Genetic characterization and genotyping of hepatitis B virus (HBV) isolates from donors with an occult HBV infection.

BACKGROUND AND OBJECTIVES: Screening of Thai blood donors has resulted in the detection of donors with an occult HBV infection (OBI), where HBsAg is undetectable, but hepatitis B virus (HBV) DNA is present in serum in low concentrations. This study was designed to determine whether the occurrence of OBI in donors was linked to the HBV genotype and possibly to mutations in the surface (S) and core (C) gene regions. MATERIALS AND METHODS: Mutations in the S and C gene regions in 48 Thai donors with OBI were mapped by sequencing. Genotyping was determined with the INNO-LiPA test and by phylogenetic analysis of sequences from the S and C genes. RESULTS: The majority of OBI samples were genotype C (81·3%) with 6·3% of samples being genotype B. In addition, two genotype I isolates were identified. Mutations in the S region (100%) were found especially in loop 1 of the major hydrophilic loop (MHL) at positions I110L, T114S, T126I and S113T, whereas mutations in the C region (65%) were within the basal core promoter region (position A1762T/G1764A) and precore region (position G1896A). CONCLUSION: The majority of OBI samples were HBV genotype C, although genotype I, which is newly emerging in Thailand, was also detected. The study demonstrated that OBI was probably not associated with a particular HBV genotype or with certain mutations in the S and C gene regions. However, mutations in the C gene region which could potentially impair viral replication and HBsAg production and potentially lead to OBI were identified.

Lowering the prophylactic platelet transfusion threshold: a prospective analysis.

The 20 x 10(9) /L threshold for prophylactic platelet transfusion may be unnecessarily high. Few prospective studies, however, in which other trigger values were tested have been published. In this study all hospitalized, thrombocytopenic adult hematology-oncology patients in our institution were prospectively evaluated daily for hemorrhage and platelet transfusion during a one year period; no patients were excluded for bleeding or infectious problems. By design, during the initial six-months (baseline period), the prophylactic platelet transfusion trigger was 20 x 10(9) /L; for the second six-months (study period) this threshold was changed to 10 x 10(9) /L. Patients studied during the two periods did not differ significantly in age, gender, diagnosis, blood or marrow transplant status, and duration of neutropenia. Compliance with the thresholds was 95.6% (baseline period) and 93.5% (study period). For patients with platelet counts under 20 x 10(9) /L, the mean use of platelet transfusions per patient per day was significantly lower in the study period (4.47) than in the baseline period (6.48; p<0.001). Both mean prophylactic (1.54/patient-day) and therapeutic (2.93/patient-day) platelet transfusions were reduced in the study period compared with the baseline period (2.26 and 4.22/patient-day, respectively). Hemorrhage was slightly reduced in the study period compared with the baseline period: major hemorrhage, 15.2% vs. 18.4% (p=0.014); minor hemorrhage, 63.6% vs. 70.1% (p<0.001). Thus, hemorrhage was not increased with the lower trigger level. A 10 x 10(9) /L prophylactic platelet transfusion threshold value is safe and effective.

Neonatal alloimmune thrombocytopenia due to anti-Nak(a).

BACKGROUND: The accurate diagnosis of neonatal alloimmune thrombocytopenia is essential in the effective treatment of potentially serious bleeding in neonates. CASE REPORT: Reported here is a case of a full-term female baby who was delivered by vacuum extraction from a gravida 1 para 1 healthy mother. She presented with generalized petechiae and bilateral cephalhematoma, which she had had since birth. At 7 hours of life, she had an upper gastrointestinal hemorrhage and was found to have severe anemia and marked thrombo-cytopenia. Coagulation screening tests were normal. The diagnosis of neonatal alloimmune thrombocytopenia was suspected, and maternal serum was collected for further study. The baby was treated with a single dose of hydrocortisone (10 mg/kg) and IVIG (400 mg/kg) while waiting for irradiated platelets from her mother. After 30 mL of a transfusion of maternal platelets, the baby's platelet count rose dramatically, from 15,000 to 162,000 per microL, and it remained stable at that level. She was discharged on the 10th hospital day in good condition. During the follow-up period of 8 months, her growth and development were satisfactorily normal, as well as her platelet count. A high-titered platelet antibody was detected in the maternal serum by use of a solid phase platelet adherence technique. RESULTS: The specificity of the platelet antibody was identified as anti-Nak(a) by the mixed passive hemagglutination test method. CONCLUSION: These findings suggested a diagnosis of NAIT caused by anti-Nak(a).

Successful bone marrow transplantation in a child with red blood cell pyruvate kinase deficiency.

We report the first successful use of BMT for the treatment of RBC pyruvate kinase (PK) deficiency in a boy who developed neonatal jaundice and severe transfusion-dependent hemolytic anemia a few months after birth. He received a BMT at the age of 5 from an HLA-identical sister who has normal PK activity after conditioning with busulfan and cyclophosphamide. The post-transplant course was uneventful. At present, 3 years after transplant, he is 8 years old and has a normal hemoglobin level and normal RBC PK activity without evidence of hemolysis. DNA analysis has confirmed full engraftment.

Purification of human hemopoietic stem cells for transplantation in Thailand.

Stem cell transplantation (SCT) has become the therapy of choice for many hematologic and immunologic disorders. At present, only 25% of patients have suitable HLA-identical donors. In an attempt to increase the donor pool for SCT in Thailand and Southeast Asia, we developed a program whereby parents and mismatched siblings can be used as donors. In this preliminary study, after granulocyte-colony-stimulating factor (G-CSF) was given to adult donors, peripheral blood stem cells (PBSC) were collected and CD34+ cells purified using a CliniMACS immunomagnetic device (Miltenyi Biotec, Germany). In seven experiments, purified CD34+ cells could be obtained from G-CSF-stimulated PBSC in large numbers (1.71 +/- 0.19 x 10(8)), with high purity (93 +/- 2.4%) and excellent recovery (64.28% - 85.62%). Immune reactive T and NK cells were adequately depleted to less than 0.2%. The purification procedure can be completed within 3 hours. In conclusion, a clinical stem cell purification program using this novel device is now established in Thailand and for the first time in Southeast Asia. This should allow further development of advanced SCT therapy including haploidentical and mismatched CD34+ SCT for patients' lacking HLA-identical donors in this region.

Exchange transfusion in severe falciparum malaria.

Malaria associated with complications or a fatal outcome is caused by Plasmodium falciparum. The mortality due to this disease is parallel to the degree of parasitemia. Successful use of exchange blood transfusion as a therapeutic adjunct for this infection was reported. The rationale for this form of therapy is based on (1) rapid reduction in parasite load by exchange transfusion, (2) removal of toxic substances and (3) reducing microcirculatory sludging. We describe here thirteen cases of severe falciparum malaria treated with infusion of quinine dihydrochloride and exchange transfusion 2,320-8,000 ml of whole blood. We observed that the greatest reduction in the average circulating infected red blood cells, from 20.7 per cent to 9.3 per cent, seemed to occur early in the first 2,000 ml of blood exchange and the parasitemia often reduced to 5.1 per cent in patients who had 4,000 ml of blood exchange. In order to reduce the initial parasitemia to 5 per cent by exchange transfusion, we suggest the volume of exchange transfusion should be 2,000 ml for average parasitemia 10 per cent, 4,000 ml for parasitemia > 20 per cent and 2,000-4,000 ml for parasitemia 10-20 per cent.

Bone marrow, peripheral blood and cord blood stem cell transplantation in children: ten years' experience at Siriraj Hospital.

Stem cell transplantations were performed in 69 children at Siriraj Hospital over a ten year period. The source of stem cells was bone marrow (60), peripheral blood (3), or cord blood (6). The diseases treated included 35 thalassemias, 11 Burkitt's lymphoma, five non-Hodgkin's lymphoma, five aplastic anemia, eight acute leukemia, and one each of neuroblastoma, severe combined immunodeficiency, Wiskott-Aldrich syndrome, myelodysplastic syndrome, and pyruvate kinase deficiency. The success rate of stem cell transplantation in Thai children varied according to the underlying diseases of the patients, ranging from 50% in acute leukemia to 100% in aplastic anemia. The outcome of stem cell transplantation in 35 thalassemic children revealed 23 (79.4%) were cured, whereas three (10.3%) remain alive with disease and the other three (10.3%) died. The incidence of graft-versus-host disease was low hen compared with that of Western countries. It is concluded that bone marrow, peripheral blood and cord blood stem cell transplantation will be the treatment of choice and will be widely used in the future to cure many hematologic and malignant disorders in children.

Reliability of automated platelet counts: comparison with manual method and utility for prediction of clinical bleeding.

The 20 x 10(9)/L (20,000/microliters) threshold for prophylactic platelet transfusion may be unnecessarily high. The widespread use of this threshold may reflect lack of confidence in the reliability of low platelet counts. We evaluated the performance of automated platelet counts and their relation to clinical bleeding. First, we prepared serial blood dilutions with "target" platelet counts from 2 to 40 x 10(9)/L. For the 48 measurements on 2 x 10(9)/L "target" dilutions, values of 1 or 2 x 10(9)/L were obtained with the Sysmex NE-8000 analyzer (mean 1.44 x 10(9)/L; SD 0.31 x 10(9)/L). Similarly, for 5 x 10(9)/L "target" counts, automated counts were 3-6 x 10(9)/L (mean 4.42 x 10(9)/L; SD 0.18 x 10(9)/L). Similar results were observed with all other "target" levels, with coefficients of variation (CV) from 6.39% to 7.71% with 10-40 x 10(9)/L "target" values. Secondly, we compared triplicate automated and manual platelet counts on thrombocytopenic patients with platelet counts from 4-30 x 10(9)/L. The triplicate automated platelet counts differed by no more than 5 x 10(9)/L among themselves, whereas the manual counts varied by as much as 30 x 10(9)/L. Mean platelet counts: automated, 14.40 x 10(9)/L (CV 10.12%); manual, 16.48 x 10(9)/L (CV 30.39%) (P = 0.038 for counts; P < 0.001 for CV). Finally, we prospectively evaluated bleeding in thrombocytopenic patients (1,809 patient-days of observation). Univariate and multivariate logistic regression analysis revealed highly significant correlations between the automated platelet count and major and minor bleeding manifestations. Thus, automated platelet counts are highly reliable and accurately predict clinical bleeding. The use of automated analyzers should facilitate improved prophylactic platelet transfusion protocols.

Potential deferral criteria predictive of human immunodeficiency virus positivity among blood donors in Thailand.

BACKGROUND: To develop deferral criteria to prevent human immunodeficiency virus (HIV) transmission by recently infected blood donors in the seronegative "window" phase, routine data on donors at a university hospital were examined for factors predicting seropositivity. STUDY DESIGN AND METHODS: Records of all 281 HIV-positive blood donors from August 1987 through September 1991 were retrospectively compared with those of 1076 randomly selected control donors matched only by year of donation. Four controls were selected for each HIV-positive donor. RESULTS: The prevalence of HIV in 102,684 donor units during the period rose from 0.02 percent in 1987 to 0.52 percent in 1991. Multivariable analysis revealed that male sex (odds ratio [OR] = 26.4), VDRL test positivity (OR = 3.0), age 21 to 30 years (OR = 2.2; referent: 16-20-year-old group), and replacement donorship (OR = 1.4; referent: voluntary donors) were independent factors significantly associated with HIV positivity among these donors (p < 0.05). Since replacement donorship cannot be avoided, only male sex, age 21 to 30 years, and VDRL test positivity were considered as potential criteria. When these findings were extrapolated to all donors in 1990 and 1991, those with all three or only two (excluding VDRL test, because the results are known only after donation) of these high-risk factors had HIV positivity probabilities of 2.2 and 1.0 percent, respectively. These probabilities were, respectively, 4.9 times (95% CI: 2.9 8.3) and 4.1 times (3.1, 5.4) the risk among other donors. However, applying such criteria would have eliminated 1.5 and 31.2 percent, respectively, of all HIV-negative donors in 1990 and 1991. The latter deferral proportion is too high to be acceptable. CONCLUSION: In Thailand, improved donor deferral criteria addressing sexual risk factors could lead to decreased probability of window-period donation, with an acceptable rate of deferral. Additional p24 antigen testing may be indicated for donors at increased risk for HIV infection, specifically, men aged 21 to 30.

PIP: Researchers compared August 1987 and September 1991 data on 281 HIV-positive blood donors, 16-59 years old, with those of 1076 randomly selected controls to determine factors associated with HIV infections they could develop simple and inexpensive deferral criteria to prevent donations from high-risk persons who may transmit HIV during the seronegative window period. The blood bank records were at Siriraj Hospital in Thailand. HIV-positive donors were significantly more likely to be men than were controls (98.9% vs. 76.3%; odds ratio = 2.64). They were also more likely to test positive for syphilis (VDRL test) (8.2% vs. 2.3%; OR = 3). A higher proportion of HIV-positive blood donors were 21-30 years old than were controls (66.2% vs. 40.5%; OR = 2.2). A greater percentage of blood donors who were asked by friends or relatives of transfused patients to donate blood (i.e., replacement donors) were HIV positive than controls (55.5% vs. 40.4%; (OR = 1.5). All these independent risk factors were significant at the 5% level. Paid donors were less likely to be HIV positive than controls (2.8% vs. 9.9%; odds ratio [OR] = 0.6). Since 40.4% of all HIV-negative donors were replacement donors, the researchers decided not to include replacement donorship in the analysis for potential deferral criteria. They used the records of 214 HIV-positive donors with all 3 other risk factors in 1990 and 1991 to determine the potential deferral criteria. The probabilities of HIV positivity of blood donors with 2 and 3 risk factors were 1 and 2.2% (respectively), which meant that they were 4.9 and 4.1 times (respectively) more likely to be HIV positive than were other donors. Using these criteria would have eliminated 1.5% and 31.2% (respectively) of all HIV-negative donors. Elimination of 31.2% is too high to be acceptable. The researchers suggest an additional p24 antigen testing for donors at high risk for HIV infection, particularly 21-30 year old men.

Acute hemodilution and autotransfusion in cardiac surgery: a comparison between rather healthy and high risk patients.

The effects of acute normovolemic hemodilution and autologous blood transfusion were studied in open heart patients, compared with rather healthy patients, NYHA class 1-2 and the high risk patients, NYHA class >2. Thirty-nine patients were involved in this study, 15 of them were identified as the rather healthy group while 24 patients belonged to the high risk group. Acute hemodilution was performed after anesthetic induction and before heparinization. Using an equal volume of polygeline 3.5 per cent (Haemaccel) to replace autologous blood removal, the number of patients who needed inotropic support to achieve normal arterial blood pressure was not significantly different between the groups. Following retransfusion of pump perfusate and autologous blood after the termination cardiopulmonary bypass, the number of patients who received additional homologous blood as well as the amount used percase were significantly less in the rather healthy patients. There was none in this group, but half of the high risk patients suffered from serious perioperative complications and one died. We conclude that this technique is safe and benefits blood conservation in rather healthy cardiac patients undergoing open heart surgery, but special precautions against risk should be considered in high risk patients.