HLA alloimmunization in patients receiving multitransfusions of red blood cells.

HLA antibodies were studied in 88 patients with chronic hemolytic anemia who received multitransfusions of red blood cells prepared by conventional (PRC-C), inverted centrifugation (LR-I) and leukocyte filter (LR-F) techniques. Their mean age was 8 years and 4 months with a duration of transfusion of 8 years. The patients were divided into five groups: group 1, receiving PRC-C (n=20); group 2, receiving LR-I (n=33); group 3, receiving LR-F (n=11); group 4, subsequently receiving LR-I and LR-F (n=10); and group 5, receiving PRC-C followed by LR-I and LR-F (n=14). The HLA class I antibodies were found in 30 out of 88 patients (34%). All were against HLA antigens commonly found in the Thai population. The patients receiving PRC-C exhibited HLA antibodies of 65%, which was significantly higher than those of patients receiving LR-I (24%) and LR-F (0%). Consequently, the transfusion reactions of fever, chill, rash and urticaria were also commonly found in patients receiving PRC-C (13.4%), which was significantly higher than patients receiving LR-I (0.4%) and LR-F (0%). The leukocyte filter technique has been shown to be effective in preventing HLA alloimmunization and transfusion reactions but the price is rather high. For the inverted centrifugation technique, only transfusion reactions were effectively prevented and the HLA alloimmunization continued to develop. A more effective and low-cost method for the removal of leukocytes should be investigated for these multitransfusion patients.

Thromboembolic complications in Thai pediatric patients.

Sixty patients with thromboembolic complications from 1987 to 1997 at the Department of Pediatrics, Ramathibodi Hospital were retrospectively studied. Twenty patients were infants and 40 patients were children and adolescents with a mean age of 18 days and 8 years, respectively. The sites of thromboembolic complications were in the central nervous system, 27.5 per cent; skin as purpura fulminans or necrotic lesions, 24.5 per cent; gangrene of the toe, finger or colon, 19 per cent; deep vein thrombosis, 16 per cent; and other sites such as heart and lungs, 13 per cent. Most of them had triggering conditions (80%) and underlying diseases (76.7%) causing thromboembolism. The low levels of either antithrombin III, protein C or protein S were found in 42 per cent (15/36). The management included administration of standard or low molecular weight heparin if not contraindicated, replacement of fresh frozen plasma 10 ml/kg twice a day and treatment of underlying and triggering conditions. The fatality rate was 15 per cent (9/60). Subsequent episodes of thromboembolism occurred in 6 patients including: pulmonary emboli in one patient with protein C deficiency who refused warfarin administration, deep vein thrombosis in 2 patients with unidentified etiology, and necrotic skin lesions in 3 patients with vasculitis who did not respond to treatment. In conclusion, a comprehensive investigation and specific treatment for patients with thromboembolic complications are emphasized in order to prevent recurring episodes.

Prevalence of factor V Leiden in Thai blood donors.

Factor V Leiden was recently found to be the most common cause of familial venous thrombosis in the European population. We have studied the prevalence of factor V Leiden by DNA analysis among 500 Thai blood donors (male 285, female 215). Their ages ranged from 18 to 60 years with a mean of 33 years and 2 months. All of them were healthy voluntary blood-donors who met the standard criteria of the American Association of Blood Banks. No history of thrombosis was found. The results revealed that factor V Leiden was not present among 1,000 chromosomes from Thai blood donors. This suggests that factor V Leiden is not the common genetic predisposing factor of venous thrombosis in the Thai population as compared to the European population.

The usefulness of X-linked polymorphic loci as gene markers to track X allele and chimerism in a post-allogeneic peripheral blood stem cell transplant patient with Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS), an X-linked recessive disorder, is characterized by progressive T-cell immunodeficiency. Laboratory findings generally demonstrate reduced response to T-cell mitogens, markedly decreased serum concentration of IgM, and thrombocytopenia with small platelet volume. Allogeneic HLA-matched sibling bone marrow transplantation (BMT) can correct this disorder. We report the usefulness of X-linked polymorphic loci to detect X-allele gene tracking among WAS siblings and chimerism between a pre- and post-allogeneic matched sibling peripheral blood stem cell transplantation (PBSCT). A 3 1/2 year old boy with clinical and laboratory findings consistent with WAS underwent allogeneic matched sibling PBSCT. We used BclI restriction fragment length polymorphism (RFLP) of intron 18 of factor VII gene and MseI RFLP of the 5' flanking region of factor IX gene to detect X-allele gene tracking among siblings and family members and chimerism in patients between pre-and post-allogeneic matched sibling PBSCT. We were able to demonstrate that determination of BclI and MseI RFLP can be employed to recognize the difference in X-allele genes between the recipient and donor for allogeneic matched sibling PBSCT. The authors also were able to demonstrate that these polymorphic loci can detect full chimerism of donor hematopoietic cells in recipient blood after allogeneic PBSCT. This finding was correlated with improvement of post-PBSCT clinical and laboratory findings. BclI and MseI RFLP associated with X-chromosome can effectively track X-allele, detect carrier state, and demonstrate the different X-allele among male siblings, and chimerism of hematopoietic cells between donors and recipients in a setting of allogeneic matched sibling BMT or PBSCT for X-linked hereditary diseases such as Wiskott-Aldrich syndrome.

Preliminary study of HLA-ABCDR antigens in CML, ANLL, thalassemia and severe aplastic anemia in Thais.

The objective of this study was to analyse human leukocyte antigen (HLA) and disease association in common blood diseases [chronic myelogenous leukemia (CML), acute nonlymphocytic leukemia (ANLL), thalassemia and severe aplastic anemia] in Thais. The subjects were patients from the Hematological Clinic, Departments of Medicine and Pediatrics, Ramathibodi Hospital who were referred for HLA typing for bone marrow transplantation (BMT) at the Histocompatibility Laboratory from March 1988 to September 1997. A total of 129 patients had complete HLA-ABC typing. The patients included 45 CML, 40 ANLL, 26 thalassemia (Thal) and 18 severe aplastic anemia (SAA). Of these, 88 patients were typed for HLA class II. The HLA class I (ABC) and II (DR, DQ) typings were performed by microlymphocytotoxicity test. It was found that HLA class I was associated with CML, ANLL and Thal, whereas, HLA class II was associated with SAA. HLA-B8 and HLA-B18 were increased in CML with R.R. values of 12.2 and 3.9, respectively, whereas, HLA-B18 was increased in ANLL with R.R. value of 4.5. In addition, HLA-DR2 and DR3 were increased in SAA with R.R. values of 3.8 and 4.8, respectively. For Thal, HLA-A2 and B46 were increased in Thal in Central Thais with R.R. values of 3.3 and 6.1, respectively, whereas, HLA-B13 was increased in Thal in Northern Thais with R.R. value of 8.5. On the other hand, HLA-B7 was absent in CML. HLA-Cw7 was decreased in CML and SAA, whereas, HLA-DR6 was decreased in ANLL and SAA. Furthermore, HLA-Cw6 was also decreased in CML, whereas, HLA-A33 and Bw4 were decreased in SAA. Although the sample size of each disease was small, the increase of HLA-DR2 was observed in SAA in Thais which was similar to other studies in different ethnic groups. These preliminary data may be useful for further study in HLA and blood disease association.

The efficacy and safety of lyophilized cryoprecipitate in hemophilia A.

This prospective study of assessing the efficacy and safety of lyophilized cryoprecipitate (LC), which was heat-treated at 60 degrees C for 25 hours, was conducted in 23 patients with hemophilia A (severe 13, moderate 9, mild 1) at the International Hemophilia Training Center, Bangkok from 1997 to 1998. A total of 223 infusions of LC were given. The status of the patients could be classified into 4 groups: group I, non-bleeding (n = 13); group II, severe bleeding requiring hospitalization (n = 9); group III, appendectomy (n = 1) and group IV, early bleeding controlled by modified home treatment (n = 200). Pharmacokinetic studies were conducted in groups I and II. The mean in vivo half-life of factor VIII clotting activity (F VIII:C) was 12.6 hours and the mean in vivo incremental recovery at baseline was 2.1 per cent/unit/kg. The mean clearance was 3.22 ml/kg/h. There was no statistically significant difference in these parameters between groups I and II (p > 0.05). The hemostasis was successfully achieved and 1 to 2 small urticarial wheals were observed in only 2 infusions. In addition, 9 out of 23 patients received LC exclusively for 1 year. None of them developed inhibitor to F VIII:C nor did any contract additional transfusion-transmitted infection except one who developed anti-hepatitis C virus seroconversion after receiving 16 bottles of LC in 4 months. Therefore, the more efficient virus-inactivation in the preparation of LC should be established.

DNA extraction and amplification of 10-day, room-temperature blood samples.

DNA was serially studied in 20 samples of buffy coat stored at room temperature. Each sample was divided into 5 equal volumes, namely D0, D3, D5, D7 and D10. DNA extraction was performed on days 0, 3, 5, 7 and 10 after blood collection. The mean ratio of OD260/OD280 of the DNA obtained from D0 to D10 ranged from 1.77 to 1.79, and the mean amounts of the DNA obtained from D0 to D10 ranged from 602 to 740 ng/ul. There were no significant differences in the mean ratio and amounts of DNA obtained among these samples (p > 0.05). Subsequently, amplification was successfully performed from this template DNA to yield products of 1.4 kb and 142 bp at the sites associated with beta globin and factor VIII genes, respectively. These findings suggest the possibility of sending blood samples for DNA analysis by mail, or no ice is required during transportation.

The correlation of transferrin saturation and ferritin in non-splenectomized thalassemic children.

Thalassemia is a public health problem in Thailand. Progressive iron overload is the life-limiting complication commonly found in thalassemic patients. The assessment of body iron stores is essential for determining the need and efficacy of iron chelation. The parameters of serum iron, total iron binding capacity (TIBC), and serum ferritin were studied in 79 children with thalassemia diseases. The ages ranged from 1 to 17 years with a mean of 7 years and 10 months. Neither of them had clinical symptoms of hepatitis. The correlation between transferrin saturation (TS = serum iron/TIBC x 100) and serum ferritin was shown in the equation of TS = 10.253 In (ferritin) (r = 0.956, p = 0.000). For example, TS = 70.83 per cent indicates serum ferritin of 1,000 ng/ml. Thus, where serum ferritin is not feasible but serum iron and TIBC are available, TS can be used to estimate the level of serum ferritin. Therefore, the assessment of iron stores and monitoring of iron chelation in thalassemic patients can be effectively achieved.

Disseminated intravascular coagulation findings in 100 patients.

A retrospective study of 100 patients with disseminated intravascular coagulation from 1993 to 1997 is reported. Forty-five patients were neonates with a mean age of 12.6 days and 55 patients were infants, children and adolescents with a mean age of 6 years and 3 months. Most of them (91.5%) had complicated underlying conditions which included congenital anomalies, prematurity, malignancy, hematological and various diseases. Additionally, every patient had triggering conditions commonly identified as gram-negative septicemia. Bleeding and thromboembolic manifestations were found in 59.4 per cent and 19.8 per cent, respectively. The laboratory findings revealed red blood cell fragmentation, 89.6 per cent and thrombocytopenia, 85.8 per cent. Natural anticoagulants were studied in a few cases and revealed low levels of antithrombin III and protein C. The prompt effective management included treatment of underlying diseases, identification and relief of triggering conditions, correction of thrombocytopenia and coagulopathy, and fully supportive care. The overall case-fatality rate was 41.6 per cent which was not correlated with age, underlying diseases, triggering conditions, manifestation of bleeding, thromboembolism or shock, and exchange transfusion. However, a significant lower case-fatality rate was found in patients with positive culture (25%) as compared to those with sepsis and negative culture (51.7%) (p = 0.044). In addition, the febrile neutropenic patients, who showed good response to the administrated granulocyte-colony stimulating factor (G-CSF), survived from the DIC.

Home treatment for patients with congenital bleeding disorders in a developing country.

Ninety-six patients with congenital bleeding disorders were enrolled in a home treatment program from 1979 to 1997. The diagnosis included severe and moderate hemophilia A and B (n = 63), mild hemophilia A (n = 18), von Willebrand disease (vWD) (n = 12) and congenital factor VII deficiency (n = 3). The median age was 9 years and the median duration of follow-up was 4 3/12 years. The home treatment was modified 4 ways: (1) Using locally-prepared single units of fresh dry plasma in the majority of the patients while mild hemophilia A and vWD patients received 1-deamino 8D-arginine vasopressin. (2) Recruiting local health personnel as the primary care providers. (3) Teaching and training patients and parents intensively. (4) Maintaining an effective control system. The heartful effort of the health personnel was not in vain; the patients learned to take good care of themselves. Twenty patients and 20 parents or relatives were able to perform venipuncture properly and no adverse effect was observed. Since the hemorrhage was treated very early, the severity and sequelae of bleeding were decreased. The utilized blood components and days of hospitalization were reduced. Impressively, the absenteeism from work or school was minimized so that the patients could enjoy a near normal life in their family, school and society.

Subcutaneous portacath utilization in pediatric oncology patients: Ramathibodi Hospital experience.

Subcutaneous portacaths (SQP) placement in 19 pediatric oncology patients were studied. Complications of SQP were evaluated. Two patients had premature SQP removal due to fungal infection and breakage, 1 for each. Two patients had catheter-related bacteremia which was resolved by antibiotic administration. Only 1 patient had occasional difficult blood drawing episodes, because the tip of catheter was inserted through external jugular vein instead of subclavian vein. There were no other serious complications except that some of them had clotted formations, which were resolved easily by urokinase administration. Long-term SQP utilization was possible in 17 of 19 patients, with the average time of 7.5 months. Few complications occurred in the group of patients studied. SQP improved quality of medical care and significantly lessened the anxiety of patients who need long-term chemotherapy treatment. Therefore, placement of the intravenous access device is feasible for pediatric oncology patients in Thailand. The patients are no longer suffering from repeated venipunctures. Although it is expensive, it is convenient and useful for some patients with relatively high socioeconomic status. It should be considered for every pediatric cancer patient who needs prolonged chemotherapy and who has affordable means.

Allogeneic bone marrow transplantation in an osteopetrosis patient: first report in Thailand.

We described the successful allogeneic matched sibling bone marrow transplantation (BMT) in a 5-year-old Thai boy in whom osteopetrosis was diagnosed on the basis of anemia, thrombocytopenia, leukoerythroblastosis, sclerotic bone, hepatosplenomegaly, and visual deficit from an encroachment of cranial nerve foramina. The preparative regimen included 4 days of busulfan 4 mg/kg/day, and 4 days of cyclophosphamide 50 mg/kg/day. Complete hematopoietic engraftment and no evidence of graft versus host disease were shown after BMT. Complete hematologic findings were corrected. His hematopoietic chimerism was changed to that of his donor. Post BMT, he has no hepatosplenomegaly. His bone radiographic findings revealed normal after BMT. Bone marrow biopsy showed normalized bone and bone marrow matrix. However, his vision remained impaired. We believe that this is the first case of successful bone marrow transplantation in an osteopetrosis patient in Thailand.

Vitamin K deficiency bleeding in Thailand: a 32-year history.

Vitamin K deficiency bleeding cases in Thailand from 1963 to 1995 were extensively studied. From 1963 to 1987 there were 499 reported cases from 10 papers including 102 cases of the authors' series. From March 1994 to April 1996, two subsequent nationwide surveys were conducted where questionnaires were sent to 714 and 732 hospitals located throughout Thailand. The responding rate was 58.2% and 67% respectively. 331 cases were found during 1988 to 1995. The total number was 830 cases of which 799 were idiopathic vitamin K deficiency in infancy (IVKDI) and 31 were secondary types. IVKDI was found exclusively breast-fed infants (92%) who did not receive vitamin K prophylaxis at birth (90%). Bleeding and pallor were the common features. The occurrence of intracranial hemorrhage was strikingly high (82%); the fatality rates was 24%. However, the fatality rate among patients receiving either 1 mg of vitamin K, intramuscularly, (17%) or 2 mg, orally, (18%) were lower than those not receiving vitamin K prophylaxis (36%). The incidence of IVKDI significantly declined to 4.2-7.8 per 100,000 births between 1988 to 1995 which was in reverse proportion to the coverage of vitamin K prophylaxis (r = -0.94, p < 0.05).

Zinc and copper status of thalassemic children.

We investigated the amount of both zinc and copper in plasma, erythrocytes and hair in 11 patients with hemoglobin H disease, 59 patients with beta-thalassemia/HbE disease and 20 patients with homozygous beta-thalassemia. Plasma and hair zinc levels were found to be much lower, but erythrocyte zinc levels were higher, in thalassemic patients than in controls. The levels of copper in both plasma and erythrocytes were higher in the patients than in the controls. The mechanism with respect to the increase of the amount of both zinc and copper in erythrocytes was not clear; this result may reflect the impairment of zinc and copper utilization in tissues in the pathogenesis of these thalassemic patients.

Eluding transfusion-transmitted HIV infection: report of two cases.

Two hemophilia A boys (FVIII: C < 1% and 2.2%), whose ages were 12 and 14 years. old, received fresh frozen plasma of 140 ml and 210 ml, respectively, in 1989. It was the 27th and 13th donation for each regular donor who was negative for anti-HIV testing. However, both donors had HIV seroconversion within 95 to 110 days after the last donation. They might have contracted HIV infection shortly after the last donation. Luckily, the two hemophiliac recipients are still in good health and negative for anti-HIV and HIV-antigen testings for 7 years.

Levels of serum interleukin-6 and tumor necrosis factor in postsplenectomized thalassemic patients.

Levels of serum interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) were studied in 34 nonsplenectomized thalassemic patients (Thal/nonsplenec), 43 postsplenectomized thalassemic patients (Thal/postsplenec), 13 splenectomized non-thalassemic patients (nonThal/ postsplenec) and 18 normal control by enzyme linked immunosorbent assay method. Serum IL-6 concentration in Thal/postsplenec was significantly increased when compared with Thal/ nonsplenec and normal volunteers (3.55 +/- 2.47 pg/ml vs 2.38 +/- 2.31 pg/ml, p = 0.036 and 3.55 +/- 2.47 pg/ml vs 2.66 +/- 0.45 pg/ml, p = 0.028, respectively). This study also demonstrated that TNF-alpha value in Thal/postsplenec was drastically increased above normal control level (15.8 +/- 4.86 pg/ml vs 9.16 +/- 2.18 pg/ml, p = 0.001) and the level was statistically significantly higher than that in Thal/ nonsplenec (15.5 +/- 4.86 pg/ml vs 9.96 +/- 5.19 pg/ml, p = 0.001). There was a trend toward increasing of cytokine levels in Thal/postsplenec with higher platelet count although no correlation was observed. This study addresses the possible role of IL-6 and TNF-alpha in the pathogenesis of reactive thrombocytosis in Thal/postsplenec.

Hemoglobin typing by high performance liquid chromatography.

To evaluate the potential application of high performance liquid chromatography or HPLC in performing hemoglobin typing, comparison between this technique and routine methods was carried out. Blood specimens from Pediatric Hematology Unit, Research Center, Faculty of Medicine, Ramathibodi Hospital were examined by these methods. The level of Hb A2, Hb E and Hb F were compared. Hb A2 level determined by HPLC and electrophoresis was statistically significantly different but correlated well. Good correlation was found between Hb A2 level from HPLC compared with microcolumn chromatography although there was a statistical difference. No difference but very good correlation was found between the level of Hb E from HPLC compared with electrophoresis. Statistical difference was encountered when Hb F level determined by HPLC was compared to that determined by Betke alkaline denaturation test. However, good correlation was observed when the level of Hb F was greater than 2.0%. In conclusion, HPLC could be an alternative way of performing hemoglobin typing provided that Hb F is 10% or more, by calculating from the equation: Alk F = (0.83 x Hb F by HPLC) - 0.98.

Osteosarcoma: a study of 130 cases.

Multidisciplinary treatment of osteosarcoma in the Faculty of Medicine Ramathibodi Hospital, Mahidol University, using preoperative intraarterial and postoperative chemotherapy, with or without local irradiation, combined with surgery and prophylactic lung irradiation provided an excellent 5 years' survival of 55 per cent, the same rate as the 9 years' survival. The survival was stable after 4.4 years. The patients with local irradiation had more tumor destruction apparent on the surgical specimen. The administration of prophylactic whole lung irradiation provided an outcome without any undesirable complication. Sixteen per cent of the cases with PLI developed lung metastasis compared to 48 per cent without PLI. The most important prognostic factor was low level of serum lactic acid dehydrogenase. The unanswered question is what is the optimal treatment for osteosarcoma?

Red cell parameters in alpha-thalassemia with and without beta-thalassemia trait or hemoglobin E trait.

Eighty-five patients who attended at Ramathibodi Hospital during November 1994 to June 1996 were investigated for thalassemia genotype, hemoglobin (Hb) typing and blood cell parameters. All patients were screened primarily for complete blood count using the Technicon H*3 automated hematology analyzer and Hb typing using the automated HPLC. Their genotypes were evaluated by in vitro gene amplification using primers for detection of common alpha-thalassemic genes found in the Thai population. We found 45 cases out of 85 were alpha-thalassemia trait with A2A typing, 10 were normal, 7 were alpha-thalassemia trait complicated by beta-thalassemia trait or HbE trait, 18 were HbH disease or HbH with Hb Constant Spring (HbH/CS), and 5 were AE Bart's disease. The alpha-thalassemia 1 trait had heterogeneity in red cell population as shown by increased red cell distribution width (RDW), the increased percent microcytic red cell (%Micro) and decreased mean cell volume (MCV). Red cell parameters in alpha-thalassemia 2 trait and HbCS trait were not significantly different from normal. The cases with coinheritance of alpha-thalassemia trait with beta-thalassemia trait or with HbE trait showed variation in their red cell parameters: one case showed less abnormal red cell parameters than those of uncomplicated alpha-thalassemia but the other two cases showed unimproved values. The homozygous alpha-thalassemia 2 showed similar red cell parameters to the alpha-thalassemia 1 trait. In conclusion, we can screen the alpha-thalassemia 1 trait and homozygous alpha-thalassemia 2 by using the simple red cell parameters such as the MCV and RDW; however, they must be confirmed for alpha-thalassemic genes. Unfortunately, red cell parameters of alpha-thalassemia 2 trait or HbCS trait were not different from those of normal subjects.

Prophylactic treatment for hemophilia A patients: a pilot study.

Prophylactic treatment with factor VIII concentrate was given to six hemophilia A boys whose factor VIII:C ranged from 1% to 3.5% at Ramathibodi Hospital. The age ranged from 11 to 16 years with the median age of 12 years old. Each patient received factor VIII concentrate twice a week in the dosage of 8-10 unit per kg for one year. During the prophylactic period, bleeding episodes seldom occurred. They did not need hospitalization. The absence from school was reduced. They became muscular from regular daily exercise. They could join the activity at school and lived a near normal life. The patients and family were very happy since they did not have to worry about bleeding. No adverse effect was found. The only constraint was the cost. It cost 180,000 baht (US$ 7,200) per year or 15,000 baht (US$ 600) per month for a 25 kg hemophiliac boy.