COAGULATION AND TRANSFUSION MEDICINE
Original Article
Age-Dependent Effect on the Level
of Factor IX
JOSEPH D. SWEENEY, MD, AND LYNNE A. HOERNIG, MS
Levels of factor IX:C and factor IX:Ag were measured in 120 healthy
subjects with an age range of 1 to 67 years. Levels of factor IX:C were
lowest in prepubertal subjects, then reached a plateau in early adult life
with a secondary increase in later adult life (>45 years). Changes in
factor IX:Ag showed a similar trend. Factor IX:Ag disproportionately
increased in early adult life, however, with a less pronounced increase in
later life, resulting in peaking of the ratio in early adult life. It is suggested that caution be exercised in interpreting low normal factor IX:C
levels in prepubertal children, and that interpretation of ratios of antigenic to coagulant activity may need to take subject age into consideration. (Key words: Factor IX; Hemophilia B; Age-related) Am J Clin
Pathol 1993; 99:687-688.
The level of blood coagulation factors are known to be influenced by variables such as age, sex, and ABO group, and by
physiologic states such as exercise and pregnancy.'-2 VitaminK-dependent factors are reduced in neonates, but are considered to attain normal values within the first year of life.3 Several
reports, however, have shown that functional levels of factor
IX (IX:C) are lower in children than adults, 45 and one study
showed an age-related increase between two groups of adult
twins of different age groups using antigenic measurements
(IX:Ag).6 A comparison with measurements of both factor
IX:C and factor IX:Ag in the same samples has not been performed in a population with a wide age range. This is important in view of the use of IX:C as an indicator of hemophilia B.7
This study measured factor IX:C and factor IX:Ag in a healthy
population with an age range of 1 to 67 years.
of 1:10, 1:20, 1:40, and 1:80. Each assay run contained a blank
(substrate plasma, with saline rather than a dilution of standard
plasma). A four-point standard curve was thus constructed.
Blank values had to be < 1 % for run validation. In each run, a
borderline abnormal control plasma with an assigned value
was included (B-Fac, George King). Test samples were diluted
1:10 and 1:20. Assay values were the mean of the two values at
each dilution. For validation of parallelism, assay values at
each dilution could not differ by more than 5%. Substrate
plasma used was from George King. This plasma was from a
patient with hemophilia B and < 1 % factor IX:C. Standard
plasmas, substrate plasmas, and abnormal control plasmas
were seronegative for anti HIV-I and HBsAg.
Chronometric assays were performed in a two channel,
photo-optic instrument (Coadata 2000, Sigma Chemical Company, St. Louis, MO), using disposable cuvettes. The activator
used was from OrganonTechnica (Durham, NC).
Factor IX:Ag was measured by enzyme immunoassay
(American Bioproducts, Parsippany, NJ). The standard
plasma used was the same as used in the factor IX:C assay, and
was arbitrarily assigned a value of 1,000 U/L. Standard plasma
in this assay was diluted 1:10, 1:20, 1:40, 1:100, and 1:200 for
construction of a standard curve. A plasma blank is also run.
Intrarun quality control was performed using a borderline low
plasma (B-Fac). Test plasmas were run in two dilutions—1:10
and 1:20—and values were mean of the two values. A linear
relationship was established between absorbance and antigen
levels. All values were expressed as U/L. Age-stratified groups
were analyzed by one-way analysis of variance, and correlations were Phearson's correlation coefficients.
METHODS A N D MATERIALS
Samples of platelet-poor plasma (PPP) for this study were
obtained as follows: For children, residual PPP was obtained
after completion of routine preoperative coagulation testing
before elective surgery. This was frozen at -70 °C within 3
hours. For adults, a venipuncture was performed after obtaining specific informed consent. PPP was prepared by centrifugation at 1500 X g for 20 minutes and was frozen as above. For
each subject, age and sex were recorded, and ABO group determined by reverse grouping.
Platelet-poor plasma samples were stored for a maximum of
1 month. Samples were then thawed at 37 °C in a water bath.
Standards for the factor IX assays were from George King
(George King, Kansas City, MO). These are frozen plasma samples prepared from a pool of donors and shipped to the laboratory in dry ice. This donor pool consists of 20-30 male and
female subjects aged 18-54.
Frozen standard plasmas were thawed in the same manner as
frozen samples. A standard curve was prepared using dilutions
RESULTS
Hemophilia Center of Western New York, Erie County Medical
Center. 462 Gricler Street. Buffalo. NY 14215.
Received February 15, 1991; accepted for publication June 12, 1992.
Address reprint requests to Joseph D. Sweeney, M.D.: Hemophilia
Center of Western New York. 462 Grider Street, 5th Floor, Buffalo.
NY 14215.
687
One hundred twenty samples from healthy subjects were
measured for factor IX:C and 104 for factor IX:Ag. The study
consisted of 63 female and 57 male subjects. The overall range
for factor IX:C was 460-2.490 U/L with a mean value of 966
and a standard deviation of 335. The range for factor IX:Ag
was 630-2,250 U/L, mean 1230, standard deviation 358. Intrarun B-Fac values (quality control) for factor IX:C assays for
the four runs in this study were as follows: 280, 310, 350, and
360 U/L. Values for the factor IX:Ag assays were 510, 550,
550, and 560 U/L. The coefficient of variation for factor IX:C
COAGULATION AND TRANSFUSION MEDICINE
688
Original Article
assays varies between 10% at low levels (300 U/L and less) and
5% in the normal ranges. For factor IX:Ag, the coefficient of
variation levels varies between 3% and 10%.
Table 1 shows the values for factor IX divided into three
arbitrary age groups. Statistically significant differences exist
between the age groups for both factor IX:C and factor IX:Ag
(P <0.01). An increase in both measures is associated with
puberty, with a disproportionate increase in factor IX:Ag observed in early adult life. A secondary increase in both factor
IX:C and factor IX:Ag is again evident in later life (arbitrarily,
> 45 years).
The correlation of factor IX:C with age was 0.58 (P <0.001),
with the regression equation IX:C = 73.7 + 0.99 (age in years).
The correlation of factor IX:Ag with age was 0.66 (P <0.001),
with the regression equation IX:Ag = 960 + 1.26 (age in years).
Ratio of lX:Ag/IX:C varied within each age group without any
consistent trend evident. This was due to the disproportionate
increase in antigen to coagulant in early adult life. The higher
mean factor IX:Ag level obtained relative to the factor IX:C
may be related to the arbitrary assignment of the 1,000 U/L to
this commercial plasma. The factor IX:C level assigned was the
assigned commercial value (890 U/L). Regardless of the absolute value, however, the relative age trend and ratio alterations
are evident. Factor IX:C correlated with Factor IX:Ag, and a
scattergram of the relationship is presented in Figure 1 (r
= 0.56, P <0.01). An analysis of the difference between males
and females and between different ABO blood groups failed to
show an effect of gender or ABO blood tvpe on either factor
IX:C or factor IX:Ag.
DISCUSSION
This report confirms the observation of an effect of age on
factor IX. 45 The single previous report of elevated factor IX:Ag
in older adult twin groups showed only slight differences in
mean values despite statistical significance.6 This report is interesting in the light of the known hemophilia B variant, hemophilia B Leyden, in which levels of factor IX increase after
puberty over 4-5 years to low normal levels. In this disease,
however, levels of antigen rise in proportion to functional activity.8 From our data, a physiologic increase in factor IX:C occurs at puberty with an apparent disproportionate increase in
factor IX:Ag, but then a plateau appears to occur early in adult
life. Our data suggest that a secondary elevation occurs in later
life. This later rise in activity is not accompanied by such a
proportionate increase in antigen, however, resulting in an antigen/coagulant ratio closer to prepubertal values. It is conceivable that the pubertal changes are related to steroid hormones,
because steroid-dependent gene expression has been implicated for the factor IX gene by Reitsma and co-workers9
TABLE 1. RELATIONSHIP OF FACTOR IX LEVELS WITH AGE
Age
Group (yr)
N
IX:C
(U/L)
IX:Ag
(U/L)
Ratio
IX:Ag/IX:C
0-15
16-44
45-67
58
43
19
800 ± 160
994 ± 250
1390 ±480
1000 ±215
1349 ±280
1700± 330
1.21 ±0.3
1.44 ±0.4
1.32 ±0.43
IX: C = factor IX coagulant activity: IX: Ag = factor IX antigen.
2490
1868
2 1245O
x
623-
0.00
0.00
560
1125
1690
2250
IX:Ag (U/L)
FIG. I. Scattergram of the relationship between 1X:C and IX:AG
The significance of these observations for the clinical laboratory lies in the interpretation of levels of factor IX:C, which could
be considered abnormal if tolerance ranges are based on values
obtained from adult samples, a common occurrence due to the
difficulty in acquiring an adequate sample size from healthy
children. Mild, unexplained prolongation of the activated partial thromboplastin time may be attributed to such a low normal value, leaving an abnormality, such as heterozygotic factor
XI deficiency, undisclosed. The occurrence of such a case
prompted the performance of this study. This is clinically significant, because such patients may exhibit abnormal bleeding.10
In addition, the alteration observed in antigen/coagulant ratio
with age is relevant for laboratories that use this ratio in assessing hemophilia B carrier status in CRM-negative hemophilia B.
REFERENCES
1. Graham JB, RizzaCR.Chediak J, etal. Carrier detection in Hemophilia A: A cooperative internation study. 1: The carrier phenotype. Blood 1986:67:1554-1559.
2. Gill JC, Endes-Brooks J, Bauer PJ. et al. The effect of ABO blood
group on the diagnosis of von Willebrand's disease. Blood
1987;69:1691-1695.
3. Manco-Johnson M. Current topics in neonatal and pediatric coagulation. In: An Educational Symposium on Hemostasis and
Thrombosis. Raritan. NJ: Ortho Diagnostic Systems, 1988, pp
32-45.
4. Dodd WJ. Moynihan AC. Benson RR, Hall CA. The value of age
and sex matched controls for coagulation studies. Br J Haematol 1975:29:305-317.
5. Simpson NE. Biggs R. The inheritance of Christmas Factor. Br J
Haematol 1962:8:191-198.
6. Orstavik HH. Magnus P. Reisner H. et al. Factor VIII and Factor
IX in a twin population: Evidence for a major effect of ABO
locus on Factor VIII level. Am J Hum Gen 1985;37:89-101.
7. Lillicrap DP. White BN. Holden JJA, Giles AR. Carrier detection
in the hemophilias. Am J Hematol 1987:26:285-296.
8. Briet E, Bertina RM. Van Tilburg NH, Veltkamp JJ. Hemophilia
B Leyden: A sex linked hereditary disorder that improves after
puberty. N Engl J Med 1982:306:788-790.
9. Reitsma PH. Mandalaki T. Rasper CK, et al. Two novel point
mutation correlate with an altered developmental expression of
blood coagulation factor IX (hemophilia b Leyden phenotype).
Blood 1989:73:743-746.
10. Balton-Maggs PHB. Young Wan-Yin B. McCraw AH, et al. Inheritance and bleeding in factor XI deficiency. Br J Haematol
1988;69:521-528.
A.J.C.P.-June 1993