British Journal of Haematology, 1979,42, 433-439.
Detection of Haemophilia A Carriers by Replicate Factor VIII Activity and Factor VIII Antigenicity Determinations URISELIGSOHN, ARIELA ZIVELIN, CHAVA PEREZ A N D MICHAELA MODAN Haernophilia Centre, Institute of Haematology and Department of Clinical Epidemiology, Chaim Sheba Medical Centre, Tel-Hashomer and Sackler Medical School, Tel-Aviv University, Israel (Received 21 September 1978; accepted for publication 15 November 1978) SUMMARY. Factor VIII (NIII) activity and antigenicity were determined on replicate fresh blood samples drawn on three separate occasions in 48 normal females and 37 obligatory haemophilia A carriers, in an attempt to improve the detection rate. N I I I activity was assayed by a one-stage method and N I I I antigenicity by the Laurell’s technique employing an anti-NIII antiserum prepared in rabbits. The mean results for the individual subjects were analysed after establishing a discriminant function. The probability of being a carrier wasdetermined on the basis oflaboratory data and family history. The probability data were translated into more simple terms applicable for genetic counselling by defining three categories: Definite carriers, doubtful carriers and definite normals. Of the obligatory carriers, 83.8% were classified definite carriers, 13.5% doubtful carriers and 2.7% as definite normals. O f the normal females, 4.2% were classified as definite carriers, 18.7% doubtful carriers and 77.1% as definite normals. Adjustment of the results according to age was found unnecessary. The precision of the determinations of fVIII antigenicity and N I I I activity in individual subjects was calculated for different numbers of replicate tests performed in the same individual. It was found that if only one test is performed, a substantial error might occur. Three replicate tests considerably diminish this error, although it is still in the range of 16-29%. For suspected carriers who fall in the doubtful category it is suggested that six or even more tests are performed. Haemophilia A is an X-linked disorder characterized by a low level of plasma factor VIII (NIII) clotting activity. As a group, the female carriersof the disease have an average low N I I I activity. However, when tested individually, the carrier state can be detected with relative certainty in only 3&50%, since the carriers’ fVIII levels overlap with the normal range (Rapaport etal, 1960; Veltkamp et al, 1968).Zimmerman etal (1971b) demonstrated that N I I I related antigen can be detected in all haemophilia A patients by an antiserum prepared in Correspondence: Ur U. Seligsohn, Haemophilia Centre, Institute of Haematology, Chaim Sheba Medical Centre, Tel-Hashomer, Israel. 0007-1048/79/0700-0433 $02.00
0 1 9 7 9 Blackwell Scientific Publications
43 3
43 4
U r i Seligsoiohn et a1
rabbits. In obligatory carriers ofhaemophilia A, Zimmerman et a1 (1971a) found that the ratio between NIII activity and NIII antigenicity was significantly lower than in normal females, and that this ratio provided a better criterion for carrier detection than N I I I activity alone. Several investigators, using various statistical methods, have reported different carrier detection rates based on measurements of N I I l activity and N I I I antigenicity (Zimmerman et al, 1971a; Meyer et al, 1975; Bouma et al, 1975; Prentice et al, 1975; Rizza et al, 1975; Elston et al, 1976; Ratnoff & Jones, 1977). A recent cooperative study conducted in three laboratories indicated that the observed differences in carrier detection rate were related to laboratory techniques rather than statistical methods (Klein ef al, 1977). In the study reported here, an attempt was made to improve the carrier detection rate by establishing a discriminant function based on measurements of fVIII activity and fVIII antigenicity in replicate blood samples of 37 obligatory carriers of haemophilia A and 48 normal females. MATERIAL AND METHODS Subjects. The following subjects were considered obligatory carriers: (1) mothers of two haemophilia A patients, (2) daughters ofhaemophilia A patients; and (3) mothers of haemophilia A patients with another relative who definitely suffers from the disease. Control subjects were females who belong to the personnel of the hospital. Blood sampling and pmrerrion. Blood was withdrawn from all subjects with plastic syringes and 21 gauge needles. I t was introduced into plastic tubes containing 1/9 vol of Ware’s anticoagulant (2 vol of 0.1 M citric acid and 3 vol of 0.1 M tri-sodium citrate). Platelet-poor plasma was obtained by double spinning a t 10 000 rpm for 10 min at 4°C. All subjects were tested on three occasions a t least a week apart. N I I I activity and fVIII antigenicity were tested on fresh plasma samples within 3 h of taking the blood. Reference normal pool plasma was obtained from 12-30 normal subjects. It was kept a t -770°C for no longer than 2 months. f v I I 1 clotting assay. A one-stage assay for N I I I was carried out by the method of Hardisty & McPherson (1962). Preparation offVIIZ antibodies. N III was partialiy purified from a commercial N I I I concentrate (Hemophil, Hyland Division, Travenol Laboratories, Costa Mesa, California) by the method of Marchesi et a1 (1972).An antiserum against the partially purified N I I I was prepared in rabbits by a previously described technique (Ekert et al, 1973). The antiserum contained 128 u/ml, as measured by a N I I I neutralization assay; one unit being the amount of antiserum neutralizing 0.5 u/ml N I I I of normal pooled plasma during 2 h a t 37°C. Measurement offvZI1 antigenicity. The measurement of N I I I antigenicity was carried out by a modification of the Laurell technique (Laurell, 1966). The height of each rocket was measured from the centre of the well to the peak of the rocket. O n each plate, two sets of four dilutions (loo%, 75%, 50%, 25%) of normal pooled plasma were applied. The mean heights of the rockets for each dilution were plotted on a semilog paper against the OO/ dilution thus forming the reference standard curve. For each test plasma sample, two or three dilutions were measured. NIII antigenicity was determined by referring the results to the standard curve. After correcting the dilution factor, the mean N III antigenicity of each test sample was expressed by per cent.
Detection of Haernophilia A Carriers
43 5 Statistical analysis. The posterior probability of each subject to belong to the carrier population was calculated according to Bayes’ theorem (Anderson, 1958).The calculation was based on the mean of three N I I I antigenicity determinations, as well as on the a priori probability of belonging to the carrier population according to the family history. Adjustment for age was done by incorporating this variable in the discriminant function. The variability within replicate tests in the obligatory carriers and normals was measured by pooled standard deviation (Sp), calculated separately for N I I I activity and fVIII antigenicity. The precision of the estimate of fVIII activity and N I I I antigenicity of an individual subject was determined by 95% confidence limits based on the Sp.The individual true value hasa 95% probability to lie within the following interval mean of n replicates = & -.SP f0.975; (N-K)
where n is the number of replicate tests performed in an individual, K is the number of examined individuals, Nis the total number of determinations performed in the K individuals. f0.97i; ( N - K ) represents the 97.5 percentile of the student’s t distribution with N-K degrees of freedom. RESULTS Thirty-seven obligatory haemophilia A carriers (aged 6-60 years) and 48 normal females (aged 22-56 years) were examined. As can be seen in Table I, there was no significant difference in fVIII antigenicity between the carriers and the normal females, while the fVIII activity was significantly lower in the carriers. The posterior probability of belonging to the carrier population was calculated, as described in the Methods section, for each of the37 obligatory carriers and 48 normal females. In order to translate the probabilities obtained into terms applicable for genetic counselling, three probability ranges were defined.: (1) Definite carrier range: included women whose posterior probability to belong to the carrier population was more than or equal to 0.50. (2) Docrbfcrl carrier range: included women whose posterior probability to belong to the carrier population was between 0.14 and 0.50. The value of 0.14 was selected on the basis of the arbitrary assumption that a false negative diagnosis is five times worse than a false positive one. (3) Dejnite normal range: included women whose posterior probability to belong to the carrier population was less than o r equal to 0.14.
TABLE 1. fVIII antigenicity and fVIII activity in normal females and obligatory carriers*
examined
‘I/ofV111antigenicity (x) Mean 1:S D (22S,)
ohjVIII activity ( y ) Mean f S D (yiS,)
Covariance o j x and y ( S x y )
48 37
110.9 f30.7 118+3* 41.7
111.6 28.1 62.0+ 23.1
638.4 558.7
No. ~~
Normal females Obligatory carriers
* Details of the raw data obtainable from the author on request.
Uri Seligsohn et a1
436 200
A
180
A
160 PIC/$:
A
140 A
-.
5
.
120
f 100 U
r
80 60 40
20
' 20
' 40 ' 60
80
100 ' 120
140
160
lb0
200
AHF like snligen %
FIG 1. A plot of NIII activity (mean of three determinations) against NIII antigenicity (mean of three determinations) in 48 normal females (A) and 37 obligatory carriers ( 0 ) .P(C/? designates the a posteriori probability to be a carrier given a specific pair of values for X (NIII antigenicity) and Y (NIII activity). The A posteriori probabilities to be carriers assuming an a priori probability of 0.5 are depicted by the lines diagonally crossing the graph. The shaded area denotes the area ofprobabilities defined as doubtful carriers. Above the shaded area is the area defined as definite normals and below is the area defined as definite carriers.
In Fig 1 the mean NIII activity of each tested subject is plotted against thc corresponding N I I I antigenicity. Also depicted are the three probability ranges defined above, assuming arbitrarily that all our tested subjects had an equal a priori probability to belong to the carrier or normal populations (the common situation in genetic counselling). The distribution of the study subjects in the three carrier definition ranges was as follows (see Table 11). O f the 48 normal females, 77.1% would have been correctly defined, while 4.2% would have been false positive and 18.7% doubtful carriers. O f the 37 obligatory carriers, 83.8% would have been correctly defined, 2.7% false negative and 13.5% doubtful carriers. Adjustment for age did not affect the distribution of the tested subjects in the three carrier definition ranges. Interestingly, in the obligatory carriers there was a significant increase of NIlI antigenicity with age (r=0.53, P
Detection of Haemophilia A Carriers by Replicate Factor VIII Activity and Factor VIII Antigenicity Determinations URISELIGSOHN, ARIELA ZIVELIN, CHAVA PEREZ A N D MICHAELA MODAN Haernophilia Centre, Institute of Haematology and Department of Clinical Epidemiology, Chaim Sheba Medical Centre, Tel-Hashomer and Sackler Medical School, Tel-Aviv University, Israel (Received 21 September 1978; accepted for publication 15 November 1978) SUMMARY. Factor VIII (NIII) activity and antigenicity were determined on replicate fresh blood samples drawn on three separate occasions in 48 normal females and 37 obligatory haemophilia A carriers, in an attempt to improve the detection rate. N I I I activity was assayed by a one-stage method and N I I I antigenicity by the Laurell’s technique employing an anti-NIII antiserum prepared in rabbits. The mean results for the individual subjects were analysed after establishing a discriminant function. The probability of being a carrier wasdetermined on the basis oflaboratory data and family history. The probability data were translated into more simple terms applicable for genetic counselling by defining three categories: Definite carriers, doubtful carriers and definite normals. Of the obligatory carriers, 83.8% were classified definite carriers, 13.5% doubtful carriers and 2.7% as definite normals. O f the normal females, 4.2% were classified as definite carriers, 18.7% doubtful carriers and 77.1% as definite normals. Adjustment of the results according to age was found unnecessary. The precision of the determinations of fVIII antigenicity and N I I I activity in individual subjects was calculated for different numbers of replicate tests performed in the same individual. It was found that if only one test is performed, a substantial error might occur. Three replicate tests considerably diminish this error, although it is still in the range of 16-29%. For suspected carriers who fall in the doubtful category it is suggested that six or even more tests are performed. Haemophilia A is an X-linked disorder characterized by a low level of plasma factor VIII (NIII) clotting activity. As a group, the female carriersof the disease have an average low N I I I activity. However, when tested individually, the carrier state can be detected with relative certainty in only 3&50%, since the carriers’ fVIII levels overlap with the normal range (Rapaport etal, 1960; Veltkamp et al, 1968).Zimmerman etal (1971b) demonstrated that N I I I related antigen can be detected in all haemophilia A patients by an antiserum prepared in Correspondence: Ur U. Seligsohn, Haemophilia Centre, Institute of Haematology, Chaim Sheba Medical Centre, Tel-Hashomer, Israel. 0007-1048/79/0700-0433 $02.00
0 1 9 7 9 Blackwell Scientific Publications
43 3
43 4
U r i Seligsoiohn et a1
rabbits. In obligatory carriers ofhaemophilia A, Zimmerman et a1 (1971a) found that the ratio between NIII activity and NIII antigenicity was significantly lower than in normal females, and that this ratio provided a better criterion for carrier detection than N I I I activity alone. Several investigators, using various statistical methods, have reported different carrier detection rates based on measurements of N I I l activity and N I I I antigenicity (Zimmerman et al, 1971a; Meyer et al, 1975; Bouma et al, 1975; Prentice et al, 1975; Rizza et al, 1975; Elston et al, 1976; Ratnoff & Jones, 1977). A recent cooperative study conducted in three laboratories indicated that the observed differences in carrier detection rate were related to laboratory techniques rather than statistical methods (Klein ef al, 1977). In the study reported here, an attempt was made to improve the carrier detection rate by establishing a discriminant function based on measurements of fVIII activity and fVIII antigenicity in replicate blood samples of 37 obligatory carriers of haemophilia A and 48 normal females. MATERIAL AND METHODS Subjects. The following subjects were considered obligatory carriers: (1) mothers of two haemophilia A patients, (2) daughters ofhaemophilia A patients; and (3) mothers of haemophilia A patients with another relative who definitely suffers from the disease. Control subjects were females who belong to the personnel of the hospital. Blood sampling and pmrerrion. Blood was withdrawn from all subjects with plastic syringes and 21 gauge needles. I t was introduced into plastic tubes containing 1/9 vol of Ware’s anticoagulant (2 vol of 0.1 M citric acid and 3 vol of 0.1 M tri-sodium citrate). Platelet-poor plasma was obtained by double spinning a t 10 000 rpm for 10 min at 4°C. All subjects were tested on three occasions a t least a week apart. N I I I activity and fVIII antigenicity were tested on fresh plasma samples within 3 h of taking the blood. Reference normal pool plasma was obtained from 12-30 normal subjects. It was kept a t -770°C for no longer than 2 months. f v I I 1 clotting assay. A one-stage assay for N I I I was carried out by the method of Hardisty & McPherson (1962). Preparation offVIIZ antibodies. N III was partialiy purified from a commercial N I I I concentrate (Hemophil, Hyland Division, Travenol Laboratories, Costa Mesa, California) by the method of Marchesi et a1 (1972).An antiserum against the partially purified N I I I was prepared in rabbits by a previously described technique (Ekert et al, 1973). The antiserum contained 128 u/ml, as measured by a N I I I neutralization assay; one unit being the amount of antiserum neutralizing 0.5 u/ml N I I I of normal pooled plasma during 2 h a t 37°C. Measurement offvZI1 antigenicity. The measurement of N I I I antigenicity was carried out by a modification of the Laurell technique (Laurell, 1966). The height of each rocket was measured from the centre of the well to the peak of the rocket. O n each plate, two sets of four dilutions (loo%, 75%, 50%, 25%) of normal pooled plasma were applied. The mean heights of the rockets for each dilution were plotted on a semilog paper against the OO/ dilution thus forming the reference standard curve. For each test plasma sample, two or three dilutions were measured. NIII antigenicity was determined by referring the results to the standard curve. After correcting the dilution factor, the mean N III antigenicity of each test sample was expressed by per cent.
Detection of Haernophilia A Carriers
43 5 Statistical analysis. The posterior probability of each subject to belong to the carrier population was calculated according to Bayes’ theorem (Anderson, 1958).The calculation was based on the mean of three N I I I antigenicity determinations, as well as on the a priori probability of belonging to the carrier population according to the family history. Adjustment for age was done by incorporating this variable in the discriminant function. The variability within replicate tests in the obligatory carriers and normals was measured by pooled standard deviation (Sp), calculated separately for N I I I activity and fVIII antigenicity. The precision of the estimate of fVIII activity and N I I I antigenicity of an individual subject was determined by 95% confidence limits based on the Sp.The individual true value hasa 95% probability to lie within the following interval mean of n replicates = & -.SP f0.975; (N-K)
where n is the number of replicate tests performed in an individual, K is the number of examined individuals, Nis the total number of determinations performed in the K individuals. f0.97i; ( N - K ) represents the 97.5 percentile of the student’s t distribution with N-K degrees of freedom. RESULTS Thirty-seven obligatory haemophilia A carriers (aged 6-60 years) and 48 normal females (aged 22-56 years) were examined. As can be seen in Table I, there was no significant difference in fVIII antigenicity between the carriers and the normal females, while the fVIII activity was significantly lower in the carriers. The posterior probability of belonging to the carrier population was calculated, as described in the Methods section, for each of the37 obligatory carriers and 48 normal females. In order to translate the probabilities obtained into terms applicable for genetic counselling, three probability ranges were defined.: (1) Definite carrier range: included women whose posterior probability to belong to the carrier population was more than or equal to 0.50. (2) Docrbfcrl carrier range: included women whose posterior probability to belong to the carrier population was between 0.14 and 0.50. The value of 0.14 was selected on the basis of the arbitrary assumption that a false negative diagnosis is five times worse than a false positive one. (3) Dejnite normal range: included women whose posterior probability to belong to the carrier population was less than o r equal to 0.14.
TABLE 1. fVIII antigenicity and fVIII activity in normal females and obligatory carriers*
examined
‘I/ofV111antigenicity (x) Mean 1:S D (22S,)
ohjVIII activity ( y ) Mean f S D (yiS,)
Covariance o j x and y ( S x y )
48 37
110.9 f30.7 118+3* 41.7
111.6 28.1 62.0+ 23.1
638.4 558.7
No. ~~
Normal females Obligatory carriers
* Details of the raw data obtainable from the author on request.
Uri Seligsohn et a1
436 200
A
180
A
160 PIC/$:
A
140 A
-.
5
.
120
f 100 U
r
80 60 40
20
' 20
' 40 ' 60
80
100 ' 120
140
160
lb0
200
AHF like snligen %
FIG 1. A plot of NIII activity (mean of three determinations) against NIII antigenicity (mean of three determinations) in 48 normal females (A) and 37 obligatory carriers ( 0 ) .P(C/? designates the a posteriori probability to be a carrier given a specific pair of values for X (NIII antigenicity) and Y (NIII activity). The A posteriori probabilities to be carriers assuming an a priori probability of 0.5 are depicted by the lines diagonally crossing the graph. The shaded area denotes the area ofprobabilities defined as doubtful carriers. Above the shaded area is the area defined as definite normals and below is the area defined as definite carriers.
In Fig 1 the mean NIII activity of each tested subject is plotted against thc corresponding N I I I antigenicity. Also depicted are the three probability ranges defined above, assuming arbitrarily that all our tested subjects had an equal a priori probability to belong to the carrier or normal populations (the common situation in genetic counselling). The distribution of the study subjects in the three carrier definition ranges was as follows (see Table 11). O f the 48 normal females, 77.1% would have been correctly defined, while 4.2% would have been false positive and 18.7% doubtful carriers. O f the 37 obligatory carriers, 83.8% would have been correctly defined, 2.7% false negative and 13.5% doubtful carriers. Adjustment for age did not affect the distribution of the tested subjects in the three carrier definition ranges. Interestingly, in the obligatory carriers there was a significant increase of NIlI antigenicity with age (r=0.53, P
![[Treatment of haemophilia with factor VIII inhibitor by giving isoagglutinin-free factor VIII concentrate (author's transl)].](/assets/img/hold.png)
