Hematology laboratory standardization: a plan for harmonization in Asia.

Hematology laboratory is generally required in the hospital. At the macroscale, hematology laboratories have served a large number of population. In Asia, more than 3,000 million people are potentially to use the hematology laboratory service, particularly the complete blood count. Since 1970s, automated technology has been introduced to Asia and as years passed by, technology diversity is increasing. However, there are considerable number of hematology laboratories that have no automated machine. They are still relied on manual technology which is still variable in spectrophotometer for hemoglobin determination, centrifuge for hematocrit and diluting pipet for cell counting. In particular, blood smear preparation and interpretation are very difficult to control for standardization from person to person and laboratory to laboratory. Different methodology and a large population in the huge geographical area in Asia, the agreement of standard criteria is greatly important. This report has shown strategy and action plan to reach the goal of hematology laboratory standardization in Asia.

Hemostatic alterations in beta-thalassemia/hemoglobin E patients.

To search for evidence of coagulation activation ex vivo, the levels of human prothrombin fragment 1+2 (F1+2) were examined in 69 beta-thalassemia/Hb E patients. Levels of protein C inhibitor (PCI) and activated protein C - PCI (APC:PCI) complex were also determined in 9 of the above patients in conjunction with protein C (PC) antigen and activity, in an attempt to detect increased consumption of PC. In mean level of F1+2, there was a statistically significant difference between normal control and post-splenectomized patients (p < 0.05) but not between normal control and non-splenectomized patients (p > 0.05). The mean levels of PC activity and PC antigen in the patients were much lower than in normal controls. However, the mean levels of PCI and the mean level of APC:PCI complex in the patients were not significantly different from those in normal controls (p > 0.05). The high level of F1+2 in post-splenectomized patients found in this study agreed well with clinical and other laboratory findings. The normal level of PC inhibitor and APC:PCI complex found in this study provided no evidence of increased consumption of protein C in thalassemia patients.

Country reports from Japan: external quality assessment scheme in Japan.

Several external quality assessment schemes (EQAS) have been conducted in Japan. Results obtained from nation-scale EQAS reveal the current quality of laboratory testing in each laboratory. The largest nation-scale EQAS in Japan is that conducted by the Japan Medical Association. The numbers of participants and of items evaluated have increased in EQAS by JMA over its history of 32 years. Improvement in inter-laboratory differences has been observed for most items in EQAS in recent decades. In 1998, about 2,500 laboratories from throughout the country participated in this surveillance, and 47 items were evaluated. The coefficient of variations for the group of all participants was less than 5% for about one third of all test items. On the other hand, very high variations over 20% were observed for 6 items. Also, inter-method differences exist for many items, which may be or may not be related to matrix effects. Retrospective evaluation of all EQAS data suggests that there is still room for improvement in inter-laboratory differences.

Field evaluation of who hemoglobin color scale in West Java.

The results obtained with a WHO hemoglobin (Hb) colour scale were evaluated in a field study in Chibubur district in Java island by comparison with hemoglobin values obtained by an automated blood cell analyzer K-800 (Sysmex. Kobe, Japan). When the color scale test was performed following the instructions for use. Hb values observed were usually higher than the values obtained by the analyzer. Thirty microl blood was loaded on the filter paper and an 60 sec waiting period was used. The sensitivity of results obtained with the color scale was 23.3% (14/60), and specificity was 96.6% (58/60). We propose an additional testing method based on our results.

Cross over placebo control trial of dilazep in beta-thalassemia/hemoglobin E patients.

An attempt was made to find better symptomatic treatment for beta-thalassemia/hemoglobin E (beta-thal/Hb E) patients in order to reduce their blood demand. Oral administration of dilazep was prescribed for these patients and a clinical trial was conducted over a 2-year period as a cross over placebo control study. Seventeen beta-thal/Hb E patients were enrolled in the study. All of them received dilazep and placebo for 10 months at different periods of time and were taken care of by the same doctor throughout the study. The blood demand of the same patients during the period of receiving dilazep with the period of receiving placebo, was 1.5 +/- 1.8 U/10 months versus 2.2 +/- 2.6 U/10 months, respectively. Thus dilazep showed a benefit in decreasing the blood demand by about 50% although the results did not reach statistical significance (p = 0.1). There was a statistical difference in hemoglobin concentration of the patients receiving dilazep compared with placebo (p = 0.038). While receiving dilazep the mean +/- SD hemoglobin level was 5.82 +/- 0.8 g/dl, significantly higher than while receiving placebo (5.66 +/- 0.9 g/dl) (p = 0.038). The liver, and renal function tests, and cardiac enzyme levels of the patients showed no significant changes throughout the study. However, one case had a problem with bleeding following tooth extraction whilst receiving dilazep and needed 1 unit of blood transfusion. In conclusion, administration of dilazep to patients with beta-thal/Hb E increased the patients' hemoglobin and reduced their blood demand with few side effects.

Thalassemic serum inhibits endothelial cell mitosis in vitro.

Human umbilical vein endothelial cells were cultured in vitro using Iscove's Modified Dulbecco's Medium (IMDM) supplemented with either pooled normal human serum, or pooled thalassemic serum, or autologous umbilical cord serum, or fetal bovine serum. The mitotic activity was determined under the inverted phase contrast microscope and the number of mitotic cells was counted. Our results showed that the mitotic cells decreased in endothelial cell culture with thalassemic serum as compared with normal human serum, autologous umbilical cord serum or fetal bovine serum. The percentage of mitotic cells decreased on day 3 in the presence of beta-thalassemia/HbE serum from both splenectomized and non-splenectomized patients as compared with normal or autologous umbilical cord serum. In the presence of alpha-thalassemic serum, a similar outcome was also observed. From this study we can conclude that the thalassemic sera might contain factors which affect the endothelial cell growth and proliferation by inhibiting mitosis in vitro.

A case-control study of acute diarrheal disease among school-age children in southern Thailand.

We conducted a case-control study of school-age children in Phatthalung, a province in southern Thailand using a questionnaire to investigate associations of children's hygiene-related behavior and hygienic conditions in their homes with acute diarrheal disease. We compared 69 acute diarrhea (less than 7 days duration) cases that attended two hospitals in Phatthalung during August 1995 to June 1996 with 69 age-, sex- and address-matched controls in primary schools who had not suffered from diarrheal disease for the past one year before August 1995. Three factors were found to be significantly associated with acute diarrheal disease: farmer or gum planter as the occupation of father [Odds ratio (OR) 6.6; 95% confidence interval (CI) 1.7-26.1, p < 0.01], installation of a refrigerator in children's homes (OR 0.2; CI 0.1-0.8, p < 0.05), and drinking untreated water (OR 2.3; CI 0.9-6.1, p < 0.1). There was no significant difference for sources of drinking water between cases and controls. Considering the data on drinking water, the results indicated that there are some problems with quality of sources of drinking water. The results also suggested that having a refrigerator could have preventive effects on acute diarrheal disease, while inadequate behavior and unhygienic environment in the homes of farmers and gum planters might be related to acute diarrheal among school-age children.

Possible evidence of endothelial cell activation and disturbance in thalassemia: an in vitro study.

Activation of vascular endothelium is considered as an important facet of inflammation, thrombosis, and vasculitis. Activated endothelial cells express a number of immunologically relevant surface markers which are not detected in dormant condition. These surface markers on endothelial cell may involve in adhesion reaction and migration of blood cell components. We demonstrated increased level of the soluble adhesion molecules in circulating blood of both alpha- and beta-thalassemic patients. These adhesion molecules are theoretically known to be released from endothelial cells. The adhesion molecules included soluble Intercellular Adhesion Molecule-1 (sICAM-1), soluble E-Selectin (ELAM-1), soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1), and von Willebrand Factor (vWF). The levels of these adhesion molecules were measured in serum from 32 thalassemic patients and 10 control healthy subjects. As compared to normal, increased sICAM-1 was found in beta-thal/HbE patients with non-splenectomy; BE-NS (p = 0.002), increased ELAM-1 in beta-thal/HbE patients with splenectomy; BE-S (p = 0.01) and HbH with Hb Constant Spring; HbH/CS (p = 0.001), and increased sVCAM-1 in BE-NS; (p = < 0.0001) and BE-S (p = 0.002). Significant increase in von Willebrand Factor (vWF), a marker for endothelial cell, was shown in BE-S (p = 0.04) as compared to normal. Adhesion molecules were also markedly demonstrated in the supernatant of in vitro culture of human vascular endothelial cell in the presence of 30% thalassemic serum, and these adhesion molecules were also detected on the surface of the cells by using the technic of laser scanning confocal microscope and direct immunofluorescence.

A double-blind placebo control trial of dilazep in beta-thalassemia/hemoglobin E patients.

Since the obtained results from the pilot study indicated that dilazep which was a membrane stabilizer would be benefit to treatment and prevention of anemia and chronic leg ulcer in beta-thalassemia/hemoglobin E (beta-thal/HbE) patients, the authors had continued the study in a second phase, ie a double blind placebo control trial. Twenty-seven beta-thal/HbE patients were recruited in the study. Eight patients who suffered from chronic leg ulcer were given dilazep. The rest of patients were given dilazep or placebo according to a randomized table. Hence, 16 patients received dilazep and 11 received placebo. When we compared the number of unit of blood transfusion, hemoglobin level, 2-3 DPG and P50 value between the dilazep and placebo groups using unpaired t-test, we found that there were no statistical differences in any of the parameters. However, when we compared the data within the group using paired t-test, there was statistical decrease in blood requirement after treatment in the dilazep group (p < 0.05). Concerning with the treatment of chronic leg ulcer, 3 in 8 patients were completely healed within 3 months, 4 in 8 patients were improved and 1 in 8 patients was not improved. There were complaints of skin itching and mild epigastric pain in placebo group but the liver function tests, kidney function tests and cardiac enzyme did not significantly change during the medication.

Pulmonary microthromboembolism by injection of sonicated autologous blood in rabbits with splenic artery ligations.

Chronic pulmonary thromboembolism (PTE) has been reported to play an important role in cardiac failure in thalassemic patients after splenectomy. However, the mechanism of PTE in these patients remains unclear. In this study, we attempted to establish an animal model of PTE seen in thalassemic patients after splenectomy. We divided New Zealand white rabbits into three groups: Group 1 was injected sonicated blood, II was injected non-sonicated blood after ligation of the splenic artery, and III was injected sonicated blood after ligation of the splenic artery. After injection of the sonicated blood, we counted the platelet number until 1 hour and the rabbits were sacrificed for histological examination. Platelets significantly decreased in number immediately after injection of the sonicated blood in Groups I and III. Many pulmonary thromboemboli composed mainly of platelets were found in Group III but not in other groups. These pathological changes seem to be partly similar to those of thalassemic patients after splenectomy. This animal model is thought to be useful to study the pathogenesis of pulmonary thromboembolism, especially in thalassemic patients after splenectomy.

Surface and total tissue factor activity of endothelial cells.

With a technic that was developed by us, we found that normal human umbilical vein endothelial cells (HUVEC) in culture characteristically had very little tissue factor (TF) activity either on the surface or in the cells which had been disrupted. In the presence of endotoxin (E. coli O26:B6), a trigger for thrombosis in septicemic patients, we could not detect an increased TF activity of HUVEC on its surface. However, an increase in TF (total TF) was detected after disruption of the cells. The increase in total TF was dose-dependent. Endotoxin at the concentration of 10 micrograms/ml caused around 5 fold increase in total TF activity compared to that of HUVEC in the absence of endotoxin.

A simple and quantitative method for the determination of tissue factor.

Tissue factor (TF), a potent initiator of the extrinsic coagulation pathway, is believed to have a critical role in thrombogenesis and haemostasis. To elucidate the role of TF in the development of various syndrome, we developed a quantitative assay method for the determination of TF using FIX complex (Profilnine) and the synthetic chromogenic substrate S-2238, all of which are commercially available. The method is simple, very sensitive, good linearity and applicable to the tissue culture plate, indicating its promising usage for the quantitation of TF activity of cells.

Detection of thalassemia genes using smeared blood film or leukocytes adhering to polysthylene fibers.

Presently genetic analyses for thalassemia types require relatively large amounts of heparinized blood (5 to 10 ml), and transport as well as degeneration of these sample is a problem in the developing world. We have developed a new method to simplify this procedure and obtain DNAs from small specimens. As experimental materials, thinly smeared blood on a glass slide or blood filtered with and adhered on polysthylene telephtalate (PST) fibers were used. These materials could be safely stored without interfering with DNA extraction for up to 3 months. The slide materials were digested with proteinase K, and DNA was extracted with Tris-EDTA-phenol:chloroform and precipitated with absolute ethanol. The PST specimens were washed with physiologic saline and treated in the same manner as described above. Products were easily amplified by PCR and digested with restriction endonucleases for beta thalassemia typing as well as for HLA-DQA1 gene typing. Results obtained by this method correlated well with previously reported incidences for thalassemia and HLA-DQA1 types in Thailand. This method can be used in the routine laboratory because it allows for stable and biosafe genetic analyses.

PF3 activity in normal subjects and beta-thalassemia trait.

Platelet factor 3 (PF3) is a platelet membrane component that plays an important role in the activation of the coagulation mechanism. Whenever platelet activation occurred, PF3 is released and participates in thrombin formation. Erythrocyte membrane fraction has also some PF3 like activity, and in abnormal erythrocyte membrane disorders, eg thalassemia, some of the membrane fraction accelerates platelet activation by increasing the PF3 activity. Formerly it was difficult to measure the PF3 activity in plasma. Recently a sensitive chromogenic test to determine the PF3 activity, which could detect the changes in PF3 activity with time, was introduced. This study was done to observe the effect of abnormal erythrocyte on platelet activation. The results obtained using the chromogenic method are the following: whole blood taken from normal subjects showed OD 0.11 +/- 0.06 at 0 minutes after blood collection and then increased significantly (p < 0.01) to 0.21 +/- 0.10 after 90 minutes, while the platelet count did not differ significantly (p > 0.05). Those results showed that there were some platelet activation after 90 minutes as seen by the increased PF3 activity, with no significant change in platelet counts. In beta-thalassemic trait subjects the PF3 activity in whole blood at 0 minutes did not differ significantly compared to the normal subjects, but after 90 minutes it was significantly higher (p < 0.01), OD 0.52 +/- 0.35. However the PF3 in platelet rich plasma at 90 minutes did not increase. The platelet count after 90 minutes was significantly decreased (p < 0.01) This result suggest that the increase in PF3 activity was caused by the role of the abnormal erythrocytes.

Automatic measurement of hemoglobin F in blood obtained from patients with hemoglobin E/E and beta-thalassemia/hemoglobin/E.

This paper presents an automated determination of hemoglobin (Hb) F in Hb E/E disease using Hi-Auto A1c. Blood specimens collected in Bangkok were frozen, and sent to Japan by air mail for the determination. The automatically determined values showed a high correlation with the values obtained by the classical alkali denaturation method. Hb E/E cases showed 4.24 +/- 1.75% of Hb F. On the other hand, Hb, Hct, MCV and MCH in the disease samples were lower than in the controls, but higher than those of beta-thalassemia/HbE disease. From the results it was concluded that Hb E/E could be differentiated from beta-thalassemia/HbE by combination of Hb F value and MCH or Hb in CBC.

Development of a new method for detection of platelet factor 3 like activity.

We asked the question, "Can thalassemic erythrocytes play some role in alteration of the hemostatic system?", because clinical examination of thalassemic patients shows symptoms and signs related to alterations in hemostatic and circulatory systems, and thalassemic erythrocytes are different from normal erythrocytes. We obtained one of the answers to the question: The erythrocytes of postsplenectomized patients of beta-thalassemia/HbE disease could stimulate their own platelets to aggregate spontaneously. To know the role of erythrocytes in platelet aggregation, we wanted to examine the effect of thalassemic erythrocytes on the coagulation system by focusing of PF3-like activity of erythrocytes, because PF3-like activity of the ghosts of erythrocytes had been reported. For the study, we tried to develop a technique that was accurate and sensitive enough to detect PF3-like activity of blood. The system we developed was the following: 1) We activated the intrinsic coagulation pathway of commercial standard plasma by ellagic acid. 2) CaCl2, a fixed amount of PF 3 and synthetic thrombin inhibitor MD 805 were added to the reaction mixture. 3) At a fixed time, thrombin activity in the mixture was measured by using S-2238 as a substrate. At full activation of the contact system by ellagic acid, the amount of thrombin formed in a certain time depended on the amount of PF3-like substances such as cephalin, freeze-thawed platelets or ghosts of erythrocytes added to the test system, indicating that PF3-like activity of those substances can be measured by the activity of thrombin generated in a fixed time.(ABSTRACT TRUNCATED AT 250 WORDS)

Thalassemic serum impairs endothelial cell growth in vitro.

Endothelial cells cultured for 3 days in the presence of hemoglobin H pooled sera had significantly decreased cell proliferation compared to those in normal serum. Inhibition was demonstrated at a concentration of 20% pooled serum in the cultured medium. Further decrease was shown in the presence of 30% pooled hemoglobin H sera. Sera from two genotypes of Hb H disease (alpha-thal 1/alpha-thal 2 and alpha-thal 1/Hb Constant Spring) had the same degree of inhibitory effect. Pooled sera from beta-thal/Hb E patients (both splenectomized or nonsplenectomized cases) had no such inhibitory effect. However, at day 4 and 5, the growth pattern relatively declined. Bilirubin at a concentration greater than 4.0 mg% in the medium 199 also caused significant decrease in cell proliferation. Since the diluted Hb H serum had bilirubin less than 4.0 mg%, the inhibitory effect of the pooled HbH serum is thus not due to effect of bilirubin. The difference between HbH and beta-thal/HbE sera in terms of inhibition of endothelial cell proliferation is the new finding that needs further investigation to explain vascularization and hemostasis in the patients of these two genotypes.

Cytoprotective effect of dilazep on hydrogen peroxide-perturbed vascular endothelial cells.

The effect of dilazep and dimethyl thiourea (DMTU) on the hydrogen peroxide-derived injury of culture pulmonary artery epithelial cells (CPAEC) was assessed by colorimetric assay of MTT formazan (MTT formazan assay). When CPAEC were treated with hydrogen peroxide, neither cell lysis nor detachment of the cells from surface of the well was observed. However, the MTT formazan formation was decreased in a time and dose dependent manner. The decrease in the formation was significantly suppressed in the presence of dilazep (0.1 to 10 microM) or DMTU (0.01 to 0.3 microM). CPAEC treated with hydrogen peroxide in the same way enhanced an activation of prothrombin, and this enhancement was significantly inhibited in the presence of dilazep (1 to 3 microM). These data indicate that dilazep exerts a cytoprotective effect against challenges of intracellular oxidant produced by hydrogen peroxide and suppresses augmented procoagulant activity of injured cells.

Size distribution curves of blood cells in thalassemias and hemoglobin H diseases.

Thalassemias and hemoglobinopathies in Thailand have been examined with a blood cell counter based on electroimpedance principle and obtained size distribution curves of red cells and platelets. Among various disorders, beta-thalassemia/hemoglobin E and homozygous hemoglobin Constant Spring showed severe anemia. Their red cell size distribution curve shifted to the left and overlapped with the platelet size distribution curve. Red cell distribution width expressed by coefficient of variation and the degree of the overlapping were stronger in beta-thalassemia/HbE than HbH. Heterozygous beta-thalassemia showed a narrow red cell distribution curve width with small standard deviation and low England's value. Although the overlapping of size distribution curves cause inaccurate red cell count and platelet count, careful observation of the size distribution curves was proved to have high diagnostic value.

Protein C and protein S deficiency in thalassemic patients.

To investigate the status of the protein C-protein S anticoagulant pathway in thalassemic patients, we measured protein C and protein S levels of plasma of 30 adults and 18 children with beta-thalassemia/HbE disease, beta-thalassemia major and HbE disease. Mean +/- 1 SD values of protein C, protein S and other coagulant proteins produced by the liver were as follows: protein C 50.4 +/- 17.2%; protein S 58.8 +/- 25.5%; antithrombin III 78.1 +/- 12.8%; PLG 86.4 +/- 18.4%; prothrombin 71.0 +/- 13.1%; factor VII 72.7 +/- 21.5%; and factor X 79.2 +/- 15.6%. Protein C and protein S levels of thalassemic patients were significantly lower than those of other coagulant proteins produced by the liver. Decrease in protein C level was stronger than that of proteins S. gamma-Carboxylated protein C levels of splenectomized patients were significantly lower than those of nonsplenectomized patients. Severe decrease of protein C and protein S may be responsible for occurrence of thrombosis in thalassemic patients.