Skip to content
How It Works
Explore
Resources Ask a Question
Who It's For
Patients
Decode results, prep for appointments, weigh options.
Caregivers & advocates
Carry the research load for someone you love.
Providers & clinicians
Resources for patients in your practice.
Log In Ask a Question
PubMed This is a summary of 21 peer-reviewed journal articles Updated
Pediatric Endocrinology

Testing and Diagnosis: From Screening to Certainty

At a Glance

A positive newborn screen for CAH using 17-OHP requires confirmatory testing because false positives are common, especially in premature babies. Doctors use advanced blood tests, electrolyte monitoring, and CYP21A2 genetic testing to confirm the diagnosis and determine disease severity.

The journey from a newborn screening result to a definitive diagnosis can be a stressful time of waiting and testing. It is important to understand that the initial Newborn Screening (NBS) is designed to be highly sensitive—meaning it is meant to catch every possible case, even if that leads to many “false alarms” (false positives) [1][2].

The First Signal: 17-OHP Screening

Newborn screening works by measuring a hormone called 17-hydroxyprogesterone (17-OHP) from a small drop of blood taken from your baby’s heel [3][4].

  • The “Backlog”: In babies with CAH, the 21-hydroxylase enzyme isn’t working, causing 17-OHP to build up in the blood like water behind a dam [3].
  • The False Positive Challenge: High levels of 17-OHP do not always mean a baby has CAH. In premature or low-birth-weight infants, 17-OHP levels are naturally higher. Additionally, the stress of birth can cause a temporary rise in these hormones [5][6][7].
  • Adjusting for Accuracy: To reduce false positives, labs often use different “cutoff” levels based on the baby’s birth weight or how many weeks they were at birth (gestational age) [8][9].

Confirmatory Testing: Building the Picture

If the screen is positive, your doctor will order more precise tests to confirm the diagnosis and determine the subtype.

1. Advanced Steroid Profiling (LC-MS/MS)

While the initial screen is a quick check, doctors use a more sophisticated method called LC-MS/MS for confirmation. This test can measure several hormones at once, including 21-deoxycortisol, which is a very specific “marker” for 21-hydroxylase deficiency [8][10][11].

2. Electrolyte Monitoring

Testing the blood for sodium and potassium levels is critical. This tells doctors if the baby is “wasting salt,” which helps distinguish the salt-wasting subtype from the simple-virilizing subtype [12][13].

3. ACTH Stimulation Test

In some cases, a doctor may perform a “stress test” for the adrenal glands. They give a small injection of ACTH (the hormone that tells the adrenals to work) and then measure how the 17-OHP levels respond. A dramatic jump in 17-OHP confirms the diagnosis [14].

Genetic Testing: The Molecular Map

Genetic testing of the CYP21A2 gene is the “gold standard” for understanding the specific cause of CAH [15][16].

  • Predicting Severity: By identifying exactly which mutations your baby has, geneticists can often predict the phenotype (the clinical severity). For example, “null” mutations, where the gene makes no enzyme at all, almost always result in the salt-wasting form [17][18].
  • Guiding Care: Knowing the genetic makeup helps your medical team tailor treatment and provides essential information for future family planning [16][19].

If genetic testing hasn’t been mentioned, you are encouraged to ask your pediatric endocrinologist if it would be helpful for your baby’s care plan [16]. Diagnosis is a multi-step process, but each piece of data helps your team provide the most accurate and safe care for your child [20][21].

To learn about how this diagnosis is managed, read Balancing the Scale: Treatment Strategies for CAH.

Common questions in this guide

What does a high 17-OHP level on a newborn screen mean?
A high 17-OHP level indicates that your baby might have CAH, but it is not a final diagnosis. High levels can also be caused by premature birth, low birth weight, or the physical stress of delivery, which can lead to a false positive result.
Why is my doctor testing my baby's sodium and potassium levels?
Doctors test sodium and potassium to check for salt-wasting, which is a life-threatening complication of severe CAH. Monitoring these electrolyte levels helps determine your baby's specific condition subtype and guides immediate medical treatment.
What is the ACTH stimulation test for CAH?
The ACTH stimulation test checks how well the adrenal glands respond to stress. A doctor gives a small injection of the ACTH hormone and measures the resulting 17-OHP levels to confirm if the 21-hydroxylase enzyme is missing or deficient.
How does genetic testing help with a CAH diagnosis?
Genetic testing looks for specific mutations in the CYP21A2 gene. Identifying the exact mutation confirms the CAH diagnosis, helps predict the clinical severity of the disease, and provides essential information for your baby's long-term care plan.

Questions to Ask Your Doctor

Curated prompts to bring to your next appointment.

  1. 1.Was the 17-OHP level on the newborn screen adjusted for my baby's birth weight and gestational age?
  2. 2.Has a second-tier test, like LC-MS/MS, been performed to look for 21-deoxycortisol?
  3. 3.Does my baby's current electrolyte panel (sodium and potassium) show any signs of salt-wasting?
  4. 4.Should we perform an ACTH stimulation test to confirm how much enzyme activity my baby has?
  5. 5.Can we order genetic testing for the CYP21A2 gene to help us understand which subtype of CAH our baby has?

Questions For You

Tap a prompt to share your answer — we'll use it plus this page's context to start a tailored conversation.

References

References (21)
  1. 1

    Review of Health Problems in Adult Patients with Classic Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency.

    Reisch N

    Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association 2019; (127(2-03)):171-177 doi:10.1055/a-0820-2085.

    PMID: 30812049
  2. 2

    [Newborn screening for congenital hypothyroidism and congenital adrenal hyperplasia: Benefits and costs of a successful public health program].

    Van Vliet G, Grosse SD

    Medecine sciences : M/S 2021; (37(5)):528-534 doi:10.1051/medsci/2021053.

    PMID: 34003099
  3. 3

    Newborn Screening for CAH-Challenges and Opportunities.

    Heather NL, Nordenstrom A

    International journal of neonatal screening 2021; (7(1)) doi:10.3390/ijns7010011.

    PMID: 33668620
  4. 4

    The First-Year Outcomes of the Nationwide Neonatal CAH Screening in Türkiye: High Rate of False Positives for 21-Hydroxylase Deficiency and a Higher Detection Rate of Non-Classical Cases

    Güran T, Yürüker E, Anık A, et al.

    Journal of clinical research in pediatric endocrinology 2025; (17(4)):488-493 doi:10.4274/jcrpe.galenos.2025.2024-9-11.

    PMID: 40405612
  5. 5

    Neonatal 17-hydroxyprogesterone levels adjusted according to age at sample collection and birthweight improve the efficacy of congenital adrenal hyperplasia newborn screening.

    Hayashi GY, Carvalho DF, de Miranda MC, et al.

    Clinical endocrinology 2017; (86(4)):480-487 doi:10.1111/cen.13292.

    PMID: 27978607
  6. 6

    Outcome of Newborn Screening for Congenital Adrenal Hyperplasia at Two Time Points.

    Eshragh N, Doan LV, Connelly KJ, et al.

    Hormone research in paediatrics 2020; (93(2)):128-136 doi:10.1159/000508075.

    PMID: 32659761
  7. 7

    Beckwith-Wiedemann syndrome mimicking the classical form of congenital adrenal hyperplasia in newborn screening.

    Martins JMES, Braga BL, Sampaio KNF, et al.

    Archives of endocrinology and metabolism 2024; (68()):e220395.

    PMID: 38427811
  8. 8

    Implementing steroid profiling by liquid chromatography-tandem mass spectrometry improves newborn screening for congenital adrenal hyperplasia in New Zealand.

    de Hora MR, Heather NL, Patel T, et al.

    Clinical endocrinology 2021; (94(6)):904-912 doi:10.1111/cen.14422.

    PMID: 33471388
  9. 9

    Combined Gestational Age- and Birth Weight-Adjusted Cutoffs for Newborn Screening of Congenital Adrenal Hyperplasia.

    Pode-Shakked N, Blau A, Pode-Shakked B, et al.

    The Journal of clinical endocrinology and metabolism 2019; (104(8)):3172-3180 doi:10.1210/jc.2018-02468.

    PMID: 30865229
  10. 10

    Congenital Adrenal Hyperplasia-Current Insights in Pathophysiology, Diagnostics, and Management.

    Claahsen-van der Grinten HL, Speiser PW, Ahmed SF, et al.

    Endocrine reviews 2022; (43(1)):91-159 doi:10.1210/endrev/bnab016.

    PMID: 33961029
  11. 11

    Newborn screening for congenital adrenal hyperplasia: Utility of liquid chromatography with tandem mass spectrometry as a secondary test.

    Tajima T

    Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology 2025; (34(1)):13-18 doi:10.1297/cpe.2024-0069.

    PMID: 39777132
  12. 12

    Congenital adrenal hyperplasia with salt-wasting crisis and arrhythmia: a case study.

    Canlas JF, Ponmani C

    BMJ case reports 2019; (12(1)) doi:10.1136/bcr-2018-227565.

    PMID: 30700462
  13. 13

    A Neonate Presenting with Severe Dehydration: A Rare Case of Congenital Adrenal Hyperplasia with Salt Losing Crisis.

    Lamichhane A, Phuyel R, Upreti M, Khadka R

    JNMA; journal of the Nepal Medical Association 2024; (62(278)):706-708 doi:10.31729/jnma.8777.

    PMID: 40655893
  14. 14

    MECHANISMS IN ENDOCRINOLOGY: Rare defects in adrenal steroidogenesis.

    Miller WL

    European journal of endocrinology 2018; (179(3)):R125-R141.

    PMID: 29880708
  15. 15

    Divergent Gender Identity in a Phenotypic Male with 46XX Karyotype Caused by a Mutation in CYP21A2 Gene with Congenital Adrenal Hyperplasia.

    Kumar KCP, Banik S, Joy P, Sahoo S

    International journal of applied & basic medical research 2024; (14(2)):134-137 doi:10.4103/ijabmr.ijabmr_473_23.

    PMID: 38912360
  16. 16

    Update on the Swedish Newborn Screening for Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency.

    Zetterström RH, Karlsson L, Falhammar H, et al.

    International journal of neonatal screening 2020; (6(3)) doi:10.3390/ijns6030071.

    PMID: 33239597
  17. 17

    Issues with the Detection of Large Genomic Rearrangements in Molecular Diagnosis of 21-Hydroxylase Deficiency.

    Concolino P

    Molecular diagnosis & therapy 2019; (23(5)):563-567 doi:10.1007/s40291-019-00415-z.

    PMID: 31317337
  18. 18

    Comparison of long-read sequencing and MLPA combined with long-PCR sequencing of CYP21A2 mutations in patients with 21-OHD.

    Lan T, Wang J, Chen K, et al.

    Frontiers in genetics 2024; (15()):1472516 doi:10.3389/fgene.2024.1472516.

    PMID: 39553475
  19. 19

    Long-Read Sequencing Solves Complex Structure of CYP21A2 in a Large 21-Hydroxylase Deficiency Cohort.

    Wang R, Luo X, Sun Y, et al.

    The Journal of clinical endocrinology and metabolism 2025; (110(2)):406-416 doi:10.1210/clinem/dgae519.

    PMID: 39049755
  20. 20

    Genetics of 21-OH Deficiency and Genotype-Phenotype Correlation: Experience of the Hellenic National Referral Center.

    Fylaktou I, Mertzanian A, Farakla I, et al.

    Current issues in molecular biology 2024; (46(10)):10696-10713 doi:10.3390/cimb46100635.

    PMID: 39451515
  21. 21

    Clinical Manifestations and Treatment Challenges in Infants and Children With Classic Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency.

    Nokoff NJ, Buchanan C, Barker JM

    The Journal of clinical endocrinology and metabolism 2025; (110(Supplement_1)):S13-S24 doi:10.1210/clinem/dgae563.

    PMID: 39836622

This page explains newborn screening and diagnostic testing for CAH for educational purposes only. Always consult your pediatric endocrinologist for interpreting your baby's specific test results.

Get notified when new evidence is published on Classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

We monitor PubMed for new peer-reviewed studies on this topic and email a short summary when something meaningful changes.