The Use of Tissue Thromboplastins of Different Origin Is a Fundamental Tool in the Initial Characterization of FVII Defects On “Factor VII Deficiency (Semin Thromb Hemost 2009;35(4):400–406)”

We read with great interest the recent article by Drs. Mariani and Bernardi[1] on factor VII (FVII) deficiency. Because of some experience in the field, we would like to take the liberty of making some comments.

We were surprised to see there was no discussion regarding the different reactivity of thromboplastins of different origin in the initial evaluation of patients with suspected or proven FVII deficiency. As a matter of fact, in a note to Table 3, it was stated that “the wide variation depends on the thromboplastin used in the clotting assay.” However, the range reported (1 to 37%) appears to be wrong because in FVII Padua, the paradigmatic defect with the R304Q mutation, ox brain–derived tissue thromboplastin yields normal values. The range, therefore, should be 1 to 90% or 1 to 100% of normal.[2] [3] Hog brain–derived tissue thromboplastins yield only slightly lower activity levels, whereas rabbit brain or human placenta or human recombinant derived reagents yield variably but constantly low levels.[2] [4]

The diagnostic role of thromboplastins of different origin is of paramount importance in FVII deficiency as first demonstrated by our group many years ago[2] [4] and subsequently confirmed by many other investigators.[5] [6] [7] [8]

In this regard it is noteworthy that the other two mutations reported in the same Table 3 as “FVII plus,” namely C310F and G331S, have not been apparently tested with ox-brain thromboplastins. The authors who studied these two mutations only used rabbit or human tissue–derived thromboplastins.[9] [10] [11] [12] Even in the article by O’Brien et al, dealing with purified R304Q FVII obtained from a single patient, only rabbit-brain and recombinant human preparations were used.[12] Due to the fact that these two mutations occur in an area very close to the R304Q site, it is likely that ox-brain thromboplastins might yield normal or near-normal values even in these cases. As a consequence, the range of 1 to 4% reported in Table 3 for these two mutations may only partially represent the picture or even be incorrect. The existence of a discrepancy between the results obtained using different tissue thromboplastins may be the first clue that one could be dealing with an abnormality or a cross-reacting material (CRM) + variant.

From available data it seems advisable to assay FVII using at least two thromboplastins, a rapid one and a slow one, namely a rabbit-brain or recombinant human and an ox-brain thromboplastin. Unfortunately, ox brain–derived thromboplastin reagents are not easily available. Until a few years ago, vials of ox-brain thromboplastin were contained in a widely used protein S assay kit (Instrumentation Laboratory, Milan, Italy). Lately, the ox-brain thromboplastin in that kit has been replaced with a rabbit-brain one, probably because the kit now contains activated protein C instead of the protein C activator obtained from Agkistrodon contortrix viper venom. However, Thrombotest, a widely used reagent in many countries, contains ox-brain thromboplastins together with adsorbed normal plasma. Therefore, Thrombotest might serve perfectly for this purpose. After all, a normal Thrombotest together with a prolonged rabbit-brain prothrombin time was the first clue that we were dealing with an abnormal FVII when we were investigating the index patient with FVII Padua many years ago.[2] In routine laboratory work, a comparison of results obtained with Normotest (rabbit-brain thromboplastins and adsorbed normal plasma) and Thrombotest, the so-called Normotest-Thrombotest discrepancy, could supply useful preliminary information about the type of FVII defect.[14] [15]

A final comment concerns the FVIIa assay. This test is rarely used. It is also cumbersome and expensive, and its significance in hematological and internal medicine clinical practice is very limited, if any. Needless to say, it may be important for special studies.[16]

Because Seminars in Thrombosis and Hemostasis is intended both for clinical and laboratory experts, we thought these comments might be of some practical interest.

REFERENCES

• 1 Mariani G, Bernardi F. Factor VII deficiency.  Semin Thromb Hemost. 2009;  35(4) 400-406
• 2 Girolami A, Fabris F, Dal Bo Zanon R, Ghiotto G, Burul A. Factor VII Padua: a congenital coagulation disorder due to an abnormal factor VII with a peculiar activation pattern.  J Lab Clin Med. 1978;  91(3) 387-395
• 3 James H L, Girolami A, Hubbard J G, Kumar A, Fair D S. The dysfunction of coagulation factor VIIPadua results from substitution of arginine-304 by glutamine.  Biochim Biophys Acta. 1993;  1172(3) 301-305
• 4 Girolami A, Sartori M T, Steffan A, Fadin M. Recombinant thromboplastins is slightly more sensitive to factor VII Padua than standard thromboplastins of human origin.  Blood Coagul Fibrinolysis. 1993;  4 497-498
• 5 Croze M, Brizard C P. Factor VII Padua 1. Another case.  Haemostasis. 1982;  11(3) 185-188
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• 7 Takamiya O, Funahashi S, Kinoshita S, Yoshioka A, Yoshioka K, Ikawa H. Factor VII activity and antigen in a patient with abnormal factor VII.  Clin Lab Haematol. 1988;  10(2) 159-165
• 8 Triplett D A, Brandt J T, Batard M A, Dixon J L, Fair D S. Hereditary factor VII deficiency: heterogeneity defined by combined functional and immunochemical analysis.  Blood. 1985;  66(6) 1284-1287
• 9 Wulff K, Herrmann F H. Twenty two novel mutations of the factor VII gene in factor VII deficiency.  Hum Mutat. 2000;  15(6) 489-496
• 10 Rodrigues D N, Siqueira L H, Galizoni A M, Arruda V R, Annichino-Bizzacchi J M. Prevalence of factor VII deficiency and molecular characterization of the F7 gene in Brazilian patients.  Blood Coagul Fibrinolysis. 2003;  14(3) 289-292
• 11 Bernardi F, Castaman G, Pinotti M et al.. Mutation pattern in clinically asymptomatic coagulation factor VII deficiency.  Hum Mutat. 1996;  8(2) 108-115
• 12 O’Brien D P, Gale K M, Anderson J S et al.. Purification and characterization of factor VII 304-Gln: a variant molecule with reduced activity isolated from a clinically unaffected male.  Blood. 1991;  78(1) 132-140
• 13 Etro D, Pinotti M, Wulff K et al.. The Gly331Ser mutation in factor VII in Europe and the Middle East.  Haematologica. 2003;  88(12) 1434-1436
• 14 Owren P A. The interrelationship between Normotest and Thrombotest.  Farmakoterapi. 1969;  25 1-13
• 15 Girolami A, Brunetti A, Patrassi G M. Normotest–thrombotest discrepancy in congenital coagulation disorders of the prothrombin complex and in coumarin-treated patients: a nonspecific phenomenon.  Am J Clin Pathol. 1977;  67(1) 57-60
• 16 Girolami A, Scandellari R, Scapin M, Vettore S. Congenital bleeding disorders of the vitamin K-dependent clotting factors.  Vitam Horm. 2008;  78 281-374