Clin Biochem, Vol. 24, pp. 505-511, 1991 0009-9120/91 $3.00 + .00 Printed in the USA. All rights reserved. Copyright © 1991 The Canadian Society of Clinical Chemists.

Serum 17oL-Hydroxyprogesterone in Infants and Children as Measured by

a Direct Radioimmunoassay Kit

ANGELINA LEE and GRAHAM ELLIS

Department of Clinical Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada

A direct 17~-hydroxyprogesterone (17-OHP) radioimmunoassay kit was used for the assay of samples from 219 infants and chil- dren. The kit was used according to the manufacturer's protocol on unextracted serum or plasma and also on reconstituted mate- rial extracted from the serum with propanol-heptane. The extrac- tion protocol recovers approximately 88% of 17-OHP. Patients were grouped as infants 3-90 days (96 subjects) or older children, adolescents and young adults 91 days-20 years (123 subjects). 17-OHP results by the direct and extraction protocols correlated but the slopes of the regression lines (0.43 and 0.63) differed in the two groups, indicating that only about 49% of the immunoreac- tive material measured by the kit in the infants was 17-OHP whereas the corresponding percentage was 72% in the older chil- dren. Despite this nonspecificity, the present antibody is much more specific for 17-OHP in the presence of neonatal plasma ste- roids than that used previously. Reference values were obtained for the two groups using the method with and without an extraction step. 17-OHP values on four untreated patients with CAH were clearly elevated at the time of diagnosis. It is recommended that when the kit is used with neonatal and infant samples, an extrac- tion step should be incorporated to enhance specificity.

KEY WORDS: reference values; infant, newborn; in- fant, child; adolescence; adult; adrenal hyperplasia, con- genital.

Introduct ion

17~-Hydroxyprogesterone is routinely measured in clinical laboratories in order to diagnose congeni- tal adrenal hyperplasia (CAH), a disorder of adre- nal steroid biosynthesis (1) that is commonly due to a mutat ion in the cytochrome P450 component (P450c21) of the enzyme 21-hydroxylase (steroid 21-monooxygenase, E.C. 1.14.99.10). The diagnosis of classical CAH is often made in the first few weeks of life. Females with CAH may be partially masculinized at birth. Sixty to seventy-five percent of children with CAH have inadequate mineralo- corticoid production and they present with severe salt wasting and dehydration, usually within the

first month after birth and rarely after 3 months. Pat ients with nonclassical CAH do not show signs of androgen excess neonatally but present as chil- dren with virilization or precocious puberty or as adolescents or adults with severe acne or men- strual irregularities, or with hirsutism or viriliza- tion. A previous widely used direct radioimmuno- assay kit from Diagnostic Products Corporation (Los Angeles, CA, USA) for assay of serum 17-OHP without an organic extraction step was reported to have two major problems. It was subject to signifi- cant interference by other steroids present in the plasma of neonates and young infants (2). Also, the kit gave unexpectedly low recoveries on quality control sera (3-5). The present study was performed to assess whether these problems were still present with a reformulated kit that became available in late 1989, and also to define reference values for children with this kit.

Patients and methods

Assays were carried out on two reference patient groups using serum or plasma samples left after routine blood tests and stored at 4 °C for up to 4 days or at - 2 0 °C up to one month prior to analy- sis for 17-OHP. Diagnostic information was ob- tained from the patient 's physician or from the case records. All patients on current or previous steroid therapy, those with panhypopituitarism, brain tumors, adrenal tumors or Cushing's disease were excluded. We grouped patients with CAH sepa- rately. Because of our previous findings with a di- rect assay from Diagnostic Products Corporation (2), the reference patients were grouped by age into two categories: infants 3 days-90 days; and older children, adolescents and young adults ages 91 days-20 years.

INFANTS (OROUP 1)

Correspondence: Graham Ellis, Ph.D., Department of Clinical Biochemistry, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G lX8.

Manuscript received March 28, 1991; accepted June 18, 1991.

The infant group (96 subjects) mainly consisted of sick hospitalized neonates and infants. Many were premature and stressed. Some had recently experienced surgery. There were 36 patients with heart malformations or other cardiac problems, 16

CLINICAL BIOCHEMISTRY, VOLUME 24, DECEMBER 1991 505

LEE AND ELLIS

with gastrointestinal diseases, 17 with respiratory distress syndrome (RDS), 6 with renal anomalies, 4 small for gestational age, 4 with spina bifida, 4 with neurological problems, 1 with congenital hy- pothyroidism and 8 with miscellaneous diagnoses. About two-thirds of the samples were taken before noon. There were 66 males and 30 females in this group.

CHILDREN OVER 3 MONTHS OLD (GROUP 2)

The majority of the 123 children in this group were ambulatory outpatients attending the Endo- crine Clinic for routine follow-up. Forty-four were males and 79 females. There were 35 patients with congenital hypothyroidism detected in the provin- cial screening program who were treated and were clinically and biochemically normal, 44 patients with various other thyroid diseases (treated and euthyroid), and 12 patients with treated precocious puberty (not CAH). Four patients had gastrointes- tinal diseases, 4 constitutional short stature, 4 neu- rological problems, 3 cardiology problems. Three had previous cancer but were well into remission. Two had delayed puberty, 2 hirsutism (but not CAH) and 10 miscellaneous illness. Samples were taken during afternoon clinics from about 90% of patients.

C A H P A T I E N T S

This group comprised 25 patients of whom 5 were previously undiagnosed. The samples from these 5 were taken at various times during the day. In CAH patients, values can vary widely dur- ing the day in treated and untreated patients (6),

whereas in normals there is a more regular diur- nal rhythm. The remaining 20 patients had been treated for several months or years. Their samples were taken at afternoon clinics.

DIRECT ASSAY

The direct assay was performed on duplicate 25 ~L aliquots as described by the manufacturer us- ing the "Coat-A-Count 17a-OH Progesterone for Serum Samples" kit (Cat. No. TKOP2) from Diag- nostic Products Corporation.

EXTRACTION ASSAY

Serum or plasma (200 ~L) was pipetted into 7 mL glass liquid scintillation vials. Two milliliters of propan-l-ol in heptane (30 mL/L) were added. The vials were t ightly capped with screw caps hav- ing an inert liner and were manual ly shaken hori- zontally in a rack for 2 min. The vials were centrifuged (capped) at 1400 × g for 10 min. From each vial, 1 mL of the propanol/heptane layer was removed and transferred to a 12 × 75 mm glass tube. To reduce vapor presure problems, the pipette was filled and gently emptied once, prior to making the accurate pipetting. The extract was dried under a flowing stream of nitrogen and the residue reconstituted in 100 ~tL of the zero calibrator that came with the kits. Duplicate 25 ~L aliquots of the reconsti tuted extracts were assayed in the same way as serum samples or standards.

Results

RECOVERY OF 17-OHP FROM CALmRATORS

The five calibrators (standards) from the kit were assayed by the extraction assay. The mean

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506 CLINICAL BIOCHEMISTRY, VOLUME 24, DECEMBER 1991

17¢x-HYDROXYPROGESTERONE IN CHILDREN

recovery was 86.8% (n = 5 for each calibrator, range 75-108%). This was a similar extraction ef- ficiency to tha t found previously, 87.6-89.2% (2).

17-OHP VALUES rN THE n~FANTS AND CHILDREN

The serum 17-OHP concentrations measured with the kit in neonates and infants 3 days to 90 days old (Group 1) are shown in Figure 1 (direct assay) and Figure 2 (extraction assay). The results of the two assays correlated but the slope was low (result by extraction assay = 0.43 × result by di- rect assay - 0.44 nmol/L, r = 0.912, Sy.= = 2.13). Values measured by the direct method on children over 90 days of age, adolescents and young adults (Group 2) are shown in Figure 3 and correspond- ing values by the extraction method in Figure 4. The values on one 4 months old outlier were not included in the statistics. (Values for this patient were 24.0 nmol/L for the direct assay and 14.9 nmol/L for the method with extraction. This pa- t ient had Down's syndrome and had recently had cardiac surgery. When tested at 1 year of age, she had a 17-OHP of 0.9 nmol/L by the direct assay.) The results again correlated and there was a higher slope for values in this group (result by ex- traction assay = 0.63 × result by direct assay - 0.00 nmol/L, r = 0.933, Sy. , , = 0.30). 17-OHP val- ues were lower in this group than in the neonatal group. Values in prepubertal children over 1 year of age tended to be lower than children during the first year of age or during puberty, but there were fewer samples on patients at this age as may be seen by inspecting Figures 3 and 4. The distribu- tions of values with age were similar for the males and females in this group. Because patients with CAH usually have markedly increased values at all ages, and because some patients had (treated)

precocious or delayed puberty and their chronolog- ical age was slightly out of phase with normal de- velopment, it was decided to pool all the data from the children over 90 days old with the adolescents and young adults. The data fitted a log Gaussian distribution when plotted as cumulative frequency on log-probability paper. The 97.5th centile was at 5.2 nmol/L for the direct assay and 3.2 nmol/L for the extraction assay.

CAH PATIENTS

Results on CAH patients who were diagnosed during the study are shown in Table 1. There was insufficient pretreatment sample from one CAH patient but the result had been diagnostic of CAH by our routine assay. Pre t reatment values in the CAH patients were markedly elevated when com- pared with results in the reference groups.

Samples from 20 CAH patients who had been treated for several months or years were tested by the direct assay and those from 12 of these patients were tested with the extraction assay. Values var- ied widely with compliance and were between 0.3 nmol/L and 111.5 nmol/L by the direct assay. The result of the two assays correlated, with a slope closer to uni ty than tha t with the other groups (re- sults by extraction assay = 0.81 × result by di- rect assay - 0.77 nmol/L, r = 0.991, sy.x = 6.07).

ASSAY OF QUALITY CONTROL MATERIAL

Quality control sera from Diagnostic Products Corporation (Cat. No. 'CON6' levels 4, 5 and 6) were assayed with each batch, with and without extraction. The coefficients of variation ranged between 8.5 and 15.3% for the direct assay and 12.9-19.9% for extraction assay (n = 19). The effici-

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CLINICAL BIOCHEMISTRY, VOLUME 24, DECEMBER 1991 507

LEE AND ELLIS

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ency of extraction assessed by the values of 17- OHP from the quality control materials over 19 batches was 58% for level 4, 70% for level 5 and 72% for level 6.

Discuss ion

INTERFERING SUBSTANCES IN SERUM FROM BABIES

Serum 17-OHP measured directly (Figure 1) or in serum extracts (Figure 2) tended to be much

higher in the group of babies 3 days to 90 days old than in the older children (Figures 3 and 4). It is known that the extraction procedure improves the specificity of the assay and that it recovers 85% to 90% of the 17-OHP (2). The correlations between the 17-OHP by the extraction and direct proce- dures had different slopes in the different groups of patients. The slopes were 0.43 in the babies' samples, 0.63 in the older children's samples, and 0.81 in the CAH patients ' samples. This suggests that there was poorer extraction of immunoassay-

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508 CLINICAL BIOCHEMISTRY, VOLUME 24, DECEMBER 1991

17a-HYDROXYPROGESTERONE IN CHILDREN

TABLE 1 Serum 17-OHP Concentrations in Patients with CAH at Diagnosis

17-OHP

Direct assay Extraction assay Patient Age (nmol/L) (nmol/L)

A 19.7 years 657 135 B 14 days >6000 5240 C 20 days 341 212 D 21 days 2377 502 D 1 day after start 22 days 98 45

of treatment *E 6 h after start 10 days 64 9

of treatment

There was insufficient serum on patient E at diagnosis to retest by the DPC method. The data on this patient as well as the repeat value for pa- tient D are included to show how rapidly values may return to normal in some patients.

able material from the babies' samples than from the other samples, i.e., there was a higher concen- tration of interfering material in these samples. The original kit from Diagnostic Products Corpora- tion gave even higher results on babies' samples than the kit with the new antibody (2). In a simi- lar group of 74 hospitalized sick neonates and in- fants 3-90 days old we found tha t 23 had values with the original kit tha t were greater than the highest standard provided with the kit (60.5 nmol/ L). In the present study, none of the infants had a value over 60.5 nmol/L. However, values by the di- rect assay (Figure 1) were significantly higher than those obtained on extracts (Figure 2).

REFERENCE VALUES

Inspection of any of the figures relating 17-OHP with age, show that there are age-related changes tha t make precise definition of 95th or 97.5th cen- tile ranges impossible without inordinately large groups of children. Samples from babies up to 1-2 days old should not be analyzed because 17-OHP crosses the placenta and the maternal circulation during the last tr imester contains higher 17-OHP than tha t of the nonpregnant woman (7). After birth, the maternal 17-OHP is eliminated within the first 2 A. A.8 h in heal thy term neonates (8-11). However, stressed or premature infants have higher values (8,12,13). Obtaining reference values in children is difficult for practical and ethical rea- sons. With infants in hospital, the use of residual serum after routine testing is the only acceptable method in our opinion. Such children are often stressed or premature and are sicker than the fe- male infant with ambiguous genitalia due to CAH who is investigated prior to a salt-losing crisis or who is not a salt loser. However, the CAH infant investigated during a salt-losing crisis may be at

greater stress than other patients on the same ward and his/her 17-OHP value should be com- pared to a reference range tha t includes stressed infants. In reviewing our patient data, we attempted to classify the infants as "stressed" or "not-stressed." The child who was very restless post-op or had very severe RDS would be classified as "stressed," in contrast to the child who was stable, well perfused and resting quietly. There was no simple relation- ship between the 17-OHP values measured by the direct or extraction methods and stress, prematu- ri ty or postnatal age, although, in general, values tended to be lower during the third month aider birth than during the first two months (Figures 1 and 2). Because the direct assay detects a group of steroid sulfates in addition to 17-OHP (14), sam- ples from babies 3 days old to 3 or perhaps 6 months old should probably be assayed by the ex- traction method. About 90% of values will be <10 nmol/L, but some stressed babies will have higher values, generally less than 30 nmol/L (Figure 2). We have not assayed cortisol in this group of ba- bies but it is probably very high because of the stress. Patients with CAH would not be expected to have very high cortisol as assayed by a specific cortisol assay. Our results are difficult to compare with those of Knudtzon et al. (15) who recently re- ported values in term and premature infants ob- tained with a commercial kit from Radioassay Sys- tems Laboratories, Inc., Carson, CA, USA. This uncertainty arises because those authors refer to "direct-assay kits" . . . "used in the present study" and imply tha t the kits were used without the "ex- traction step prior to assay" tha t the manufacturer recommends for sera from "pregnant women and newborns."

Samples on children over 90 days or perhaps 6 months old, adolescents and young adults could be assayed by the direct method and most samples (excluding tha t from the 4 month old outlier)

CLINICAL BIOCHEMISTRY, VOLUME 24, DECEMBER 1991 509

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taken during the afternoon had 17-OHP concentra- tions <7 nmol/L (by inspection of Figure 4). In pre- pubertal children, values are generally <4 nmol/L. Plotting data from the whole group as cumulative frequency on log-probability paper gave 97.5th cen- tiles of 5.2 umol/L for the direct method and 3.2 nmol/L by the extraction method. It would proba- bly be good practice to reassay by the extraction method any samples from older children not previ- ously diagnosed as CAH if the result is elevated by the direct method.

It should be recognized that the data represent "reference" values rather than strictly "normal" values (16). Most of the patients were attending the endocrine clinic, and about half were euthyroid patients with previous thyroid abnormality. Sev- eral had early or delayed puberty and their 17-OHP values could perhaps have better related to their bone matur i ty (bone "age") rather than chronological age. But this information was not available for most patients. Despite this, our re- sults with the direct assay in the group over 90 days old are similar to those of several other stud- ies in which organic extraction and/or chromatog- raphy were used prior to RIA in order to improve specificity for 17-OHP (8,17).

VALUES IN PATIENTS WITH C A H

The infant patients who presented with CAH during this study had high values for 17-OHP by the extraction method, and also had high concen- trations of interfering substances. Pat ient E was already in the normal range for 17-OHP although the sample was taken only 6 h after the start of treatment. Pat ient A, a 19.7-year-old female, also had high concentrations of interfering substances as well as 17-OHP. The diagnostic sensitivity of the assay was enhanced in this patient because of its analytical nonspecificity. We speculate that this could also happen in other children and adult pa- tients, but we have no other data.

With other children over 6 months old and with other (extraction-type) assays we have encountered patients with borderline elevation of 17-OHP, e.g., 12-30 nmol/L. Samples taken on other occasions from the same patients had much higher values and the patients were subsequently shown to have nonclassical CAH on the basis of ACTH stimula- tion tests or their HLA type and that of other af- fected family members. 17-OHP values should always be interpreted in relation to the patient 's clinical presentation.

EXTRACTION OF 1 7 - O H P FROM STANDARDS AND QUALITY CONTROL SERA

eries" from quality control materials (DPC sera # 4,5,6) were 58-72% (assessed as extracted value/di- rect value x 100%). This suggests that some qual- ity control sera such as serum #4 (at 58% re- covery) m a y contain more interfering substances that contribute to the direct assay result. This may also explain in part some of the recent controversy about alleged poor recovery and the suitabili ty of direct 17-OHP kits for routine use (3,4). Cross-re- actants react differently from 17-OHP and have different dose response curves. The dose response of the cross-reactants is affected by the addition of authentic 17-OHP and the combined response is not additive when compared to the 17-OHP dose response curve. This leads to poor recovery in that matrix (2). Assays that efficiently extract 17-OHP from the major cross-reactants show better recov- eries. It is not clear from our study whether the re- sults of direct and extraction assays will agree bet ter with pooled sera from CAH patients in place of quality control sera, as Middle has suggested (4). In our study, there was a good correlation and a slope of 0.81 between the extracted result and the direct result in the treated CAH patients. However, the untreated patients showed much higher "17-OHP" by the direct assay compared with the extraction assay (Table 1). Although most of these were neonates who might be expected to show discrepancies, this also applied to the 19.7- year-old female. Precursors of 17-OHP and their sulphates are important cross-reactants in direct assays in babies (14) and they are increased in un- treated CAH (18). These interferences may add di- agnostic sensitivity to direct assays in untreated CAH.

Direct assays should probably be used cautiously in the at tempted classification of heterozygotes for CAH by taking measurements of 17-OHP before and after an ACTH stimulation test. The increases in normals and some heterozygotes can be small (19), and we speculate that cross-reactants in di- rect assays could give a reduced response by falsely elevating the baseline prior to stimulation, or possibly an overestimated response if interfer- ing steroids or steroid sulphates were produced af- ter ACTH stimulation. The cut-off value is often assessed as the 17-OHP increment at 1 h after ACTH (19) and it may need revision for direct as- says. Perhaps gene probes (20,21) will reduce the usage of this st imulation test for heterozygote detection.

Acknowledgements

We wish to thank Diagnostic Products Corporation for funding this project and their Technical Director, Dr. Pe- ter Bodlaender, for helpful discussions. We thank staff in the Endocrine Clinic and on the wards for helping us obtain the clinical information.

The 17-OHP values on extracts of the calibrators supplied with the kits were 75-108% of those as- sayed directly. However, the corresponding "recov-

References

1. New MI, White PC, Pang S, Dupont B, Speiser PW. The adrenal hyperplasias. In: Scriver CR, Beaudet

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AL, Sly WS, Valle D, eds. The metabolic basis of in- herited disease. 6th ed. Pp. 1881-1917. New York: McGraw-Hill, Inc., 1989.

2. Makela SK, Ellis G. Nonspecificity of a direct 17a- hydroxyprogesterone radioimmunoassay kit when used with samples from neonates. Clin Chern 1988; 34: 2070-5.

3. Read GF, Walker RF, Ford P, Riad-Fahmy D. Exter- nal quality assessment in small schemes: the case of 17-a-hydroxyprogesterone. Ann Clin Biochem 1990; 27:276 (Lett.).

4. Middle J. Reply to: external quality assessment in small schemes: the case of 17-a-hydroxyprogesterone. Ann Clin Biochem 1990; 27:277-8 (Lett.).

5. Bodlaender P. Reply to: external quality assessment in small schemes: the case of 17-a-hydroxyprogester- one. Ann Clin Biochem 1990; 27:278 (Lett.).

6. Frisch H, Parth K, Schober E, Swoboda W. Circa- dian patterns of plasma cortisol, 17-hydroxyproges- terone, and testosterone in congenital adrenal hyper- plasia. Arch Dis Child 1981; 56: 208-13.

7. Florensa E, Harrison R, Johnson M, Youssefnejadian E. Plasma 20a-dihydroprogesterone, progesterone and 17-hydroxyprogesterone in normal human pregnancy. Acta Endocrinol 1977; 86:63 A. A.0.

8. Sippell WG, Dtirr HG, Bidlingrnaier F, Knorr D. Plasma levels of aldosterone, corticosterone, ll-deoxy- corticosterone, progesterone, 17-hydroxyprogesterone, cortisol and cortisone during infancy and childhood. Pediatr Res 1980; 14: 39-46.

9. Gddo B, Visser HKA, Degenhart HJ. Plasma 17-OH- progesterone in fullterm and preterm infants at birth and during the early neonatal period. Horm Res 1981; 15: 65-71.

10. Doerr HG, Sippell WG, Versmold HT, Bidlingmaier F, Knorr D. Plasma mineralocorticoids, glucocorti- coids and progestins in premature infants: longitudi- nal study during the first weeks of life. Pediatr Res 1988; 23: 525-9.

11. Hughes IA, Riad-Fahmy D, Griffiths K. Plasma 17

OH-progesterone concentrations in newborn infants. Arch Dis Child 1979; 54: 347-9.

12. Murphy JF, Joyce BG, Dyas J, Hughes IA. Plasma 17-hydroxyprogesterone concentrations in ill newborn infants. Arch Dis Child 1983; 58: 532-4.

13. Doerr HG, Versmold HT, Bidlingmaier F, Sippell WG. Adrenocortical steroids in small-for-gestational- age term infants during the early neonatal period. Pediatr Res 1989; 25: 115-8.

14. Wong T, Ellis G. Partial characterization of sub- stances in neonatal plasma that interfere with 17a- hydroxyprogesterone assays. Clin Chem 1990; 36: 1150 (Abst.).

15. Knudtzon J, Markestadt T, Aakvaag A, Bergsjti P. Elevated 17-hydroxyprogesterone levels in prema- ture infants. Acta Paediatr Scand 1991; 80: 96-97.

16. Lindblad B, Alstrt~m T, Bo Hansen A, et al. Recom- mendation for collection of venous blood from chil- dren, with special reference to production reference values. Scand J Clin Lab Invest 1990; 50: 99-104.

17. Hughes IA, Winter JSD. The application of a serum 17-OH-progesterone radioimmunoassay to the diag- nosis and management of congenital adrenal hyper- plasia. J Pediatr 1976; 88: 766-73.

18. Shimozawa K, Saisho S, Yata J-I, Kambegawa A. Serum 17-hydroxypregnenolone and 17-hydroxypreg- nenolone sulfate concentrations in patients with con- genital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocrinol Japon 1988; 35: 11-18.

19. Fi~t J, Gueux B, Gourmelen M, et al. Comparison of basal and adrenocorticotropin-stimulated plasma 21- deoxycortisol and 17-hydroxyprogesterone values as biological markers of late-onset adrenal hyperplasia. J Clin Endocrinol Metab 1988; 66: 659-67.

20. Strachan T. Molecular pathology of congenital adre- nal hyperplasia. Clin Endocrinol 1990; 32: 373-93.

21. Azziz R, Wells G, Acton RT, Zacur HA. Pseudogene/ functional gene ratio in late-onset 21-hydroxylase- deficient adrenal hyperplasia. Am J Obstet Gynecol 1990; 162: 633-8.

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