Separate Reference Intervals Research Articles

Comparison of Blood Aminotransferase Methods for Assessment of Myopathy and Hepatopathy in Florida Manatees (Trichechus manatus latirostris)

Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate aminotransferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 micromol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement, indicating that separate reference intervals should be established for serum and plasma. Finally, storage of these enzymes at -70 degrees C for 1 mo resulted in up to a 25% decrease in enzymatic activity in manatee plasma.

Normal blood cells reference intervals of healthy adults at the Gaza Strip—Palestine

Hematological parameters are affected by different factors that include age, sex, smoking, ethnicity, and environmental altitude. It has been justified that each population must establish its own normal reference intervals to be used in clinical assessments and interpretations. Hematological reference intervals for adults from the Gaza Strip-Palestine have never been addressed. Therefore, this study was designed and aimed at the establishment of normal blood cells reference intervals for healthy adults at the Gaza Strip-Palestine. This study involved 89,491 apparently healthy individuals (from both sexes and from the different governorates of the Gaza Strip) who were referred to the Thalassemia Central Laboratory during the period from September 2000 until February 2008. Complete blood counts were performed. Subjects were categorized into subgroups according to gender, smoking habit, and age (15-18, 19-45, and >45 years old). For each subgroup, descriptive and comparative statistical analysis was performed for hematological parameters. The results showed substantial differences between males and females, between smokers and nonsmokers, and between the different age groups. Moreover, reference intervals derived from our population are markedly shifted downward as compared with Western European and American populations. It was concluded that separate and region-specific reference intervals based on gender, smoking, and age for the Palestinian population at the Gaza Strip should be generalized for clinical laboratories and clinical practitioners, which could help in interpreting laboratory hematological tests more specifically, and potentially develop the quality of medical care provided to patients.

Biochemical reference intervals for sex hormones with a new AutoDelfia method in aged men

Our aim was to establish sex hormone reference intervals measured with a new AutoDelfia immunoassay method for aged men free of medication and/or conditions known to influence sex hormone levels. The reference population consisted of 466 individuals between 64 and 97 years (mean 72 years) and a mean body mass index (BMI) of 26.9 kg/m(2). Because age correlated significantly with most sex hormones studied, we calculated reference intervals for three age groups (64-69, 70-74 and > or =75 years). In clinical practice, single ranges can be used for men aged 64 years or over for testosterone, estradiol and follicle-stimulating hormone (FSH) with the AutoDelfia method. For free testosterone and luteinizing hormone (LH), separate reference intervals should be used for men aged 64-74 years and those aged 75 years or over. For sex hormone-binding globulin, two separate reference intervals by age (64-69 and > or =70 years) are also needed for aged men. LH and FSH reference ranges should be judged with caution, because they may be too high due to cases of subclinical hypogonadism included in the reference population.

Holotranscobalamin and Total Transcobalamin in Human Plasma: Determination, Determinants, and Reference Values in Healthy Adults

We developed microbiological assays (MBAs) to identify determinants and to establish reference values for cobalamin bound to transcobalamin [holotranscobalamin (holoTC)] and total TC in plasma. We captured holoTC with magnetic beads with TC antibodies and used a conventional MBA for cobalamin measurements. Total TC was determined as holoTC after TC was saturated with cyanocobalamin. The new assays were compared with published methods. Determinants and reference values were determined in 500 blood donors, ages 18-69 years. Determination of cobalamin, holoTC, and TC by MBA required <150 microL. HoloTC and TC by MBA correlated with holoTC by RIA (r = 0.95) and TC by ELISA (r = 0.79), respectively. Between-day CVs for holoTC and total TC were 4%-9%. Women had lower holoTC than men, but only at age < or = 45 years. In multivariate regression analyses, holoTC was positively associated with age (in women only), creatinine (in men only), and plasma concentrations of total TC, folate, and cysteine, but inversely correlated with homocysteine and methylmalonic acid. For all study participants, total TC was associated with holoTC and number of TCN2 766C alleles; in female participants only, total TC was also associated with age, homocysteine, and cysteine. Reference values were 670-1270 pmol/L for TC and 42-157 pmol/L for holoTC, but they differed according to age and sex. Our MBAs for TC and holoTC required low plasma volume and performed acceptably compared with other methods. Determinants of holoTC and TC differed between men and women and according to age. Separate reference intervals for holoTC should be considered in younger women.

Clinical biochemistry of pregnant and nursing mares

Pregnancy and lactation result in increased metabolic demands. Although homeostatic mechanisms function to keep substances in blood at relatively constant levels, some changes in the concentrations of routine clinical chemistry analytes are likely to occur. The purpose of this study was to determine what physiological changes occur in serum clinical biochemistry analytes in pregnant and nursing mares, and to determine whether the changes were substantial enough to warrant separate reference intervals for pregnant or lactating horses. Forty-two Quarter Horse, Thoroughbred, Saddlebred, Standardbred and Morgan mares were entered into the study while pregnant. They were bled once each month through birth. Studies were continued on 20 mares until their foals were weaned. Test results were tabulated by time before or after birth. Serum biochemistry values were determined by standard methods using automated analyzers. Test results were analyzed using the Kruskal-Wallis 1-way ANOVA on ranks. If a significant difference was found (P<.05), Dunn's multiple comparison procedure was performed on all pairs. Results from pregnant and nursing mares also were compared with a reference group of 19 open, nonlactating mares. Serum triglyceride, potassium, creatinine, and total bilirubin concentrations were lower during lactation than during pregnancy. Serum calcium concentration also was slightly decreased at 2 time points during lactation. Triglyceride concentration was highest during midgestation, while bilirubin and creatinine values increased, and potassium and calcium remained constant during pregnancy. Serum urea concentration also remained constant during pregnancy but increased during lactation. Serum phosphate concentration was lowest during midgestation and highest during lactation. Total CO2 values were highest, and anion gaps were lowest, during midgestation. No significant differences were found in serum albumin, globulin, albumin:globulin ratio, total protein, or glucose values. When compared with the reference group of open mares, serum triglyceride, potassium, bilirubin, and total CO2 concentrations were lower, and anion gap was higher in horses that were nursing. Although most biochemical values remained relatively constant, significant differences were observed during pregnancy and lactation. Changes in the concentrations of triglycerides, potassium, bilirubin, total CO2, and anion gap during lactation were substantial enough to warrant separate reference intervals for lactating horses.

Performance Characteristics of an HPLC Assay for Urinary Albumin

Microalbuminuria is a marker of diabetic nephropathy and cardiovascular risk. Immunoassays underestimate the amount of intact albumin present in urine. The purpose of this study was to evaluate a new urinary albumin assay that uses size exclusion high-performance liquid chromatography (HPLC). We determined the limit of detection, linearity, imprecision, a comparison with an immunoturbidimetric assay, and pediatric and adult reference intervals. The limit of detection was 3.4 mg/L. The assay was linear from 4 to 240 mg/L. Total imprecision was less than 10% from 16 to 206 mg/L. Comparison of the albumin/creatinine ratio by HPLC with an immunoturbidimetric method showed positive proportional bias, which decreased with increasing concentrations of albumin. Nonparametric reference intervals were 22 to 250 mg/g for girls, 20 to 130 mg/g for boys, 14 to 62 mg/g for women, and 10 to 37 mg/g for men. This HPLC assay for urinary albumin shows acceptable performance and quantifies albumin species that are not detected by immunoassay. Separate reference intervals for children and adults seem necessary.

Performance Characteristics of an HPLC Assay for Urinary Albumin

Microalbuminuria is a marker of diabetic nephropathy and cardiovascular risk. Immunoassays underestimate the amount of intact albumin present in urine. The purpose of this study was to evaluate a new urinary albumin assay that uses size exclusion high-performance liquid chromatography (HPLC). We determined the limit of detection, linearity, imprecision, a comparison with an immunoturbidimetric assay, and pediatric and adult reference intervals. The limit of detection was 3.4 mg/L. The assay was linear from 4 to 240 mg/L. Total imprecision was less than 10% from 16 to 206 mg/L. Comparison of the albumin/creatinine ratio by HPLC with an immunoturbidimetric method showed positive proportional bias, which decreased with increasing concentrations of albumin. Nonparametric reference intervals were 22 to 250 mg/g for girls, 20 to 130 mg/g for boys, 14 to 62 mg/g for women, and 10 to 37 mg/g for men. This HPLC assay for urinary albumin shows acceptable performance and quantifies albumin species that are not detected by immunoassay. Separate reference intervals for children and adults seem necessary.

Laryngopharyngeal Reflux: Trends in Diagnostic Interpretation Criteria

Laryngopharyngeal reflux (LPR) is becoming recognized as a clinical entity with a variety of presentations distinct from those of gastroesophreflux disease (GERD). However, much uncertainty remains as to what is considered pathologic versus physiologic reflux. The aim of the study was to determine the normal range of pharyngeal reflux (PR) occurring in healthy adults based on pH-monitoring parameters utilized in the DeMeester scoring system for GERD. We have reviewed the current pool of prospective literature examining ambulatory dual-channel pH-monitoring study data derived from hypopharyngeal proximal probes in normal adults. From our review we have identified trends in several monitoring parameters based on the DeMeester scoring system for GERD. Our discussion recognizes and accepts the limitations imposed by small sample sizes and the number of healthy individuals that would be required to determine the general adult physiologic range of PR. We also explore the possible need for separate normal PR reference intervals based on age or gender disparities. Additional discussion and the summary address future directions for LPR research notably, (1) identification of the most appropriate research paradigm for LPR (i.e., pH 4 vs. 5), (2) establishing reproducibility for the appropriate LPR research paradigm, and (3) complementary modalities to ambulatory dual-channel pH monitoring for the study of acid and nonacid bolus movement within the esophagus

Effect of Ethnicity on Reference Intervals

The clinical chemist desiring to set up appropriate reference intervals must consider race or ethnicity as a possible factor on which to subdivide an interval. The effect of ethnicity on various analyte values has not been systematically studied (1)(2). The advent of National Health and Nutrition Examination Survey (NHANES) data (3) has made it possible to determine the effect of ethnicity on reference interval estimation and whether separate reference intervals should be derived for each ethnic group. The Third National Health and Nutrition Examination Survey (NHANES III) contains data for 33 994 persons ages 2 months and older who participated in the survey. Clinical chemistry measurements were made on several analytes, and health status was determined by a physician. In this report, we will use only individuals with the “Excellent”rating of health status as recommended by the IFCC (4) and NCCLS (5). The issue of whether to use separate reference intervals has been addressed by Harris and Boyd (6). Their recommendation, which is used by the NCCLS (5), is that two groups should be combined into a single group unless their means and/or SDs exceed appropriate predetermined thresholds. By treating the known healthy individuals as a “gold standard”, the effect of the addition of ethnicity on these estimators will be examined. We used a previously described outlier detection scheme (7) to reduce the effect of atypical observations on these estimators. The NHANES population was obtained from NHANES 3x Ver. 1.21 (3). The data are the result of a complex survey design involving stratification and clustering that yielded individual weights for each observation. However, for our purposes in this report, we will treat the individuals as coming from a random sample, i.e., individual weights …

Reference values for serum biochemical parameters in free-ranging harp seals.

The harp seal (Phoca groenlandica) is one of the most important predators in the Northeastern Atlantic ecosystem. Establishing biochemical reference intervals is important for evaluating the health status of harp seals kept in captivity and for evaluating the effects of environmental changes on the health of populations in the wild. The purpose of this study was to determine reference values for serum biochemical parameters in wild adult harp seals using readily available current methods. Blood samples were obtained from 14 adult female harp seals and 9 suckling pups on the pack ice of the Greenland Sea in early March 1998. Seven seals were humanely killed on the ice by permission of the Norwegian Directory of Fisheries and in conjunction with several other research projects. The seals were sampled within 15 minutes postmortem. Remaining seals were captured alive and sampled via the extradural intravertebral vein. Serum biochemical parameters were measured using a Technicon Axon analyzer and included electrolytes (sodium, chloride, potassium, magnesium, and calcium), substrates (free fatty acids, triglycerides, fructosamine, and glucose), end products (urea and uric acid), and proteins (total protein, globulins, and albumin). Serum protein electrophoresis also was done. Data were tested for normality and reference limits were calculated as mean +/- 1.96 X SD. Results between groups were compared using 2-tailed t-tests. Serum levels of glucose and triglycerides were lower, but serum levels of urea were higher in dead animals than in animals that were captured alive. Serum levels for 7 of 17 parameters were significantly different in pups compared with adults. Separate reference intervals were calculated for adult seals and seal pups. Both sampling method and age should be considered when evaluating the results of future analysis of serum parameters in wild and captive harp seals.

Serum Cystatin C as a Marker of Kidney Dysfunction in an Elderly Population

The prevalence of end-stage renal disease is increasing worldwide. Because nephropathy induced by type 2 diabetes accounts for most of the increase, a growing proportion of the patients are elderly. As preventive and renoprotective interventions are available, early identification of nephropathy is crucial, and there is a growing demand for a clinically convenient and reliable marker of renal function. Serum creatinine is widely used as a marker of the glomerular filtration rate (GFR), but the influence of muscle mass and, hence, the considerable interindividual variability limit its usefulness, especially in elderly individuals (1). Screening for microalbuminuria is used in assessing incipient nephropathy in diabetic patients, but the intraindividual variation and the need for repetitive urine sampling make it impractical in a geriatric setting. Serum cystatin C has been claimed to be a more sensitive indicator of GFR than serum creatinine (2)(3). It is unaffected by muscle mass, and it has been reported that cystatin C, unlike creatinine, might be able to mirror the involutional decrease in GFR that occurs with ageing (4)(5). Separate reference intervals for the elderly have been proposed in a few studies (5)(6)(7)(8), but the usefulness of cystatin C as a marker of renal function has not been extensively examined in large elderly populations. In a cross-sectional epidemiologic study, we compared serum cystatin C, serum creatinine, …

Biological variation of retinoids in man

This investigation was undertaken to assess biological variation, especially the within-subject variations of all- trans retinoic acid, 13- cis retinoic acid and retinol in human serum. Diurnal variation and variation over a week, a month and a year were studied in 11 males (aged 21 - 54 years) and 17 females (aged 22 - 63 years), all subjectively healthy. We found no diurnal variation with the exception of all- trans retinoic acid, which had maximal concentrations at noon irrespective of food intake. Seasonal variations were marginal. Both all- trans and 13- cis retinoic acids had fairly high within-subject (13.1% and 12.6%, respectively) and between-subject coefficients of variation (15.9% and 21.0%, respectively), while the within-subject CV of retinol was less (5.6%, with a between-subject CV of 21.1%). Thus, the indices of individuality were <1 for all retinoids. The critical differences between two consecutive samples were <40% for the retinoic acids and <20% for retinol. Women had higher all- trans retinoic acid concentrations in serum (5.1 nmol/L vs. 4.5 nmol/L), lower 13- cis retinoic acid concentrations (4.5 nmol/L vs. 5.5 nmol/L) and lower retinol concentrations in serum (2.1 µmol/L vs. 2.5 µmol/L) than men. Thus, samples for retinoid determinations should be drawn in the morning and evaluated using separate gender reference intervals.

What Is Normal?

ABSTRACT.For most quantities measured in clinical medicine, healthy subjects produce a range of values which overlaps the range found in patients with disease, so that there is no sharp distinction between normal and abnormal. Furthermore, the word “normal” is ambiguous and potentially misleading. The “normal range” has therefore been superseded in clinical laboratory work by the 95% reference interval, which includes values from 95% of the healthy population. The probability of a healthy person producing a value outside the 95% reference interval is 0.05, or one in twenty. Determining a reliable reference interval requires a representative sample of at least 120, and preferably 200, subjects from the healthy population, and many more may be needed if the distribution of values is asymmetrical. Subjects should be chosen according to clearly defined criteria. The possibility of correlation with age, height, etc. should be considered, and the reference interval calculated by a method appropriate to the data distribution. The common practice of defining the normal range as (sample mean ± 2 standard deviations) is not applicable to non-Gaussian distributions, which should either be transformed to Gaussian form, or the 95% reference interval should be calculated non-parametrically by determining the 2.5 and 97.5 percentiles. Where a test comprises two or more measurements (latency and amplitude of an evoked potential, for example) the separate reference intervals must be adjusted so that the combined reference region includes 95% of healthy subjects.

Standards of laboratory practice: evaluation of fetal lung maturity

In this standard of laboratory practice I recommend guidelines for fetal lung maturity (FLM) testing. If possible, obtain a 10-mL uncontaminated sample by amniocentesis. Keep the amniotic fluid at 4 degrees C and mix well before testing. If centrifugation is required, strictly adhere to the protocol. Most laboratories should offer a rapid test, such as fluorescence polarization, phosphatidylglycerol, or foam stability index, daily on both a routine and emergency basis. Requests for lecithin/sphingomyelin ratio may be referred to a reference laboratory. Communicate immediately the results of any FLM test to the ordering location. The report should contain the result, sample contamination, and reference information. Separate reference intervals for diabetic patients are not recommended.

Statistical criteria for separate reference intervals: race and gender groups in creatine kinase

Previously published data confirming differences in creatine kinase (EC 2.7.3.2) among various race and gender subgroups in the Los Angeles area have been re-examined with use of recently proposed statistical criteria for defining separate reference intervals. Results indicate that one criterion may be too lenient, whereas another is clearly too restrictive in suggesting the need for separate intervals. Further experience with other analytes in both large and small population samples would be helpful.

Lactate dehydrogenase isoenzyme-1 in serum for detection of peri-operative myocardial infarction after cardiac surgery.

We prospectively studied changes in serum lactate dehydrogenase isoenzyme-1 (LD-1, EC 1.1.1.27) in 99 consecutive patients after either coronary artery bypass grafting (CABG, n = 61), isolated cardiac-valve replacement (n = 24), or the two procedures combined (n = 14); 86 of these had no clinical evidence of peri-operative myocardial infarction (MI). Blood was sampled immediately after surgery and at 6-h intervals for up to 42 h thereafter. LD-1 was isolated by using the LD M-subunit antiserum. Samples from the non-MI patients were used to establish the reference intervals for LD-1. By 24 h after surgery, mean serum LD-1 values were higher (P less than 0.001) in non-MI patients who underwent isolated valve replacement (222 +/- 74 U/L) or combined CABG and valve replacement (266 +/- 58 U/L) than in 50 non-MI patients who underwent CABG alone (134 +/- 42 U/L). Separate reference intervals were determined for CABG and other patients at each sampling time. By 24 h after operation, LD-1 exceeded these reference intervals in the 10 CABG and two combined-procedure patients in whom other evidence of MI was present. Measurement of LD-1 24 to 42 h after cardiac surgery appears to be a useful test for the diagnosis of perioperative MI.

Clinical acceptance of a multi-test reference region for biochemical-panel results.

Results of a panel of six biochemical tests on a patient's specimen were mathematically combined into a "six-test signal strength" (STSS) value. This value indicated the overall extent of change from physiological results, and it was calculated in a way that ensured that a STSS value less than or equal to 1 occurred in 95% of apparently healthy people. STSS was reported with the test results for hospital inpatients during a four-month trial period. Doctors requested a repeat of the panel less often when a low STSS was reported, even if some test results were outside their separate reference intervals. Clinicians expressed differing opinions about its usefulness, some finding that a high STSS value had saved them from overlooking abnormal results, others not finding the value to be any practical advantage. Using a multi-test normal region resolves a statistical dilemma, while compounding the problem of knowing what results really mean.

Comparing multivariate and univariate subject-specific reference regions for blood constituents in healthy persons.

We examined the comparative behavior of subject-specific multivariate and univariate reference regions, using both computer-generated data and serial (semi-annual) measurements of selected analytes in subjects from a large health-maintenance program. Univariate studies under both homeostatic and random-walk time-series models were helpful in defining expected results, but only the homeostatic model was used in multivariate as well as univariate forms. Analysis of the computer-generated data and the real biochemical series produced similar findings, which showed the multivariate subject-specific reference region to be much more conservative than corresponding univariate intervals. That is, a multidimensional point of p correlated observations is quite likely to lie within the individual's multivariate reference region (based on past observation vectors), even when one or more of the observations lie outside their separate reference intervals for that individual. One consequence of this high specificity against univariate false positives in a large surveillance program is a higher than expected proportion of positive multivariate vectors in which none of the values lie outside their univariate ranges. Thus, although the development of multivariate reference regions should be encouraged, they should be used in conjunction with, not instead of, univariate ranges.