Blur, Band 33, Seite 97-102 (1976) University of Padua Medical School, Institute of "Semeiotica Medica", Padua
An Immunological Investigation of Factor VIII Associated Antigen in Combined Factor V and Factor VIII Deficiency Antonio Girolami, Nicoletta Borsato, Giovanni Patrassi and Antonio Sticchi Summary The behavior of factor VIII associated antigen of three patients with combined factor V and factor VIII deficiency has been evaluated in several immunological systems. Factor VIII associated antigen resulted to be normal or higher than normal in all three patients in the radial immunodiffusion and in the electroimmunoassay systems. In the bidimensional electrophoresis system only one factor VIII precipitate was evident and such factor VIII precipitate showed the sam~ electrophoretic mobility as normal factor VIII antigen. These findings firmly establish the fact that the factor VIII defect in congenital combined factor V and factor VIII deficiency is of the hemophilia type. Zusammenfassung Das Verhalten Faktor VIII-assoziierten Antigens yon drei Patienten mit kombiniertem Faktor V- und Faktor VIII-Mangel wurde in verschiedenen immunologischen Tests untersucht. Faktor VIII-assoziiertes Antigen erwies sich in tier radialen Immundiffusion und im Elektroimmunoassay bei allen drei Patienten-als normal oder erh6ht. In der zweidimensionalen Elektrophorese ergab sich nur ein Faktor VIII-Pr~izipitat; es zeigte dieselbe elektrophoretische Mobilit/it wie normales Faktor VIII-Antigen. Diese l~rgebnisse sichern die Annahme, dab der Faktor VIII-Defekt bei kongenitalem kombiniertem Faktor V- und Faktor VIII-Mangel vom H~mophilie-Typ ist.
Key words: Factor VIII defect, congenital combined factor V and factor VIII deficiency, hemophilia.
The immunological investigation of congenital coagulation disorder has received considerable attention in recent years. As a result of these studies, congenital coagulation disorder due to abnormalities of fibrinogen, factor tI, factor IX and factor X, have already been recognized and extensively studied [1,4,7,9~24,30]. The description of these variants has markedly spurred the interest towards congenital coagulation disorders posing new classification problems and interesting considerations [28]. The introduction of satisfactory anti factor VIII antiserum has allowed also a clearcut differentiation between hemophilia A and yon Willebrand disease [3,8,25~31] and, possibly, a satisfactory identification of the carriers [2j31]. Eingegangen am1.9. 1975
98
A . Girolami, AT. Borsalo, G. Patrassi and A . Sticchi
Congenital c o m b i n e d factor V and factor V I I I is an extremely rare coagulation disorder. O n l y about 20 satisfactorily d o c u m e n t e d cases have been described so far in the w o r l d literature. A normal factor V I I I antigen in one patient with this peculiar disorder was reported b y Zimmermann et al. in 1969 [31]. T w o years ago we have confirmed this finding in another patient [15]. H o w e v e r no extensive study on factor V I I I associated antigen in this rare condition has been carried out so far. T h e unusual o p p o r t u n i t y to study in recent years three unrelated patients with this disorder [13,17,19] p r o m p t e d this report. Materials and Methods Agar and agarose as supplied by Behringwerke Laboratories, Marburg, Germany. Tris buffer (High resolution buffer) as supplied by Gelman Instrument Company, Ann Arbour, Michigan, USA. Reference normal plasma as supplied in lyophilized form by Behringwerke Laboratories. Pooled normal plasma as obtained from at least 6 healthy subjects of both sexes. Anti-factor VIII associated protein serum as supplied by Behringwerke Laboratories. Lot no. 2 was used in all experiments. Fresh or deep frozen plasma of three patients with combined factor V and factor VIII deficiency. The factor VIII activity in these patients was 14, 12 and 8~o, respectively. The factor V activity was 18, 55 and 14% respectively. Deep frozen hemophilia A plasma as obtained from a severe hemophiliac (factor VIII < 1% of normal). Deep frozen plasma of a patient with yon Willebrand disease. The factor VIII activity in this patient was 10% whereas the factor VIII antigen was 9%. Immunoelectrophoresis as been carried out according to a modification [14] of the Scheidegger's method [29]. Immunodiffusion studies were carried out according to the Ouchterlony's method [26], as previously reported [14]. A quantitative evaluation of factor VIII antigen was carried out according to the Laurell's electroimmunoassay [22], as previously reported [14]. Radial immunodiffusion factor VIII assay was carried out according to a modification [14] of the method proposed by Mancini et al. [23]. Cross-over electrophoresis or electrosyneresis was carried out according to a modification of the method of Bussard [5] which was already adapted by us [10,11] to coagulation studies. Bidimensional immunoelectrophoresis was carried out according to a modification [14] of the method proposed by Clarke and Freeman [6].
Results O n standard immunoelectrophoresis and immunodiffusion no b a n d or precipitate was seen b u t this was identical to the findings with p o o l e d normal plasma or with hemophilia A plasma. O n electroimmunoassay factor V I I I associated antigen resulted to be normal or above normal in all these patients (Fig. 1). T h e average values f o u n d were 125, 145, 160, respectively (Table 1). O n radial immunoassay a faint b u t still legible ring was evident in all three cases. T h e values observed in the three patients were 95, 115, 130, respectively: These values were smaller as c o m p a r e d to those observed in the electroimmunoassay system but there was still a g o o d correlation between the t w o series of values.
An immunological investigalion of factor VIII Factor V activity
99
Factor VIII activity
Factor VIII antigen (Electroimmunoassay)
Patient I (G. G.)
18
14
125
Patient II (S. A.) Patient III (P. N.)
55 14
12 8
145 160
Table 1: Factor V, factor VIII levels and factor VIII associated antigen immunological assay (in % of normal) in our three patients. The levels reported are the average of at least three determinations carried out on different occasions.
Fig. 1: Electroimmunoassay (Laurell) in our three patients with combined factor V and factor VIII deficiency (Wells 3, 4 and 5). Wells 6, 7, 8 and 9 refer to 1 : 1, 1 : 2, 1 : 4, 1 : 8 dilutions of pooled normal plasma. Well 1 and well 2 contain hemophilia A and yon Willebrand disease plasma, respectively.
Fig. 2: Bidimensional immunoelectrophoresis of plasma with combined factor V and factor VIII deficiency (left) and of pooled normal plasma (right). Only one factor VIII associated antigen peak is evident; furthermore the electrophoretic mobility is the same in both instances. A similar pattern was obtained in hemophilia A plasma.
100
A . Girolami, 2V. Borsato, G. Palra*d and A. St#chi
On cross-over electrophoresis (electrosyneresis) a single precipitate was seen, usually up to the 1 : 8 dilution, and the pattern was identical to what observed in pooled normal plasma or in hemophilia A plasma. On bidimensional immunoelectrophoresis, only one factor VIII band was evident. Furthermore the factor VIII associated antigen showed the same mobility as normal or hemophilia A factor V I I I associated antigen (Fig. 2).
Discussion Factor V I I I antigen in combined factor V and factor VIII deficiency is normal. In every assay system, no difference was noted from normal factor V I I I associated antigen. The fact that no band or precipitate became visible in standard immunoelectrophoresis and in immunodiffusion has no significance. Similarly negative results were obtained with normal and hemophilia A plasma. These negative results are surely due to the poor sensitivity of these two methods to the small amounts of antiserum and of plasma used as compared in the other methods [14]. In this regard it is to be remembered that we used untreated plasma. Other authors obtained satisfactory results but used ethanol concentrates or cryoprecipitates [3,25,31]. A similar discrepancy between standard immunoelectrophoresis and crossover electrophoresis or bidimensional electrophoresis was noted for factor X [16]. The best results were obtained using the electroimmunoassay and the bidimensional electrophoresis. The radial immunodiffusion appeared less satisfactory because only light precipitation rings were evident. These findings confirm our previous observations on the subject [14]. Our three patients had all factor V I I I associated greater than 100%. This has been a consistent finding over a four years period for the first patient and over a one year period for the remaining two patients. Our normal range is 60 to 160 and therefore the values observed are still within normal limits. Only in one occasion we have obtained a value of about 200% in our third patient. The average level is definitely higher than the average value found in normal subjects. The significance of this finding is probably trifle since it is known that in hemophiliacs factor V I I I associated antigen is often greater than normal. The normal mobility observed in our patients on bidimensional immunoelectrophoresis is of interest in view of the fact that recently in some yon Willebrand variant cases an abnormal mobility of the factor V I I I associated antigen was demonstrated
[21,27]. The results obtained by us suggest both that a homogeneous factor V I I I population is present in the patients with combined factor V and factor V I I I defect and also that the mobility of their factor V I I I associated antigen is normal. These studies lend support to the assumption that in congenital factor V and factor V I I I deficiency the defect, as far as factor V I I I is concerned, lies in a defective activation. It is probably secondary to the lack of autosomal gene controlling factor V I I I activation. No sure information is so far available for factor V. The problem will be clarified when satisfactory anti-factor V antiserum will be available.
A n immunological investigation of factor VIII
101
It is likely however that the same is true for this factor too. I n this case the autosomal gene would seem to be able to control both factor V and factor VIII activation. A defect in activation with a discrepancy between biological and antigen protein level was recently demonstrated also for combined factor V I I and factor VIII deficiency [18]. I n this latter case it was also sPeculated that the defect might be due to the lack or malfunctioning of an autosomal gene controlling both factor V I I and factor V I I I activation. This study was supported in part by a grant from the C.N.R. (grant CT 74.00189.04).
R e f orences :
1. Beck E. A., Carache P. & Jackson D.: A new inherited coagulation disorder caused by an abnormal fibrinogen (Fibrinogen Baltimore). Nature 208, 143 (1965). 2. Bennett B. & Ratnoff O. D.: Detection of the carrier state for classic hemophilia. New Eng. f. Med. 288, 342 (1973). 3. Bouma B. N., yon Mourik J. A., Wiegorinck Y., Sixma J. J. & Mochter J. A. : Immunological characterisation of antihaemophilie factor A related antigen in Hemophilia A. Scand. f. Haemat. 11, 184 (1973). 4. Brown P. E., Hongie C. & Roberts M. R. : The genetic heterogeneity of hemophilia B. DgewE.ng.]. Med. 283, 61 (1970). 5. Bussard A.: Description d'une technique combinant simultan6ament l'electrophorese et la precipitation immunologique dans un gel: l'electrosyn6r&se. Biochem. biophys. Acta 34, 258 (1959). 6. Clarke H. G. M. & Freeman T. : Quantitative immunoelectrophoresis of human serum proteins. Clin. Sci. 35, 403 (1968). 7. Denson K. W., Biggs R. & Mannucci P. M. : An investigation of three patients with Christmas disease due to an abnormal type of factor IX. ar. din. Path. 21, 160 (1968). 8. Denson K. W. : The detection of factor VIII like antigen in hemophilic carriers and in patients with raised levels of biologically active factor VIII. Brit. J. t-Iaemat. 24, 451 (1973). 9. Girolami A., Molaro G., Lazzarin M., Scarpa R. & Brunetti A.: A new congenital hemorrhagic condition due to the presence of an abnormal factor X (factor X Friuli). Study of a large kindred. Brit. J. tIaemat. 19, 179 (1970).
10. Girolami A., Sticchi A. & Bareggi G.: Cross-over electrophoresis (electrosyneresis) visualization of the abnormal factor X (factor X Fruili). J. lab. din. Med. 80, 740 (1972). 11. Girolami A., Bareggi G. & Fioretti D. : Different cross-over electrophoresis mobility of factor X Friuli and coumarininduced abnormal factor X. Haemoslasis 1, 229 (1972/73). 12. Girolami A., Bareggi G., Brunetti A. & Sticchi A.: Prothrombin Padua: a new congenital dysprothrombinemia. J. Lab. din. Med. 84, 654 (1974). 13. Girolami A., Brunetti A. & De Marco L. : Congenital combined factor V and factor VIII deficiency in a male born from a brother-sister incest. Blur 28, 33 (1974). 14. Girolami A., Sticchi A., Barbui T. & Bareggi G. : Factor VIII immunological assay. An evaluation of several methods using whole plasma. Blur 29, 309 (1974). 15. Girolami A. & Bareggi G. : Normal factor VIII antigen level in combined factor V and factor VIII congenital deficiency. Acta I-Iaemat. 52, 362 (1974). 16. Girolami A., Bareggi G. & Borsato N. : Factor X Friuli : an immunological study in plasma and in serum using several methods. Blur 30, 203 (1975). 17. Girolami A., Gastaldi G., Patrassi G. M. & Galletti A. : Congenital combined deficiency of factor V and factor VIII. A report of a further case with some considerations on the hereditary transmission of this disorder. Acta t-Iaemat. 55, 234 (1976). 18. Girolami A., Venturelli R., Virgolini L. & Burul A. : Congenital combined factor VII and factor VIII deficiency. A
102
A . Girolami, N. Borsalo, G. Patrassi and A . Sticchi
distinct coagulation disorder due to the "lack" of an autosomal gene controlling both factor VII and factor VII1 activation? Acta Haemat. 55, 181 (1976). 19. Girolami A., Violante N., Brunetti A. & Celia G. : Combined deficiency of factor V and factor VIII. Report of another case. Blur (Submitted for publication). 20. Josso F., Monasterio de Sanchez J., Lavergne J. M., Menach6 D. & Soulier J. P.: Congenital abnormality of the prothrombin molecule (factor II) in four sillings. Prothrombin Barcelona. Blood 38, 9 (1971). 21. Kernoff P. B. A., Grusan R. & Rizza C. R.: A variant of factor VIII related antigen. Brit. J. Haemat. 26, 435 (1974). 22. Laurell C. B. : Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biocb. 15, 45 (1966). 23. Mancini G., Carbonara O. & Heremans J. F. : Immunochemical quantitation of antigens by single radial immunodiffusion. Immunoch. 2, 235 (1965). 24. Menach6 D. : Constitutional and familiar abnormal fibrinogen. Thromb. Diath. Haem. Suppl. 13, 173 (1963). 25. Meyer D., Lavergne J. N., Larrieu M. J. & Josso F. : Cross-reacting material in
congenital factor VIII deficiencies (hemophilia A and yon Willebrand disease). Thromb. Res. I, 183 (1972). 26. Ouchterlony O. : Immunodiffusion and immunoelectrophoresis in Weir D. N. (Editor): Handbook of experimental Immunology. Pag. 655, Blackwell Sci. Pub., Oxford 1967. 27. Peake I. R., Bloom A. L. & Giddings J. C. : Inherited variants of factor-VIIIrelated protein in yon Willebrand's disease. New Eng.J. Med. 291, 113 (1974). 28. Ratnoff O. D. : The molecule basis of hereditary clotting disorders. In progress in Hemostasis and Thrombosis fEd. Spaet M. M.) pag. 39, Grune and Stratton, 1972. 29. Scheidegger J. J. : Une micro-methode de l'immuno-electrophor&se. Internal. Arch. Allergy 7, 103 (1955). 30. Shapiro S. S., Martinez J. & Holburn R. H. : Congenital dysprothrombinemia: an inherited structural disorder of human prothrombin. J. din. Invest. 48: 2251 (1969). 3I. Zimmerman Th. S., Ramoff O. D. & Powell A. E. : Immunological differentiation of classic hemophilia (factor VIII deficiency) and yon Willebrand's disease. J. din. Invest. 50, 244 (1971).
Author's address: Prof. A. Girolami, University of Padua Medical School, Institute of "Semeiotica Medica", Via Ospedale Civile, 105 Padua (Italy).
An Immunological Investigation of Factor VIII Associated Antigen in Combined Factor V and Factor VIII Deficiency Antonio Girolami, Nicoletta Borsato, Giovanni Patrassi and Antonio Sticchi Summary The behavior of factor VIII associated antigen of three patients with combined factor V and factor VIII deficiency has been evaluated in several immunological systems. Factor VIII associated antigen resulted to be normal or higher than normal in all three patients in the radial immunodiffusion and in the electroimmunoassay systems. In the bidimensional electrophoresis system only one factor VIII precipitate was evident and such factor VIII precipitate showed the sam~ electrophoretic mobility as normal factor VIII antigen. These findings firmly establish the fact that the factor VIII defect in congenital combined factor V and factor VIII deficiency is of the hemophilia type. Zusammenfassung Das Verhalten Faktor VIII-assoziierten Antigens yon drei Patienten mit kombiniertem Faktor V- und Faktor VIII-Mangel wurde in verschiedenen immunologischen Tests untersucht. Faktor VIII-assoziiertes Antigen erwies sich in tier radialen Immundiffusion und im Elektroimmunoassay bei allen drei Patienten-als normal oder erh6ht. In der zweidimensionalen Elektrophorese ergab sich nur ein Faktor VIII-Pr~izipitat; es zeigte dieselbe elektrophoretische Mobilit/it wie normales Faktor VIII-Antigen. Diese l~rgebnisse sichern die Annahme, dab der Faktor VIII-Defekt bei kongenitalem kombiniertem Faktor V- und Faktor VIII-Mangel vom H~mophilie-Typ ist.
Key words: Factor VIII defect, congenital combined factor V and factor VIII deficiency, hemophilia.
The immunological investigation of congenital coagulation disorder has received considerable attention in recent years. As a result of these studies, congenital coagulation disorder due to abnormalities of fibrinogen, factor tI, factor IX and factor X, have already been recognized and extensively studied [1,4,7,9~24,30]. The description of these variants has markedly spurred the interest towards congenital coagulation disorders posing new classification problems and interesting considerations [28]. The introduction of satisfactory anti factor VIII antiserum has allowed also a clearcut differentiation between hemophilia A and yon Willebrand disease [3,8,25~31] and, possibly, a satisfactory identification of the carriers [2j31]. Eingegangen am1.9. 1975
98
A . Girolami, AT. Borsalo, G. Patrassi and A . Sticchi
Congenital c o m b i n e d factor V and factor V I I I is an extremely rare coagulation disorder. O n l y about 20 satisfactorily d o c u m e n t e d cases have been described so far in the w o r l d literature. A normal factor V I I I antigen in one patient with this peculiar disorder was reported b y Zimmermann et al. in 1969 [31]. T w o years ago we have confirmed this finding in another patient [15]. H o w e v e r no extensive study on factor V I I I associated antigen in this rare condition has been carried out so far. T h e unusual o p p o r t u n i t y to study in recent years three unrelated patients with this disorder [13,17,19] p r o m p t e d this report. Materials and Methods Agar and agarose as supplied by Behringwerke Laboratories, Marburg, Germany. Tris buffer (High resolution buffer) as supplied by Gelman Instrument Company, Ann Arbour, Michigan, USA. Reference normal plasma as supplied in lyophilized form by Behringwerke Laboratories. Pooled normal plasma as obtained from at least 6 healthy subjects of both sexes. Anti-factor VIII associated protein serum as supplied by Behringwerke Laboratories. Lot no. 2 was used in all experiments. Fresh or deep frozen plasma of three patients with combined factor V and factor VIII deficiency. The factor VIII activity in these patients was 14, 12 and 8~o, respectively. The factor V activity was 18, 55 and 14% respectively. Deep frozen hemophilia A plasma as obtained from a severe hemophiliac (factor VIII < 1% of normal). Deep frozen plasma of a patient with yon Willebrand disease. The factor VIII activity in this patient was 10% whereas the factor VIII antigen was 9%. Immunoelectrophoresis as been carried out according to a modification [14] of the Scheidegger's method [29]. Immunodiffusion studies were carried out according to the Ouchterlony's method [26], as previously reported [14]. A quantitative evaluation of factor VIII antigen was carried out according to the Laurell's electroimmunoassay [22], as previously reported [14]. Radial immunodiffusion factor VIII assay was carried out according to a modification [14] of the method proposed by Mancini et al. [23]. Cross-over electrophoresis or electrosyneresis was carried out according to a modification of the method of Bussard [5] which was already adapted by us [10,11] to coagulation studies. Bidimensional immunoelectrophoresis was carried out according to a modification [14] of the method proposed by Clarke and Freeman [6].
Results O n standard immunoelectrophoresis and immunodiffusion no b a n d or precipitate was seen b u t this was identical to the findings with p o o l e d normal plasma or with hemophilia A plasma. O n electroimmunoassay factor V I I I associated antigen resulted to be normal or above normal in all these patients (Fig. 1). T h e average values f o u n d were 125, 145, 160, respectively (Table 1). O n radial immunoassay a faint b u t still legible ring was evident in all three cases. T h e values observed in the three patients were 95, 115, 130, respectively: These values were smaller as c o m p a r e d to those observed in the electroimmunoassay system but there was still a g o o d correlation between the t w o series of values.
An immunological investigalion of factor VIII Factor V activity
99
Factor VIII activity
Factor VIII antigen (Electroimmunoassay)
Patient I (G. G.)
18
14
125
Patient II (S. A.) Patient III (P. N.)
55 14
12 8
145 160
Table 1: Factor V, factor VIII levels and factor VIII associated antigen immunological assay (in % of normal) in our three patients. The levels reported are the average of at least three determinations carried out on different occasions.
Fig. 1: Electroimmunoassay (Laurell) in our three patients with combined factor V and factor VIII deficiency (Wells 3, 4 and 5). Wells 6, 7, 8 and 9 refer to 1 : 1, 1 : 2, 1 : 4, 1 : 8 dilutions of pooled normal plasma. Well 1 and well 2 contain hemophilia A and yon Willebrand disease plasma, respectively.
Fig. 2: Bidimensional immunoelectrophoresis of plasma with combined factor V and factor VIII deficiency (left) and of pooled normal plasma (right). Only one factor VIII associated antigen peak is evident; furthermore the electrophoretic mobility is the same in both instances. A similar pattern was obtained in hemophilia A plasma.
100
A . Girolami, 2V. Borsato, G. Palra*d and A. St#chi
On cross-over electrophoresis (electrosyneresis) a single precipitate was seen, usually up to the 1 : 8 dilution, and the pattern was identical to what observed in pooled normal plasma or in hemophilia A plasma. On bidimensional immunoelectrophoresis, only one factor VIII band was evident. Furthermore the factor VIII associated antigen showed the same mobility as normal or hemophilia A factor V I I I associated antigen (Fig. 2).
Discussion Factor V I I I antigen in combined factor V and factor VIII deficiency is normal. In every assay system, no difference was noted from normal factor V I I I associated antigen. The fact that no band or precipitate became visible in standard immunoelectrophoresis and in immunodiffusion has no significance. Similarly negative results were obtained with normal and hemophilia A plasma. These negative results are surely due to the poor sensitivity of these two methods to the small amounts of antiserum and of plasma used as compared in the other methods [14]. In this regard it is to be remembered that we used untreated plasma. Other authors obtained satisfactory results but used ethanol concentrates or cryoprecipitates [3,25,31]. A similar discrepancy between standard immunoelectrophoresis and crossover electrophoresis or bidimensional electrophoresis was noted for factor X [16]. The best results were obtained using the electroimmunoassay and the bidimensional electrophoresis. The radial immunodiffusion appeared less satisfactory because only light precipitation rings were evident. These findings confirm our previous observations on the subject [14]. Our three patients had all factor V I I I associated greater than 100%. This has been a consistent finding over a four years period for the first patient and over a one year period for the remaining two patients. Our normal range is 60 to 160 and therefore the values observed are still within normal limits. Only in one occasion we have obtained a value of about 200% in our third patient. The average level is definitely higher than the average value found in normal subjects. The significance of this finding is probably trifle since it is known that in hemophiliacs factor V I I I associated antigen is often greater than normal. The normal mobility observed in our patients on bidimensional immunoelectrophoresis is of interest in view of the fact that recently in some yon Willebrand variant cases an abnormal mobility of the factor V I I I associated antigen was demonstrated
[21,27]. The results obtained by us suggest both that a homogeneous factor V I I I population is present in the patients with combined factor V and factor V I I I defect and also that the mobility of their factor V I I I associated antigen is normal. These studies lend support to the assumption that in congenital factor V and factor V I I I deficiency the defect, as far as factor V I I I is concerned, lies in a defective activation. It is probably secondary to the lack of autosomal gene controlling factor V I I I activation. No sure information is so far available for factor V. The problem will be clarified when satisfactory anti-factor V antiserum will be available.
A n immunological investigation of factor VIII
101
It is likely however that the same is true for this factor too. I n this case the autosomal gene would seem to be able to control both factor V and factor VIII activation. A defect in activation with a discrepancy between biological and antigen protein level was recently demonstrated also for combined factor V I I and factor VIII deficiency [18]. I n this latter case it was also sPeculated that the defect might be due to the lack or malfunctioning of an autosomal gene controlling both factor V I I and factor V I I I activation. This study was supported in part by a grant from the C.N.R. (grant CT 74.00189.04).
R e f orences :
1. Beck E. A., Carache P. & Jackson D.: A new inherited coagulation disorder caused by an abnormal fibrinogen (Fibrinogen Baltimore). Nature 208, 143 (1965). 2. Bennett B. & Ratnoff O. D.: Detection of the carrier state for classic hemophilia. New Eng. f. Med. 288, 342 (1973). 3. Bouma B. N., yon Mourik J. A., Wiegorinck Y., Sixma J. J. & Mochter J. A. : Immunological characterisation of antihaemophilie factor A related antigen in Hemophilia A. Scand. f. Haemat. 11, 184 (1973). 4. Brown P. E., Hongie C. & Roberts M. R. : The genetic heterogeneity of hemophilia B. DgewE.ng.]. Med. 283, 61 (1970). 5. Bussard A.: Description d'une technique combinant simultan6ament l'electrophorese et la precipitation immunologique dans un gel: l'electrosyn6r&se. Biochem. biophys. Acta 34, 258 (1959). 6. Clarke H. G. M. & Freeman T. : Quantitative immunoelectrophoresis of human serum proteins. Clin. Sci. 35, 403 (1968). 7. Denson K. W., Biggs R. & Mannucci P. M. : An investigation of three patients with Christmas disease due to an abnormal type of factor IX. ar. din. Path. 21, 160 (1968). 8. Denson K. W. : The detection of factor VIII like antigen in hemophilic carriers and in patients with raised levels of biologically active factor VIII. Brit. J. t-Iaemat. 24, 451 (1973). 9. Girolami A., Molaro G., Lazzarin M., Scarpa R. & Brunetti A.: A new congenital hemorrhagic condition due to the presence of an abnormal factor X (factor X Friuli). Study of a large kindred. Brit. J. tIaemat. 19, 179 (1970).
10. Girolami A., Sticchi A. & Bareggi G.: Cross-over electrophoresis (electrosyneresis) visualization of the abnormal factor X (factor X Fruili). J. lab. din. Med. 80, 740 (1972). 11. Girolami A., Bareggi G. & Fioretti D. : Different cross-over electrophoresis mobility of factor X Friuli and coumarininduced abnormal factor X. Haemoslasis 1, 229 (1972/73). 12. Girolami A., Bareggi G., Brunetti A. & Sticchi A.: Prothrombin Padua: a new congenital dysprothrombinemia. J. Lab. din. Med. 84, 654 (1974). 13. Girolami A., Brunetti A. & De Marco L. : Congenital combined factor V and factor VIII deficiency in a male born from a brother-sister incest. Blur 28, 33 (1974). 14. Girolami A., Sticchi A., Barbui T. & Bareggi G. : Factor VIII immunological assay. An evaluation of several methods using whole plasma. Blur 29, 309 (1974). 15. Girolami A. & Bareggi G. : Normal factor VIII antigen level in combined factor V and factor VIII congenital deficiency. Acta I-Iaemat. 52, 362 (1974). 16. Girolami A., Bareggi G. & Borsato N. : Factor X Friuli : an immunological study in plasma and in serum using several methods. Blur 30, 203 (1975). 17. Girolami A., Gastaldi G., Patrassi G. M. & Galletti A. : Congenital combined deficiency of factor V and factor VIII. A report of a further case with some considerations on the hereditary transmission of this disorder. Acta t-Iaemat. 55, 234 (1976). 18. Girolami A., Venturelli R., Virgolini L. & Burul A. : Congenital combined factor VII and factor VIII deficiency. A
102
A . Girolami, N. Borsalo, G. Patrassi and A . Sticchi
distinct coagulation disorder due to the "lack" of an autosomal gene controlling both factor VII and factor VII1 activation? Acta Haemat. 55, 181 (1976). 19. Girolami A., Violante N., Brunetti A. & Celia G. : Combined deficiency of factor V and factor VIII. Report of another case. Blur (Submitted for publication). 20. Josso F., Monasterio de Sanchez J., Lavergne J. M., Menach6 D. & Soulier J. P.: Congenital abnormality of the prothrombin molecule (factor II) in four sillings. Prothrombin Barcelona. Blood 38, 9 (1971). 21. Kernoff P. B. A., Grusan R. & Rizza C. R.: A variant of factor VIII related antigen. Brit. J. Haemat. 26, 435 (1974). 22. Laurell C. B. : Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biocb. 15, 45 (1966). 23. Mancini G., Carbonara O. & Heremans J. F. : Immunochemical quantitation of antigens by single radial immunodiffusion. Immunoch. 2, 235 (1965). 24. Menach6 D. : Constitutional and familiar abnormal fibrinogen. Thromb. Diath. Haem. Suppl. 13, 173 (1963). 25. Meyer D., Lavergne J. N., Larrieu M. J. & Josso F. : Cross-reacting material in
congenital factor VIII deficiencies (hemophilia A and yon Willebrand disease). Thromb. Res. I, 183 (1972). 26. Ouchterlony O. : Immunodiffusion and immunoelectrophoresis in Weir D. N. (Editor): Handbook of experimental Immunology. Pag. 655, Blackwell Sci. Pub., Oxford 1967. 27. Peake I. R., Bloom A. L. & Giddings J. C. : Inherited variants of factor-VIIIrelated protein in yon Willebrand's disease. New Eng.J. Med. 291, 113 (1974). 28. Ratnoff O. D. : The molecule basis of hereditary clotting disorders. In progress in Hemostasis and Thrombosis fEd. Spaet M. M.) pag. 39, Grune and Stratton, 1972. 29. Scheidegger J. J. : Une micro-methode de l'immuno-electrophor&se. Internal. Arch. Allergy 7, 103 (1955). 30. Shapiro S. S., Martinez J. & Holburn R. H. : Congenital dysprothrombinemia: an inherited structural disorder of human prothrombin. J. din. Invest. 48: 2251 (1969). 3I. Zimmerman Th. S., Ramoff O. D. & Powell A. E. : Immunological differentiation of classic hemophilia (factor VIII deficiency) and yon Willebrand's disease. J. din. Invest. 50, 244 (1971).
Author's address: Prof. A. Girolami, University of Padua Medical School, Institute of "Semeiotica Medica", Via Ospedale Civile, 105 Padua (Italy).
