OBJECTIVETo compare the characteristics of immunomagnetic beads and hespan precipitation for isolation of mononuclear cells (MNCs) from umbilical cord blood and try to find a better isolation method for MNCs.

METHODSFifteen umbilical cord blood samples from healthy parturiens were collected between December 2007 and March 2008. MNCs were isolated using hespan precipitation and CD133 immunomagnetic beads, respectively. MNCs were identified using the surface marker CD34 by flow cytometry on the 30th of primary culture. Growth conditions and morphologic changes of primary cells were observed by an inverted microscope.

RESULTSThe number of MNCs from umbilical cord blood isolated by hespan precipitation (15.23 +/- 4.30 x 10(6)/mL) was significantly greater than that by CD133 immunomagnetic beads (0.066 +/- 0.027 x 10(6)/mL) (p<0.05). The MNCs isolated by hespan precipitation suspended at the culture medium and their growth was slow after passage. The growth of MNCs isolated by CD133 immunomagnetic beads was kept in a good condition. The CD34 positive rate of MNCs isolated by hespan precipitation and immunomagnetic beads was 10.1% and 0.5%, respectively.

CONCLUSIONSThe hespan precipitation is an effective method for MNCs isolation from human umbilical cord blood, but with a cell growth condition below the mark. The MNCs isolated by CD133 immunomagnetic beads are in a high purity quotient.

AC133 Antigen ; Antigens, CD ; immunology ; Antigens, CD34 ; analysis ; Cell Separation ; methods ; Chemical Precipitation ; Fetal Blood ; cytology ; Glycoproteins ; immunology ; Humans ; Hydroxyethyl Starch Derivatives ; chemistry ; Immunomagnetic Separation ; methods ; Infant, Newborn ; Leukocytes, Mononuclear ; cytology ; Peptides ; immunology

BACKGROUND: The aberrant, leukemia-associated antigen expression patterns allow us to discriminate leukemic blasts from normal precursor cells. Our major goal was to determine a guideline for the detection of minimal residual disease using CD20+/CD34+ and myeloid Ag+/CD19+ combination in the bone marrow of acute leukemia in complete remission (CR) after chemotherapy. METHODS: Bone marrow samples from 117 patients with acute leukemia in complete remission after chemotherapy and from 22 healthy controls were immunophenotyped by triple staining and measured by flow cytometry. RESULTS: The CD20+/CD34+ cells in the large lymphocyte gate (R1) ranged from 0% to 3.24% (0.8+/-0.82%, P=0.000) in CD20+/CD34+ B-lineage ALL CR (N=31), from 0.03% to 4.2% (0.7+/-0.83%, P=0.000) in CD20-/CD34- B-lineage ALL CR (N=66), from 0.1% to 0.96% (0.45+/-0.32%, P=0.016) in T-ALL CR (N=10), and from 0.02% to 0.48% (0.18+/-0.15%, P=0.776) in AML CR (N=10). The CD13,33+/CD19+ cells in R1 gate ranged from 0% to 2.69% (0.37+/-0.48%, P<0.001) in CD13,33+/CD19+ B-lineage ALL CR (N=31), from 0% to 1.8% (0.31+/-0.28%, P<0.001) in CD13,33-/CD19+B-lineage ALL CR (N=65), from 0.02% to 0.64% (0.29+/-0.22%, P=0.071) in T-ALL CR (N=9), and from 0% to 0.17% (0.07+/-0.09%, P=0.341) in AML CR (N=3). CONCLUSIONS: Using an immunophenotypic method for the detection of early relapse or minimal residual disease of B-lineage ALL bone marrow in CR after chemotherapy, different cutoff values should be applied according to antigen combination and gating. When the proportion of aberrant antigen combination was less than 5% in large lymphocyte gate, the results should be interpreted with caution.
Acute Disease ; Antigens, CD/*metabolism ; Antigens, CD19/metabolism ; Antigens, CD20/metabolism ; Antigens, CD34/metabolism ; Antigens, Differentiation, Myelomonocytic/analysis/metabolism ; Bone Marrow Cells/*classification/metabolism ; Flow Cytometry ; Hematopoietic Stem Cells/classification/metabolism ; Humans ; Immunophenotyping ; Leukemia/*diagnosis/drug therapy ; Leukemia, Myeloid, Acute/diagnosis/drug therapy ; Neoplasm, Residual ; Remission Induction ; Tumor Markers, Biological/immunology

This study was purposed to investigate the expansion and hematopoietic reconstitution capability of CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria (PNH) by using BALB/c nude mice so as to provide experimental basis for clinical anto-BMT or auto-PBHSCT in patients with PNH. CD34(+)CD59(+) cells were selected from the bone marrow mononuclear cells in normal persons and PNH patients by immunomagnetic positive double sorting and were engrafted sublethally irradiated BALB/c nude mice. The human CD45(+) cells in bone marrow, spleen and peripheral blood of recipient mice were detected by flow cytometry and DNA assay. The results showed that the CD34(+)CD59(+) cells in PNH patient group and normal person group could expanded ex vivo, but ex vivo expansion capability of CD34(+)CD59(+) cells in PNH patient group at day 7 seemed inferior to that in normal control. While CD34(+)CD59(+) cells of PNH patients and normal persons were transfused into recipient mice, the human CD45(+) cells could be detected in bone marrow, spleen and peripheral blood at 6 weeks after transfusion, but there was no statistical difference in counts of CD45 cells between 2 groups. It is concluded that CD34(+)CD59(+) cells from PNH patients may keep characteristics of normal hematopoietic stem cells, and possess ability to expand ex vivo and support hemopoiesis.
Animals ; Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; immunology ; CD59 Antigens ; analysis ; Female ; Hematopoiesis ; physiology ; Hematopoietic Stem Cell Transplantation ; Hemoglobinuria, Paroxysmal ; pathology ; Humans ; Immunomagnetic Separation ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Transplantation, Heterologous ; Whole-Body Irradiation

OBJECTIVETo study the clinical and pathologic characteristics of poorly differentiated cutaneous angiosarcoma of scalp.

METHODSEight cases of poorly differentiated cutaneous angiosarcoma of scalp were enrolled into this study. The clinical manifestations and histopathologic features were analyzed. Immunohistochemical study for CD31, CD34, factor VIII-related antigen, vimentin, AE1/AE3, CAM5. 2, epithelial membrane antigen and carcinoembryonic antigen was performed.

RESULTSThe mean age of the patients was 69 years. The male-to-female ratio was 5 : 3. The tumor manifested clinically as bruise-like lesion in early phase, indurated erythematous plaque accompanied by nodules, ulcerations and bleeding in advanced phase. Histologically, the tumor was composed of solid sheets of undifferentiated spindle cells which were not easily recognizable as vascular in origin. Nuclear atypia was always present. The tumor cells in all of the 8 cases strongly expressed CD31, factor VIII-related antigen and vimentin. Weak expression of CD34, AE1/AE3 and CAMS. 2 was noted in 2, 4 and 4 cases, respectively. The staining for epithelial membrane antigen, carcinoembryonic antigen and S-100 was negative. Conclusions Angiosarcoma needs to be excluded by histologic examination whenever bruise-like and erythematous lesions occurring on scalp skin of elderly patients. The endothelial origin of the tumor cells can be confirmed with immunostaining for CD31, CD34 and factor VIII-related antigen.

Aged ; Aged, 80 and over ; Antigens, CD34 ; immunology ; Biomarkers, Tumor ; analysis ; Cell Adhesion Molecules ; Cell Differentiation ; Endothelium ; metabolism ; Female ; Hemangiosarcoma ; immunology ; Humans ; Male ; Middle Aged ; Platelet Endothelial Cell Adhesion Molecule-1 ; immunology ; Scalp ; pathology ; Skin Neoplasms ; immunology ; metabolism ; pathology ; Vimentin ; analysis

Epithelioid hemangioendothelioma is a rare vascular origin tumor which usually occurs in soft tissues, liver, and lung. It usually affects adult women and presents as multiple hepatic nodules with mainly peripheral distribution. It is difficult to diagnose and treat because of non-specific clinical manifestations and findings on the imaging study. Moreover, pathological misdiagnosis is common. We report a case of this rare tumor that was detected incidentally. Final diagnosis was based on histological evidence. A 52-years old man suffered from right upper quadrant abdominal pain for 3 months, and was initially misdiagnosed as a metastatic carcinoma. Physical examination revealed superior cervical lymphadenopathy with mild hepatomegaly. Finally, hepatic epithelioid hemangioendothelioma was diagnosed on the basis of positive immunohistochemical staining for factor VIII, CD34, and VEGF. Our case highlights the importance of a histological diagnosis to avoid misdiagnosis.
Antigens, CD34/analysis/immunology ; Carcinoma/secondary ; Diagnosis, Differential ; Factor VIII/analysis/immunology ; Hemangioendothelioma, Epithelioid/*diagnosis/pathology ; Humans ; Immunohistochemistry ; Liver Neoplasms/*diagnosis/pathology ; Male ; Middle Aged ; Positron-Emission Tomography

The aim of study was to analyze the clinical, biological features, treatment outcome and prognosis of mixed-lineage acute leukemia (MAL). 48 MAL patients diagnosed according to European Group of International Leukemia (EGIL) scoring system were retrospectively analyzed and the analysis results were compared with that from 68 cases of AML and 61 cases of ALL. The results showed that the incidence of MAL in acute leukemia was 9.6%. Morphologically, the subtypes of M(1) and M(2) were predominant in AML, while L(2) in ALL. The median of white blood cell count in MAL was significantly higher than that of non-mixed-lineage cases (AML and ALL) observed during the same period (P < 0.05). The incidences of enlargement of liver, spleen and lymphonodes in MAL were higher than those in AML. The difference was significant (P < 0.01) and was not significant compared with those in ALL (P > 0.05). Coexpression of myeloid and B lymphoid antigens in MAL patients was predominant, its rate was 70.9%. The coexpression rate of T lymphoid and myeloid antigens was 20.8%, coexpression of B, T lymphoid and myeloid antigens was 8.3%. CD34 was expressed in 79.2% of MAL cases, it was higher than those expressed in AML (54.4%) and ALL (52.5%) (P < 0.01), which suggests that MAL might originate from malignant transformation of hematopoietic stem cells. Cytogenetic analysis revealed normal and abnormal karyotypes in 32.1% and 67.9% of MAL cases respectively. In MAL Ph chromosome abnormality incidence was 25% and was significantly higher than that in AML group (0%) (P < 0.01), but was not statistical defference with that in ALL group (16.7%) (P > 0.05). The completed remission rate of MAL was 38.1%, lower than CR rate in AML (70.8%) and ALL (72.2%) respectively (P < 0.01). Treatment outcomes were negatively related to the expression of CD34 antigen and Ph chromosome. It is concluded that MAL is supposed to be originated from stem cells, coexpression of lymphoid/myeloid antigens is the major feature of MAL which accompanies many poor prognosis factors and lower CR rate. Appropriate chemotherapeutic strategy should be further searched.
Adolescent ; Adult ; Antigens, CD34 ; analysis ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Child ; Female ; Humans ; Immunophenotyping ; Karyotyping ; Leukemia, Biphenotypic, Acute ; drug therapy ; immunology ; pathology ; Male ; Middle Aged ; Philadelphia Chromosome ; Prognosis ; Retrospective Studies ; Young Adult

A 46-year-old woman was found to have a huge liver mass that was detected by abdominal ultrasonography. Abdominal CT and MRI showed a 10 cm-sized, encapsulated mass occupying the anterior segment of the right hepatic lobe. Extended right hemihepatectomy was performed and pathological examination revealed fibroblast-like spindle cells within dense deposits of collagen. On immunohistochemical staining, these spindle tumor cells showed an intense CD34 immunoreactivity. The patient is alive without evidence of tumor recurrence 7 months after the resection. Solitary fibrous tumor is a very rare neoplasm found in the liver parenchyma, and it has been reported in less than 30 patients in the English literature. We present here the first such case in Korea.
Antigens, CD34/analysis/immunology ; Female ; Humans ; Liver Neoplasms/*diagnosis/pathology/surgery ; Magnetic Resonance Imaging ; Middle Aged ; Solitary Fibrous Tumors/*diagnosis/pathology/surgery ; Tomography, X-Ray Computed ; Tumor Markers, Biological/analysis/immunology

To evaluate the significance of FCM in minimal residual disease (MRD) detection, the immunophenotyping and leukemia-associated immunophenotypes (LAIP) of leukemia cells from 273 adult and 142 childhood patients with B lineage acute lymphoblastic leukemia (B-ALL) were detected by four to six antibody combinations of 4-color CD45/SSC gating multiparametric flow cytometry (FCM). The results showed that the B-ALL patients could be classified into 4 subtypes based on different expression CD34 and CD10: subtype I (CD34(+)/CD10(-)), subtype II (CD34(+)/CD10(+)), subtype III (CD34(-)/CD10(+)), subtype IV (CD34(-)/CD10(-)). The LAIP was observed in 100% and 92% patients of subtype I and subtype II, respectively, whereas only 79.2% in subtype III. The incidence of LAIP in total B-ALL cases was 90% by using the antibodies detected in this investigation. There was no significantce different for incidence of LAIP between adult and pediatric patients. LAIP was observed in 77.6% of patients by labeling only CD34/CD10/CD19/CD45 4-color antibody combination. It is concluded that in 90% of childhood and adult B-ALL patients LAIP can be found, which suits MRD detection by multiparameter flow cytometry.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD34 ; analysis ; B-Lymphocytes ; immunology ; Burkitt Lymphoma ; classification ; immunology ; pathology ; Cell Lineage ; Female ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Male ; Middle Aged ; Neoplasm, Residual ; diagnosis ; Neprilysin ; analysis ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; classification ; immunology ; pathology

Ex vivo expanded human bone marrow CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria (PNH) were transplanted into BALB/c mice in order to investigate their proliferation ability and reconstruction of hemopoiesis, and to lay the groundwork for clinical ABMT/APBSCT in PNH patients. CD34(+)CD59(+) cells were selected from the bone marrow mononuclear cells in PNH patients by using immunomagnetic positive double sorting. Sublethally irradiated BALB/c mice were transplanted with CD34(+)CD59(+) cells enriched from bone narrow of PNH patients. The results showed that human CD45(+) cells were detected in the bone marrow, spleen and peripheral blood of the nude mice by flow cytometry and DNA analysis at 6 weeks post-transplant. Blood routine indicators of nude mice were found to recover to some extent, but did not fully recover. It is concluded that ex vivo expanded bone marrow CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria could keep their biological characteristics and ability to reconstruct hemopoiesis in irradiated BALB/c mice.
Animals ; Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; immunology ; CD59 Antigens ; analysis ; Female ; Hematopoietic Stem Cell Transplantation ; methods ; Hemoglobinuria, Paroxysmal ; pathology ; therapy ; Humans ; Immunomagnetic Separation ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Transplantation, Heterologous

This study was aimed at exploring the immunophenotypic features of blasts in patients with myelodysplastic syndromes (MDS). Four-color flow cytometry using conventional and secondary gating strategies was used to immunophenotype blasts and the CD34 positive cells in bone marrow nucleated cells of 29 patients with MDS. The results showed: (1) with progression of MDS from RA/RAS, RAEB to RAEB-T, the proportion of CD34(+) cells were gradually increased from 8.0%, 46.4% to 76.8% (P < 0.05); (2) using CD45 vs SSC gating strategy, with the transformation of RA/RAS, RAEB to RAEB-T, the expression of HLA-DR, CD13, CD33, CD117 were also gradually increased (P < 0.05), and the expression of CD15 was gradually decreased (P < 0.05); (3) using CD45 vs CD34 gating strategy, the expression of HLA-DR, CD13, CD33, CD117 on blasts were higher by secondary gating method than those by conventional gating (P < 0.05). However, there were no significant difference (P > 0.05) in the expression of above-mentioned antigens on CD34(+) cells among different MDS subtypes. It is concluded that conventional gating method can reflect MDS progression from RA/RAS, RAEB to RAEB-T, and secondary gating strategy may accurately reflect the biological features of blasts in MDS. Abnormal expression of CD34 is related to the immaturity level and heterogeneity of blast cells, which is beneficial to the diagnosis of clinically suspected MDS incapable of diagnosing with morphology and cytogenetics.
Adult ; Aged ; Antigens, CD34 ; analysis ; Bone Marrow Cells ; immunology ; pathology ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Male ; Middle Aged ; Myelodysplastic Syndromes ; immunology ; pathology