Familial discrepancy between the one-stage and two-stage factor VIII methods in a subgroup of patients with haemophilia A.

A higher result for plasma factor VIII:C measured by the one-stage as compared with the two-stage method has been described in some patients with haemophilia A or with von Willebrand's disorder. We used both methods to measure FVIII:C in 95 patients with haemophilia A. The results were equivalent in all 21 patients with severe haemophilia (16 families) and in 45 of the patients with mild or moderate haemophilia (18 families). However, the results were discrepant (FVIII:C by one-stage assay 2-7-fold higher than by two-stage assay) in the other 29 patients with mild or moderate haemophilia (12 other families). For each patient with discrepant FVIII:C results the classification was the same for all other affected members of his family. In some families with haemophilia A the gene defect leads to a discrepancy between the one-stage and two-stage FVIII:C results and may be more widespread than previously recognized.

An increase in phosphatidic acid in the absence of changes in diacylglycerol in human platelets stimulated with ADP.

1. ADP caused an increase in radioactivity of phosphatidic acid but not diacylglycerol in human platelets labelled with [3H]arachidonic acid. 2. The radioactivity of phosphatidic acid was significantly increased 10 sec after adding 10 microM ADP and this increase did not depend on production of thromboxane A2. 3. Thrombin (1 U/ml) caused an increase in both diacylglycerol and phosphatidic acid, the latter being much greater than that caused by ADP. 4. The results confirm that ADP stimulates phosphatidic acid production and suggest that a weak stimulus of the phosphatidyl inositol cycle, such as ADP, does not cause accumulation of diacylglycerol.

A dose-ranging study of the antiplatelet effect of enteric coated aspirin in man.

Enteric coated aspirin was given to eight human volunteers in escalating doses (20, 40, 60, 80, 100 mg daily), each dose being given over two weeks. In addition, to measure the maximum effect of aspirin, each volunteer was given two single doses of 600 mg of soluble aspirin. At the end of each dosing interval we measured platelet aggregation and thromboxane formation in response to four aggregating agents and to whole blood coagulation. The doses of aspirin required to inhibit platelet aggregation in response to various stimuli were: for collagen 60-80 mg, for adenosine diphosphate and adrenaline 60 mg, and for arachidonate 40 mg. For maximum inhibition of thromboxane formation the doses were: for collagen greater than 100 mg, for adenosine diphosphate and adrenaline 60 mg, for arachidonate 80 mg, and for whole blood coagulation 100 mg. Different aspirin doses are required to inhibit the responses to different stimuli. Furthermore, for some stimuli, inhibition of thromboxane generation may require more aspirin than is required for inhibition of aggregation. The clinical implications of these findings are uncertain since we do not know which stimuli are important in arterial thrombosis in man.

Storage of platelets for tests of platelet function: effects of temperature on platelet aggregation, platelet morphology and liberation of beta-thromboglobulin.

We have studied the effect of temperature on platelets during storage for tests of platelet function. Aliquots of PRP were stored at constant pH at 37 degrees C, room temperature and 4 degrees C. At intervals up to five hours, samples were taken for estimation of platelet shape, plasma levels of beta-thromboglobulin and 14C-serotonin, and assessment of platelet aggregation in response to a range of concentrations of ADP and collagen. When PRP was stored at 37 degrees C there was a gradual decrease in the aggregation response during the period of storage. At room temperature the decrease was slower but the response to ADP often increased dramatically before decreasing; at this temperature there was pronounced liberation of beta TG while there was none at 37 degrees C. Platelets stored at 37 degrees C were smooth and elliptical when examined by electron microscopy, but those stored at room temperature showed partial loss of discoid shape and formation of some pseudopodia. Storage at 4 degrees C was associated with total loss of discoid shape and formation of many large pseudopodia. Light transmission studies also showed loss of discoid shape at room temperature and 4 degrees C. We conclude that storage at 4 degrees C or at room temperature causes platelet activation. To avoid this PRP should be stored at 37 degrees C prior to tests of platelet function.

Destruction of ristocetin cofactor by coagulation at 4 degrees C.

Ristocetin cofactor (VIIIR:RCo) and factor VIII-related antigen (VIIIR:Ag) were measured in anticoagulated and non-anticoagulated blood incubated at 4 degrees C, room temperature (RT) or 37 degrees C for 24 hours. A marked decrease in VIIIR:RCo, to almost undetectable levels, and a smaller decrease in VIIIR:Ag occurred when whole blood clotted at 4 degrees C. These changes were slight or absent when blood clotted at RT or 37 degrees C. VIIIR:RCo lost at 4 degrees C was not recoverable by further incubation at 37 degrees C but the less-marked loss of VIIIR:Ag was partially recovered. In blood which had clotted at 4 degrees C there was a change in the electrophoretic profile of VIIIR:Ag on crossed immunoelectrophoresis: there was more anodal migration of the VIIIR:Ag peak, consistent with a decrease in the mean molecular size. Further experiments showed that the decrease in VIIIR:RCo during coagulation at 4 degrees C preceded the decrease in fibrinogen levels. In cell-free plasma VIIIR:RCo also decreased markedly when coagulation occurred at 4 degrees C. The results show that loss of VIIIR:RCo occurs when blood is allowed to clot at 4 degrees C: this is not due to cryoprecipitation and does not require the presence of blood cells. The data suggest that it is probably caused by plasma proteases activated early in the coagulation pathway.

Storage of platelets for tests of platelet function: comparison of two methods of pH control.

A comparison was made of two methods to control the pH of platelet-rich plasma (PRP) stored for tests of platelet function. Citrated PRP at 37 degrees C was maintained at pH 7.3-7.4 by incubation either in a controlled CO2/air environment or in a plastic syringe from which all air was expelled. At intervals over 2-5 hours platelet aggregation induced by ADP and collagen was measured. Plasma beta-thromboglobulin (beta TG) was assayed to assess liberation of beta TG from platelets during storage. Platelet aggregation responses were more stable when PRP was stored in a syringe. Liberation of beta TG from platelets did not occur in this system, but did occur in the CO2 system in many experiments. The differences between the two systems were not due to the lower pO2 levels in the syringe, but were probably related to the presence of an air/liquid interface in the CO2 system. The syringe system of storage is a simple method of pH control which offers better preservation of platelet function than a controlled CO2/air environment.

Detection of haemophilia carriers: multivariate analysis compared with discriminant analysis using up to five factor VIII variates.

Multivariate analysis is potentially superior to the linear discriminant analysis which is commonly used to identify carriers of haemophilia. Our aim was to compare these two statistical methods, and to find which factor VIII variates most effectively partitioned carrier and normal subjects. In this study we assayed one- and two-stage factor VIII coagulant activity, factor VIII related antigen by electroimmunoassay and by fluoroimmunoassay, and ristocetin co-factor in 50 normal females and 50 carriers of haemophilia. From the results we calculated multivariate ellipses which circumscribed the normal and the carrier populations, and we displayed these on the monitor of a microcomputer. These ellipses separated the two populations better than linear discriminants calculated on the same data. Multivariate analysis correctly identified 94% of the carriers whereas discriminant analysis correctly identified only 84%. Discriminant analysis gave poorer results because the statistical assumption of equal variance was breached, whereas the assumption of multivariate normality was upheld. Of the five factor VIII variates, two-stage factor VIII coagulant activity and factor VIII related antigen by electroimmunoassay correctly identified the most subjects. Ristocetin co-factor did not improve the diagnostic ability, either when in lieu of or when added to factor VIII related antigen.

The effects of age, gene source and familial severity on Factor VIII in normals and haemophilia carriers: analysis by multiple regression.

There are conflicting views on the effects of age, gene source and familial severity on levels of factor VIII in carriers of haemophilia. Different workers have found that factor VIII increases with age, is higher in paternal carriers, and is higher in carriers from families with more severe haemophilia. Other workers have disagreed with these findings. In this study we explored some of the causes of this conflict. We measured factor VIII related antigen and factor VIII coagulant activity on 40 normal females and 48 carriers, and analysed the results by multiple regression and analysis of covariance. Our results indicated that both factor VIII coagulant activity and factor VIII related antigen increased with age, but were unaffected by the familial severity of haemophilia or whether the defective gene came from the mother or the father. We found that the conflicting reports of previous authors were due to high inter-correlations of the studied variables.

Storage of platelets for tests of platelet function: effects of pH on platelet aggregation and liberation of beta-thromboglobulin.

Platelet aggregation responses are influenced by conditions of storage of platelet-rich plasma (PRP). The aim of the present study was to further define the necessity for pH control during storage of PRP for tests of platelet function. Aliquots of citrated PRP were maintained at different pH levels by alteration of the CO2 content of the atmosphere in an incubation chamber. At intervals over 2-2 1/2 hours, plasma beta-thromboglobulin and 14C-serotonin were measured as well as platelet aggregation induced by ADP and collagen. At each time a dose response curve was studied for aliquots stored at each pH level. When two aliquots were maintained at different pH levels in the range 6.85-7.90, there was a significant increase in aggregation at the higher pH, even when the pH difference was as small as 0.2 units. In this range, pH did not influence the rate of deterioration of the aggregation response, but when pH was above 8.0, there was marked deterioration of the response. Increased pH was associated with an increase in plasma levels of beta-thromboglobulin and 14C-serotonin, which was more marked when pH was above 8.0. It appears that increases in pH are harmful to platelets and even small pH changes should be avoided during storage of platelet-rich plasma for tests of platelet function.

Effect of EDTA and PGE1 on beta-thromboglobulin liberation from platelets.

The aim of this study was to evaluate the effect of PGE1 and EDTA on liberation of beta-thromboglobulin (beta TG) from platelets in vitro. Liberation of beta TG was followed in citrated blood at room temperature for 120 minutes after venesection. PGE1 reduced beta TG liberation, and maximal inhibition was attained by concentrations greater than 2 X 10(-6)M. EDTA induced the efflux of beta TG. This EDTA-induced efflux was delayed but not prevented by PGE1 and by citrate; it was not found at 0-4(0)C. Therefore the use of EDTA to prevent beta TG liberation during sampling for in vitro or in vivo studies depends heavily on modifying factors such as PGE1 and low temperature, and on the time taken to process samples. Its effectiveness must be in some doubt where the platelets may be sufficiently stimulated to overcome these modifying influences, or where handling of samples is less than optimal.

Platelet counts in stored donor blood.

Platelets were counted in eight units each of one- and three-day-old blood. Counts were done both before and after the blood had passed through a standard 170 micron filter. In the one-day-old blood, platelet counts were within the normal range. The mean count was 237,000 platelets/microlitre. Platelet counts on three-day-old blood were lower, but generally still within the normal range. The mean count was 183,000/microlitre. Only a few platelets were retained by the filter in the transfusion set; about 90% of platelets passed the filter in both the one- and three-day-old blood. It appears that whole blood, anticoagulated with citrate/phosphate/dextrose (CPD), and stored under Australian blood bank conditions retains platelets in sufficient numbers for at least the first three days to be clinically significant. However, it remains to be determined whether satisfactory platelet activity can be expected during this time.

Stability of diluted heparin sodium stored in plastic syringes.

The stability of heparin diluted in 0.9% sodium chloride injection and stored in plastic syringes for a three-week period was studied. Heparin activity was assayed by the activated partial thromboplastin time (APTT) method. Heparin sodium (25,000 units/ml) was diluted to 500 units/ml and stored in 50-ml polypropylene syringes. Concentrations were compared in two brands of syringes stored at room temperature in the dark. In another experiment controlled for order-related assay errors, heparin was stored in one brand by syringe at either 0-4 degrees C or room temperature. There was a statistically decrease in heparin activity over three weeks in both syringes and at both 0-4 degrees C and room temperature. However, the overall drop in activity was only about 8%. Analysis of covariance confirmed significant regression with time at both temperatures. An unexpected finding was that heparin at 500 units/ml consistently assayed higher than this value. A study of the effect of glass and plastic showed that when heparin was diluted into either a glass or plastic container, there was significantly less heparin activity in the glass containers within two hours. One possible explantation for this phenomenon is absorption of heparin to glass surfaces. It was concluded that heparin can be stored in polypropylene syringe for up to three weeks without refrigeration. However, once diluted, heparin should not be stored in glass containers.