Establish Reference Intervals Research Articles

Hematological and plasma biochemical profile of two species of freshwater stingrays from the Amazon.

The evaluation of hematological and plasma biochemical parameters and the subsequent establishment of reference intervals facilitate the diagnosis of the health status of animals. This work aimed to determine the blood parameters of wild specimens of the stingrays Potamotrygon motoro and Potamotrygon orbignyi from the lower Solimões River region, Amazonas, Brazil. One hundred forty-one stingrays were captured, 92 specimens of P. motoro and 49 of P. orbignyi, of both sexes and at different stages of development. No effect of sex was observed on the blood parameters of juvenile animals for both species. P. motoro neonates presented a distinct hematological and biochemical profile, with significantly lower hematocrit values, hemoglobina, number of erythrocytes, mean corpuscular hemoglobin concentration, monocytes, plasma glucose, total proteins, albumin, and globulin. On the other hand, total cholesterol and urea levels were significantly higher in this same group compared to juveniles of the same species. Comparison between species revealed lower values of triglycerides and total cholesterol in P. orbignyi of both sexes. The results obtained are pioneering for these Amazonian species in white water environments and will serve as a basis for evaluating the health status of wild stingrays. Thus, from the analysis of the blood of the P. motoro and P. orbignyi stingrays, it was possible to observe good health conditions.

Standard tests of haemostasis do not predict elevated thromboelastographic maximum amplitude, an index of hypercoagulability, in sick dogs.

To compare standard tests of haemostasis in hypercoagulable and normocoagulable sick dogs as identified by thromboelastography. We compared prothrombin time, activated partial thromboplastin time, D-dimers, packed red blood cell volume and platelet counts in client-owned sick dogs with hypercoagulable (n = 36) and normocoagulable (n = 35) states as identified by thromboelastography maximum amplitude above or within the established reference interval, respectively. Seventy-one dogs were included. Thirty-six dogs were hypercoagulable and 35 were normocoagulable on thromboelastography. Using multivariable linear models, prothrombin time, activated partial thromboplastin time and D-dimers were not associated with hypercoagulability. Platelet counts and packed red blood cell volume were significantly associated with hypercoagulability. Prothrombin time, activated partial thromboplastin time and D-dimers were not useful in the identification of hypercoagulability in dogs as defined by thromboelastography.

Age- and Sex-Dynamic Fluctuations and Reference Intervals for Alkaline Phosphatase Among the Spanish Population.

Interpretation of alkaline phosphatase (ALP) activity is essential for the diagnosis of certain diseases. ALP changes during life and may vary between different populations. To establish reference intervals (RIs) and percentile charts for ALP activity in the Spanish population through a multicentric observational study and to compare the RIs to those defined in other countries. A total of 662 350 ALP measurements from individuals ages 0 to 99 years from 9 Spanish tertiary care centers collected between 2020 and 2022 were analyzed. This study is the largest published in the literature to date. Continuous percentile charts for ALP according to sex and age were established which can be used as RIs. Higher levels are reached during the first weeks of life. In puberty, a differential evolution is observed in both sexes, reaching a peak at 10 to 13 years of age in boys and remaining stable in girls at this age. Significant differences were also observed in adults, higher in men between ages 20 and 49 years and between ages 50 and 79 years in women, as reported in some countries. ALP activity follows an age- and sex-dependent fluctuation with geographic differences. It is important to have appropriate reference values for each population in order to allow for a correct diagnostic interpretation and early diagnosis of diseases related to ALP abnormalities.

Establishing reference intervals for common hematology test parameters from apparently healthy geriatrics in Asella town, Southeast Ethiopia, 2020: a community-based cross-sectional study

BackgroundReference intervals are an important method tool for identifying abnormal laboratory test results. Complete blood count reference values are useful to interpret complete blood count (CBC) results and make clinical decisions, but these values have not been established for geriatrics in Asella town. Therefore, this study aimed to establish reference intervals (RIs) for complete blood count (CBC) parameters from geriatric participants/subjects in Asella town, Southeast Ethiopia.MethodsA community-based cross-sectional study was conducted from December 2019 to May 2020. An interviewer-administered questionnaire was used to collect data on sociodemography and other characteristics from 342 eligible geriatric participants. Weight, height, and vital signs were measured, and 8 mL of blood sample was collected. Screening tests such as HIV, HBsAg, HCV, syphilis, stool examination, and urinalysis were performed. The hematological parameter was measured using a Sysmex kx-21 hematology analyzer. The data were analyzed using SPSS version 21 software. The non-parametric independent Kruskal–Wallis test and Wilcoxon rank-sum test (Mann–Whitney U test) were used to compare the parameters between age groups and genders. The 97.5 and 2.5th percentile were the upper and lower reference limit for the population.ResultsAccording to the study’s findings, the reference intervals of red blood cell, white blood cell, platelet count, hemoglobin (HGB), and hematocrit (HCT) in male geriatrics were 3.8–5.85 × 1012/L, 3.1–9.66 × 109/L, 115.8–353 × 109/L, 12.4–17.76 g/dL, and 35.06–50.2%, respectively. The respective values for women were 3.94–5.48 × 1012/L, 3.13–8.4 × 109/L, 137.5–406 × 109/L, 12.5–16.4 g/dL, and 36.09–48.2%. Most of the hematological parameters showed significant differences between the two genders (p value <0.05).ConclusionAccurate gender and age-specific reference intervals are crucial in managing patient health. The current study offers essential CBC hematological parameters that can assist clinicians in interpreting laboratory results and can improve healthcare quality in the geriatric population. Therefore, it is more relevant to use the current RIs in the geriatric set-up.

Establishment of reference interval for Hemoglobin A1C and other Hemoglobin subfractions for healthy Saudi adults

Establishment of reference interval for Hemoglobin A1C and other Hemoglobin subfractions for healthy Saudi adults

Establishment of reference intervals for serum and salivary cortisol in Kano, Nigeria

Establishment of reference intervals for serum and salivary cortisol in Kano, Nigeria

Updated Reference Intervals for Alanine Aminotransferase in a Metabolically and Histologically Normal Population

Background and AimsThis study aims to reevaluate upper reference limit (URL) for alanine aminotransferase (ALT) by considering the changing epidemiology of major liver diseases. We employed histological and metabolic parameters in Asian living liver donors. MethodsWe performed a retrospective analysis of 5,455 potential living liver donors from 2005 to 2019. Participants were screened for hepatitis B, C, HIV, and alcohol use. Histologically and metabolically healthy participants were assessed using the Prati criteria (body mass index <23 kg/m2, triglyceride ≤200 mg/dL, fasting glucose ≤105 mg/dL, total cholesterol ≤220 mg/dL). The updated ALT-URL was determined as the 95th percentile among participants without hepatic steatosis and who met the Prati criteria. ResultsThe median age was 30 years, with a male predominance (66.2%). Among 5,455 participants, 3,162 (58.0%) showed no hepatic steatosis, with 1,553 (49.1%) meeting both the criteria for no steatosis and the Prati criteria for metabolic health. The updated URL for ALT in these participants was 34 U/L for males and 22 U/L for females, which was significantly lower than conventionally accepted values. Using this revised ALT-URL, 72.8% of males with ALT levels ≥34 U/L and 55.0% of females with ALT levels ≥22 U/L showed signs of steatosis, while 32.7% of males and 22.2% of females met the criteria for metabolic syndrome. ConclusionsOur study provided the newly established reference intervals for ALT levels in a metabolically and histologically verified Asian population. The proposed URL for ALT are 34 U/L and 22 U/L for males and females, respectively.

A comprehensive analysis of the immune system in healthy Vietnamese people

Lifestyle, diet, socioeconomic status and genetics all contribute to heterogeneity in immune responses. Vietnam is plagued with a variety of health problems, but there are no available data on immune system values in the Vietnamese population. This study aimed to establish reference intervals for immune cell parameters specific to the healthy Vietnamese population by utilizing multi-color flow cytometry (MCFC). We provide a comprehensive analysis of total leukocyte count, quantitative and qualitative shifts within lymphocyte subsets, serum and cytokine and chemokine levels and functional attributes of key immune cells including B cells, T cells, natural killer (NK) cells and their respective subpopulations. By establishing these reference values for the Vietnamese population, these data contribute significantly to our understanding of the human immune system variations across diverse populations. These data will be of substantial comparative value and be instrumental in developing personalized medical approaches and optimizing diagnostic strategies for individuals based on their unique immune profiles.

Reference intervals for bone marrow cells in juvenile and young adult cats bone marrow cytology.

The current bone marrow (BM) reference intervals (RI) are based on a limited number of cats. Age-related changes in BM variables might be important, possibly affecting the interpretation of the results. Establish BM cytologic reference intervals (RIs) and association of age and sex on these findings, in healthy juvenile and young adult cats. BM aspirates of cats deemed healthy based on history and clinical, CBC, serum chemistry findings, and negative retrovirus serology were obtained and examined cytologically. The examination included a 1000-nucleated differential cell count and cell morphologic assessment. RIs were calculated using parametric, robust, and nonparametric methods. The cytologic findings were examined for associations with sex and age. The study included 40 cats (females, 22 [55%]; males, 18 [45%]) with a median age of 1.5 years (range 0.5-5). Most calculated RIs were similar to those previously reported. BM plasma cell and monocyte counts were weakly and positively correlated with age (rs, .312 and .373, respectively; P < .05). Metarubricytes were higher in females (mean, 25.1%; SD, 6.0%) than males (mean, 21.2%; SD, 6.0%; P < .05). The BM differential cell counts determined in this study can serve as RIs for cats aged 0.5-5 years.

CLSI-based verification and de novo establishment of reference intervals for common biochemical assays in Croatian newborns.

This study aimed to examine whether the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) reference intervals for 19 commonly used biochemical assays (potassium, sodium, chloride, calcium, magnesium, inorganic phosphorous, glucose, urea, creatinine, direct and total bilirubin, C-reactive protein (CRP), total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) and lactate dehydrogenase (LD)) could be applied to the newborn population of one Croatian clinical hospital. Reference interval verification was performed according to the CLSI EP28-A3c guidelines. Samples of healthy newborns were selected using the direct a posteriori sampling method and analyzed on the Beckman Coulter AU680 biochemical analyzer. If verification wasn't satisfactory, further procedure included de novo determination of own reference intervals by analyzing 120 samples of healthy newborns. After the first set of measurements, 14/19 tested reference intervals were adopted for use: calcium, inorganic phosphorous, glucose, urea, creatinine, total bilirubin, CRP, total protein, albumin, AST, ALT, GGT, ALP and LD. A second set of samples was tested for 5 analytes: potassium, sodium, chloride, magnesium and direct bilirubin. The verification results of the additional samples for sodium and chloride were satisfactory, while the results for potassium, magnesium and direct bilirubin remained unsatisfactory and new reference intervals were determined. The CALIPER reference intervals can be implemented into routine laboratory and clinical practice for the tested newborn population for most of the analyzed assays, while own reference intervals for potassium, magnesium and direct bilirubin have been determined.

Clinical Impact of New Reference Intervals for the Roche Prolactin II Immunoassay.

The Roche prolactin immunoassay is used throughout the world. It reports higher values than the Siemens immunoassay but the manufacturer-defined reference intervals are similar. Patient results are often above the Roche upper limit but within the Siemens interval, causing diagnostic confusion. Establish new reference intervals for the Roche and Siemens prolactin immunoassays. We established new reference intervals for the Roche and Siemens immunoassays using 374 specimens from healthy outpatients. We performed chart review for unnecessary testing and treatment for 298 patients in a 6-month period with at least 1 Roche prolactin value above the manufacturer-defined upper limit and below our new upper limit. The new upper limit for the Roche assay was 37.8 ng/mL (females) and 22.8 ng/mL (males). The manufacturer-defined limits were 23.3 ng/mL and 15.2 ng/mL, respectively. New intervals for the Siemens assay matched the manufacturer. No cases of clinically significant pathophysiologic prolactin excess were identified in patients with values between the manufacturer-defined upper reference limit and our new Roche upper limit. Unnecessary further evaluation in these patients included 459 repeat prolactin measurements, 57 macroprolactin measurements, 39 magnetic resonance imaging studies, and 28 endocrine referrals. Eleven patients received dopamine agonists. The minimum cost of excess care using Medicare reimbursement rates was $34 134, with substantially higher amounts billed to patients and their insurance providers. Adoption of new upper reference limits for the Roche prolactin assay of 37.8 ng/mL (females) and 22.8 ng/mL (males) would not delay diagnosis or necessary intervention in patients with clinically significant pituitary tumors but would reduce unnecessary evaluation in patients without pathophysiologic prolactin excess.

Verification of bile acid determination method and establishing reference intervals for biochemical and haematological parameters in third-trimester pregnant women.

The aims of this study were to verify the bile acids (BA) method and to establish reference intervals (RIs) for bile acids (BA) and biochemical and haematological parameters in Croatian pregnant women. BA spectrophotometric method verification was performed on Siemens Atellica Solution CH 930 automated analyser using Sentinel reagent. Stability, precision, trueness, linearity, and RIs, as well as lipemia interference were tested according to CLSI guidelines. BA, biochemical, and haematological parameters were measured in serum (BA, biochemical) and whole blood (haematological) samples of fasting healthy third-trimester pregnant women from Croatia (n=121). The establishment of the RIs was done a priori according to the CLSI EP28-A3C:2010 guideline. Selected reference individuals' data were analysed using parametric, non-parametric, and robust methods. Stability study showed that BA are stable in serum samples for 2days at 20 °C, 14days at 4-8 °C, and 22days at-20 °C. The precision study and adult RIs verification met the criteria. Linearity was verified for the concentration range of 3.5-172.1 μmol/L whereas the lipemia interference test showed a positive bias (%) in BA concentration. The determined reference limits generally exhibited better precision for haematological parameters, being lower than the upper recommended value 0.2, unlike biochemical parameters. Haematological parameters showed notable differences between pregnant and non-pregnant women, while many biochemical parameters' RIs remained similar. Only ALT and GGT showed lower non-comparable RI upper limits in the population pregnant women. Spectrophotometric BA method showed satisfactory performance and all examined parameters were within the set criteria. Moreover, RIs for key biochemical and haematological parameters, including BAs, have been established for the first time in the population of Croatian pregnant women.

Reference intervals for thyroid function from the fifth to seventh day of life in twin-pregnancy preterm neonates: an 8-year retrospective study.

The immature and developing hypothalamic-pituitary-thyroid axis leads to different levels of thyroid function in twin neonates, including free thyroxine (FT4), free triiodothyronine (FT3), and thyroid stimulating hormone (TSH) levels. No reference intervals for twins have been established until now. To compensate for this lack, we collected data and established this standard across different gestational ages (GAs) and sexes. A total of 273 pairs of neonates admitted to the NICU in Southeast China from 2015 to 2022 were included. Each pair was divided into Neonate A (relatively heavy birth weight (BW)) and Neonate B (relatively light BW). Their thyroid functions were analyzed to establish reference intervals and comparisons were made stratified by GA and sex. The FT3, FT4, and TSH reference intervals in twin neonates with a GA of 26-36 weeks were as follows: Neonate A and B: 3.59 ± 0.99 and 3.57 ± 1.00 pmol/L; Neonate A and B: 17.03 ± 5.16 and 16.77 ± 5.29 pmol/L; and Neonate A and B: 4.097 ± 3.688 and 4.674 ± 4.850 mlU/L, respectively. There were significant differences between serum FT3 and FT4 reference intervals and GA (p < 0.05). The serum FT3 and FT4 reference intervals for male neonates were lower than those for female neonates in the 29-32-week group (p < 0.05). This was the first study, to our knowledge, to establish reference intervals for thyroid function in twin neonates from the fifth to seventh day of life, which will be beneficial for the diagnosis and management of congenital hypothyroidism.

Determination of Reference Interval for Serum Homocysteine, Magnesium, and Uric Acid from Tertiary Care Hospital Data By the Bhattacharya Method

Abstract Introduction: Direct ways of establishing reference intervals for analytes are time-consuming and costly. Bhattacharya analysis is an indirect way to mathematically derive reference intervals from the results of analytes in a hospital database and is practically a simple and inexpensive alternative to direct methods. Methods: Data of serum uric acid, magnesium, and plasma homocysteine levels measured in the department of clinical biochemistry over 3 years were extracted from the hospital database along with the age, gender, and location of the patients. Bhattacharya analysis was performed by identifying underlying Gaussian distribution within the non-Gaussian distribution of the patient data by log transformation of the distribution of the data. Reference intervals for each of the analytes were calculated from the identified Gaussian distribution. Results: The reference interval for serum uric acid was 1.49–8.97 mg/dL in men and 2.13–6.14 in women, serum magnesium in adults was 1.19–3.21 mg/dL, and plasma homocysteine in adults was 4.6–21 μg/dL. Conclusion: Using Bhattacharya analysis, we were able to derive reference intervals for analytes from our hospital data.

Serum Biochemistry Parameters of the Endangered Miranda's Donkey Breed: Reference Intervals and the Influence of Gender and Age.

Miranda's donkey is an endangered, autochthone breed from Northern Portugal. Understanding the physiological and pathological conditions of Miranda's donkey is crucial for the conservation of the breed. Our study aimed to establish reference intervals (RIs) for blood biochemistry parameters and to evaluate the influence of gender and age on these parameters. Blood samples from 75 clinically healthy animals were analyzed for 21 blood biochemistry parameters using Respons® 920 and Start® 4-Diagnostica-Stago. RIs were calculated according to the ASVCP guidelines, utilizing Reference Advisor software V. 2.1 and the statistical program SPSS version 29 to analyze the effects of gender and age. Significant gender-related differences (p < 0.05) were observed in cholesterol, chlorine, creatinine (CREA), glucose (GLU), sodium, and triglycerides (with higher values in females) and in aspartate aminotransferase, creatine phosphokinase (CK), gamma-glutamyl transferase, potassium, magnesium (Mg), and urea (with higher values in males). Age-related differences (p < 0.05) were noted for alkaline phosphatase, CK, fibrinogen, GLU, and phosphorus (higher in young animals) and for globulin, Mg, and total protein (higher in adults). The RIs described here are invaluable for assessing and monitoring the health status of individual animals and herds. Our study highlights the importance of considering gender and age in the interpretation of biochemical parameters, providing critical insights for the conservation and management of this endangered breed.

Establishing non-fasting reference values for plasma lipids levels based on age, sex, and puberty stage in a French-Canadian pediatric population

BackgroundDyslipidemias, including familial hypercholesterolemia (FH), are a significant risk factor for cardiovascular diseases. FH is a genetic disorder resulting in elevated levels of low-density lipoprotein cholesterol (LDL-C) and an increased probability of early cardiovascular disorders. Heterozygous familial hypercholesterolemia (HeFH) is the most common form, affecting approximately 1 in 250 individuals worldwide, with a higher prevalence among the French-Canadian population. Childhood is a critical period for screening risk factors, but the recommendation for non-fasting screening remains controversial due to a lack of specific reference values for this state. This study aims to establish reference values for lipid levels in non-fasting children from Sherbrooke, Quebec, Canada, that will be specific for sex, age, and pubertal stages.MethodsBlood samples and corresponding anthropometric data were collected from 356 healthy children aged from 6 to 13. They were categorized either into two age groups: Cohort 6–8 and Cohort 9–13, or into pubertal stages. Reference values, specifically the 2.5th, 5th, 10th, 50th, 90th, 95th, and 97.5th percentiles were determined using the CLSI C28-A3 guidelines.ResultsLipid profiles did not significantly differ between sexes, except for higher levels of high-density lipoprotein (HDL-C) in boys within Cohort 6–8. HDL-C levels significantly increased, while LDL-C and non-HDL-C levels significantly decreased in both sexes with age. Non-fasting age- and pubertal stages-specific reference values were established.ConclusionThis study established reference intervals for lipid markers in non-fasting state within the pediatric French-Canadian population. These findings could be used in dyslipidemia screening in daily practice.

Establishment of Reference Intervals of Blood Parameters Among the Healthy Afghan Population.

This study was designed and conducted to validate the reference values of hematological parameters for healthy adult male and female residents of Kabul city, Afghanistan. In this cross-sectional study, the samples were collected according to a non-random sampling method. Blood samples were collected from students and employees of Kabul University. The study included 166 males and 125 females, aged 18-45 years. The selection and exclusion of participants were carried out according to a questionnaire and the assessment of serum ferritin and vitamin B12 levels. Candidates with lower serum ferritin and vitamin B12, a history of chronic disease, females with menstruation or pregnancy, and those with chronic abdominal pain were excluded. Reference ranges for all blood parameters were determined by a non-parametric method. The determined reference values were compared between males and females by the Z-test. Reference intervals for hemoglobin (4.5-6.3 g/dL for males and 3.66-5.67 g/dL for females) and hematocrit (36.23-55.93% for males and 30.20-53.86% for females) were significantly (p<0.05) higher in males. No significant (p<0.05) differences were observed between the reference intervals for the red blood cell count. Therefore, we conclude that the commonly used reference intervals should be revised for the Afghan population, as our findings indicated higher reference values for the hemoglobin and hematocrit indices.

Validation of a LC-MS/MS method for establishing reference intervals and assessing diagnostic performance of urinary free cortisol and cortisone

Background24-h urinary free cortisol (UFF) is recommended for screening of Cushing's syndrome (CS), a rare disease characterized by apparent cortisol and cortisone excess. We aimed to validate a simple LC-MS/MS method for accurate measurement of UFF and urinary free cortisone (UFE), establishment of reference ranges, and evaluation of performance for CS diagnosis. MethodsUrine samples were processed using solid-phase extraction cartridges, followed by elution with methanol and acetonitrile. Analysis was performed via tandem mass spectrometry, utilizing multiple reaction monitoring and electrospray ionization source in positive ion mode. ResultsThe assay displayed excellent linearity (r > 0.99) in the range of 0.05–100 ng/mL for cortisol and 0.25–500 ng/mL for cortisone, with lower limits of quantification (LLOQ) at 0.05 ng/mL for cortisol and 0.25 ng/mL for cortisone. The obtained results for intra-day and inter-day imprecision for both analytes were within the acceptable range of less than 10 %. The trueness values for both compounds were also within the acceptable limit of 15 %. No significant matrix effects or carry over observed in our method. The reference intervals of UFF, UFE and UFF:UFE ratio were 7.01–45.66 µg/24-h, 27.97–139.21 µg/24-h and 0.17–0.56, respectively. UFF > 56.75 µg/24-h showed 100 % specificity and 100 % sensitivity for CS diagnosis, which was superior to UFF:UFE ratio. ConclusionsWe developed and validated a sensitive LC-MS/MS method to detect UFF and UFE. Our data indicate that UFF measured by the current LC-MS/MS assay exhibited high diagnostic performance for CS.

Development and validation of a simple, fast and sensitive liquid chromatography-tandem mass spectrometry method to establish reference intervals for 24-h urinary free normetanephrine, metanephrine and methoxytyramine

ObjectiveTo develop and validate a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect urinary free metanephrines and methoxytyramine, establishing reference intervals. MethodsUrine samples were diluted with isotope internal standard solution, then analyzed directly using tandem mass spectrometry with multiple reaction monitoring measurement and electrospray ionization source in positive ion mode. Analytical parameters including linearity, lower limit of quantitation, imprecision and accuracy of the method were evaluated. The reference intervals for urinary catecholamine metabolites were established by analyzing 24-h urine samples collected from 81 apparently healthy volunteers. ResultsThe analytical times for MN, NMN, and 3-MT were at 2.79, 2.80, and 2.74 min, respectively. The method displayed excellent linearity (r > 0.99) in the range of 1-1000 ng/mL, with lower limits of quantification (LLOQ) at 0.50 ng/mL for MN and NMN, and 0.25 ng/mL for 3-MT. The method's intra-day and inter-day imprecisions were less than 8 %. The method recovery ranged from 96.8% to 105.8 % for MN, 89.7%–106.4 % for NMN, and 93.5%–106.2 % for 3-MT. No carry-over was observed during the analysis of all analytes. The LC-MS/MS method was used to establish reference intervals in 24-h urine samples from 81 apparently healthy volunteers. There was no association of sex with urinary free metabolites. ConclusionThis study established a novel, fast and sensitive LC-MS/MS method for determining urinary free catecholamine metabolites, which could facilitate screening and diagnosis for catecholamine-related tumors more conveniently and quickly.

Reference intervals for serum 11-oxygenated androgens in children.

Classic androgens such as dehydroepiandrosterone, androstenedione, and testosterone are generally measured for diagnosis and treatment monitoring in children and adolescents with hyperandrogenism, as can occur in congenital adrenal hyperplasia, premature pubarche, or polycystic ovarian syndrome. However, adrenally-derived 11-oxygenated androgens also contribute to the androgen pool and should therefore be considered in clinical management. Nevertheless, paediatric reference intervals are lacking. Therefore, we developed a serum assay to establish reference intervals for four 11-oxygenated androgens in addition to four classic androgens. Reference interval study for serum 11-oxygenated androgens in children. We developed and validated a sensitive LC-MS/MS assay and quantified eight serum androgens, including four 11-oxygenated androgens, in serum of 256 healthy children (aged 0-17 years old). An age-dependency for all androgens was observed, and therefore we divided the cohort based on age (prepubertal (n=133; 94 boys, 39 girls) and pubertal (n=123; 52 boys, 71 girls)) to compute reference intervals (2.5th - 97.5th percentiles). In the prepubertal group, there was no significant sex-difference for any of the measured androgens. In the pubertal group, androstenedione, testosterone, and dihydrotestosterone showed a significant difference between boys and girls. In contrast, adrenal androgens dehydroepiandrosterone, 11-hydroxyandrostenedione, 11-ketoandrostenedione, 11-hydroxytestosterone, and 11-ketotestosterone did not. We developed and validated an assay for 11-oxygenated androgens, in addition to four classic androgens and established reference intervals. This enables a comprehensive evaluation of serum androgen status in children with clinical symptoms of hyperandrogenism.