Application of indicators for quality improvement in the coagulation laboratory.

Indicators are tools that measure work performance and serve as a guide to improve the quality of laboratories. Seven Indicators for quality improvement have been established in our coagulation laboratory. They are :- 1). percentage of pre-analytical problems, 2). personnel competency scores, 3). results of external quality assessment, 4). % coefficient of variation (CV) of control materials, 5). unit cost, 6). percentage of reports within determined time, and 7). percentage of customers who were satisfied. The percentage of preanalytical error gradually decreased from 1.8% in April 2001 to 0.8% in June 2001 as a result of co-operation between the coagulation laboratory and the wards. Since there is no system to check personnel competency at a national level in Thailand, we set up a program for testing personnel competency in our department by asking every technician to take a written and practical laboratory examination. The scores achieved by our personnel ranged from 40 to 90%. For those who achieved scores of lower than 70%, we limited their responsibilities and organized a training program for them. In order to check our laboratory's accuracy, we are enrolled in the WHO International External Quality Assessment Scheme (IEQAS) in Blood Coagulation and have been since 1987. The survey results indicated that most of our laboratory tests were within consensus including our homemade ELISA tests for protein C, protein S and vWF antigen. The percent CVs of control materials used for the internal daily control for every test were analyzed. They ranged from 2.3 for normal APTT to 11.4 for the low level of free protein S in plasma. The unit cost for each test was analyzed to determine the cost-effectiveness of the laboratory. We set the goal for the turn around time for emergency coagulation tests to be within an hour and the percentage of reports within this time was 91.6% in August 2001. The last indicator was the percentage of satisfied customers, which gave an indication of the quality of all Out Patient Department (OPD) services performed by our department. We sent 400 questionnaires to doctors, nurses and patients in OPD asking their opinion of both the technical services and the behavior of our technicians. The percentage satisfaction of our customers concerning services offered to OPD was lower than 50%. We plan to improve the last 2 indicators by expanding the space of the OPD/emergency laboratory and reorganizing the service system. All indicators mentioned above have helped to improve the quality of our laboratory greatly.

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.

Standardization of coagulation tests.

A lot of attempts have been made to standardise both activated partial thromboplastin time (APTT) and prothrombin time (PT). Only the standardization of PT has been successfully implemented while the standardization of APTT is still underway. The PT test is a common method for monitoring oral anticoagulant therapy. Owing to the variable response of the thromboplastins and the different ways of reporting, PT results obtained from patients treated with oral anticoagulants have not been interchangeable between laboratories. In 1977, the World Health Organization (WHO) designed a batch of human brain thromboplastin as the first international reference preparation (IRP) for thromboplastin and a calibration system was proposed in 1982, based on the assumption that a linear relationship exists between the logarithm of the PT obtained with the IRP and test thromboplastins. This calibration model is used to standardize the reporting of the PT by converting the PT ratio observed with the local thromboplastin into an International Normalized Ratio (INR). The INR system is being adopted by an increasing number of hospitals in many countries. With the increasing use of the INR system, a number of problems have been identified with the INR system. The most serious one is that the ISI of a thromboplastin depends on the coagulometer used. Besides, a number of investigators have noted that the ISI value provided by the manufacturer for each new batch of thromboplastin reagent may be incorrect and the use of inappropriate control plasma can lead to erroneous INR calculations. Four solutions have been proposed to solve the problems of the INR system as follows: (a) the local system calibration with lyophilized plasma calibrants with assigned manual PT determined in terms of the relevant IRP for thromboplastin; (b) the use of a mean normal prothrombin time (MNPT) obtained with the coagulometer to derive the prothrombin ratio: (c) PT standardization by means of the procedure using plasma calibrants: and (d) selection of sensitive thromboplastin with low ISI values. The INR system has been adopted in Thailand since 1984. There are 3 steps in the implementation as follows: (a) preparation of National Reference Thromboplastin; (b) selection of high sensitive thromboplastin; and (c) optimal therapeutic range for Thai patients. The anticoagulant effect of heparin is usually monitored by the APTT, a test that is sensitive to the inhibitory effects of heparin on thrombin, factor Xa. and factor IXa. However, the type of clot detection system, the contact activator; and the phospholipid composition of the reagent affect the APTT response. In 1995. ISTH/ICSH proposed a calibration model for APTT standardization. As the problem showed a great similarity to PT standardization, the same model of calibration was applied but no international reference preparation for the APTT is yet available. In 1998. van den Besselaar et al proposed a lyophilized APTT reagent comprising synthetic phospholipids and colloidal silica as a candidate IRP for the APTT.

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.

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.

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.

Difference in pattern of erythropoietin response between beta-thalassemia/hemoglobin E children and adults.

Thalassemia is one of the most common genetic disorders in Thailand. The thalassemic patients have many pathophysiologic changes secondary to chronic anemia. During these last few years there have been many trials to cure or improve the anemic condition in thalassemia by using various agents, including erythropoietin (EPO). Thus it is very important to understand the EPO response to different degree of anemia in the thalassemic patients. In this study we evaluated the EPO status in 53 beta-thalassemia/HbE patients, from 4-61 years old, by enzyme-linked immunosorbent assay. The results showed that the levels of EPO in beta-thalassemia/HbE patients were much higher than in normal control subjects: mean +/- SE = 527 +/- 183.20 and 3.45 +/- 0.47 mIU/ml respectively. The reverse correlation between the levels of EPO and hematocrit (r = -0.704) was also observed. There was also a tendency to have higher levels of EPO in beta-thal/HbE children than in adults, although this was statistically insignificant. The observed versus predicted levels of EPO (log O/P ratio) showed that most patients had good EPO response to the degree of anemia. However, inappropriate decrease of EPO response was observed in 8/40 adult patients. The EPO levels in these patients were not correlated with any physical or laboratory studies, including kidney function. We thus propose that if EPO is to be considered as one of the alternative treatment to the thalassemic patients, in the future, it may benefit only the patients with low EPO levels.

Effect of different oral anticoagulant intensities on prothrombin fragment 1 + 2 in Thai patients with mechanical heart valve prostheses.

The minimal intensity of oral anticoagulant required for antithrombotic protection in patients with a mechanical heart valve is still debatable, and that of the Westerner may not be directly applied to Thai patients. Our preliminary clinical review suggested that International Normalized Ratio (INR) 2-3 might be enough but it needs further supporting evidence. Therefore, we studied the effect of different anticoagulant intensities, expressed as INR, on the in vivo coagulation activation by measuring prothrombin fragment 1 + 2 (F1 + 2) in 116 patients with mechanical heart valve replacements. The patients had received warfarin for not less than one month with different intensities. The mean +/- S.D. of F1 + 2 level in 30 normal controls was 0.7 +/- 0.17 nmol/L. After excluding two outliers, the maximum linear correlation between INR and F1 + 2 was -0.658 (p < 0.001) when only patients whose intensities were lower than INR3 were taken into account. Adding more data from the patients having higher intensities decreased the correlation coefficient. The patients were subsequently classified by INR values in the range INR 1.1-1.9, 2-3 and 3.1-4.2. The F1 + 2 in each group was 0.6 +/- 0.30, 0.28 +/- 0.13 and 0.24 +/- 0.13 nmol/L respectively. The F1 + 2 in the first group did not differ from normal (p = 0.119) but was higher than the others (p = 0.000). The latter two groups had no difference between them (p = 0.112). Hence, from the laboratory point of view, we did not see additional benefit in the reduction of thrombin activation by the anticoagulant intensities higher than the range INR 2-3. The evidence supported that this therapeutic range might be enough for Thai patients with mechanical heart valves.

A comparative study of coumadin and aspirin for primary cardioembolic stroke and thromboembolic preventions of chronic rheumatic mitral stenosis with atrial fibrillation.

The comparative study of the efficacy of coumadin and aspirin in primary cardioembolic stroke prevention of chronic rheumatic heart disease (mitral stenosis) with atrial fibrillation was conducted at Siriraj Hospital, Mahidol University, Bangkok, Thailand. Seventy-nine patients were enrolled in the trial. Allocation of patients into coumadin or aspirin groups depended upon the patients' choice. Nineteen patients were given coumadin at the adjusted dosage to maintain the therapeutic range of International Normalised Ratio between 1.5-3. Sixty patients were given aspirin at the fixed dosage of 75 mg per day. Six patients were lost to follow-up over the 3 yr period; four in the aspirin group and 2 in the coumadin group. There were three patients with nonfatal cardioembolic stroke in the aspirin group but none in the coumadin group after three years of follow-up. Six patients had mitral valve replacement during the study (i.e. three patients in each group). There were complications in 12 patients, 10 in the aspirin (16.6 per cent) and 2 in the coumadin (10.5 per cent) group. The complications in coumadin group were minor bleeding over the thigh in one patient and generalised ecchymosis over the whole body in one other. In the aspirin group, the complication was gastrointestional symptoms, mainly epigastric pain, but no frank bleeding was observed. Primary prevention of cardioembolic stroke in chronic rheumatic heart disease was found to be more effective with coumadin than aspirin. Our study does not support the use of aspirin in primary prevention of cardiac embolism in chronic rheumatic heart disease.

Thai national reference preparation in blood coagulation. IV. Home made reference plasma for von Willebrand's factor assay.

Two lots of home made reference plasma: FVIII R:Ag 30/8/88 and FVIII R:Ag 18/10/88 were prepared by lyophilization of pooled normal human plasma. Modification of J. Cejka's technique was used to determine FVIII R:Ag. This technique was tested for reliability i.e. precision, reproducibility and sensitivity. The concentration of FVIII R:Ag, determined by calibration against the 1st British Standard for FVIII R:Ag, human 66/355, which was established by National Institute for Biological Standard and Control (NIBS & C), London, the WHO International Laboratory for Biological Standard, were respectively 1.058 and 1.023 Ag units/ml for FVIII R:Ag 30/8/88 and FVIII R:Ag 18/10/88, respectively. The precision of the procedure and the accuracy of FVIII R:Ag concentration of both lots were verified by using them as standard curve to determine FVIII R:Ag in 4 unknown plasma samples, supplied by the UK Reference Laboratory for Anticoagulant Reagent & Control; WHO Collaborating Center for Quality Assessment, in Blood Coagulation Testing for International Quality Control Survey in Blood Coagulation. The results were very satisfactory. The coefficient of variation was between 2.22-5.47 per cent when compared with other 29 laboratories around the world. These home made reference preparation are stable at least up to 30 months at -70 degrees C, and can be applied for calibration of unknown sample instead of the 1st British Standard for FVIII R:Ag, human 66/355.

Preoperative autologous blood collection and haemodilution with gelatin solution (gelifundol).

Autologous blood collection and haemodilution with gelatin solution had an effect on the decrease in red blood cells, haemoglobin, haemotocrit, fibrinogen and platelets; however, this technique had no effect on coagulograms, platelet function and haemostasis. In conclusion, this technique is suitable and possibly practical in obtaining sufficient blood for elective surgical patients and is without any undesirable side effects.

Bernard-Soulier syndrome: a case report.

An adopted Thai girl has been followed at Children's Hospital, Bangkok, since she was 8 months old. The diagnosis of Bernard-Soulier syndrome was made, based on the clinical features of easy bruising, purpura, petechial hemorrhages and recurrent epistaxis. The abnormal laboratory tests included giant platelets with dark stained granules, mild to moderate thrombocytopenia, prolonged bleeding time, absence of ristocetin induced agglutination but normal ristocetin cofactor, factor VIII coagulant activity and von Willebrand factor antigen. These findings suggested the absence of glycoprotein Ib (GPIb) on the platelet membrane. The genetic transmission can not be evaluated in this patient.

Laboratory assessment of thrombolytic therapy in acute myocardial infarction.

Hemostatic profiles and cardiac enzymes were studied in 55 acute myocardial infarct (AMI) patients to assess SK and rt-PA therapy. Hypofibrinogenemia occurred 85% in SK group and 55% in rt-PA group with high FDP and D-Dimer, indicating systemic fibrinogenolysis and local crosslinked fibrin clot lysis. The incidence of bleeding in SK and rt-PA groups combined with anticoagulants were the same but lower in rt-PA with antiplatelet. The mean FDP was significantly higher in the bleeding group (p < 0.01). Cardiac enzymes: CK, CK-MB peak values indicated reperfusion were 26.6%, 60% and 90% in conventional, SK and rt-PA therapy, respectively. Early and late occlusion did not occur either in SK or rt-PA followed by anticoagulants. Late occlusion was found in patients treated with rt-PA and antiplatelet. Mortality rate was 20% in conventional therapy.

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)

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.

Increase in spontaneous platelet aggregation in beta-thalassemia/hemoglobin E disease: a consequence of splenectomy.

Clinical symptoms related with disturbances of the circulatory system are often observed in beta-thalassemia/hemoglobin E (beta-thal/HbE) patients after splenectomy. Pulmonary thrombosis is one of the important contributing factors. However, the pathogenesis of this phenomenon was not known. Previous studies on platelet functions were controversial as platelet-rich plasma (PRP) was employed for all of the studies. By centrifugation, most of the hyperactive platelets were excluded before platelet aggregation tests were performed. Besides, the role of red cells related to platelet aggregation was not investigated. In this study, a platelet function test was designed to avoid these two handicaps of previous work as mentioned, by using whole blood from 15 normal and 40 beta-thal/HbE patients (15 nonsplenectomized and 25 splenectomized) to study spontaneous platelet aggregation. The principle of the test was to evaluate platelet number in whole blood by electronic platelet counter at time 0 (45 minutes after blood collection) and this number was used as 100% of free unaggregated platelets. Then the same specimen of whole blood was incubated at 37 degrees C with continuous stirring by magnetic stirrer in an aggregometer for 8 minutes; at 1 minute intervals free unaggregated platelets were evaluated and calculated as a percentage of the initial control value. The results indicated increased spontaneous platelet aggregation in whole blood of post-splenectomized beta-thal/HbE patients. The residual free platelet number were 24% at 8 minutes after incubation. Effects of red blood cells on spontaneous platelet aggregation were studied by mixing autologous beta-thal/HbE red cells obtained from splenectomized and non-splenectomized patients with platelet rich plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of PF3 availability in whole blood from volunteers and beta-thalassemia/HbE patients: a promising method for prediction of thrombotic tendency.

The platelet factor 3 (PF 3) plays a very important role in activation of coagulation factors and is regarded to be available during activation of platelets. However, membrane fraction of erythrocytes is also shown to have PF 3-like activity, suggesting that the abnormal erythrocytes may accelerate the activation of platelet by forming thrombin on their abnormal membrane or by way of other factors of the abnormal erythrocytes, and may increase the availability of PF 3 in whole blood (WB). To examine this hypothesis, we developed a method for determination of PF 3 activity, because the method now available for the PF3 determination could not detect changes in PF 3 activity with time. The principles of our method were as follows: 1) The reaction system was adjusted so that the amount of thrombin generated in a fixed reaction time correlates with the amount of PF 3. 2) To avoid inhibition of thrombin activity by antithrombin III, a synthetic thrombin inhibitor, MD 805, was added to the system and the activity of thrombin generated was measured by synthetic thrombin substrate S-2238 using A405 as an indicator of the availability of PF3. The results obtained by the method were the following: WB taken from volunteers showed A405 of 0.12 +/- 0.02 at 30 minutes after blood collection and then the A405 increased to 0.27 +/- 0.03 at 90 minutes. However, one volunteer showed the value of 0.59 at 90 minutes, though the value at 30 minutes was 0.16. The platelet number in his WB did not change during the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Long term oral anticoagulant therapy after heart valve prostheses at Siriraj Hospital.

One hundred and forty-six cases of cardiac valvular prostheses in the Department of Surgery, Siriraj hospital, were studied retrospectively for the effect of long-term oral anticoagulant therapy. Warfarin sodium was given to 119 patients after operation, 5 cases discontinued therapy and 27 cases received no anticoagulant at all due to loss of follow-up. One stage prothrombin time was used as laboratory control. The advocated therapeutic range for commercial rabbit brain thromboplastin was 1.35-2.2 P.T. ratio. The incidence of thromboembolism was 8.9 per 100 patients - year in the nontherapeutic group, and was 0.6 per 100 patients - year in the therapeutic group (p less than 0.05). Bleeding complications was 26.9 per cent. These were 34 minor-, 11 major-and 2 fatal bleeding episodes. One bleeding manifestation was found in the nontherapeutic group, the etiology was not recorded. The mean dose of warfarin sodium in the thromboembolic group was 2.5 mg/day, this gave a therapeutic ratio of less than 1.4. In the bleeding group that had P.T. ratio not exceeding 2.2, the mean dose was 3.37 mg/day; and 5.23 mg/day when P.T. ratio was higher than 2.2 (p less than 0.01). Therefore, it seems justifiable to conclude that the appropriate mean dose should be over 2.5 mg/day and less than 3.37 mg/day. However, regular blood test to determine the appropriate daily dose for each individual patient is obligatory, as patients might be more or less sensitive to the drug than the average and drug requirement varies from time to time even in the same individual owing to many factors.(ABSTRACT TRUNCATED AT 250 WORDS)