BACKGROUND: Although the bone marrow transplantation is now recognized as an effective therapy for some hematologic and malignant diseases, HLA-matched related donor is limited. Human umbilical cord blood contains sufficient numbers of hematopoietic stem cells to provide long-term engraftment in the unrelated recipient with several advantages. The number of hematopoietic stem cells is an important factor for the better result in transplantation. The purpose of this study is the development of technique for the collection, separation and cryopreservation of the cord blood mononuclear cells for transplantation. METHODS: Forty-four cord bloods were collected during an uneventful delivery from the Kyungpook National University Hospital and Hana Hospital, Taegu, Korea from May to June, 1997. The mononuclear cell separation with Ficoll-Hypaque(Lymphocyte separation medium, Bionetics, USA) or red cell sedimentation with 3% gelatin(Sigma, USA) or 6% hydroxyethyl starch(HES; Fresenius, Germany) was done and the numbers and viabilities were compared with hemocytometry(Technicon H-3 RTX System, Irland). The viabilities of separated cells by different cell separators in the L-15 medium(Gifco, USA) containing 2% FBS(Gifco, USA) according to time were compared. The viabilities of separated cells by different cell separators in the L-15 medium(Gifco, USA) containing 17% FBS according to time and temperature were compared. The cell viabilities according to the different cell separating, freezing(direct, program-A or program-B) and thawing(rapid or slow) methods were compared. The method of direct freezing is putting the cells into the liquid nitrogen chamber(-196oC) directly. The method of program-A is lowering the temperature as 1oC/min to -90oC by programmed cell freezer(Cryomed 1010, USA) and then putting the cells into the liquid nitrogen chamber. The method of program-B is lowering the temperature as 1oC/min, to -4oC and 25oC/min, to -40oC, and raising as 15oC/min, to -12oC, and lowering again as 1oC/min, to -40oC and 10oC/min, to -90oC by programmed cell freezer and then putting the cells into the liquid nitrogen chamber. The method of rapid thawing is thawing the freezed cells in the 37oC incubator and the method of slow thawing is in the room temperature of 20oC. The functional aspect of stem cell after cryopreservation was performed with cell culture in the methylcellulose medium(Sigma, USA) and GM-CSF(LG Pharmacy, Korea) in the 5% CO2 incubator(37oC; Forma, USA). The statistical analysis was done with Analysis of Variance Procedure and T-test. RESULTS: The mean volume of cord blood was 121 mL(80~155 mL) and was no difference with maternal and fetal conditions, but the large volume could be obtained with rapid collection from early ligation of cord. The hemoglobin was 14.2+/-1.6 g/dL, hematocrit 46+/-6%, red blood cell 4.1+/-0.4x106/microL, platelet 236+/-82x103/microL and total white blood cell 11,893+/-4,010/microL with neutrophil 54.5+/-11.5%, eosinophil 2.6+/-1.3%, basophil 1.4+/-1.1%, lymphocyte 31.7+/-12.0% and monocyte 8.5+/-2.8%. The removal of red blood cell was 98.3% with F-H, 80.7% with gelatin and 54.3% with HES(P<0.0001). The removal of platelet was 64.8% with F-H, 85.2% with gelatin and 80.0% with HES. The removal of mononuclear cell was 73.3+/-15.4% with F-H, 99.1+/-22.4% with gelatin and 94.2+/-22.8% with HES(P<0.0001). The removal of polymorphonuclear cell was 91.0% with F-H, 42.3% with gelatin and 35.5% with HES(P<0.0001). The viabilities of separated cells by gelatin or HES in the L-15 medium containing 2% FBS for 18~24 hours were low compared to that by F-H and these differences were resolved with raising the concentration of FBS up to 17%. The program-B freezing(59.4%) was the best method in recovery of cells compared to the direct(36.9%) or program-A freezing(41.6%). But no differnces were observed according to the different cell separating or thawing methods. The colony formation from cryopreserved cell was observed at 7 th day of cell culture. CONCLUSION: In summary, the large volume of cord blood could be obtained with rapid collection after ligation of cord. The best way of mononuclear cell separation was the method with 3% gelatin among three the program-B freezing was the best way in recovery of cells after thawing these will be important factors to raise the success rate in hematopoietic stem cell transplantation.
Basophils ; Blood Platelets ; Bone Marrow Transplantation ; Cell Culture Techniques ; Cell Separation* ; Cell Survival ; Cryopreservation* ; Daegu ; Eosinophils ; Erythrocytes ; Fetal Blood* ; Freezing ; Gelatin ; Gyeongsangbuk-do ; Hematocrit ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Humans* ; Incubators ; Korea ; Leukocytes ; Ligation ; Lymphocytes ; Methylcellulose ; Monocytes ; Neutrophils ; Nitrogen ; Pharmacy ; Stem Cells ; Tissue Donors

BACKGROUND: Development of hematopoietic growth factor made it possible to treat anemia and granulocytopenia following intensive chemotherapy and for thrombocytopenia, recently found thrombopoietin(TPO) is being applied experimentally in several countries. The megakaryocyte colony assay can assess the effect of TPO on the thrombocytopenia resulted from cancer chemotherapy or hematopoietic stem cell transplantation. In vitro colony assay procedures for detecting human erythroid and granulocyte macrophage progenitors have been in widespread use for many years. However, reproducible assay methods for human megakaryocyte progenitors have lagged considerably behind especially in Korea. Duration platelet recovery following transplantation depends on the origin of the hematopoietic cells. Usually thrombocyte recovery is delayed following cord blood stem cell transplantation because of the small amount of cells administered. This study was carried out to investigate and establish the megakaryocyte colony assay of hematopoietic stem cells obtained from the various origin of the hematopoietic stem cells with or without TPO. METHOD: Mononuclear cells of bone marrow, peripheral blood and cord blood were collected following Ficoll density gradient centrifugation and megakaryocyte colony assay was done using MegaCultTM(Stem Cell Tech. Inc., Canada). After liquifying the agarose, mononuclear cells were added and then agarose and cell mixture were dispersed into the two wells of the chamber slide. These slides were incubated for 18~21 days at 37oC, 5% CO2. The megakaryocyte colonies were detected by staining of the cells with a primary antibody to the GPIIb/IIIa antigen, secondary antibody, alkaline phosphatase and Evans Blue in order. Changes of CD34 and GPIIb/IIIa positive cells were also analysed in flask culture using flow cytometry. RESULTS: CD34 positive cells were most abundant in the mononuclear cells of the bone marrow, meanwhile the number of CFU-GM and megakaryocyte colony were greater in the mononuclear cells of the cord blood. After administration of TPO, the cell number of megakaryocyte colony was increased dose dependently, but CFU-GM colony did not show any response to TPO. With flask culture, the cell number was decreased with or without TPO. However adding GM-CSF, IL3 and TPO to cord blood mononuclear cell, the number of the cord blood mononuclear cells was increased on the 5 th day. The amount of CD34 positive cells was increased dose dependently to TPO in one of two cord blood and one peripheral blood. The amount of GPIIb/IIIa positive cells was increased dose dependently to TPO following incubation of all the mononuclear cells. CONCLUSION: This study revealed successful result of megakaryocyte colony assay using MegaCultTM in various kinds of mononuclear cells and suggested that TPO was useful for CFU-mega colony formation. The amount of GPIIb/IIIa positive cells was increased with TPO in the flask culture. Therefore TPO could be useful for assessment of CFU- mega, and could be applied for the in vivo and in vitro expansion of megakaryocytes and platelets.
Agranulocytosis ; Alkaline Phosphatase ; Anemia ; Blood Platelets ; Bone Marrow ; Cell Count ; Centrifugation, Density Gradient ; Cord Blood Stem Cell Transplantation ; Drug Therapy ; Evans Blue ; Fetal Blood ; Ficoll ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; Granulocyte-Macrophage Progenitor Cells ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells* ; Humans ; Korea ; Megakaryocyte Progenitor Cells ; Megakaryocytes* ; Sepharose ; Thrombocytopenia

Purpose: Long-term culture- initiating cells(LTC-IC) are stem cells that have the capacities of long-term engraftment and helping to establish hematopoietic microenvironment. For evaluation of the LTC-IC, we measured the counts and function with multidimentional flowcytometry in long-term culture media. METHODS: Samples were obtained from umbilical cord blood, leukapheresis products and bone marrow(BM). LTC-IC were counted with flowcytometric analysis using anti- CD34, anti-CD38, and anti-HLA-DR antibodies at 0, 5, and 8 weeks. Cell adhesion molecule related with stem cell were evaluated with flowcytometric analysis also using anti-VCAM-1(CD106) and anti-VLA-4(CD49d) at 0 and 8 weeks. RESULTS: The proportion of CD34+/CD38- cell from fractionated mononuclear cells at 0 week were 0.46%, 0.044%, and 0.038% for BM, leukapheresis products, and umbilical cord blood respectively and then rapidly decreased at 5 weeks, but still persisted at 8 weeks in all three groups. The proportion of CD34+/HLA-DR- cells was the same tendency to CD34+/CD38-. VCAM+ expression rate from fractionated CD34+ cells at 0 and 8 weeks were 67.3% and 40.2% for BM and 64.1% 44.2% for umbilical cord blood but it was very low 31.2% and 5.1% for leukapheresis products. VLA-4+ expression rate for fractionated CD34+ cells at 0 and 8 weeks were similar tendency to VCAM+ cells. CONCLUSION: This study suggest that the count of LTC-IC decreased with time but still persisted until 8 weeks. Umbilical cord blood including BM help to establish the hematopoietic microenvironments.
Antibodies ; Cell Adhesion ; Culture Media ; Fetal Blood ; Leukapheresis ; Stem Cells

BACKGROUND: Peripheral blood stem cell (PBSC) transplantation has been widely used to support the hematopoietic recovery after high dose chemotherapy in patients with advanced malignancies. The procedure of PBSC collection in pediatric patients is similar to that in adults, but needs the 'fine tuning' of the volume shift of each procedure and the technical factors to achieve specific target goals. This article provides our experience with fourteen collections of PBSC from five patients less than 25 kg in weight. METHODS: Patient's diagnoses were 2 stage IV neuroblastoma, 1 non-Hodgkin's lymphoma, 1 stage IV Ewing sarcoma, and 1 stage IV rhabdoid tumor of kidney. Their age ranged from 2 to 7 years old. Collections were performed using COBE Spectra or CS3000 plus that had been primed with leukoreduced, irradiated red blood cells. Patients underwent large volume leukapheresis. Radial artery was used as draw line and subclavian vein was used as return line. The blood to ACD ratio was 24:1 with 3000 units of heparin added to each 500 mL of ACD, in addition, heparin (1000 units) was added to collection bag when performed with COBE Spectra. Simultaneously, calcium chloride solution was dripped into an another venous line. During one course of large volume leukapheresis, about 5,000 mL of blood (>three total blood volume) were processed at a flow rate of 25~35 mL/min. RESULTS: The mean of total WBCs in collected components per procedure was 5.9+/-2.9x109 (3.0-10.5x109) with yield of 3.6+/-2.0x108 per kg of body weight. The mean of total CD34+ cells was 5.2+/-4.5x106 per kg (1.6-14.6x106/kg) for each collection. The patients tolerated well during the procedure without any apparent symptoms related to anemia or volume deficit or overload. CONCLUSION: In children weighing less than 25 kg, peripheral blood progenitor cell collection can be safely and efficiently performed with continuous flow blood cell separators, primed with red cells and additional heparin anticoagulation.
Adult ; Anemia ; Blood Cells ; Body Weight ; Calcium Chloride ; Child ; Diagnosis ; Drug Therapy ; Erythrocytes ; Heparin ; Humans ; Kidney ; Leukapheresis* ; Lymphoma, Non-Hodgkin ; Neuroblastoma ; Peripheral Blood Stem Cell Transplantation ; Radial Artery ; Rhabdoid Tumor ; Sarcoma, Ewing ; Stem Cells* ; Subclavian Vein

BACKGROUND: This study was undertaken to investigate the frequency, causes, and outcome of second malignancies(SM) following treatment of childhood cancer in Korea. METHODS: The Korean Society of Pediatric Hematology-Oncology(KSPHO) reviewed the records of patients who developed SM during the period of 1981-1997 through nationwide search. RESULTS: Twenty four cases were collected, among which 7 AML, 5 osteosarcoma and 5 ALL were observed. Fifteen of them were boys, and 9 girls (1.7:1). Familial cancer was registered in 5 cases among direct relatives. No interrelationship between first and SM was observed except in 2 retinoblastoma patients who developed osteosarcoma. The SM developed in a period of 8 to 144 months (mean:55 months) after the initiation of treatment for the first malignancy. Sixteen cases had radiotherapy for the first malignancy, and in 6 of them the SM developed in the irradiated area. Fifteen patients were treated with alkylating agents, 12 received anthracyclines and 7 received etoposide. They survived 1 to 110 months (mean:15 months) after development of the SM. Sixteen patients are dead, 3 currently free of disease, and 5 alive with disease. CONCLUSION: AML, osteosarcoma and ALL were most prevalent SM in Korean children. The mean latent period was 55 months, and showed poor mean survival period of 15 months. Radiotherapy seems to be a significant risk factor for the development of SM, but more cases are needed to assess the actual risk of certain chemotherapeutic agent. For this purpose, KSPHO continues to collect the cases of SM and to follow up the registered patients.
Alkylating Agents ; Anthracyclines ; Child ; Drug Therapy ; Etoposide ; Female ; Follow-Up Studies ; Humans ; Korea ; Neoplasms, Second Primary* ; Osteosarcoma ; Radiotherapy ; Retinoblastoma ; Risk Factors

BACKGROUND: 'Eight drugs in a day' was one of the widely used regimen in medulloblastoma. Result of treatment of this regimen and comparison between pre-RT chemotherapy and post-RT chemotherapy were presented. METHODS: Medical records of children who were diagnosed as medulloblastoma and treated with 8 in 1 therapy in Seoul National University Children's Hospital from January 1986 to June 1997 were reviewed. RESULTS: 1) Forty nine cases(male: 30, female: 19) were analyzed. The age at diagnosis was between 3 months and 15 years 3 months and median age was 7 years 10 months. 2) The T stage by Chang classification revealed T1(2%), T2(26%), T3a(9%), T3b(56%), and T4(7%) in 43 cases. M stage revealed M0(46%), M1(15%), M3(37%), and M4(2%) in 41 cases. The surgical results revealed gross total resection(36%), near total resection(18%), subtotal resection(38%), and partial resection(9%). 3) The 5-yr disease free survival(DFS) rate of all tumors was 53%. There was no difference in DFS about sex, age, pathology, T stage, M stage, and surgical result. 4) Difference of 5-year DFS between pre-RT chemotherapy group(53%) and post-RT chemotherapy group(83%) was significant[-2Log(LR), P=0.023], excluding the patient relapsed or too young(below 18 month, who had more chemotherapy to delay radiation) to be received radiation therapy. CONCLUSIONS: DFS in post-RT chemotherapy was better than pre-RT chemotherapy. As medulloblastoma has the property to confine in CNS system, prior treatment with radiation as a intensive local therapy may be more effective than the systemic chemotherapy. Progression or relapse were the main causes of treatment failure. Result of treatment may be improved by more intensive therapy.
Child ; Classification ; Diagnosis ; Drug Therapy* ; Female ; Humans ; Medical Records ; Medulloblastoma* ; Pathology ; Recurrence ; Seoul ; Treatment Failure

BACKGROUND: Wilms tumor, the most common primary malignant renal tumor of childhood, has relatively good prognosis among solid tumors occurring in childhood because of the improved operation skill, proper selection of chemotherapeutic agents, and combined radiotherapy on the primary and distant metastatic sites after nephrectomy. However, successful therapy has been associated with long-term toxicity occurring years or decades later. So it is important to identify and understand the possible late effects of treatment for Wilms tumor. METHODS: We reviewed 73 cases of Wilms tumor who were diagnosed and treated from Mar., 1983 to Nov., 1996 and calculated the survival rate. In addition, 42 cases were investigated to see the late effects of treatment for Wilms tumor since Feb., 1993, especially with predesigned indices such as blood pressure, blood urea nitrogen(BUN), serum creatinine(S-Cr), 24 hour urine protein/creatinine(24 HU Pr/Cr), 24 hour urine microalbumin(24 HU MA), 24 hour urine beta2-microglobulin/creatinine(24 HU beta2-MG/Cr). RESULTS: Overall survival rate of Wilms tumor was 86% in 5 years. There was significant difference in survival between stage I, II, III group and stage IV, V group(90% vs 72%, P=0.032), and survival rate of favorable histology group was higher than that of unfavorable histology group(92% vs 73%, P=0.043). Of the 42 cases in our study of late effects, no patient has presented significant late sequelae causing morbidity, and there were one case of transient hypertension, three cases of microalbuminuria, three cases of proteinuria, one case of asymptomatic microscopic hematuria, one case of radiation hepatitis and two cases of nut-cracker syndrome. No abnormality was noted in BUN, S-Cr, or 24 HU beta2-MG/Cr. CONCLUSION: Wilms tumor in our center have an excellent prognosis and no significant late sequelae that might affect long-term morbidity were found. But thorough and further longer follow-up is mandatory to understand the possible late effects fully, so that cope with properly and improve the quality of life.
Blood Pressure ; Follow-Up Studies ; Hematuria ; Hepatitis ; Humans ; Hypertension ; Nephrectomy ; Prognosis ; Proteinuria ; Quality of Life ; Radiotherapy ; Survival Rate* ; Urea ; Wilms Tumor*

BACKGROUND: Tuberculosis(Tbc) has been known to be prevalent and associated with high mortality in adult patient with cancer due to cancer itself or immunosuppressive therapy but to be rare in children with cancer. Little about Tbc in children with cancer has been reported in this country. To investigate the incidence, treatment, timing of diagnosis, clinical symptoms and response to the treatment of Tbc in children with cancer, this study was undertaken. METHODS: From 1983 until 1997, 252 children treated for cancer at the Department of Pediatrics of Yeungnam University Hospital were studied for developing pulmonary Tbc while receiving anticancer therapy by reviewing the medical records. The incidence, onset, clinical symptoms and the response to the therapy of pulmonary Tbc in these children with cancer were studied. RESULTS: Among 252 children with cancer, 8(3.2%) children developed pulmonary Tbc which included 5 of 128(3.9%) children with ALL, 3 of 25(12%) children with malignant lymphoma. These 3 malignant lymphoma included 2 of total 3(66.7%) Hodgkin's lymphoma and 1 of 1(100%) lymphoepithelioma of the study population. Incidence of pulmonary Tbc in children with cancer per 100 person year for the first year was 2.9, for the second year was 1.4. Among 8 children with pulmonary Tbc, five were male and 3 were female with male to female ratio of 1.67:1. Mean age of them was 13.2(5~18) years old. The onset of pulmonary Tbc was average 7.1(2~14) months after starting anticancer therapy. No one had pulmonary Tbc on diagnosis of cancer. Coughing, sputum, cold sweating, mild fever, loss of appetite, weight loss were the clinical symptoms on diagnosis of pulmonary Tbc. Chest X-ray showed findings compatible with the active pulmonary Tbc in all cases but sputum examination for acid fast bacilli by direct smear, culture or polymerase chain reaction were all negative. They were treated with isoniazid and rifampin for average 22.7+/-7.2(16.5~23.6) months with combination of streptomycin or kanamycin for first one month. All patients started to show signs of improvement clinically within several days and radiologically within few weeks after starting anti-Tbc therapy and eventually recovered from pulmonary Tbc completely. CONCLUSIONS: It seems to be important to recognize that incidence of pulmonary Tbc is higher in children with cancer, especially in those with leukemia and malignant lymphoma, especially Hodgkin's whose cellular immunity is suppressed, than immunocompetent children. Thus when these patients shows symptoms of coughing, sputum, cold sweating, mild fever, fatigue, loss of appetite or weight loss, chest X-ray and studies for Tbc are indicated for prompt diagnosis and treatment for pulmonary Tbc in children with cancer.
Adult ; Appetite ; Child* ; Cough ; Diagnosis ; Fatigue ; Female ; Fever ; Hodgkin Disease ; Humans ; Immunity, Cellular ; Incidence ; Isoniazid ; Kanamycin ; Leukemia* ; Lymphoma* ; Male ; Medical Records ; Mortality ; Pediatrics ; Polymerase Chain Reaction ; Rifampin ; Sputum ; Streptomycin ; Sweat ; Sweating ; Thorax ; Tuberculosis, Pulmonary* ; Weight Loss

BACKGROUND: Cancer is the second cause of childhood death following accident, and leukemia is the most frequent childhood cancer in Korea. For the desirable control of childhood leukemia, of which the mortality is still high, the basic data for the incidence has a great importance. This is the second report from the data during 1991~1995 following the first one that analyzed the data from 328 cases of childhood leukemia during 1981~1990 in the same area, Pusan city, Korea. METHODS: The data were obtained from 138 new cases(84 males and 54 females from 0 to 15 years old) of childhood leukemia who had been living in the city of Pusan and were admitted to the 4 university hospitals and 11 general hospitals from 1991 to 1995. The cases were confirmed by CBC and bone marrow examination. RESULTS: The crude annual incidence rate per 100,000 population varied 1.50~5.30, 2.59~6.00 and 1.58~2.61 in the age group of 0~4 years, 5~9 years and 10~14 years, respectively. The standardized annual incidence rate per 100,000 population varied from 2.05 to 3.46(male 2.96~4.89, female 0.98~3.57). Sex ratio(male to female) was 1.58:1, 1.44:1, and 1.82:1 in total cases, ALL and AML, respectively, while incalculable in CML. By the major types of childhood leukemia, the cases were composed of 105 ALL (76.1%), 31 AML(22.5%), 2 CML(1.4%). CONCLUSION: It was concluded that the annual incidence rate of childhood leukemia per 100,000 population in Pusan city during 1991~1995 was similar to that of previous report during 1981~1990, while the proportion of ALL had tendency to increase up to that of United States, in contrast to the low proportions of ALL in the previous reports.
Bone Marrow Examination ; Busan* ; Female ; Hospitals, General ; Hospitals, University ; Humans ; Incidence* ; Korea* ; Leukemia* ; Male ; Mortality ; United States

BACKGROUND: The purpose of our investigation was to evalute response to therapy and prognosis according to clinical and pathologic stage. METHODS: Fourteen children who had LCH were diagnosed between Jan., 1987 and Dec., 1996. Of the 14 patients, 3 patients with only skin involvement were excluded from this study. The medical records of 11 patients were reviewed for organ involvement at diagnosis, treatment, disease course, and late sequelae. Results: 1) As for the clinical stages, 4 patients were in stage I, 4 in stage II, one in stage III, and 2 in stage IV. As for the pathologic stages, four had monostotic disease, 2 polyostotic disease and 5 multisystemic disease. 2) All six patients with monostotic and polyostotic disease had a complete response to the therapy and remain alive free of disease. Three of the five with multisystemic disease had a complete response to the therapy, while two had a progression of disease. 3) Sequelae were observed in 3 patients including 2 with diabetes insipidus and 1 with hepatic and pulmonary dysfunctions. 4) The median follow-up time from diagnosis for all patients was 3.5 years. The Kaplan-Meier estimate of survival rate is 91%, with estimated EFS of 81.8% at 5 years. CONCLUSION: The clinical outcome of LCH patients with single system involvement including monostotic and polyostotic diseases were favorable and all remains alive free of disease. Multisystemic disease was associated with several unfavorable prognostic features including diagnosis at age <2 year, dysfunction of involved organs and advanced clinical stage.
Child ; Diabetes Insipidus ; Diagnosis ; Follow-Up Studies ; Histiocytosis* ; Humans ; Kaplan-Meier Estimate ; Medical Records ; Prognosis ; Skin ; Survival Rate