultured and maintained in supplemented Dulbecco’s modified Eagle’s medium.MSC immunophenotype of cultures were tracked along increasing passages for positivity to CD106,Sca-1 and CD44 and negativity to CD45,CD11b and MHC classⅡ.Differentiation capacity of MSC towards osteogenic and adipo-genic lineages were also assessed. RESULTS:MSC were successfully cultured from bone marrow of all 3 strains,albeit differences in the temporal expression of certain surface antigens.Their differentiation into osteocytes and adipocytes were also observed. MSC from all 3 mouse strains demonstrated a shift from a haematopoietic phenotype(CD106-CD45+CD11b+Sca-1low)to typical MSC phenotype(CD106+CD45-CD11b-Sca-1high)with increasing passages. CONCLUSION:Information garnered assists us in the decision of selecting a mouse strain to generate MSC from for downstream experimentation.

High frequency of CD34+CD38-/low immature leukemia cells is correlated with unfavorable prognosis in acute myeloid leukemia

AIM To evaluate the importance of the CD34+CD38-cell population when compared to the CD34+CD38+/low and CD34+CD38+/high leukemic cell sub-populations and to determine its correlations with leukemia characteristics and known prognostic factors, as well as with response to therapy and survival.METHODS Two hundred bone marrow samples were obtained at diagnosis from 200 consecutive patients with newly diagnosed acute myeloid leukemia(AML) were studied between September 2008 and December 2010 at our Institution(Hematology Department, Lyon, France). The CD34/CD38 cell profile was analyzed by multiparameter flowcytometry approach using 8 C panels and FACS CANTO and Diva software(BD Bioscience).RESULTS We analyzed CD34 and CD38 expression in bone marrow samples of 200 AML patients at diagnosis, and investigated the prognostic value of the most immature CD34+CD38-population. Using a cut-off value of 1% of CD34+CD38-from total "bulk leukemic cells" we found that a high(> 1%) level of CD34+CD38-blasts at diagnosis was correlated with advanced age, adverse cytogenetics as well as with a lower rate of complete response after induction and shorter disease-free survival. In a multivariate analysis considering age, leukocytosis, the % of CD34+ blasts cells and the standardized cytogenetic and molecular risk subgroups, a percentage of CD34+CD38-leukemic cells > 1% was an independent predictor of DFS [HR = 2.8(1.02-7.73), P = 0.04] and OS [HR = 2.65(1.09-6.43), P = 0.03].CONCLUSION Taken together, these results show that a CD34/CD38 "backbone" for leukemic cell analysis by multicolour flowcytometry at diagnosis provides useful prognostic information.

Mode of delivery by an ulcerative colitis mother in a case of twins: Immunological differences in cord blood and placenta

AIM To understand the effects of delivery mode on the immune cells frequency and function in cord blood and placenta.METHODS We evaluated immunological differences in cord blood and placental tissues for a case of twins one of which delivered vaginally while the other delivered by caesarian section(C-section). Cord blood mononuclear cells were isolated and placenta tissues were processed for cell isolation. Immune phenotyping was performed by flow cytometry methods following staining for T cells, natural killer(NK) cells, monocytes, neutrophils and CD71^+erythroid cells in both cord blood and placenta tissues. In addition, fetal calprotectin of twins was measured 12 wk after birth.RESULTS We found lower percentages of immune cells(e.g. T cells, monocytes and neutrophils) in the cord blood of C-section delivered compared to vaginally delivered newborn. In contrast, percentages of monocytes and neutrophils were > 2 folds higher in the placental tissues of C-section delivered newborn. More importantly, we observed lower percentages of CD71^+ erythroid cells in both cord blood and placental tissues of C-section delivered case. Lower CD71^+ erythroid cells were associated with a more pro-inflammatory milieu at the fetomaternal interface reflected by higher expression of inhibitory receptors on CD4^+ T cells, higher frequency of monocytes and neutrophils. Furthermore, type of delivery impacted the gene expression profile in CD71^+erythroid cells. Finally, we found that C-section delivered child had > 20-fold higher FCP in his fecal sample at 12 wk of age.CONCLUSION Mode of delivery impacted immune cells profile in cord blood/placenta. In particular frequency of immunosuppressive CD71^+ erythroid cells was reduced in C-section delivered newborn.

STUDIES OF THE CHARACTERISTIC FEATURES OF KI-1 POSITIVE NON-HODGKIN'S LYMPHOMA

The clinical histopathological and immuno-phenotypic features in 5 patients with Ki-1 positive non-Hodgkin's lymphoma (NHL) were studied. When firstseen, 4 patients presented enlargement of superficiallymph nodes, with skin lesions in 2 patients. Two patientsin stage Ⅳ with fever, hepato-splenomegaly and bonemarrow invasion, died. Histologically, the tumor cellsshowed diffused or patchy hyperplasia. The cells wererelatively large in size, rich in basophilic or slightlyeosinophilic cytoplasm with irregular-shaped nuclei,prominent nucleoli, and distinct anaplasia andpleomorphism. Some of the cells looked very much likethe Reed-Sternberg cells. Multinucleated giant cells wereseen. Immunophenotypically, all the cells were CD30 (Ki-1) and CD25 (IL-2 receptor) positive but CD15 (Leu M1)negative. Thus, the 5 patients with Ki-1 positive NHLwere all of T cell type.

Immunophenotype and Ultrastructure of B-cell Lymphoproliferative Disorder with Cytoplasmic Projection

To identify the knowledge of rare lymphoproliferative disorder, the clinical and biological features of three kinds of lymphoproliferative disorders with cytoplasmic projections were compared The clinical manifestations, ultrastructure and immunophenotype were analyzed The results showed that hairy cell leukemia (HCL), splenic lymphoma with villous lymphocyte (SLVL) and hairy cell leukemia-variant (HCL-V) had some common characters including splenomegaly, peripheral blood and bone marrow infiltration by villous lymphocyte and B lymphocyte immunophenotype; but these three disorders had specific features respectively It was concluded that overall analysis of clinical and laboratory features might be contributive to the differential diagnosis of these three disorders

Flow Cytometric Diagnosis of Acute Leukemia and Aberrant Antigen: Sohag University Experience

Background: Multiparameter flow cytometry is the most important method for the lineage assignment and maturational analysis of acute leukemias (AL) cells. The multi parametric immunophenotyping analysis allows the detection of aberrant antigen expression and the analysis of heterogeneity and clonality of malignant cells in AL. The aim of the work is to study the immunophenotypes of blasts from patients with AL and determine the frequency of aberrant markers. Subjects and Methods: Retrospective study to analyze immunophenotypic data of de novo 144 AL patients who were diagnosed in Clinical Pathology Department, Sohag University. Results: We found that 61.8% of AL patients were classified as acute myeloblastic leukemia (AML) while 38.9% classified as acute lymphoblastic leukemia (ALL). The commonest FAB subtype in AML group was AML-M2 (31.8%) followed by M4-M5 27.3%. As regard ALL, there were 85.7% with B-ALL and 14.3% with T-ALL. The aberrancy expressions were found in 66 of AL cases (45.8%), CD7 was the most commonly expressed lymphoid antigen in AML (25%), CD13 was the most commonly expressed myeloid antigen in ALL (39.3%). Conclusion: The multi parametric immunophenotyping analysis of AL is sufficient for diagnosis and classification of leukemia. The frequencies of

Evaluation of TransFix^(■) Mediated Stabilisation of Adipose-Derived Stromal Vascular Fraction for Delayed Flow Cytometry Analysis

The increasing implementation of multicentre studies has led to a need for the optimization of a method that allows for accurate post-hoc analysis of patient biological samples. Assessment of total cell number, viability and immunophenotype can present logistical challenges which can be aided by batch processing. The increased sample storage time that this requires necessitates the use of reagents to preserve cellular integrity, viability and immunophenotype. TransFix is a stabilising reagent that has been developed for the preservation of cell numbers and cell marker expression in peripheral whole blood for up to ten days. This study investigated the use of TransFix reagent for the preservation of the stromal vascular fraction (SVF) of collagenase digested adipose tissue. It was demonstrated that TransFix was suitable for accurately measuring nucleated SVF cell numbers for up to seven days as well as back calculating original cell viability. It also stabilised three CD markers commonly used to identify populations within SVF (CD90, CD31 and CD45) for up to seven days. There was no significant difference between the number of CD90, CD31 and CD45 positive cells after stabilisation at Day 7 compared to Day 0 unstabilised samples. The results suggest that TransFix can be used to preserve a biological mixed cell population from human adipose-derived SVF for up to seven days for accurate post-hoc analysis.

Blastic Plasmacytoid Dendritic Cell Neoplasm:Progress in Cell Origin,Molecular Biology,Diagnostic Criteria and Therapeutic Approaches

Blastic plasmacytoid dendritic cell neoplasm(BPDCN)is a rare hematological malignancy characterized by recurrent skin nodules,an aggressive clinical course with rapid involvement of hematological organs,and a poor prognosis with poor overall survival.BPDCN is derived from plasmacytoid dendritic cells(pDCs)and its pathogenesis is unclear.The tumor cells show aberrant expression of CD4,CD56,interleukin-3 receptor alpha chain(CD 123),blood dendritic cell antigen 2(BDCA 2/CD303),blood dendritic cell antigen 4(BDCA4)and transcription factor(E protein)E2-2(TCF4).The best treatment drugs are based on experience by adopting those used for either leukemia or lymphoma.Relapse with drug resistance generally occurs quickly.Stem cell transplantation after the first complete remission is recommended and tagraxofusp is the first targeted therapy.In this review,we summarize the differentiation of BPDCN from its cell origin,its connection with normal pDCs,clinical characteristics,genetic mutations and advances in treatment of BPDCN.This review provides insights into the mechanisms of and new therapeutic approaches for BPDCN.

Individualized leukemia cell-population profiles in common B-cell acute lymphoblastic leukemia patients

Immunophenotype is critical for diagnosing common B-cell acute lymphoblastic leukemia (common ALL) and detecting minimal residual disease. We developed a protocol to explore the immunophenotypic profiles of common ALL based on the expression levels of the antigens associated with B lymphoid development, including IL-7Rα (CD127), cytoplasmic CD79a (cCD79a), CD19, VpreB (CD179a), and sIgM, which are successive and essential for progression of B cells along their developmental pathway. Analysis of the immunophenotypes of 48 common ALL cases showed that the immunophenotypic patterns were highly heterogeneous, with the leukemic cell population differing from case to case. Through the comprehensive analysis of immunophenotypic patterns, the profiles of patient-specific composite leukemia cell populations could provide detailed information helpful for the diagnosis, therapeutic monitoring, and individualized therapies for common ALL.