Simple and accurate bedside diagnostic kit for determining haemophilia A and B: a revised version

Abstract

Apart from the bleeding manifestations, the diagnosis of haemophilia is confirmed by laboratory testings ranging from simple screening to sophisticated procedures. Venous clotting time (VCT) is a simple laboratory investigation that can be performed in every level of hospital service. The VCT is prolonged in patients with severe coagulation factor deficiency and will be shortened if the deficient factor is added. The VCT of haemophilia A patients will be shortened if factor VIII is added, similarly, with haemophilia B patients if factor IX is added. A diagnostic kit using plasma from patients with haemophilia B and A as the source of factor VIII and factor IX in the mixing VCT has been previously reported 1. We report a revised version of the diagnostic kit. It consisted of three glass test tubes (13 × 100 mm) marked at 2.0 mL and labelled in order. The amount of 1.2 units of factor VIII or factor IX concentrate reconstituted in 15 mcl of sterile water was added into the second and third tubes, respectively. Then these factor concentrates were freeze dried in a Virtis lyophilizer and the dry powder was kept in the properly capped test tubes at 4°C until use. The procedure started with the reconstitution of the lyophilized factor VIII and factor IX concentrate in the second and third tubes with 15 mcl or one drop of sterile water immediately before use. Ten millilitre of whole blood was drawn from the studied subjects using the two-syringe technique. The first stopwatch was switched on when the blood was drawn into the second syringe. An amount of 2.7 ml of whole blood mixed with 0.3 ml of citrate buffer was used for laboratory testing. Then 2 ml of whole blood was put into the first, second and third tubes, consecutively. The whole blood and the reconstituted factor concentrate in the second and third tubes were thoroughly mixed by titling the tubes up and down five times. The time used for whole blood alone to have completed clotting was recorded from the first stopwatch, whereas the time used for each whole blood sample mixed with factor VIII concentrate and factor IX concentrate to have completed clotting was recorded from two separated stopwatches. The revised kit using factor concentrate was more convenient than that of the previously reported kit using citrate plasma from severe haemophilia B and A patients as the source of factor VIII and factor IX, respectively. It resulted in a simplified procedure as calcium chloride was not required in the test. The procedures of adding calcium chloride and one step of mixing were omitted as shown in Table 1. The kit was applied to 35 patients with haemophilia (A = 27, B = 8), and 22 normal healthy volunteers as normal controls. The median ages of patients and normal controls were 13 years (interquartile range 8–16) and 25.5 years (interquartile range 21.8–29.8), respectively. The patients with haemophilia were classified as a severe degree (deficient factor <1%), moderate degree (deficient factor 1–5%) and mild degree (deficient factor >5–40%). Four severe haemophilia A patients exhibited inhibitor. Three patients had low inhibitor of 1.3, 4.4 and 4.4 BU, whereas one patient had high inhibitor of 50 BU. Only one severe haemophilia B patient had inhibitor of 4.6 BU. All patients abstained from blood component or factor concentrate administration for at least 5 days before participating in the study. The normal controls had no personal or family history of bleeding disorders and did not take any medication. Coagulation tests included levels of factor VIII clotting activity (FVIII:C), factor IX clotting activity (FIX:C) and inhibitor to FVIII:C and FIX:C was determined by standard methods 2, 3 in every subject. The median levels of FVIII:C and FIX:C among the normal controls were 110% (interquartile range 99–130%) and 96% (interquartile range 90–115%), respectively. The results of the VCT of whole blood alone and the correction of VCT after adding factor VIII and factor IX concentrates among haemophilia and normal controls are shown in Table 2 (excluding one haemophilia A patient with high inhibitor). The VCT of whole blood alone was significantly prolonged in haemophilia A patients with severe and moderate degrees compared with those of mild degree (P = 0.037). On the contrary, some haemophilia B patients with severe and moderate degrees had a slightly prolonged VCT, whereas some of them had a significantly prolonged VCT similar to those of haemophilia A patients. However, both haemophilia A and B patients with mild degree had minimally elevated VCT which was slightly more prolonged than those of normal controls. Subsequently, 34 haemophilia patients' VCTs were corrected to the normal range of less than 15 min after adding factor VIII or factor IX concentrate accordingly, no matter whether the VCT of whole blood alone was prolonged or minimally elevated. One severe haemophilia A patient with high inhibitor of 50 BU. He had markedly prolonged VCT which could not be normalized after adding factor VIII concentrate. The status of haemophilia A and B could be accurately diagnosed for the remaining 34 patients. Patients with haemophilia A had a prolonged or minimally elevated VCT which normalized after adding factor VIII concentrate in the second tube. Vice versa, patients with haemophilia B had a prolonged or minimally elevated VCT which normalized after adding factor IX concentrate in the third tube. The correction of VCT expressed as time and percentage of correction after adding factor VIII concentrate in patients with haemophilia A was significantly shortened and higher than those after adding factor IX concentrate with P values of 0.0001. Similarly, patients with haemophilia B also had significantly shortened VCT and higher percentage of correction after adding factor IX concentrate compared with those after adding factor VIII concentrate with P values of 0.012. The median time corrections after adding factor VIII concentration for haemophilia A or factor IX concentrate for haemophilia B among severe, moderate and mild degrees were not significantly different with P values ranging from 0.50 to 0.95. The extent of correction of VCT among patients with initial prolonged VCT was higher than those with minimally elevated VCT with P values of 0.002 for haemophilia A patients and 0.25 for haemophilia B patients. However, the sample size of haemophilia B patients was rather small. The revised kit is useful to determine the accurate status of haemophilia A and B patients in developing countries who have not yet been treated. However, some patients previously treated could not actually know their definite diagnosis. The health personnel are still able to use this kit to determine the status of haemophilia A and B even though they have low inhibitor titre of less than 5 BU. It reflects that the amount of added factor concentrate is sufficient to overcome the low inhibitor titre to obtain normalized VCT. This diagnostic kit is an additional tool for determining the deficient state of factor VIII or factor IX at bedside. It is simple and useful especially in developing countries where the confirmation test of activated partial thromboplastic time and specific factor assay is not immediately available. The diagnostic kit can be transported with ice to different levels of health care services. The shelf life of the lyophilized factor concentrate at 4°C is 1 year. Importantly, the physicians, nurses and medical personnel can perform this test by following easy-to-understand instructions. This work was supported by the Thailand Research Fund – Senior Research Scholar 2006 (AC). The authors stated that they had no interests which might be perceived as posing a conflict or bias.