David B. Endres

Limitations of direct estradiol and testosterone immunoassay kits.

Estradiol (E2) and testosterone (T) are biologically active hormones that serve as important diagnostic markers in serum of premenopausal and postmenopausal women and in men. These hormones are measured frequently by immunoassay in clinical laboratories and the test results are used in the diagnosis and treatment of patients. For measuring the hormones by immunoassay, most laboratories utilize commercially available reagents that are packaged in the form of a kit and are used either in an automated instrument or manually. However, both the diagnostic kit manufacturer and testing laboratory seldom thoroughly validate the assay methods generated with these kits. This deficiency may lead to unreliable test results that could affect clinical evaluation and treatment of patients. The purpose of the present study was to assess the reliability of immunoassays that quantify serum E2 and T levels with commercial diagnostic kits. The data generally show wide differences in the apparent levels of each hormone in a given sample obtained with kits from different manufacturers. This was especially true when measuring postmenopausal E2 and T levels. However, a purification step, which included organic solvent extraction, prior to radioimmunoassay (RIA) of E2 gave values that compared well with those obtained by conventional RIA (with preceding extraction/chromatographic steps). Our results point out the importance of more thoroughly validating assays performed with commercial immunoassay kits, especially with respect to sensitivity and specificity, prior to their use for measuring hormone levels in patient samples.

Hypothalamic-pituitary-adrenal activity among Armenian adolescents with PTSD symptoms.

This study evaluated basal levels and responsiveness to exercise of plasma adrenocorticotropic hormone (ACTH), and serum thyroid stimulating hormone (TSH), growth hormone (GH) and cortisol among adolescents from two differentially exposed groups 61/2 years after the 1988 earthquake in Armenia. Severity of total PTSD and Category C and D symptoms were negatively correlated with baseline cortisol. Preexercise ACTH was significantly lower, and preexercise TSH higher, among adolescents with more exposure. Depressive symptoms were negatively correlated with baseline cortisol and positively with TSH. Mean GH, TSH, and cortisol levels in both groups fell within normal limits. The pre- to postexercise increase in GH, TSH, and cortisol suggests that exercise challenge may be useful in the field investigation of neurohormonal activity among traumatized individuals.

Investigation of hypercalcemia

Hypercalcemia is a relatively common clinical finding. Primary hyperparathyroidism, hypercalcemia associated with malignancy and chronic renal failure (with calcium and vitamin D metabolite treatment or tertiary hyperparathyroidism) are the most common causes. Less common causes of hypercalcemia include vitamin D-related (granulomatous diseases, lymphoma, vitamin D intoxication), other endocrine (thyrotoxicosis), medications (milk-alkali, thiazides, lithium) and other causes (immobilization, familial hypocalciuric hypercalcemia). The clinical laboratory is central to the diagnosis and differential diagnosis of hypercalcemia. Its role has expanded from measuring routine chemistry tests such as total calcium, phosphate, creatinine and alkaline phosphate to include quantification of ionized calcium, parathyroid hormone (PTH) and vitamin D metabolites. In spite of this progress, the diagnosis and differential diagnosis of hypercalcemia can be significantly improved by: 1) increasing the availability and utilization of ionized calcium since total and corrected calcium are often inaccurate; 2) establishing more accurate reference intervals for parathyroid hormone by excluding individuals who are vitamin D insufficient or deficient; and 3) harmonizing intact PTH immunoassays.

Comparison of fresh frozen serum to proficiency testing material in College of American Pathologists surveys: α-fetoprotein, carcinoembryonic antigen, human chorionic gonadotropin, and prostate-specific antigen

CONTEXT Most proficiency testing materials (PTM) contain an artificial matrix that may cause immunoassays to perform differently with this material than with clinical samples. We hypothesized that matrix effects would be reduced by using fresh frozen serum (FFS). OBJECTIVE To compare the performance of an FFS pool to standard PTM for measurement of alpha-fetoprotein, carcinoembryonic antigen, human chorionic gonadotropin (hCG), and prostate-specific antigen (PSA). DESIGN One FFS specimen and 4 different admixtures of PTM were distributed in the 2003 College of American Pathologists K/KN-A (for alpha-fetoprotein, carcinoembryonic antigen, hCG, and total and free PSA) and C-C (hCG only) Surveys. PARTICIPANTS The number of laboratories that participated in the surveys varied from a low of 288 (free PSA, K/KN-A Survey) to a high of 2659 (hCG, C-C Survey). MAIN OUTCOME MEASURES Method imprecision and method bias were compared between the FFS specimen and the standard PTM specimen with the closest value. Method imprecision was determined by calculating the coefficients of variation for each method and for all methods combined. Bias was defined as the proportional difference between peer-group mean and the median of all method means. RESULTS The FFS specimen gave significantly higher imprecision than PTM for the analytes alpha-fetoprotein, carcinoembryonic antigen, total PSA, and free PSA. For hCG, no substantial imprecision differences were observed in both surveys. Bias was significantly greater for the alpha-fetoprotein, carcinoembryonic antigen, and total PSA assays and significantly lower for the hCG and free PSA assays when comparing the FFS with the PTM. CONCLUSIONS Fresh frozen serum did not provide consistently lower imprecision or bias than standard PTM in a survey of commonly ordered tumor markers.

Absence of hyperparathyroidism in severe liver disease

SummaryElevated serum levels of intact parathyroid hormone (PTH) have been reported in severe versus mild biliary cirrhosis. The aim of this study was to determine whether hyperparathyroidism was present in severe liver disease on the basis of the inability of the liver to catabolize the hormone. Because biologic activity resides in the amino terminal, and amino terminal PTH determinations have not been routinely made in liver disease, it is possible that hyperparathyroidism was previously missed in these patients. Accordingly, we obtained fasting blood from 11 patients with severe liver disease and 8 age-matched controls. We measured intact, amino terminal, and mid-region PTH, vitamin D metabolites, bone gamma carboxyglutamic acid protein (BGP), ionized calcium, phosphorus, magnesium, and liver function tests. Serum levels of PTH were normal with all assays and 1,25(OH)2D levels were not elevated. These findings argue against the possibility that hyperparathyroidism plays a role in the pathogenesis of hepatic osteodystrophy.

Serum 25-hydroxyvitamin D deficiency; a risk factor for chronic kidney disease in ambulatory indigent patients.

OBJECTIVE To assess whether 25-hydroxyvitamin D (25[OH]D) deficiency is a risk factor for chronic kidney disease (CKD) in ambulatory indigent patients. METHODS Data for all serum 25(OH)D concentrations measured during 2010 in our ambulatory nondialysis-dependent patients were analyzed along with CKD-related parameters. Patients were stratified into groups based on 25(OH)D levels of <10, 10 to 19, 20 to 29, and ≥30 ng/mL. CKD was defined by estimated glomerular filtration rate (eGFR; Chronic Kidney Disease-Epidemiology Collaboration [CKD-EPI] equation) and abnormal urine protein to creatinine ratios. CKD-associated parameters included serum parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25[OH]2D), alkaline phosphatase, albumin, corrected calcium, and total CO2 levels. RESULTS A total of 2,811 patients had 25(OH)D levels measured. Patients with 25(OH)D levels <10 ng/mL had significantly increased relative risk (RR) of an eGFR <15 mL/min/1.73 m2 (RR, 4.0), an eGFR of 15 to 29 mL/min/1.73 m2 (RR, 2.6), urine protein to creatinine ratio >3.5 g/g (RR, 5.6), and serum PTH >100 pg/mL (RR, 2.8) compared to patients with a 25(OH)D level ≥30 ng/mL. Patients with 25(OH)D levels of 10 to19 ng/mL had significantly increased RR of a urine protein to creatinine ratio >3.5 g/g (RR, 4.8) and serum PTH >100 pg/mL (RR, 1.5) compared to patients with 25(OH)D levels ≥30 ng/mL. CONCLUSION 25(OH)D deficiency (<10 ng/mL) was associated with reduced eGFR, nephrotic-range proteinuria, and increased PTH levels in our population of ambulatory urban indigent patients.

Vitamin D deficiency in urban indigent patients in Southern California.

OBJECTIVE To determine the prevalence of 25-hydroxy (OH) vitamin D deficiency in ambulatory and hospitalized patients from a large urban county medical center in Southern California, and assess the effects of season, ethnicity, age, location of care, and comorbidities on prevalence. METHODS Data for all serum 25(OH)-D2 and -D3 concentrations measured during 2010, along with associated demographic characteristics and comorbidity data, were analyzed. 25(OH) D concentrations were measured using liquid chromatography-tandem mass spectrometry. RESULTS Of 210,695 patients, serum 25(OH) D concentrations were measured for 3,276 (1.6%), 78% of whom were Hispanic, 69% female, 14% hospitalized, and 86% ambulatory. Median patient age was 54 years. Prevalence of 25(OH) D <10 ng/mL was 6.5% overall, 5.5% in Hispanics, 6.7% in Asians, 15.5% in African Americans, and 8.9% in whites. Prevalence was significantly higher in African Americans than in Hispanics (relative risk (RR): 2.79), males (RR: 2.07), hospitalized patients (RR: 4.96), and winter (RR: 1.34). Prevalence of 25(OH) D <20 ng/mL was 35% overall, 34% in Hispanics, 32% in Asians, 49% in African Americans, and 33% in whites, and was significantly higher in African Americans than Hispanics (RR: 1.45), males (RR: 1.32), hospitalized patients (RR: 2.02), and younger patients (RR: 1.21, age ≤30; 1.16, age 31-50) versus those age 51 to 70 years, and in winter (RR: 1.21). CONCLUSION Our study estimated the prevalence of 25(OH) D deficiency and identified at-risk patient groups in Southern California; 25(OH) D deficiency should be suspected, diagnosed, and adequately treated to improve the health status in at-risk urban indigent patient populations.