In order to explore the factors that affect CD62p expression in platelet during the whole course of cryopreservated platelets preparation, CD62p expression of platelet was evaluated by flow cytometry assay. The whole course of cryopreservated platelets preparation in order included whole blood collection, centrifugation for fresh platelet-rich plasma preparation, addition of dimethyl sulfoxide, and freeze in -80 degrees C refrigerator and thaw in 38 degrees C water bath. Result showed that the CD62p expressed slowly from whole blood collection to addition of dimethyl sulfoxide, but expressed abruptly after freeze and thaw and it occupied 82 per cent of the whole expression. It was concluded that the whole blood collection, centrifugation for fresh platelet enriched plasma preparation and addition of dimethyl sulfoxide were optimized in the whole course, but the damage to platelets in the whole course of cryopreservation could not be avoided. It suggests that improved techniques are needed to reduce the damage to cryopreservated platelets.
Blood Platelets ; metabolism ; Blood Preservation ; methods ; standards ; Cryopreservation ; methods ; standards ; Flow Cytometry ; Humans ; P-Selectin ; biosynthesis

Flowcytometer has been flourishing since the passed decade in our nation. Many general hospitals use it for routine tests. One of the main usage is immunophenotyping for hematological malignancies. Flowcytometric immunophenotyping (FCM-IM) facilitates accurate diagnosis in both leukemias and lymphoid proliferative disorders (LPDs). It becomes an indispensable criteria in the diagnosis of hematological malignancies. It may also offer additional therapeutically relevant information. Typical patterns of immunophenotype in leukemias and certain types of LPDs are illustrated in figure 1 and figure 2. But variation or overlapping appears often. In order to reach a definitive diagnosis and to have a common interpretation between laboratory to laboratory the following items need not to be ignored: 1. Direct staining and multi-color or multi-parameter flow cytometric analysis is necessary. 2. The phenotype of abnormal cells should be determined on as pure a population as possible therefore CD45/SSC gating is critical for isolating the blast population (abnormal cells). CD45/SSC gating is much better than FSC/SSC gating especially when percentage of blast is low in the sample. Else, each subtype of leukemia has its special CD45/SSC pattern. 3. The phenotype of this certain population should be described clearly but it is not necessary to count the percentage of each marker. 4. > 20% for positive threshold used in indirect staining method may be confused in multi-color direct staining method. 5. Hybrid leukemia diagnosed by FCM only should be in caution since the divergence of antigen/antibody, the lineage infidelity of antigen expression and the biological characteristics of the leukemic cells. Flowcytometer is not a cell counter. "It provides a pattern of information that must be interpreted by a knowledgeable professional in the context of the clinical situation".
Antigens, CD ; analysis ; Diagnostic Techniques and Procedures ; Flow Cytometry ; Humans ; Immunophenotyping ; Leukemia ; classification ; pathology ; Reference Standards

Lymphocyte subset analysis is widely used in clinical laboratories, and more than two levels of daily QC materials are required for reliable results. Commercially available, expensive QC materials have short shelf lives and may not be suitable in resource-poor settings. We compared different methods for preparing homemade QC material, including fixation with 1%, 2%, or 4% paraformaldehyde (PFA); freezing with 10% dimethylsulfoxide (DMSO), 0.1% bovine serum albumin-phosphate buffered saline, or after ethanolic dehydration; and using cryopreservation temperatures of -20℃, -80℃, or -196℃. We found an optimal experimental condition, which is 'fixation with 4% PFA, freezing with 10% DMSO, and storage at 80℃'. To evaluate long-term stability of QC materials prepared in this optimal condition, two levels of QC materials (QM1 and QM2) were thawed after 30, 33, 35, 37, 60, 62, 64, and 67 days of cryopreservation. Lymphocyte subset was analyzed with BD Multitest IMK kit (BD Biosciences, USA). QM1 and QM2 were stable after 1-2 months of cryopreservation (CV <3% for CD3, CD4, and CD8 and 5-7% for CD16/56 and CD19). We propose this method as an alternative cost-effective protocol for preparing homemade internal QC materials for lymphocyte subset analysis in resource-poor settings.
Cryopreservation ; Cryoprotective Agents/chemistry ; *Flow Cytometry/standards ; Lymphocyte Subsets/*cytology ; Quality Control ; Reagent Kits, Diagnostic ; Time Factors

No abstract available.
Antigens, CD34/*metabolism ; Automation ; Blood Cells/*cytology/metabolism ; Cell Count/*instrumentation/standards ; Flow Cytometry/standards ; Hematopoietic Stem Cells/*cytology/metabolism ; Humans

BACKGROUND: Detection methods for ABO antibody (Ab) titers vary across laboratories, and the results are different depending on the method used. We aimed to compare titer values using different detection methods for the measurement of ABO Ab titers. METHODS: For ABO Ab detection, pooled group A or B red blood cells (RBCs) were reacted with each of 20 sera from blood groups A, B, or O without dithiothreitol treatment. The room-temperature (RT) incubation technique and the indirect antiglobulin test (IAT) were used in the tube test and gel card test. Flow cytometry (FCM) was performed by using anti-IgM and anti-IgG Abs. RESULTS: Regardless of the blood groups tested, the FCM assay with anti-IgM showed the highest titer compared to the tube test and gel card test with RT incubation in both. The tube test with IAT showed a higher titer than the gel card test with IAT (Gel-IAT) or FCM with anti-IgG in blood group A and B, while Gel-IAT showed the highest titer relative to the other tests, only for the anti-A Ab in blood group O. CONCLUSIONS: There were significant differences in the titers depending on the detection method used, and each method showed a different detection capacity for each ABO Ab depending on the ABO blood group tested. Therefore, caution should be exercised in interpreting ABO Ab titer results, taking into consideration the detection method used and the blood group.
ABO Blood-Group System/*immunology ; Adult ; Aged ; Agglutination Tests/instrumentation/*standards ; Antibodies/*analysis ; Antibodies, Anti-Idiotypic/analysis ; Erythrocytes/chemistry/metabolism ; Female ; *Flow Cytometry ; Humans ; Male ; Middle Aged ; Temperature

In order to find a method suitable for purifying large amount of CD34(+) cells, from 5 cases who accepted autologous peripheral blood stem cell transplantation, CD34(+) cells were collected and enriched by using Isolex 300i (Nexell). Phenotypes were detected by flow cytometry and the biological viability were assayed by the colony-forming experiments and cell expansion experiment in vitro. The results showed that the number of mononuclear cells first collected was about (3.5 - 6.0) x 10(10) and (0.55 - 1.2)% of cells were CD34 positive. The number of positive production was about (2.0 - 3.0) x 10(8); the CD34(+) cells purity was (75 - 85)% and the yield was (40 - 65)%. The CD34(+) cells of positive production could expand up to 2 - 3 times when cultured with SCF + IL3 + FL + TPO + EPO in vitro. The results of colony-forming experiments demonstrated that the CD34(+) cells collected has enough colony-forming ability. All results showed the enriched CD34(+) cells with biological viability. In conclusion, the CD34(+) immunomagnetic isolation apparatus Isolex300i is suitable to clinical application for a large amount of CD34(+) cell enrichment.
Antigens, CD34 ; immunology ; Colony-Forming Units Assay ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans ; Immunomagnetic Separation ; instrumentation ; methods ; standards ; Neoplasms ; blood

No abstract available.
Adult ; Aged ; Aged, 80 and over ; Blood Chemical Analysis/instrumentation ; Blood Platelets/*cytology/physiology ; Female ; Flow Cytometry/*instrumentation/standards ; Humans ; Male ; Middle Aged ; Platelet Count/*instrumentation/standards ; Reference Values ; Republic of Korea ; Young Adult

OBJECTIVETo establish a flow cytometric internal standard method for counting platelet-derived microparticles (PMPs) and to study its clinical significance.

METHODSPMPs suspension (platelet poor plasma, PPP) was extracted by gradual centrifugation. According to the size of PMPs, 3 microm and 0.8 microm latex beads were used as internal standards for the quantitation. PMPs were counted by adjusting flow cytometric discrimination and voltage of forward scatter and side scatter.

RESULTSIn 30 healthy donors, the average concentration of resting PMPs was (1.2 x 10(5) +/- 5.7 x 10(4))/ml and that of activated PMPs was (1.6 x 10(6) +/- 9.1 x 10(5))/ml. Compared with healthy donors, PMPs mean value was significantly higher (P < 0.001) in 18 patients with coronary artery disease, 12 with acute cerebral infraction and 23 with chronic renal failure [the average PMPs concentration, (6.1 x 10(5) +/- 2.5 x 10(5))/ml, (6.8 x 10(5) +/- 3.4 x 10(5))/ml and (5.9 x 10(5) +/- 3.1 x 10(5))/ml respectively]. However, no significant difference in PMPs concentration was observed in 25 patients with acute leukemia and severe thrombocytopenia during the aplastic phase after chemotherapy [(1.3 x 10(5) +/- 6.1 x 10(4))/ml, (P > 0.05)].

CONCLUSIONSPMPs is a useful indicator in monitoring platelet activation, and plays an important role in thrombotic disease. By flow cytometric internal standard method, PMPs can be counted rapidly and accurately, which may be very helpful in interlaboratory comparative studies.

Adolescent ; Adult ; Aged ; Blood Platelets ; chemistry ; ultrastructure ; Cell Membrane ; chemistry ; ultrastructure ; Child ; Child, Preschool ; Coronary Disease ; blood ; Flow Cytometry ; methods ; Humans ; Kidney Failure, Chronic ; blood ; Middle Aged ; Particle Size ; Platelet Activation ; Platelet Count ; Platelet Membrane Glycoproteins ; analysis ; Reference Standards

OBJECTIVE: To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation. METHODS: Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 ^-/-, arhgef10 ^+/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively. RESULTS: WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 ^-/- zebrafish had more severe symptoms than arhgef10 ^+/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis. CONCLUSION Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.
Animals ; Annexin A5 ; Apoptosis ; Blotting, Western ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Cells, Cultured ; Flow Cytometry ; Genotype ; Humans ; In Situ Hybridization ; Larva/physiology* ; Phenotype ; RNA/isolation & purification* ; Real-Time Polymerase Chain Reaction/standards* ; Rho Guanine Nucleotide Exchange Factors/metabolism* ; Sincalide/analysis* ; Spectrophotometry/methods* ; Zebrafish/physiology*