Studies on the inheritance and nature of hemophilia BM

Studies on the Inheritance of Hemophilia J. J. TWOMEY,M.B., J. CORLESS, Houston, Texas

M.D.,

L.

El Paso, Texas

THORNTON,

and Nature B,* M.D.

and C. HOUGIE,

M.D. i

Seattle, Washington

The family of two brothers who have hemophilia B,, namely, factor IX deficiency and a concomitant early one-stage inhibitor, was studied. Both defects were demonstrable at levels compatible with a heterozygous state in the mother and maternal grandmother. Normal factor IX and the inhibitor shared similar physicochemical properties which included temperature and pH stability, and chromatographic and density gradient characteristics. It is suggested that both defects are inherited as a single defect which is genetically distinct from classic Christmas disease. The inhibitor, whose activity demonstrated species specificity, is probably an altered inactive form of factor IX and is found in plasma from about 14 per cent of patients with Christmas disease.

TUDIESon hemophilia and von Willebrand’s disease [I,21 indicate that factor VIII deficiency can result from more than a single genetic defect. There is also evidence to suggest that Christmas disease (synonyms: hemophilia B, PTC deficiency and congenital factor IX deficiency) may be genetically heterogenous. Fantl et al. [3] described a substance in plasma from some patients who have Christmas disease but not in others that inactivated factor IX antibodies. A patient studied by Stocker [4] was considered to have a congenital inhibitor rather than a lack of factor IX. This inhibitor had adsorption properties similar to factor IX. Christmas disease has also been associated with deficiencies of other vitamin K dependent factors [5,6]. However, concomitant deficiencies of other clotting factors in Christmas disease may be spurious, as these results may reflect an effect of factor IX deficiency upon the assay rather than actual deficiency of other factors [7]. These reports also do not exclude chance coexistence of genetically unrelated defects.

A new type of Christmas disease in which factor IX deficiency is associated with an inhibitor of one-stage prothrombin times has recently been identified [8,9]. The one-stage abnormality was considered an inhibitor on the basis of the following evidence: (1) Prothrombin times remained prolonged after one part of patient plasma was diluted in nineteen parts of normal plasma. (2) The logarithmic relationship between prothrombin times and percentage concentrations of normal plasma mixed with patient plasma was not linear. A linear relationship would be expected if patient plasma lacked a clotting factor. [IO]. (3) The inhibitor could be removed from patient plasma by adsorption with AI(O We have called this new syndrome hemophilia B,, M bein? the surname initial of the affected family that we studied [8]. This report presents further studies on hemophilia B,,. Coagulation and biochemical genetic studies on the M kindred are described. Some physicochemical properties of the onestage inhibitor present in M patient plasma

S

* From the Hematology Section, Veterans Administration Hospital and Baylor University College of Medicine, Houston, Texas: the Department of Medicine and Pediatrics, William Beaumont General Hospital, El Paso, Texas; and the Department of Pathology, University of Washington, Seattle, Washington. A part of this work appeared in abstract form in J. Clin. Invest., 46: 1125, 1967. Requests for reprints should be addressed to J. J. Twomey, M.B., Chief, Hematology Section, Veterans Administration Hospital, 2002 Holcombe Boulevard, Houston, Texas 77031. Manuscript received February 6, 1968. + Present address: Department of Pathology, University of California, San Diego, California. 372

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Hemophilia

i- 3

B,r--

II (1c~)ml~-etl with those of factor Ix in normal inlaid la. Factor I)\:deficient plasma from unreiated patients was also surveyed to determine ,.Ile incitlence of llemophilia B,, in Christmas

I

IiSZiS?.

1&1od was obtained by venipuncture through ZOgaugcb, disposable, nonsiliconized needles into clean, dry glassware using 0.1 RI trisodium citrate for anticoagulant, except for prothrombin times, for which 0.1 M sodium oxalare was used. Recalcification during clotting studies was with 0.025 M calcium chloride except for thrombotests (Nyegaard, Oslo, Norway) and rabbit brain one-stage prothrombin times (Warner Chilcott Laboratories, Richmond, Calif.). Thrush commercial reagents include calcium. All dilutions of plasma for coagulation studies were made rzith 0.05 nl imidazole (glyoxaline) buffer, pH 7.3. Kaolin partial thromboplastin time determinations (KPTT) (Hyland Laboratories, Los Angeles, Calif ) were performed as described by Proctor and Rapaport [II], using cephalin prepared from rabbit brain [12J. Standard methods were used for onestage prothrombin times, Stypven@ times [I3J, thrombotests [14] and fibrinogen quantitation 1151. Cephalin (Warner Chilcott Laboratories, Richmontl. Calif.) was ;added to Stypven time test systems. The factor VIII assay of Langdell et al. [Id] was lnodified by the addition of kaolin [II]. Onestage methods [17] were used to assay other clotting factors. A saline extract of human brain was used in the assays of factor VII and prothrombin. Prothrombin times were repeated, substituting saline extracts of fresh animal brain obtained from different species and human brain obtained at autopsy for tlte rabbit brain-calcium commercial reagent. In experiments in which the physicochemical properties of the inhibitor in patient plasma were compared with those of factor IX in normal plasma, the inhibitor could not be titrated in elution fractions because of dilution. The inhibitor, therefore, was measured from its effect upon one-stage prothrombin times in a test system containing ox brain, nornlal human plasma diluted 1 to 3 in buffer and test sample 181. In these experiments the fraction containing the highest level of factor IX was arbitrarily recorded as 100 per cent. Temperature stability was compared using 1 in 5 dilutions of test plasma in buffer; 0.4 ml. aliquots of test plasma titrated with 0.1 N hydrochloride or 0.1 N sodium hydroxide were used to compare pH St;*Ililitl. Sephadex@ G-200 (Pharmacea Fine Chemicals,‘Piscataway, N. J.) columns (31 by 1 cm.; 2.5 ml. flow rate per hour) were equilibrated with imida7ole buffer, pH 7.3, to which 1.3 gm. per cent sodium chloride had been added (0.22 RI final moIaritl). Sucrose was dissol\,ed in imida7ole huffcr VOL.

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1969

q

: Factor IX Deficient,

(--:

Corner

n

:

of Factor

Expremng IX Deficiency

4

3

2

I

inhibitor ond

(Mole)

Inhibitor

(Femle)

Deceased 1:K.. 1.

<;ct~e;~lo~v

of ttlc, Yl

family.

(pH 7.3) and -l.8 ml. 01 a IO to 20 per cent density gradient was prepared using a Buchler apparatus. A 0.4 ml. aliquot of a 1 to 2 saline dilution of test plasma was layered on to the density gradient. The tubes were then placed in a precooled SW39 (Spinco) Rotor and centrifuged in a Spinco Model L ultracentrifuge at 39,000 r.p.m. for seventeen hours, at which time the bottom of each tube was pierced with a needle and the contents collected in nine fractions. Red cell glucose-6-phosphate dehydrogenase (G-6PD) activity was measured using a modification [18J of Glock and McLean’s method [13J-normal range 9.44 to 13.55 moles reduced triphosphopyridine nucleotide (TPNH) per milliter of cells. G6-PD electrophoresis was performed as described by Kirkman and Hendrickson [20J with hemoglobin concentration adjusted to 0.2 gm. per 100 ml. Specific antiserum was used for Xga blood grouping. RESULTS

Clinical

and

Laboratory

Diagnosis.

Two

brothers (IV-~ and IV-~, Fig. 1) from the family under study required medical attention because of severe bleeding diatheses. An alleged identical twin (IV-~, Fig. 1) of the younger brother died from intracranial bleeding at the age of two years. There was no history suggestive of a bleeding diathesis among other members of the kindred. Pertinent coagulation studies are listed on Table I. The affected brothers’ greatly prolonged KPTTs, which were shortened by adding equal parts of normal serum but not by adding Al(OH), adsorbed plasma. supports the diagnosis of Christmas disease-as indicated by factor IX assays.

374

Hemophilia

BMTABLE

PERTINENT

COAGULATION

STUDIES

Twomey et al. I ON MEMBERS

OF THE

M FAMILY

I-2 II-2

IV-2

Study KPTT (sec.) Prothrombin Thrombotest Stypven time Factor assays

time (sec.) (sec.) (sec.) ( yO)

IX VII s V

Prothrombin Fibrinogen (mg. %)

IV-3

III-3

grandmother

.,.

.,.

‘54

‘41’

Patient

126 19 90 6.5

102 17 124 6.5

38 14 47

<2 70 99 >lOO

<2 75 80 >lOO >lOO 640

50 >lOO

63

>;oo

:::

...

.

35,

TABLE SPECIFICITY

OF THE

ONE-STAGE

IV-~ Half Brother

111-h

Father 42 14 39

40 13 41

.,.

>lOO >lOO 98

92 65 85

:::

>;00

Control 42 13 40 7

40-200 70-130 70-160 >250

.

in equal parts with normal plasma. It is therefore evident that these boys are deficient in factor IX and have a second defect involving the initial stages of one-stage clotting systems. Our studies did not directly implicate known extrinsic system clotting factors. The reasons summarized at the beginning of this paper indicate that the one-stage defect is caused by an inhibitor. Species Specificity of the Inhibitor. The greater sensitivity of the thrombotests than prothrombin times to the presence of this inhibitor was due to the use of ox rather than rabbit brain as the source of tissue extract [8]. One-stage studies were made on both siblings, two unrelated control patients who have Christmas disease, and five normal control subjects using saline extracts of brain obtained from six species, including man (Table II). The control patients’ thrombotest times were normal, although their factor IX assays were

The factor IX cont’ent of a mixture containing equal parts of normal and patient (IV-~, Fig. 1) plasma was 52 per cent and did not alt’er significantly after two hours’ incubation at 37”c., indicating that the patients’ plasma did not contain an inhibitor of factor IX. Prolongation of their prothrombin times (using rabbit brain) was not nearly as marked as the gross prolongation of their thrombotests. The siblings’ one-stage defect was not corrected with parenterally administered vitamin K and was not associated with overt liver dysfunction, as indicated by normal serum glutamic oxalacetic transaminase, alkaline phosphatase and thymol turbidity determinations. Normal Stypven times support the normal assays of factor x, v, prothrombin and fibrinogen, Factor VII assays were within normal limits, and equal parts of factor VII deficient and patients’ plasmas gave comparable thrombotest times to those obtained when patient plasma was mixed

SPECIES

Grandmother

Patient

Mother

Great-

II

INHIBTOR

ON PROTHROMBIN

Brain Thromboplastin

TIMES

(SEC.)

Used

Sheep

Rat

Man

ox

Dog

Pig

IV-~ (Fig. 1) rv-3 (Fig. 1) Control r Control n

85 62.7 38.4 38.2

48.2 46.2 34.5 37.7

49.2 45.0 34.0 33.6

44.5 40.5 29.3 29.1

45.9 46.5 35.5 40.2

34 30.5 30.2 30.3

Normal controls Mean SD.&

38.0 1.7

35.8 1.7

35.7 2.9

29.1 1.36

34.7 3.9

34.6 3.4

Patient

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(1 per cent and less than 2 per cent, respectively. l’rolonged clotting times were recorded for both siblings with each tissue extract except lmman brain, which gave normal results. The significance of this observation is presently not clear. Ox brain produced the longest times. Eoth control (factor IX deficient) patients had normal prothrom.bin times using tissue extracts from all six species. Phvsicochemical Properties of the Inhibitor and Factor IX. The inhibitor and factor IX were measured in hemophilia B, and normal plasma, respectively, after a half hour’s incubation at different temperatures (Fig. 2). Similar studies were performed after thirty minutes of incubation over a wide pH range at 23”~. (Fig. 3). The results indicate that the inhibitor and normal factor IX have similar ranges of temperature and pH stability. Both were inactivated following incubation for thirty minutes .u 56”~. and pH 3. Tire inhibitor and factor IX were compared by Sephadex G-200 filtration and by 10 to 20 per cent sucrose gradient ultracentrifugation. Total recovery of factor IX activity from chromatographic eluates of normal plasma was about 33 per cent; there was 50 per cent recovery from sucrose gradients. Figures 4 and 5 indic.ate that the inhibitor and normal factor CChave similar filtration and centrifugational properties, which suggests that both substances are of approximately similar molecular size. Genetic Studies. The results are listed in Table I. The siblings’ mother and maternal grandmother (III-~ and 11-2, Fig. 1) had borderline levels of factor IX (50 and 63 per cent, respectively) and slightly but significantly prolonged thrombotests (forty-seven seconds and fifty-four seconds, respectively)-the normal range for the thrombotest is thirty-five to fortyfive seconds. Their one-stage defects were more obvious when prothrombin times using ox brain were measured in systems in which a 1 to 5 dilution of test plasma was mixed in equal parts with a 1 in 3 dilution of normal pkiSJna in imidazole buffer [8]. Factor IX assays and one-stage studies were within normal limits for the maternal greatgrandmother, father, and maternal half brother (1-2, III-~, IV-~, Fig. 1) of the propositi. The status of other known characteristics inherited in sex-linked fashion were studied in both M homozygotes because their coagulaVOL

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1969

01, 0

I

IO

I

20

I

30

I

40 EC

OC.

FIG. 2. Temperature stability of factor IX from normal plasma (A) and the one-stage inhibitor from M homozy gote plasma (0) .

3. pH stability of factor IX from normal plasma (A) and the one-stage inhibitor from M homozygote plasma (a) . FIG.

2

6

IO TUBE

18 14 NUMBER

22

26

FIG. 4. Factor IX and inhibitor activities measured on Sephadex G-200 column eluate fractions from normal and M homozygote plasmas.

Fraction

number

Fm. 5. Sucrose density gradient of factor IX from normal plasma (A) and the inhibitor from M homozygote plasma (0).

Hernophilia

376

B,- - 7’zuome~el a/.

TABLE In STUDIESOF KNOWNX CHROMOSOME PHENOTYPICFUNC’TION ON BOTH M FAMILY HOMOZYGoTES P.S.M.

Study

Xga blood group + 12.43 G-6-PD assay (MlO-9TPNH/ml.) B G-6-PD electrophoresis Normal Deutan color vision >lOO Factor VIII assay (70 normal plasma)

M.M.M. + 12.15 B Normal >lOO

tion defect differed from that ia classic Christmas disease. Both siblings’ red cells demonstrated Xga antigenicity and quantitatively and electrophoretically normal G-6-PD (Table III). The older boy had normal deutan color vision on testing with Dvorine plates; his younger brother could distinguish red from green threads. Factor VIII #assayswere normal. Epidemiologic Survey. Patients with congenital factor IX deficiency from eleven kindreds were surveyed to determine the approximate incidence of hemophilia B,,. It is evident from the data in Table IV that the thrombotest is insensitive to factor IX deficiency but yielded results similar to one-stage prothrombin ti,mes using tissue extract derived from ox brain. Both the thrombotest and ox brain proTABLE Iv HEMOPHILIABP AMONGPATIENTSWITH CHRISTMAS DISEASE FROM ELEVEN KINDREDSSTUDIEDBY THE AUTHORS

Kindred 1 (propositi) a b 2* 3t 4t 5 6 7 8 9 10 11 Control

Factor IX (% Activity)

<2 <2 <2 <2 <2 <2 <2 <2 <2 <2 6 10

Thrombotest (sec.) 124 90 122 110 82 51 46 42 41 42 42 39 38-46

Ox-Brain Prothrombin Time (sec.) 132 97 192 158 127 45 43 42 40 36 37 37 36-52

* Patient of G. R. Gray, M.D., Vancouver General Hospital, Vancouver, B. C. 7 Selected patients of C. K. Kasper, M.D., University of Southern California, Los Angeles, Calif.

thrombin times clearly indicate the presence of hemophilia BA1in five patients from four of the eleven kindreds that we studied. The borderline thrombotest on the fifth kindred’s patient was not supported by a prolonged ox brain prothrombin time. Eighteen more unrelated patients who were studied by C. K. Kasper, M.D., at the University of Southern California, did not have hemophilia Bhl. Thus, hemophilia B,, was demonstrated in patients with Christmas disease in four of twenty-nine kindred. COMMENTS It is generally accepted that the reaction product(s) of factor VII and tissue extract activate factor x which then, in the presence of phospholipid and factor v, activates prothrombin [21]. The factors v, VII, x and prothrombin assays on both M propositi were normal. Alternatively, factor x can be activated by Russell’s viper venom. The propositi’s normal Stypven times provides additional evidence that the abnormality occurs proximal to the involvement of factor x. In the presence of normal factor VII activity, the only reasonable hypothesis based on currently accepted theory of extrinsic clotting is that the inhibitor interferes with the reaction between factor VII and brain. Our studies indicate that tissue activity is intimately involved in the abnormality, and suggest that tissue extracts derived from different species may participate differently in the initial steps of one-stage clotting systems. Factor IX deficiency is not associated with a concomitant one-stage inhibitor in classic Christmas disease. Borderline levels of factor IX activity and slightly prolonged thrombotests, as well as one-stage prothrombin times using ox brain tissue extract in dilute normal plasma [8], indicate that low grade manifestations of both abnormalities were present in grandthe siblings’ mother and maternal mother. These findings suggest that the affected siblings inherited both factor IX deficiency and the inhibitor in sex-linked recessive fashion (Fig. 1). Failure to detect intermediate levels of factor IX or inhibitor in their maternal ,greatgrandmother, in the absence of a known bleeding diathesis in her spouse, is at variance with this pattern of inheritance. There are a numAMERICAN

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IXY of possible ~explanations for this discrepXII(‘): (1) The maternal grandmother may be 111~’illegitimate claugllter of an aff’ected male 01’ rc presents the point of genetic mutation : ‘21. (2) The maternal greatgrandmother may icrve transmitted both defects in occult fashion. \orn~al and slightly depressed levels of factor I\ arcs found among obligatory female carriers I)L c,lassic Christmas disease occurring in a I)imotlal distribution 1233. This inconsistent (‘xprt,ssion of heterozygosity may reflect limitations of currently available assay technics in cl::tec ting slight abnormalities. It is also t oml)atible with the Lyon hypothesis of sexlinks tl inheritance [24] which postulates that orle sex chromosome in each female cell is repl.c.sellted as a functionally defective Barr body. I‘he percentage of sex chromosomes carrying tlefec ti1.e genes that are randomized as Barr I)odics may be c:ritical for phenotypic expresbion of subtle abnormalities associated with IleterozygoL]s expression of sex-linked recessive tlyfet ts S~IC-has hemophilia B,,. Siiuilar ph)sicocllemical properties of nol~n~al factor IX and the one-stage inhibitor stlpport bioch:rnical genetic evidence that an :IS~O(iation exists between factor IX deficiencv ;tnd the inhibitor. These physicochemica’l similarities include adsorption with aluminum hydroxide [8], temperature and pH stability, and also chroma tographic and density gradient char‘lcteristics. Although these properties are indi\.idually shared by numerous biologic ~nol~~cules, the over-all findings suggest that normal factors IS and the inhibitor are molecularly si:nilar. The inhibitor may, in fact, be an altrred factor IS molecule, and as such be inherited as the same genetic defect in these patients that causes factor ix deficiency. Hemophilia B,, appears to be genetically distinct from rlassic Christmas disease. Classic Christmas disease has not been linked genotypit.ally with X chromosome loci that regulate ?(%I blood group antigenicity, G-6-PD or Iremophilia A [25] but may be proximal to a locus regulating deutan color vision [26]. We l~erca unable to demonstrate linkage between the tlefective hemophilia BII gene and dysfunction of other known sex-linked traits. Demonstration of an altered factor IX molecule has profound implications for the inIleritance and pathophysiology of Christmas tliseilse. It substantiates the suggestion of Fantl

ct al. [J] that hemophilia 1~ is heterogcncous. Detct tioll ol’ this molecule depends U~OII its ability to inhibit one-stage clotting tests. Other hemophilioitl disorders may also result Iro111 inolec ular ;Ilteration analogous to the h(~noglobinopathies but currently lack identification. Oui, lindings, however, do not rule out the widely held concept that an inherited lntk of factor activity results from a failure of factor synthesis [2$] in some instances of Christmas disease. identification of an inherited I~OIIantibody inhibitor of coagulation is a10 :I lnost unusual observation [28]. There is now substantial evidence [29,30], which is supported by the present study, that Owrcn’s thrombotest is insensitive to factor IX deficiency. However, our results and those of others [?I] indicate that thrombotes1.s in some patients who have Christmas disease ale considerably prolonged. We believe that prolonged thrombotests in some patients who have Christmas disease represent the presence of hemophilia I)>, and relates to the use of ox brain in the reagent. This choice of tissue extract fortuitously makes the thrombotest useful in the diagnosis of hemophilia BJI. Our preliminary survey, when combined with the c’xperiences of Gray and Kasper, suggests that about 1.I per cent of kindreds expressing Christmas disease have hemophilia B,,. Simple one-stage screening studies that use ox brain [8] should also prove valuable in detecting felnale carriers of hemophilia BII. ADDENDUM Since this manuscript was submitted for publication, Roberts et al. [32] have identified a substance in plasma from 10 per cent of patients who have Christmas disease that inactivates a circulating factor IX inhibitor in a fashion similar to normal factor IX. Their findings substantiate an earlier report by Fantl et al. [?I. This substance, which appears to be genetically determined, has an incidence among patients who have Christmas disease which approximates our estimate of the incidence of hemophilia B,. Denson et al. [33] have also identified a substance in plasma from patients whose laboratory data are compatible with the diagnosis of hemophilia B,,, that is antigenically indistinguishable from normal factor IX.

Hemophilia

&a-Twomey

Acknowledgment: We are indebted to C. K. Kasper, M.D., University of Southern California, Los Angeles, California, and G. R. Gray, M.D., Vancouver General Hospital, Vancouver, B. C., who have generously provided us with plasmas from their patients who have Christmas disease. We are also indebted to W. L. Long, M.D., University of Texas, Austin, Texas, for performing the G-6-PD studies; to Doctors R. R. Race and R. Sanger, Lister Institute, London, England, for Xg* blood grouping; and Mrs. Wilma Hudson and Mrs. Polly Schmidt, respectively, for valuable technical and secretarial assistance provided.

et nl.

14. OWEN, P. A. Thrombotest; a new method for controlling anticoagulant therapy. Lance& 2: 754, 1959. 15. PARFENTJEV,I. A., JOHNSON,M. L. and CLIFF~OI\‘, E. E. The determination of plasma fibrinogen by turbidity with ammonium sulfate. Arch. Biothem., 46: 470,1953.

16. LANGDELL,R. D., WAGNER,R. H. and BIUNKHOUS, K. M. Effect of antihemophilic factor on one-stage clotting tests: a presumptive test for hemophilia and a simple one-stage antihemophilic factor assay procedure. J. Lab. & Clin. Med., 41: 637, 1953. 17. DENSON,K. W., B~r:c:s,R. and MACFARLANE,R. G. Treatment of Haemophilia and Other Coagulation Disorders, p. 337. Philadelphia, 1966. F. A. Davis Co. 18. LONG, W. K., KIRKMAN,H. N. and SUTTON,H. E. Electrophoretically slow variants of glucose-6REFERENCES phosphate dehydrogenase from red cells of Ne1. BARROM’, E. M., HEINDEL,C. C., ROBERTS,H. R. and groes. J. Lab. & Clin. Med., 65: 81, 1965. GRAHAM,J. B. Heterozygosity and homozygosity 19. GLOCK, G. E. and MCLEAN, P. Further studies on in von Willebrand’s disease. Proc. Sot. Exber. the properties and assay of glucose 6-phosphate I Biol. & Med., 188: 684, 1965. dehydrogenase and 6-phosphogluconate dehydro2. PRENTICE,C. R. M. and RATNOFF, 0. D. Genetic genase of rat liver. Biochem. j., 55: 400, 1953. disorders of blood coagulation. Sem. Hematol., 4: 20. KIRKMAN,H. N. and HENDRICKSON, E. M. Sex-linked 93, 1967. electrophoretic difference in glucose-6-phosphate 3. FANTL,P., SAWERS,R. J. and MARR, A. G. Investigadehydrogenase. Am. J. Human Genet., 15: 241. tion of haemorrhagic disease due to beta-pro1963. thromboplastin deficiency complicated by a spe21. BARTON,P. G., JACKSON,C. M. and HANAHAN,D. J_ cific inhibitor of thromboplastin formation. AusRelationship between factor v and activated factralasian Ann. Med., 5: 163, 1956. tor x in the generation of prothrombinase. Nn4. STOCKER,H. A congenital inhibitor against factor ture, 214: 923, 1967. IX. Thromb. et Diath. Haemorrh., 14: 346, 1965. 22. IKKALA,E. Haemophilia. A study of its laboratory, 5. VERSTRAETE, M., VERMYLEN,C. and VANDENBROUCKE, clinical, genetic and social aspects based on known J. Hemophilia B associated with a decreased fachemophiliacs in Finland. Scandinav. J. Clin. & tor VII activity. Am. J. M. SC., 243: 20, 1962. Lab. Invest., 12 (supp.): 46, 1960. 6. OWEN, C. A., JR., AMUNDSEN, M. A., THOMPSON,J, H., 23. BARROW,E. M., BULLOCK,W. R. and GRAHAM, J. B. JR., SPITTELL,J. A., JR., BOWIE,E. J. W., STILWELL, A study of the carrier state for plasma thromboG. G., HEWLETT,J. S., MILLS, S. D., SAUER,W. G. plastin component (PTC, Christmas factor) deand GAGE, R. P. Congenital deficiency of factor ficiency, utilizing a new assay procedure. j. Lab. & VII (hypoconvertinemia); critical review of literaClin. Med., 55: 936, 1960. ture and report of three cases, with extensive 24. LYON, M. F. Gene action in the X-chromosome of pedigree study and effect of transfusions. Am. J. the mouse (Mus musculus L.) Nature, 190: 372, Med., 37: 71, 1964. 1961. 7. SHANBERGE, J. N. and MATSLJOKA, T. Studies regard25. HARRISON, J. F. Haemophilia, Christmas disease and ing the effect of foreign-surface contact on the the Xg blood groups; observations based on the one-stage prothrombin time determination. haemophiliacs of Birmingham. Brit. J. Haemat., Thromb. et Diath. Haemorrh., 15: 442, 1966. 10: 115, 1964. 8. HOUCIE, C. and TWOMEY,J. J. Haemophilia B,: a 26. WALL, R. L., MCCONNELL, J. L. and MOORE, 1). new type of factor IX deficiency. Lancet, 1: 698, Christmas disease, duetan color blindness, and the 1967. Xg” blood group in the Amish. J. Lab. & Clin. 9. GRAY, G, R., TEASDALE,J. M. and THOMAS, J. W. Med., 64: 1015, 1964. Hemophilia B,. Canad. M. A. J., 98: 552, 1968. 27. ROBERTS,H. R., GRO& G. P., WEBSTER,W. P., 10. MARGOLIS,J. and BRUCE, S. An experimental apDEJANOV,I. I. and PENICK,G. D. Acquired inhibiproach to the kinetics of blood coagulation. Brit. tors of plasma factor IX; a study of their inducj. Haemat., 10: 513, 1964. tion, properties and neutralization. Am. I. M. SC., 11. PROCTOR,R. R. and RAPAPORT,S. I. The partial 251: 43, 1966. thromboplastin time with kaolin: a simple screen28. MARGOLIUS, A., JR., JACKSON, D. P. and RATNOFF, ing test for first stage plasma clotting factor de0. D. Circulating anticoagulants: a study of 40 ficiencies. Am. J. Clin. Path., 36: 212, 1961. cases and a review of the literature. Medicine, 40: 12. BELI,, W. N. and ALTON,H. G. A brain extract as a 145, 1961. substitute for platelet suspensions in the thrombo29. QUICK, A. J. and HUSSEY,C. V. Comparison of the plastin generation test. Nature, 174: 880, 1954. thrombotest with the one-stage prothrombin 13. MIALE, J. B. Laboratory Medicine-Hematology, time. New England J. Med., 265: 1286,196l. 2nd ed., p. 858. St. Louis, 1962. C. V. Mosby Co. AMERICAN

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70. 2 )I.I.I\~(.I.K, \V. Comparative study of control metb ads for anticoagulant therapy. In: Thrombosis and Anticoagulant Therapy, p. 42. Edited by \Valkcr. W. Baltimore, 1961. Williams & Wilkins co. 31. hIDo, I’., DEKSO,N, K. \\‘. and BKGS, R. The thrombotest wagent :and Christmas disease. Lancel, 2: 522.

1963.

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32. ROBIKIS. 1% K., GRIZZLE, J. E.. M~~.I~IIR. \V. i). and I’I:NIcli, C;. II. Genetic \alMlls ol hemophilia 13: detection by means of a specific P’I‘C inhibitor. I. Cliw. Invest., 4T: 360. l!Ni% 33. 111 ASOX, IL W. E., BIGGS, R. and MANNUCCI, P. VI. An investigation of three patients with Christmas disease due to an abnormal tape of factor IX. J. Clin. Path., 21: 160. 1968,