Improved hemophilia A carrier detection by DDAVP stimulation of factor VIII

Comment: Many readers will find this paper, especially the Results section, to be rather heavy going. Nevertheless, the improved accuracy in identifying carriers of hemophilia A is of considerable importance and will allow better genetic counseling of young female relatives of males with hemophilia.--J.M.G.

Improved hemophilia A carrier detection by DDAVP stimulation of factor VIII II'e studied the differential increase in FVIIIc attd FVIII R:dg after the intravenous infusion o f O.30 r DD,,| liP in 20 obligate hemophilia ,4 carriers and in 20female controls. FVIIlc increased in carriers (59.5 • 23.1 to 137.5 -4- 45.9) and in controls (98.0 • 20,7 to 259.9 • 57.4) (P < 0.001), but the magnitude o f the FVIIIc increase in carriers was less than that in controls b)" 51.9% (P < 0.001). FVIII R:Ag increased comparably in carriers (105.2 • 30.4 to 171.9 -4- 25.4) attd controls (92.1 + 33.0 to 165.2 • 20.6). Using the post-DDA VP instead o f the standard FVIIIc/FVlll R:Ag ratio, hemophilia carrier detection was increased f r o m 85% (with 10% false positive and 20% false negative assignments) to 95% (with 5% false positive attd 5% false negative assigmnents). Toxicity associated with DDA VP infusion correlated linearly with doses >10.5 +. 1.3 pg/m 2 (P < 0.02) and with total doses >17.0 +_ 4.5 lag (P < 0.02). The use o f DDA VP improves carrier detection in factor VIII-deficient hemophilia. (J PL'OIATR i04:718, 1984)

Nathan L. Kobrinsky, M.D., Catherine M. Watson, B.N., Mary S. Cheang, M.Math., and Agnes J. Bishop, M.D. Winipeg, Man., Canada

TIlE ACCURATE ASSIGNMENT OF GENOTYPE to possible carriers of hemophilia A remains a major problem? Present carrier detection methods rely on discordance between factor VIII coagulant and factor VIII related antigen concentrations, as assessed by the FVIIIc/FVIll R:Ag ratio? Using this method to classify carriers, 86.7% correct assignments with 7.7% false positives and 25.0%

From the Department o f Pediatrics and the Computer Department for the tlealth Sciences, Faculty o f Medicine. University o f Manitoba; the Departments o f Pediatrics attd Nursing. Children's Ilospital o f 1Vimdpeg; attd the Manitoba Cancer Treatment and Research Foundation. 9Supported by the Manitoba Chapter o f the Canadian ttemophilia Society attd b)' the Children's llospital o f Winnipeg Research Fottndation. Presente'd in part at the ,-lnnual Meeting o f the Society for Pediatric Research, Washington. D.C.. May 4, 1983. Reprint requests: Nathan L. Kobrinsk.l', M.D.. Manitoba Cancer Treatment attd Research Foundation. I00 Olivia St., IVinnipeg. Man.. Canada R3E OV9.

7 18

The Journal o f P E D I A T R I C S

false negatives have been reported. 3 Classification rates as low as 73% have been reported in earlier series? A more accurate method of carrier detection would be of considerable value. It has been recently shown that DDAVP, a synthetic derivative of vasopressin, produces a marked though tranDDAVP FVlllc FVIII R:Ag FVIII:RCoF 6-oxo-PGF~, PG 12

l-Desamino-8-o-arginine vasoprcssin Factor VIII coagulant activity Factor VIII related antigen [:actor Vlll ristocetin cofactor Prostagladin 6-oxo-F~ Prostacyclin

sient increase in FVIIIc and FVIII R:Ag after intravenous infusion in healthy subjects?7 In mild or moderately affected patients with hemophilia A (>5% FVIIIc) the absolute increase in FVIIIc is limited, though the proportionate increase is maintained?"~~~ In severely affected patients (<5% FVIIIc), the FVIIIc response is virtually

Volume 104 Number 5

absent." In contrast, the FVIII R:Ag response is normal in both groups. Based on these observations, we hypothesized that carriers of hemophilia A may similarly demonstrate a limited FVIIIc but intact FVIII R:Ag response to intravenously administered DDAVP and that this discordant response might improve carrier detection. We describe the FVIIIc and FVIII R:Ag responses to intravenously administered DDAVP in a group of obligate carriers and healthy female controls. Further, we evaluate the discriminant potential of DDAVP-induced FVIIIc and FVIII R:Ag perturbation in hemophilia A carrier detection.

Hemophilia A carrier detection

7 19

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METHODS Subjects. Twenty obligate (mean age 35.8 ___ 13.9 years, 9 range 6 to 62 years) and five possible (mean age 29.0 +-16.7 years, range 11 to 57 years) carriers of factor Vlll~leficient hemophilia and 20 healthy female controls (mean age 32.2 - 8.1 years, range 2I to 46 years) were the subjects of the present study. Obligate carriers had either ( 1) father, (2) a son and at least one other relative (brother or maternal uncle), or (3) two or more sons with hemophilia. Possible carriers had either (1) an extensive maternal family history of hemophilia, (2) one affected son but no other known affected relatives, or (3) one affected brother with or without other affected relatives (excluding sons). Subjects with known hypertension or other systemic illnesses, as well as those receiving any medication, were excluded from study. Recent use of acetylsalicylates or use of orally administered estrogens did not disqualify subjects, but this information was recorded. Approval for the study was obtained from the University of Manitoba, Faculty of Medicine, Committee for the Use of Human Subjects in Research. All subjects were aware of the experimental nature of the investigations performed and gave informed consent in accordance with the Declaration of Helsinki. DDAVP administration. The DDAVP (0.30 pg/kg) (courtesy Ferring, A.B., Malmo, Sweden) was diluted to a final concentration of 0.5 /~g/ml in physiologic saline solution and infused intravenously over 20 minutes. Blood pressure was monitored over 1 hour; urine specific gravity determinations were performed before and I hour after DDAVP administration. A symptomatic toxicity score was assigned according to the following scale: O, no toxicity; !, mild toxicity (facial flushing); 2, moderate toxicity (facial flushing, transient lightheadedness, headache); 3, severe toxicity (facial flushing, prolonged [several hours] or marked [requiring analgesia] headache or backache). Blood collection and plasma preparation; Blood samples were collected by venipuncture through a 21-gauge needle

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0 160 200 3()0 FACTORVIIIRELA'W=D ANTIGEN(%of Normal) Fig. I. Changes in FVIII R:Ag and FVIIIc with DDAVP infusion in obligate carriers of hemophilia A and in female controls. Solid DDAVP response lines indicate changes in (FVIll R:Ag, FVIIlc) after intravenous infusion of 0.30 /~g/kg DDAVP for each of 20 obligate carriers of hemophilia A (top) and 20 healthy female controls (bottom). Dashed identity lines indicate FVIIIc/ FVlll R:Ag = 1.

before infusion and 60 minutes after the infusion was completed. Nine parts blood were added to plastic tubes containing one part acid citrate (two parts 0.1M citric acid and three parts 0ol M sodium citrate). Platelet-poor plasma was obtained after centrifugation at 2000• g for 10 minutes at 4~ and used fresh or kept frozen at - 2 0 ~ until tested. Assay methods. FVIIIc was assayed in fresh samples by a one-stage method based on the partial thromboplastin

7 20

Kobrinsky et al.

The Journal of Pediatrics May 1984

Table I. Response of FVIIIc and FVIII R:Ag to DDAVP infusion in obligate carriers of hemophilia A and in female controls

Variable FVIIIc Pre-FVIIle Post-FVIIle AFVIIIc A|:VIIIc/prc-FVIIIc [:VIII R:Ag Prc-FVIII R:Ag Post-FVIII R:Ag AFVIII R:Ag AFVIII P,:Ag/Prc-FVIII R:Ag

[

Controls

Carriers

98.0 259.9 162.0 1.71

+__20.7 __. 57.4 __. 50.1 "+ 0.60

59.5 137.5 78.0 1.41

___23.1 + 45.9 _+ 36.6 _ 0.76

92.1 165.2 73.2 1.01

--. 33.0 "4- 20.6 + 17.6 -+. 0.74

105.2 _ 30.4 171.9 -+- 25.4 66.6 --. 17.8 0.75 +-- 0.52

[

P <0.001 <0.001 <0.001 NS NS NS NS NS

Results expressedas mean • SD. NS, No significantdifferencebetweencarrier and conlrolgroups.

time using factor VIII-deficient plasma (Pacific llemostasis) as substrate. ~-"FVIIIe concentrations were expressed as percent of pooled normal plasma (nine male and 14 female donors) assigned a value of 100%. FVIII R:Ag was assayed by quantitive immunoelectrophoresis t3 using a commercial monospecific antiserum (Clottimmune-AHG-associated protein, Behringwerke). Statistical methods Univariate attalysis. Pre-FVlllc, post-FVlllc, preFVIII R:Ag, post-FVIII R'Ag, and various mathematical functions of these four variables were compared between obligate carriers and controls using independent t tests. When it could not be assumed that the variances of the two groups were equal, separate rather than pooled variance estimates were used. Multivariate analysis. Discriminant function analysis ~4~ was performed individually and on subsets of the above variables to maximize the differences between obligate carriers and controls. Some of the variables had to be transformed to meet the assumption of homogeneous variances in the two groups. For the most predictive variables and subsets, individual subjects were assigned to either carrier or control groups, and the probability of membership calculated according to the proximity of their discriminant scores from the group centroids (or points of maximum clustering). RESUI~TS

DDAVp response. In general, DDAVP response lines between (pre-FVlll R:Ag, pre-FVlllc) and (post-FVlll R:Ag, post-FVlllc) were above the identity line (FVIIIc/ FVII! R:Ag I) for controls and below the identity line for carriers (Fig. 1). Control response lines were steeper (AFVIIIc/AFVIII R:Ag) and longer (AFVIIIc: + AFVIII R:Ag") ~- than carrier response lines. Carriers of mild and severe hemophilia were not differ-

ent in pre- and post-FVlllc, pre- and post-FVlll R:Ag, AFVIIIc, and AFVIII R:Ag. FVIlIc. Before DDAVP infusion, FVIIIc was lower in obligate carriers than in controls (P < 0.001). After infusion, although FVIIIc increased in both groups (P < 0.001), the level achieved by carriers was lower than that in controls by 47.1% (P
Volume 104 Number 5

Hemophilia A carrier detection

721

Table II. Accuracy of hemophilia A carrier detection using various mathematical functions of FVIIIc and FVIII R:Ag with and without DDAVP Variable

Controls

Carriers

Correct assignment (%)

Post-(FVIllc/FVIII R:Ag) Pre-(FVIIIc/FVIII R:Ag) AFVilIc [AFVIIIc2-AFVIil R:Ag2]~ In[(AFVIIIc/FVIII R:Ag)] AFVIIIc/Pre-FVIIIc

1.58 __. 0.31 1.16 __. 0.34 162.0 -L-_50.1 179.3 +__47.1 0.78 __+ 0.36 1.71 _+_ 0.60

0.81 __. 0.30 0.60 + 0.23 78.0 _ 36.6 104.6 __. 34.9 0.08 _ 0.44 1.41 +_. 0.76

95.0 85.0 80.0 80.0 77.5 67.5

Results expressed as mean "4- SD. Assignments based on discriminate functions calculated for each variable.

The most powerful predictor was found to be post( F V I l l c / F V I I I R:Ag), described by the discriminant function: D2, = 3.289 • post-(FVIIIc/FVlll R:Ag), - 3.932 where D2~ represents the discriminant score for subject i with a post-(FVlllc/FVlll R:Ag) ratio of post-(FVlllc/ FVllI R:Ag)I. Again, individual discriminant scores were calculated and subjects assigned to either control or carrier groups based on their distance from the group centroids (__. i.261). Control subjects were correctly assigned in 19 of 20 cases (5% false positives). Carriers were correctly assigned in 19 of 20 cases (5% false negatives). Overall correct assignment was 38 of 40, or 95%. Combinations of individual variables did not improve on the predictive power of post-(FVlllc/FVlll R:Ag) alone (Fig. 2).

Assignment of possible carriers. Group assignments and probability scores for five possible hemophilia carriers were calculated using traditional (DI) and DDAVP derived (D2) functions. Assignments were the same in four of the five subjects. In one, the assignment was changed from carrier ( P = 0 . 6 4 ) to noncarrier ( P = 0 . 8 5 ) . In two others, although assignment to the noncarrier group was unchanged, the probability of correct assignment was increased from P = 0.83 to P > 0.99 in one and from P = 0.69 to P = 0.91 in the other. In the remaining two subjects, both assignment and probability of correct assignment were unchanged. Side effects. In general, the DDAVP infusion was well tolerated. Fifteen subjects (33.3%) were completely asymptomatic. Thirty (66.7%) developed facial flushing during or shortly after the infusion. Nine (20.0%) developed transient lightheadedness or a mild headache. Five (11.1%) developed significant side effects including prolonged or severe headache or (in one patient) backache. In this latter group, symptoms lasted as long as 48 hours.

Toxic effects did not correlate with age or clinical group. Although all subjects received the same weight-adjusted dose (0.30 tag/kg), toxicity did linearly correlate with total dose (P < 0.02) and with body surface area adjusted dose (P < 0.02). Subjects with severe toxic effects received a mean total dose of 24.0 +__8.7 tag or a surface area adjusted dose of 12.6 _+ 2.9 tag/m 2. Asymptomatic subjects received a mean total dose of 17.0 +_ 4.5 tag or a surface area adjusted dose of 10.5 +- 1.3 tag/m 2. A slight decrease in both systolic (P <0.005) and diastolic (P < 0.001) blood pressure with no net change in pulse pressure was observed immediately after infusion (i15__+ 16/73___ 11 mg Hg to 111 ___ 18/67 ___ 12 mm Hg.). At 1 hour after infusion, both systolic (P = 0.012) and diastolic (P = 0.001) pressures remained decreased compared with preinfusion levels (1 ! 1 _+_ 16/70 +__ 10 mm llg.), although the diastolic pressure tended toward baseline (P = 0.036), with a resultant slight narrowing of pulse pressure (P = 0.034). Urine specific gravity increased from 1.017 _+ 0.007 before to 1.021 + 0.006 an hour after DDAVP infusion (P < 0.001). Clinically evident oliguria was not observed, however, in any of the 45 subjects studied. Potential confounding effects. No confounding effects on either DDAVP response or toxicity could be attributed to age or to the ingestion of drugs (specifically, acetylsalicylates or estrogens). DISCUSSION Our study has demonstrated a limited increase in FVIlIc but a normal increase in FVIII R:Ag after intravenous infusion of 0.30 tzg/kg DDAVP in obligate carriers of hemophilia A compared with increases in healthy female controls. Furthermore, this discordant response, reflected by the post-FVlllc/FVlll R:Ag ratio, has been shown to improve carrier detection of hemophilia A. DDAVP response. The increase in FVIIIc and FVIII R:Ag observed after DDAVP infusion has been presumed

7 22

Kobrinsky et al.

The Journal of Pediatrics May 1984

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Carriers Controls Carriers Controls without DDAVP with DDAVP Fig. 2. Changes in discriminant score with and without DDAVP infusion in obligate carriers of hemophilia A and in female controls. Discriminant scores (a) without DDAVP calculated from a diseriminant function using pre-(FVIIIe/FVIII R:Ag) as the predictor of carrier status. Discriminant scores with DDAVP calculated from a discriminant function using post-(FVIilc/ FVIII R:Ag) as the only pi-edictor of carrier status. Group centroids or points of maximum clustering are indicated by a horizontal line ( ). Scores <0 indicate assignment to the carrier group. Scores >0 indicate assignment to the control group. False positive (ll) and false negative (A) assignments decrease with DDAVP.

to be related to release from endogenous stores rather than to de nero synthesis? 7 This hypothesis is supported by the rapidity of the response ~s and by the development of tachyphylaxis with repeated use. tg':~ It follows, therefore, that after maximal stimulation, AFVIII R:Ag and postFVIII R:Ag'should reflect total FVIII R:Ag stores, and AFVIIIe and post-FVIIle should reflect total FVIIIc stores. Both should be relatively unaffected by ambient submaximal stimulating factors such as adrenalin, 2Lzz corticosteroids,z3 exercise,24'z~ or stress) 5 Accordingly, the completely normal response of FVIII R:Ag in both carriers and controls suggests that FVIII R:Ag stores are normal in both groups. This result is

consistent with the observation that, unlike FV~IIIc,~FVIII R:Ag is unaffected in hemophilia A. 26 Hemophilia A is an X:linked disorder. According to the Lyon hypothesis,27 random inactivation of the X chromosome should result, on average, in a 50% decrease in FVIIle total body stores in obligate carriers. If AFVIIIc and post-FVIIIe do in fact reflect total end6genous FVIIIe stores, they should be decreased by 50% compared with values in healthy controls. Furthermore, in view of the normal FVIII R:Ag response, the A F V I I I c / F V I I I R'Ag ratio should be similarly decreased by 50%. The latter conditional requirements have been confirmed: the decrease in AFVIIIc (51.9%), post-FVllle (47.1%), and A F V I I I c / F V I I I R:Ag (48.5%) were not different (by X2 goodness of fit) from the theoretical 50%. Carrier detection Without DDA VP. The accuracy of carrier assignment in our study using the standard F V I I I c / F V I I I R:Ag ratio was comparable to that of other reported series; overall correct assignment was 85%, with 10% false positive and 20% false negative results. In 1977, Klein et al. 2s reported the results of a cooperative study for hemophilia A carrier detection. They found correct assignments to vary from 72% to 92%, with 5% to 24% false positive and 72% to 24% false negative results in different laboratories examining coded samples drawn from the same women at the same time. These differences were related more to laboratory technique than to different types of statistical analysis? In 1978, Reisner et a l ) evaluated the accuracy of the FVIII R:Ag to ristocetin cofactor ( F V I I I c / F V I I I : R C o F ) ratio with the standard F V I I I e / F V I I I R:Ag ratio. They found that both were equally predictive, with 86.7% overall correct assignments and 7.7% false positive and 25% false negative results. With DDAVP. Discordance between FVIIIc and FVIII R:Ag in obligate carriers compared with controls is the basis for the discriminant potential of F V I I I e / F V I I I R:Ag in hemophilia A detection. After DDAVP infusion, this discordance was significantly amplified and the discriminant potential thereby increased. By discriminant function analysis, p o s t - ( F V l l l c / F V l l l R:Ag) was found to improve carrier detection, compared with pre-(FVIIIc/FVIII R:Ag); using the latter predictor, overall correct assignment was 95%, with 5% false positive and 5% false negative results. The major role for DDAVP in improving carrier detection appears to be in identifying those individuals with a "normal" FVIIIc level but limited total FVIIIe stores. This principle of evaluating total FVIIIc and FVIII R:Ag stores rather than ambient concentrations should apply regardless of the agent used to provoke release. On this basis, it

Volume 104 Number 5

would be predicted that adrenalin stimulation may be equally effective. Adrenalin-induced stimulation of FVIIIc and FVIII R:Ag has been recognized for some time3 * The importance of minimizing these effects (by controlling environmental factors and by repeated testing) to obtain a basal level of FVIIIc for carrier detection has been stressed? 5 Limited FVIIIe but normal FVIII R:Ag responses to adrenalin stimulation in patients with hemophilia A have been demonstrated39 This limited FVIIIe response has been recommended as a means of distinguishing certain patients with mild hemophilia from those with yon Willebrand disease?~ Regardless of the agent used to provoke release, there are theoretical limitations inherent in any carrier detection method based on the principle of FVIIIc stimulation: those carriers who, because of the random nature of X inactivation, have a very small percentage of expressed (i.e., inactive) hemophilia FVIIIc genes, would not be identified.27 The power of any discriminant function should be confirmed by demonstrating the function's ability to assign membership correctly to an independent group of unknowns. Because of limited numbers of obligate carriers, this check was not possible in our institution. Side effects. Doses of DDAVP of 0.30/zg/kg, 26'~1 0.40 ,ug/kg, ~~ and 0.50/~g/kg t9 have been used by different investigators. The dose we used was based on an observed plateau of FVIIIc dose response in normal subjects at 0.30 ~ug/kg when incremental doses from 0.10 tzg/kg to 0.40 vg/kg are used? Side effects reported with the use of intravenously administered DDAVP include facial flushing, subclinical antidiuresis, and mild cardiovascular changes. One investigator has reported water retention, hyponatremia, and headache with dosage of 0.50 vg/kg every 12 hours. ~9 Despite the low dosage schedule that we used, significant side effects were observed. Although all subjects received the same weight-adjusted dose, the severity of side effects correlated linearly with body surface area adjusted dose and with total dose. We therefore recommend a dosage schedule of l0 tzg/m', with a maximum dose of 25 /zg, to minimize toxicity in future studies. Water retention and hyponatremia are clearly related to the potent antidiuretic hormone effect of DDAVP. The cause of other side effects, however, is less clear. Belch et al) ~have speculated that the facial flushing observed with intravenous infusion results from the stimulation of PGI, production by vascular endothelium. They have demonstrated a two- to fourfold increase in PGI2 (as measured by the stable metabolite 6-oxo-PGF~,) in vitro using 10."M to 10-gM DDAVP in a rabbit aorta assay. They have

ltemophilia A carrier detection

723

further demonstrated that the facial flushing observed with DDAVP infusion in vivo could be abolished by prior treatment with 600 mg acetylsalicylate, a potent inhibitor of PGIv In our study, facial flushing was not abolished in the two subjects who had received acetylsalicylates; however, the dose ingested (300 mg) may have been inadequate. Whether other side effects of DDAVP infusion, including cardiovascular effects, lightheadedness, headache, and backache, are related to stimulation of PGI2 production (and hence potentially blocked by acetylsalicylate) remains to be seen. Our patients given acetylsalicylates did not experience severe symptoms. Furthermore, there was no demonstrable effect on FVIIIc or FVIII R:Ag release. The potential use of acetylsalicylates in decreasing toxic effects requires further study. We thank Linda tteinemann, R.T., Cathy Ulicki, R.T., and Patricia Wingert, R.T., for technical assistance; Lori Dcvlin for preparation of the manuscript; the nursing staffs of the Manitoba Cancer Treatment and Research Foundation Outpatient Clinic and of the Children's Hospital of Winnipeg Emergency Room; and all participants in the study.

REFERENCES 1. Graham JB: Genetic counselling in classic hemophilia A. N Engl J Med 296:996, 1977. 2. Akhcmetcli MA, Aledort LM, Alexaniats S, et ah Methods for the detection of hemophilia carriers: A memorandum. Bull WHO 55:675, 1977. 3. Reisncr tIM, Katz HZ, Goldin LR, Barrow ES, Graham JB: Use of a simplevisual assay of Willebrand factor for diagnosis and carrier identification. Br J llaematol 40:339, 1978. 4. Rizza CR, Rhymes IL, Austen DEG, Kernoff PBA, Aroni SA: Detection of carriers of hemophilia: A "blind' study. Br J tlaematol 30:447, 1975. 5. Manucci PM, Canciani NiT, Rota l., Donovan BS: Response to factor Vlll/von Willebrand factor to DDAVP in healthy subjects and patients with hemophilia A and yon Willebrand's disease. Br J itaematol 47:283, 1981. 6. Takahashi H, Itoh M, Kobayashi I, Sakuragawa N, Shibata A: Properties of the factor ViII after DDAVP (l-desamino8-D-argininevasopressin) infusion in normal subjects. Tokohu J Exp Med 132:133, 1980. 7. Prowsr CV, Sas G, Gader AMA, Cort J}|, Cash JD: Specificity of the factor VIII response to vasopressininfusion in man. Br J Haematol 41:437, 1979. 8. Mannucci PM, Ruggcri ZM, Pared FI, Capitanio A: IDesamino-8-argininc vasopressin: A new pharmacological approach to the management of hemophilia and yon WiIlebrand's disca~. Lancet 1:869, 1977. 9. DDAVP in hemophilia [editorial]. Lancet 1:889, 1977. 10. Manucci PM, Rugged ZM, Pareti FI, Capitanio A: DDAVP in hemophilia [letter]. Lancet 2:1171, 1977. I1. Manucci PM, Pareti FI, ttomberg L, Nilsson IM, Ruggeri ZM: Studies on the prolonged bleeding time in yon Wille-

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brands's disease. J Lab Clin Med 88:662, 1976. 12. Wilson WJ, Ingram GIC, Hills M: The use of kaolin or contact product in the one-stage assay for factor VIII. Coagulation 4:113, 1971. 13. Laurell CB: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 15:45, 1966. 14. Elston RC, Graham JB, Miller CH, Reisner HM, Bouma BN: Probabilistic classification of hemophilia A carriers by discriminant analysis. Thromb Res 8:683, 1976. 15. Graham JB: Genotype assignment (carrier detection) in the hemophilias. In Rizza CR, editor: Congenital coagulation disorders. Clin Haematol 8:i 15, 1979. 16. Cooley WW, Lohnes PR: Multivariate data analysis. New York, 1971, John Wiley, p 243. 17. Garcia VV, Coppola R, Mannucci PM: The role of the spleen in regulating the plasma levels of factor Vlll/von Willebrand's factor after DDAVP. Blood 60:1402, 1982. 18. Mannucci PM, Aberg M, Nilson 1M, Robertson B: Mechanism of plasminogen activator and factor Vlll increase after vasoaetive drugs. Br J Haematol 30:81, 1975. 19. Lowe G, Pettigrew A, Middleton S, Forbes CD, Prentice CRM: DDAVP in hemophilia [letter]. Lancet 2:614, 1977. 20. Boulton FE, Smith A: DDAVP and cryoprecipitate in mild hemophilia [letter]. Lancet 2:535, 1979. 21. Kitaguchi H, Hirata M, Funahara Y, Tada K, Okamoto S: Effect of vasoactive agents on factor Vlli release from perfused isolated dog leg. Thromb Res 16:159, 1979. 22. Britton BJ, Giddings JC, Brooks L, Bloom AL: Fibronolytic, factor VIII and pulse rate responses to repeated adrenaline infusion followed by haemorrhage. Thromb Haemost 37:527, 1977.

The .lournal o f Pediatrics May 1984 23. Ozsolu S, Tugue K, Cotelli 1, K ~ i o g l u H, Urkmex M, Gursel T: Effect of intravenous adrenalin infusion and corticosteroid treatment in patients with yon Willebrand's disease. Acta Haematol 64:289, 1980. 24. Prentice CRM, Forbes CD, Smith SM: Rise of factor VIII after exercise and adrenaline infusion, measured by immunological and biological techniques. Thromb Res 1:493, 1972. 25. Stibbe J: Effect of exercise on F VIII-complex: Proportional increase of ristocetin cofactor (yon Willebrand factor) and FVIII-AGN, but disproportional increase of FVIII-AHF. Thromb Res 10:163, 1977. 26. Ludlam CA, Peakel R, Allen N, Davies BL, Furlong RA, Bloom AI~: Factor VIII and fibrinolytie response to desamino-8-o-arginine vasopressin in normal subjects and dissociate response in some patients with haemophilia and von Willebrand's disease. Br J Haematol 45:449, 1980. 27. Graham JB, Barrow ES, Elston RC: Lyonization in hemophilia: A cause of error in direct detection of heterozygous carriers. Ann NY Acad Sci 28:482, 1976. 28. Klein HG, Aledort LM, Bouma BN, Hoyer LW, Zimmerman TS, DeMets DL: Cooperative study for the detection of the carrier state of classic hemophilia. N Engl J Med 296:959, 1977. 29. Eyster ME, Dranchak M, Haverstick J, Rogers JS: Absence of VIiI-AHF response to adrenalin in hemophilia A. Am J Haematol 3:I93, 1977. 30. Eyster ME, Ballard ZO, Prager D: Comparison of factor Vlll levels after adrenalin in classic hemophilia and yon Willebrand's disease (vWd). Thromb Haemost 39:657, 1978. 31. Belch JJF, Small M, McKenzie F, et al: DDAVP stimulates prostacyclin production. Thromb Haemost 47:122, 1982.