Calculated Reference Intervals Research Articles

Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey.

In a collaboration between the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) and the Canadian Health Measures Survey (CHMS), we determined reference value distributions using an a priori approach and created a comprehensive database of age- and sex-stratified reference intervals for clinically relevant hematologic parameters in a large household population of children and adults. The CHMS collected data and blood samples from 11 999 respondents aged 3-79 years. Hematology markers were measured with either the Beckman Coulter HmX or Siemens Sysmex CA-500 Series analyzers. After applying exclusion criteria and removing outliers, we determined statistically relevant age and sex partitions and calculated reference intervals, including 90% CIs, according to CSLI C28-A3 guidelines. Hematology marker values showed dynamic changes from childhood into adulthood as well as between sexes, necessitating distinct partitions throughout life. Most age partitions were necessary during childhood, reflecting the hematologic changes that occur during growth and development. Hemoglobin, red blood cell count, hematocrit, and indices (mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration) increased with age, but females had lower hemoglobin and hematocrit starting at puberty. Platelet count gradually decreased with age and required multiple sex partitions during adolescence and adulthood. White blood cell count remained relatively constant over life, whereas fibrinogen increased slightly, requiring distinct age and sex partitions. The robust dataset generated in this study has allowed observation of dynamic biological profiles of several hematology markers and the establishment of comprehensive age- and sex-specific reference intervals that may contribute to accurate monitoring of pediatric, adult, and geriatric patients.

Age- and sex-specific dynamics in 22 hematologic and biochemical analytes from birth to adolescence.

Pediatric laboratory test results must be interpreted in the context of interindividual variation and age- and sex-dependent dynamics. Reference intervals as presently defined for separate age groups can only approximate the age-related dynamics encountered in pediatrics. Continuous reference intervals from birth to adulthood are not available for most laboratory analytes because of the ethical and practical constraints of defining reference intervals using a population of healthy community children. We applied an indirect method to generate continuous reference intervals for 22 hematologic and biochemical analytes by analyzing clinical laboratory data from blood samples taken during clinical care of patients. We included samples from 32 000 different inpatients and outpatients (167 000 samples per analyte) from a German pediatric tertiary care center. Measurements were performed on a Sysmex-XE 2100 and a Cobas Integra 800 during clinical care over a 6-year period. The distribution of samples considered normal was estimated with an established indirect statistical approach and used for the calculation of reference intervals. We provide continuous reference intervals from birth to adulthood for 9 hematology analytes (hemoglobin, hematocrit, red cell indices, red cell count, red cell distribution width, white cell count, and platelet count) and 13 biochemical analytes (sodium, chloride, potassium, calcium, magnesium, phosphate, creatinine, aspartate transaminase, alanine transaminase, γ-glutamyltransferase, alkaline phosphatase, lactate dehydrogenase, and total protein). Continuous reference intervals capture the population changes in laboratory analytes during pediatric development more accurately than age groups. After local validation, the reference intervals provided should allow a more precise consideration of these dynamics in clinical decision making.

CLSI-based transference of the CALIPER database of pediatric reference intervals to Beckman Coulter DxC biochemical assays

BackgroundThe CALIPER program has established a comprehensive database of age- and sex-stratified pediatric reference intervals for over 85 common biochemical markers, largely using the Abbott ARCHITECT assays. To allow a broader application of the CALIPER database, we examined transference to 36 Beckman Coulter Synchron Unicel DxC800 assays, based on the CLSI C28-A3/EP9-A3 guidelines. MethodsPatient sample comparisons were performed for 36 biochemical assays using 200 serum specimens obtained from pediatric patients on the Abbott ARCHITECT ci8200 and the Beckman Coulter DxC800. For each analyte, R2 values were calculated to assess the quality of correlation between the platforms. Statistical criteria used to assess transferability included a) regression analysis to create the equation of the line of best fit, b) standardized residual, c) Bland–Altman, and d) quantile–quantile plots. Transferred reference intervals were further verified by analyzing serum samples from 100 healthy children from the CALIPER cohort on the Beckman Coulter system. ResultsThe reference intervals for most of the assessed analytes were transferable to Beckman Coulter assays (31 out of 36 studied) and the newly calculated reference intervals were verified through analysis of CALIPER reference samples (28 out of 31). Eighteen assays demonstrated excellent correlation (R2≥0.95), and 13 assays showed strong correlation (0.77≤R2≤0.94). ConclusionThe current study allowed successful transference of a large number of biochemical markers from the CALIPER database to assays on the Beckman Coulter DxC800 platform. Transference should facilitate broader application of CALIPER reference intervals at pediatric centers using DxC biochemical assays.

Thyroid function in pregnancy: what is normal?

Gestational thyroid dysfunction is common and associated with maternal and child morbidity and mortality. During pregnancy, profound changes in thyroid physiology occur, resulting in different thyroid-stimulating hormone (TSH) and free thyroxine (FT4) reference intervals compared to the nonpregnant state. Therefore, international guidelines recommend calculating trimester- and assay-specific reference intervals per center. If these reference intervals are unavailable, TSH reference intervals of 0.1-2.5 mU/L for the first trimester and 0.2-3.0 mU/L for the second trimester are recommended. In daily practice, most institutions do not calculate institution-specific reference intervals but rely on these fixed reference intervals for the diagnosis and treatment of thyroid disorders during pregnancy. However, the calculated reference intervals for several additional pregnancy cohorts have been published in the last few years and show substantial variation. We provide a detailed overview of the available studies on thyroid function reference intervals during pregnancy, different factors that contribute to these reference intervals, and the maternal and child complications associated with only minor variations in thyroid function. There are large differences in thyroid function reference intervals between different populations of pregnant women. These differences can be explained by variations in assays as well as population-specific factors, such as ethnicity and body mass index. The importance of using correct reference intervals is underlined by the fact that even small subclinical variations in thyroid function have been associated with detrimental pregnancy outcomes, including low birth weight and pregnancy loss. It is therefore crucial that institutions do not rely on fixed universal cutoff concentrations, but calculate their own pregnancy-specific reference intervals.

Biochemia Medica's editorial policy on authorship.

Computer- intensive resampling/bootstrap methods are feasible when calculating reference intervals from non-Gaussian or small reference samples. Microsoft Excel® in version 2010 or later includes natural functions, which lend themselves well to this purpose including recommended interpolation procedures for estimating 2.5 and 97.5 percentiles. The purpose of this paper is to introduce the reader to resampling estimation techniques in general and in using Microsoft Excel® 2010 for the purpose of estimating reference intervals in particular. Parametric methods are preferable to resampling methods when the distributions of observations in the reference samples is Gaussian or can transformed to that distribution even when the number of reference samples is less than 120. Resampling methods are appropriate when the distribution of data from the reference samples is non-Gaussian and in case the number of reference individuals and corresponding samples are in the order of 40. At least 500-1000 random samples with replacement should be taken from the results of measurement of the reference samples.

Reference intervals data mining: no longer a probability paper method.

To describe the application of a data-mining statistical algorithm for calculation of clinical laboratory tests reference intervals. Reference intervals for eight different analytes and different age and sex groups (a total of 11 separate reference intervals) for tests that are unlikely to be ordered during routine screening of disease-free populations were calculated using the modified algorithm for data mining of test results stored in the laboratory database and compared with published peer-reviewed studies that used direct sampling. The selection of analytes was based on the predefined criteria that include comparability of analytical methods with a statistically significant number of observations. Of the 11 calculated reference intervals, having upper and lower limits for each, 21 of 22 reference interval limits were not statistically different from the reference studies. The presented statistical algorithm is shown to be an accurate and practical tool for reference interval calculations.

Establishment of trimester-specific thyroid stimulating hormone and free thyroxine reference interval in pregnant Chinese women using the Beckman Coulter UniCel™ DxI 600.

Thyroid-stimulating hormone (TSH) and free thyroxine (FT4) reference intervals are essential for screening and diagnosing thyroid dysfunction during pregnancy. The aim of this study was to establish method- and trimester-specific TSH and FT4 reference intervals in pregnant Chinese women using the Beckman Coulter UniCel™ DxI 600. A cross-sectional dataset analysis was performed. A total of 3507 participants were recruited, and 2743 were eligible for analysis to set reference intervals. TSH, FT4, and thyroid peroxidase antibody (TPOAb) levels were analyzed with the Beckman Coulter UniCel™ DxI 600 Access® immunoassay system. Ranges between the 2.5th and 97.5th percentiles were defined as reference intervals for TSH and FT4. The calculated reference intervals for the first, second, and third trimesters were TSH: 0.06-3.13, 0.07-4.13 and 0.15-5.02 mIU/L, respectively, and FT4: 8.72-15.22, 7.10-13.55 and 6.16-12.03 pmol/L, respectively. Our reference intervals for TSH and FT4 are distinct from the ranges reported in the DxI 600 instruction manual and previously reported data, confirming the importance of method-specific reference intervals.

Thromboelastography in healthy dairy cows

Thromboelastography is a whole blood-based coagulation assay that can be used to investigate hypocoagulability and hypercoagulability, as seen with thromboembolic diseases and disseminated intravascular coagulation. Numerous coagulopathies due to different causes are reported in cows. The objective was to establish reference intervals for thromboelastography using the TEG 5000 (Haemonetics GmbH, Munich, Germany) with citrated whole blood samples and kaolin activation in dairy cows and to investigate possible thromboelastographic changes between cows in different lactation periods. An additional objective was to test the stability of samples for up to 100h. Sixty blood samples from healthy Holstein-Friesian cows were examined. The samples were allocated to 3 different lactation groups (≤30 d postcalving, 31–99 d postcalving, ≥100 d postcalving). Thromboelastography was performed by using the TEG 5000 analyzer with citrated whole blood samples with kaolin activation. The calculated reference intervals were as follows: reaction time=2.2 to 6.2min, coagulation time=0.8 to 2.0min, angle α=58.2 to 81.8°, maximum amplitude=64.3 to 89.2mm, and clot rigidity=9.2 to 41.2 dyn/cm2. The 3 different lactation groups showed no significant differences in TEG parameters. No significant difference was seen in samples stored for up to 48h at room temperature, which indicates that delays in processing samples, such as those arising during transit, are not an issue.

Plasma biochemical and PCV ranges for healthy, wild, immature hawksbill (Eretmochelys imbricata) sea turtles

In recent years, the use of blood chemistry as a diagnostic tool for sea turtles has been demonstrated, but much of its effectiveness relies on reference intervals. The first comprehensive...

Haematological reference intervals in a multiethnic population.

IntroductionSimilar to other populations, full blood count reference (FBC) intervals in Malaysia are generally derived from non-Malaysian subjects. However, numerous studies have shown significant differences between and within populations supporting the need for population specific intervals.MethodsTwo thousand seven hundred twenty five apparently healthy adults comprising all ages, both genders and three principal races were recruited through voluntary participation. FBC was performed on two analysers, Sysmex XE-5000 and Unicel DxH 800, in addition to blood smears and haemoglobin analysis. Serum ferritin, soluble transferrin receptor and C-reactive protein assays were performed in selected subjects. All parameters of qualified subjects were tested for normality followed by determination of reference intervals, measures of central tendency and dispersion along with point estimates for each subgroup.ResultsComplete data was available in 2440 subjects of whom 56% (907 women and 469 men) were included in reference interval calculation. Compared to other populations there were significant differences for haemoglobin, red blood cell count, platelet count and haematocrit in Malaysians. There were differences between men and women, and between younger and older men; unlike in other populations, haemoglobin was similar in younger and older women. However ethnicity and smoking had little impact. 70% of anemia in premenopausal women, 24% in postmenopausal women and 20% of males is attributable to iron deficiency. There was excellent correlation between Sysmex XE-5000 and Unicel DxH 800.ConclusionOur data confirms the importance of population specific haematological parameters and supports the need for local guidelines rather than adoption of generalised reference intervals and cut-offs.

Validity of establishing pediatric reference intervals based on hospital patient data: A comparison of the modified Hoffmann approach to CALIPER reference intervals obtained in healthy children

ObjectivesTo compare pediatric reference intervals calculated using hospital-based patient data with those calculated using samples collected from healthy children in the community as part of the CALIPER study. MethodsHospital-based data for 13 analytes (calcium, phosphate, iron, ALP, cholesterol, triglycerides, creatinine, direct bilirubin, total bilirubin, ALT, AST, albumin and magnesium), measured on the Vitros 5600, collected between 2007 and 2011 were obtained. The data for each analyte were partitioned by age and gender as previously defined by the CALIPER study. Outliers in each partition were removed using the Tukey method. The cumulative distribution function (cdf) was then determined for each analyte value following which, the inverse cdf values of a standard Gaussian distribution were calculated. The analyte values were plotted against the inverse cdf of the standard Gaussian distribution. Piece-wise regression determined the linear portion of the resulting graph using the statistical software R. Linear regression determined an equation for the linear portion in each partition and reference intervals were calculated by extrapolating to identify the 2.5th and 97.5th centiles in each partition based on the inverse cdf values (which would correspond to the values −1.96 and 1.96 of the Gaussian distribution). Using the 90% confidence intervals for the reference intervals defined by CALIPER and the Reference Change Value (RCV) as the criteria, these calculated reference intervals were compared to those reported previously by CALIPER. Reference samples were also measured on the Vitros 5600 analyzer in an attempt to validate the calculated reference intervals. ResultsIn general, the reference intervals calculated from hospital-based data were generally wider than those calculated by CALIPER. None of the reference intervals calculated using the Hoffmann approach fell completely within the 90% confidence intervals calculated by CALIPER. ConclusionsThese results suggest that calculating pediatric reference intervals from hospital-based data may be useful, as a guide, in some cases but will likely not replace the need to establish reference intervals in healthy pediatric populations.

Reference interval studies: what is the maximum number of samples recommended?

Little attention has been paid to the maximum number of specimens for reference interval calculation, i.e., the number of specimens beyond which there is no further benefit in reference interval calculation. We present a model for the estimation of the maximum number of specimens for reference interval studies based on setting the 90% confidence interval of the reference limits to be equal to the analyte reporting interval. Equations describing the bounds on the upper and lower 90% confidence intervals for logarithmically transformed and untransformed data were derived and applied to determine the maximum number of specimens required to calculate a reference interval for 12 common chemistry and hematology analytes. Maximum sample sizes ranged from 126 to 18,171 and depended on the standard deviation of the population, any transformation involved and on the chosen reporting interval. This paper demonstrates the importance of the influence of reporting interval on reference intervals. Using this technique can reduce the cost of determining a reference interval by identifying the maximum number of specimens required.

Establishing Reference Intervals for Bone Turnover Markers in the Healthy Shanghai Population and the Relationship with Bone Mineral Density in Postmenopausal Women

The reference ranges of bone turnover markers (BTMs) were important during the treatment of osteoporosis, and the associations with bone mineral density (BMD) were controversial. The aim of this study was to establish the reference ranges of N-terminal procollagen of type l collagen (P1NP), osteocalcin (OC), and beta C-terminal cross-linked telopeptides of type I collagen (β-CTX) in Shanghai area and to investigate the relationships between BTMs and BMD in postmenopausal women. 2,799 subjects recruited in Shanghai City were measured BTMs to establish the reference ranges. Additional 520 healthy postmenopausal women were also measured BTMs, these women measured BMD in addition. BTMs were measured using the Roche electrochemiluminescence system. We used the age range of 35 to 45-year-olds to calculate reference intervals. The reference range of OC was 4.91 to 13.90 ng/mL for women and 5.58 to 16.57 ng/mL for men, P1NP was 13.72 to 32.90 ng/mL for women and 16.89 to 42.43 ng/mL for men, and β-CTX was 0.112 to 0.210 ng/mL for women and 0.100 to 0.378 ng/mL for men. BTMs significantly negatively correlated with lumbar spine and femoral and total hip in postmenopausal women (Betastd = −0.157 ~ −0.217, P < 0.001). We established the normal reference ranges of P1NP, OC, and β-CTX in the Shanghai area. This study also found that BTMs correlated with BMD and suggested that BTMs were the key determining factors of early BMD decreases.

Reference intervals for organically raised sheep: Effects of breed, location and season on hematological and biochemical parameters

Hematological and biochemical tests are widely used for assessment of health status and diagnosis of disease in domestic animals. They are almost indispensible in health management of organic flocks, but environmental conditions and food supply can be highly variable in organic farming, which may cause levels of many analytes to lie outside the published reference intervals. We investigated key hematological and biochemical parameters in organically raised sheep in Croatia. Different (mostly indigenous) breeds raised in various geographic regions were evaluated throughout the year. We identified key parameters that varied under different conditions using classification based on machine learning. Our findings suggest that location (which determines climate and food availability) has a profound effect on hematological and biochemical values in organically raised sheep. The effect of location was more pronounced than that of breed and season. The results clearly show the merit of establishing specific reference intervals for each flock, especially in organic farming. We demonstrate feasibility of this approach by successful calculation of reference intervals from a small sample (30 individuals) using robust statistical methods.

A generalised Box–Cox transformation for the parametric estimation of clinical reference intervals

Parametric methods for the calculation of reference intervals in clinical studies often rely on the identification of a suitable transformation so that the transformed data can be assumed to be drawn from a Gaussian distribution. In this paper, the two-stage transformation recommended by the International Federation for Clinical Chemistry is compared with a novel generalised Box–Cox family of transformations. Investigation is also made of sample sizes needed to achieve certain criteria of reliability in the calculated reference interval. Simulations are used to show that the generalised Box–Cox family achieves a lower bias than the two-stage transformation. It was found that there is a possibility that the two-stage transformation will result in percentile estimates that cannot be back-transformed to obtain the required reference intervals, a difficulty not observed when using the generalised Box–Cox family introduced in this paper.

Plasma total adiponectin levels in pediatrics: Reference intervals calculated as a continuous variable of age

ObjectivePlasma total Adiponectin (tAN) is a novel biomarker with interesting potentialities in pediatrics. Age-related intervals are needed for the correct interpretation of results. In this study, we calculated the reference values for tAN using a large number of results derived from pediatric patients. Design and methodstAN was determined by ELISA in more than 4000 samples and the results collected in the Laboratory Information System were used for the calculation of reference intervals. ResultstAN reference values for the different age intervals were obtained and age related tAN reference intervals were calculated. Some differences were observed in males and females. In obese children, no correlation between age and tAN could be observed. On the contrary, significant relations were observed in the total group of patients and in healthy control subjects. For this reason, the parameters of the equations to calculate tAN reference values using age as a continuous variable were defined. ConclusionThe use of continuous tAN reference intervals, calculated on age, should be considered a useful laboratory tool, at least in the pediatric population, for those biomarkers whose reference values have been shown to change from birth to the late adolescence.

Cerebrospinal fluid cathepsin B and S

Cathepsins are increased in the brain of elderly animals. We investigate the presence of cathepsin B and S in human cerebrospinal fluid (CSF) plasma and the associations with cystatin C, age and sex. We measured cathepsin B and S concentrations in CSFs from 118 persons, undergoing elective surgical procedures, with ELISA. Both cathepsin B and cathepsin S were positively correlated with age. No correlation was observed between cathepsin B or S and length, height or body mass index. Both cathepsin B and S were positively correlated to the cystatin C concentration in CSF. Calculated reference intervals were 4,893-17,636 pg/mL for cathepsin B and 2,681-11,459 pg/mL for cathepsin S. Elderly individuals had significantly higher levels of both cathepsin B (r s = 0.38, p = 0.00002) and cathepsin S (r s = 0.35, p = 0.0001) in CSF.

Reference interval computation: which method (not) to choose?

BackgroundWhen different methods are applied to reference interval (RI) calculation the results can sometimes be substantially different, especially for small reference groups. If there are no reliable RI data available, there is no way to confirm which method generates results closest to the true RI. MethodsWe randomly drawn samples obtained from a public database for 33 markers. For each sample, RIs were calculated by bootstrapping, parametric, and Box-Cox transformed parametric methods. Results were compared to the values of the population RI. ResultsFor approximately half of the 33 markers, results of all 3 methods were within 3% of the true reference value. For other markers, parametric results were either unavailable or deviated considerably from the true values. The transformed parametric method was more accurate than bootstrapping for sample size of 60, very close to bootstrapping for sample size 120, but in some cases unavailable. ConclusionsWe recommend against using parametric calculations to determine RIs. The transformed parametric method utilizing Box-Cox transformation would be preferable way of RI calculation, if it satisfies normality test. If not, the bootstrapping is always available, and is almost as accurate and precise as the transformed parametric method.

Reference intervals of extended erythrocyte and reticulocyte parameters

Optical analysis of erythrocytes can provide information on the hemoglobin concentration and content of reticulocytes and mature erythrocytes. Such parameters have proven clinical utility in anemia diagnosis and therapy monitoring. For interpretation, reliable reference ranges are needed. The aim of this study was to establish reference intervals for extended erythrocyte and reticulocyte parameters as measured with the Abbott CELL-DYN Sapphire hematology analyzer. Secondary aims were to study sample stability and to investigate gender- and age dependency of the reference ranges. Extended RBC parameters were measured in routine samples of a primary health care laboratory. The study cohort included 8161 samples of unique individuals, which were analyzed using Bhattacharya statistics. As a comparison, reference intervals were calculated in a subset of individuals without iron depletion. The majority of erythrocyte and reticulocyte parameters were normally distributed, allowing calculation of reference intervals. Only for hypo- and hyperchromic erythrocytes non-parametric statistics had to be used. The reference range for mean cellular hemoglobin content of reticulocytes (MCHr) was 28.5-34.5 pg (1.77-2.14 fmol) in the entire study group and 26.0-35.1 pg (1.60-2.17 fmol) in the non iron-depleted subgroup. No differences between sexes were found. Most parameters showed significant age effects in children and adolescents. Reference intervals have been established for extended RBC and reticulocyte parameters for the CELL-DYN Sapphire. Gender effects could not be demonstrated and age effects were of limited size, except for individuals younger than 18 years. Extended RBC parameters are stable for at least 6 h after blood collection.

Consensus: a framework for evaluation of uncertain gene variants in laboratory test reporting

Accurate interpretation of gene testing is a key component in customizing patient therapy. Where confirming evidence for a gene variant is lacking, computational prediction may be employed. A standardized framework, however, does not yet exist for quantitative evaluation of disease association for uncertain or novel gene variants in an objective manner. Here, complementary predictors for missense gene variants were incorporated into a weighted Consensus framework that includes calculated reference intervals from known disease outcomes. Data visualization for clinical reporting is also discussed.