Bone marrow morphology and karyotyping are effective ways to diagnose myelodysplastic syndromes. However, there are still many patients without these two abnormalities. Recently, there are considerable amount of evidence showing that flow cytometric immunophenotyping is a good approach to detect the abnormal differentiation and maturation of different cell lines and maturation stages of bone marrow cells in MDS patients. The highly reproducible results allow to distinguish MDS from other non-clonal hematocytopenia disorders and have a diagnostic advantage especially in case of low-risk MDS. Meanwhile, it showed a good correlation with the morphology and karyotyping, and with the International Prognostic Scoring System (IPSS) playing a prognostic role. This article reviewed recent advances of research in this field such as the application of FCM in MDS detection, the correlation of FCM parameters with morphologic and cytogenetic diagnosis of MDS as well as the relationship of parameters with typing and prognosis of MDS and so on.
Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Myelodysplastic Syndromes ; diagnosis ; immunology

OBJECTIVESTo evaluate the specificity of three-color flow cytometry in childhood acute lymphoblastic leukemia (ALL) immunophenotyping.

METHODSImmunophenotyping was performed by three-color flow cytometry analysis using CD(45)/SSC gating.

RESULTSThe percentage of blasts was correlated better with leukemic cell count compared with that of FSC/SSC, and the false positive results were low. Among eighty six cases of ALL, 95.3% was B-ALL, in which common-ALL and Pro-B-ALL were 76.8% and 6.1%, respectively, and 2.3% was T-ALL. CD(34)(+) and myeloid-associated antigen expression were observed in 57.0% and 34.9% of the cases, respectively, among which Pro-B-ALL was the commonest. CD(33) was more commonly expressed than CD(13) in Pro-B-ALL cases, but no difference in the expression between these two antigens in other subtypes.

CONCLUSIONGating of CD(45)/SSC eliminated effection of normal cells to blasts in bone marrow, with which the immunophenotyping results were more reliable.

Antigens, CD ; analysis ; Child ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology

This study was purposed to investigate the immunophenotypic characteristics in multiple myeloma (MM) cells and their significance. Thirty three cases of MM and 12 cases of reactive plasmacytosis (as control group) were enrolled in the study. The expressions of surface antigens in MM cells were detected with flow cytometry by using direct immunofluorescent technique and gating method of CD38/SSC and were confirmed with morphologic observation of myeloma cells. The results indicated that the proportion of myeloma cells detected by morphologic examination was 6.0% - 76.0%. With CD38/SSC gating method, a cluster of CD38 bright positive cells could be detected in their scatter plot, the proportion ranged from 0.99% to 57.54%. Most phenotype of MM was 38(st+)CD138(+)CD19(-)CD56(+) (78.8%). While the expressions of CD20, CD33, CD117, HLA-DR were seen in some MM patients, the positive rates were 12.1%, 15.2%, 30.3%, 9.1%, respectively; the expression of other antigens was negative. cκ or cλ monoclonal restriction was detected in 27 cases (81.8%) of MM, both cκ and cλ in the remaining cases of MM was negative. It is concluded that detecting the immunophenotype of MM patients by flow cytometry with CD38/SSC gating method and basing on the heterogeneity of cell antigens can discriminate myeloma cells from normal plasma cells, which provides evidence for targeted therapy and prognosis evaluation.
Adult ; Aged ; Female ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Male ; Middle Aged ; Multiple Myeloma ; immunology

OBJECTIVE: To establish 10-color fluorescent antibody combination panels for the detection of minimal residual disease (MRD) of acute myeloid leukemia (AML) in our laboratory and discuss the value of clinical application. METHODS: According to the antigen expression characteristics of leukemia cells of incipient AML patients, MRD in bone marrow were detected by multiparameter flow cytometry, and the test results were compared with both bone marrow cell morphology and PCR results, then 10-color fluorescent antibody combination panels in our lab for MRD detection was determined. RESULTS: The immunophenotypic characteristics of 392 incipient patients with AML in the First Affiliated Hospital of Zhengzhou University were analyzed, among them 357 (91.07%) cases showed abnormal immunophenotypes, which mainly included cross-lineage expression, cross-stage expression, deficiency of antigen expression or abnormal antigen intensity and other abnormal expression. The 10-color fluorescent antibody combination panels established according to abnormal immunophenotypic characteristics of leukemia cells were applied for detecting MRD in 156 patients with AML, the positive rate (43.6%) was higher than 26.8% of morphology, and the results were highly consistent with PCR detection results (96.49%), moreover, the recurrence rate of MRD positive patients (86.96%) was significantly higher than 5.75% of MRD negative patients. Therefore, this method could truly reflect the load of leukemia cells and prompt change of disease condition. CONCLUSION Multiparameter flow cytometry can detect various abnormal immunophenotypes of AML. The 10-color fluorescent antibody combination panels in our lab based on the characteristics of antigens expression in leukemia cells can well detect MRD of leukemia cells, so as to predict relapse and provide basis for clinical treatment.
Bone Marrow ; Flow Cytometry/methods* ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute/diagnosis* ; Neoplasm, Residual/diagnosis*

OBJECTIVEAcute leukemia (AL) is one of the most common malignant diseases in children. AL immunophenotypes are known to be benefit to the predictable prognoses and specific therapy. Recently, the accuracy of AL immunophenotyping was dramatically improved with the application of the flow cytometry, the new monoclonal antibodies, the improvement of the gating strategies and the multi-parameter analytic techniques. The aim of this study was to evaluate the value of multi-color flow cytometry in the immunophenotyping of acute leukemia in children.

METHODSThree- or four-color flow cytometry and CD(45)/Side Scatter (SSC) gating were used to analyze the surface and cytoplasmic (Cy) antigen expressions in 222 successive cases of childhood acute leukemia.

RESULTSCells from 222 cases of children with acute leukemia were analyzed. Based on the diagnostic criterion proposed by European Group for the Immunological Characterization of Leukemia (EGIL), four categories could be identified: the undifferentiated type accounted for 0.9%, acute myeloid leukemia (AML) 35.1%, acute lymphoblastic leukemia (ALL) 55.9%, and mixed lineage AL 8.1%. Of 124 patients with ALL, 94 patients (75.8%) were classified as B lineage and 30 patients (24.2%) T lineage ALL. Antigen aberrant expressions were found in AML (24.4%), B lineage ALL (36.2%) and T lineage ALL (30.0%). CD(7) was the most commonly expressed lymphoid antigen in AML (12.8%), followed by CD(19) (6.4%) and CD(2) (5.1%). CD(13) was the most commonly expressed myeloid antigen in ALL (18.5%), followed by CD(15) (11.3%), CD(11b) (6.5%) and CD(33) (4.3%). CD(117) and CD(56) presented in 73.3% and 38.0% cases of AML, respectively, but were generally absent on blast cells of ALL. CyCD(22), CyCD(3) and CyMPO were specifically expressed in B lineage, T lineage and myeloid lineage leukemia, respectively, and the first two could be more sensitively detected than they were on cell membrane surface.

CONCLUSIONSMulti-color flow cytometry is a reliable technique in the diagnosis, differential diagnosis and classification of acute leukemia in children.

Acute Disease ; Adolescent ; Child ; Child, Preschool ; Female ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Infant ; Leukemia ; classification ; diagnosis ; immunology ; Male

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated from umbilical cords and are therapeutically used because of their ability to differentiate into various types of cells, in addition to their immunosuppressive and anti-inflammatory properties. Fetal bovine serum (FBS), considered as the standard additive when isolating and culturing MSCs, has a major limitation related to its animal origin. Here, we employed a simple and economically efficient protocol to isolate MSCs from human umbilical cord tissues without using digestive enzymes and replacing FBS with umbilical cord blood serum (CBS). METHODS AND RESULTS: MSCs were isolated by culturing umbilical cord pieces in CBS or FBS supplemented media. Expansion and proliferation kinetics of cells isolated by explant method in the presence of either FBS or CBS were measured, with morphology and multi-differentiation potential of expanded cells characterized by flow cytometry, RT-PCR, and immunofluorescence. MSCs maintained morphology, immunophenotyping, multi-differentiation potential, and self-renewal ability, with better proliferation rates for cells cultured in CBS compared to FBS supplement media. CONCLUSIONS: We here present a simple, reliable and efficient method to isolate MSCs from umbilical cord tissues, where cells maintained proliferation, differentiation potential and immunophenotyping properties and could be efficiently expanded for clinical applications.
Animals ; Fetal Blood* ; Flow Cytometry ; Fluorescent Antibody Technique ; Humans* ; Immunophenotyping ; Kinetics ; Mesenchymal Stromal Cells* ; Methods* ; Multipotent Stem Cells ; Umbilical Cord*

This study was aimed to establish the technique of fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) used to smear of bone marrow, so as to develop a new technique for detection of the molecular cytogenetic abnormalities in multiple myeloma (MM). By using the bone marrow smear as the carrier and the anti-CD138 antibody linked with FITC, direct fluorescence staining was applied to mark plasma cells (PCs) and differences were compared in the proportion of both PCs marked by fluorescence staining and PCs detected in morphology. At the same time, the chromosome 8 centromere probe was used in interphase fluorescence in situ hybridization (I-FISH) for detection of the chromosome 8 abnormalities in PCs marked by fluorescence staining. The results showed that there was no significant difference between the proportions of both PCs marked by fluorescence staining and PCs detected in morphology on smear (p>0.05). 4 out of 9 patients (44%) had the chromosome 8 abnormalities, including 3 cases with -8 (33%) and one case with +8 (11%). It is concluded that the FICTION technique on the basis of bone marrow smear is characterized by convenience, specificity and accuracy. Therefore, it can be used for molecular cytogenetic research in MM.
Aged ; Bone Marrow ; pathology ; Chromosome Aberrations ; Female ; Humans ; Immunophenotyping ; In Situ Hybridization, Fluorescence ; methods ; Male ; Middle Aged ; Multiple Myeloma ; genetics ; pathology

This study was aimed to distinguish abnormal cells and to diagnose hematologic diseases through recognizing antigen expression pattern and percentage of peripheral blood cells in normal elderly men. Antigen expression of blast cells, granulocytes, monocytes, lymphocytes, nucleated red blood cells and plasma cells was detected by seven-color flow cytometry in a total of 88 peripheral blood samples from normal elderly men, aged median 82 years old, from 70 to 98 years. Groups were divided according to age, region and underlying diseases, and the percentages of different subgroup cells were examined to confirm whether the differences were significant or not. The results showed that the median proportion of CD34(+) blast cells in peripheral blood from normal elderly men were 0.017% (0.015%-0.020%), with high expression of HLA-DR, CD33, CD13 and CD117, low expression of myeloid antigens, such as CD15, CD11b and CD16, while lymphoid antigens were seldom positive, including CD7, CD19 and CD56. Dim-expression of CD38 was found in peripheral blood blast cells, CD38(dim)+/- cell percentage in blast cells was 61.36% ± 18.26%. In the differentiation and development of granulocytes, CD16(-), CD13(+) CD16(+) (intermediate) and CD16(+) (strong) CD13(+) cells appeared in sequence from immature to mature granulocytes, whose median proportions in nuclear cells were 0.04%, 0.30% and 61.30%, respectively. The percentages of immature monocytes, such as CD64(+) CD14(-) and HLA-DR(+) CD11b(-) cells, were from 0.00% to 0.10% and from 0.07% to 0.68%, separately. No significant differences were found between different subgroups (P > 0.05). It is concluded that the immunophenotypic characteristics and referential percentages of CD34(+) blast cells, granulocytes and monocytes with different development stages in peripheral blood from normal elderly men are recognized, which can help to discriminate abnormal cells.
Aged ; Aged, 80 and over ; Antigens, CD ; metabolism ; Blood Cells ; immunology ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Leukocyte Count ; Male

The aim of this study was to establish the standard protocols for isolating and enriching hematopoietic stem/progenitor cells (HSPC) from human placenta tissue (PT). Single-cell suspension from of human PT was prepared by mechanical method combined with collagenase digestion. Mononucleated cells (MNC) derived from PT were separated by hydroxyethyl starch (6% HES), then the three cell subsets of different immunophenotypes (CD34(-), CD34(+)CD38(-), CD34(+)CD38(+)) contained in MNC were isolated by Magnetic Activated Cell Sorting (MACS). The cell immunophenotype of each sorting steps was analyzed by flow cytometer (FCM). The cell enrichment and recovery rate of each sorting step were calculated. The results showed that MNC could be harvested up to (12.30 +/- 3.51) x 10(8) from a single-cell suspension of human PT by mechanical method and collagenase digestion, no significant difference existed as compared with umbilical cord blood (UCB) initial sample [(8.86 +/- 5.38) x 10(8)], but the percentage of CD34(+) cells in MNC of human PT was (3.93 +/- 2.31)%, much higher than that in UCB [(0.44 +/- 0.29)%] (P < 0.001). recovery rate of MNC and CD34(+) cells from PT after separation with 6% HES were (45.3 +/- 11.7)% and (51.1 +/- 9.8)%, respectively. After MNC being sorted by MACS, the enrichment and recovery rate of CD34(+) cells in CD34(+) group were (73.4 +/- 14.1)% and (52.7 +/- 11.7)% respectively. It is concluded that the protocols established here for isolating and enriching hematopoietic stem/progenitor cells from human placenta can acquire HSPC with high abundance, enrichment and viability and may be a useful reference of isolating methods for future related study.
Antigens, CD34 ; analysis ; Cell Proliferation ; Cell Separation ; methods ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans ; Immunophenotyping ; Placenta ; cytology

OBJECTIVETo analyze the immunophenotype and leukemia associated immunophenotype (LAIP) of leukemia cells from patients with acute myeloid leukemia (AML) in minimal residual disease (MRD) detection.

METHODSFour-color multiparametric flow cytometry (FCM) with CD45/SSC gating was used to determine the expression of the following antibodies of CD7, CD117, CD33, CD34, CD10, CD19, CD56, CD38, CD13, CD14, CD64, CD9, CD16, CD2, CD5, CD11b, CD123, HLA-DR in 610 AML patients and 20 normal bone marrow (NBM) samples.

RESULTSThe mean percentages of CD34+ and CD117+ side scatter low (SSC(low)) cells in NBM mononuclear cells (BMMNCs) were (0.35 +/- 0.15)% and (0.76 +/- 0.31)%, respectively. The majority (84% -95%) of CD34+ SSC(low) cells co-expressed CD13, CD33, CD38, CD117 and HLA-DR. 33% and 20% of CD34+ SSC(low) cells were CD15+ and CD9, respectively. Only a small proportion (< 10%) of CD34 SSC(low) cell co-expressed CD11b, CD56, CD19 and CD64, while co-expressed CD7 was 12%. In CD117 SSC(low) cells, the relative proportions of CD19+, CD11b+, CD56+ and CD7+ were less than 10%, while CD9+ was 14%. CD38+ and HLA-DR+ were 87% and 91%, respectively. The expressions of CD15, CD34, CD13 and CD33 on CD117 SSC(low) cells were between 30% and 50%. In AML patients, most cases were CD117+ (95.08%), CD38+ (94.74%), CD9+ (84.93%), CD33+ (84.60%), HLA-DR+ (77.23%) and CD13 (75.25%). The proportions of CD64+ and CD34+ were 64.41% and 59.51%, and that of CD15 and CD11b+ were 43.06% and 22.07%, respectively. 86.39% of AML patients were found to have at least one LAIP, the highest incidence being in AML-M1 and M3 subtypes and the lowest in AML-M4Eo subtype. The cross-lineage antigen and asynchronous antigen expression were the most frequent aberrant phenotypes. CD7, CD19 and CD56 expressing on CD34+ cells were major cross-lineage antigen. For asynchronous antigen expression, CD34+ CD64+, CD117+ CD11b+ , CD34+ CD38(-/dim) and CD34+ HLA-DR(-/dim) were seldom expressed on normal BMMNCs (about 0.01%), and the logarithm difference between AML and NBM was larger than 3.0, being the more sensitive LAIP.

CONCLUSIONMRD detection by multiparameter flow cytometry can be applied to more than 80% of AML patients.

Adult ; Female ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; immunology ; Male ; Middle Aged ; Neoplasm, Residual ; diagnosis