HPLC detection of fetal blood in meconium: improved sensitivity compared with qualitative methods.

We describe an HPLC-based method for the detection and quantification of fetal hemoglobin in stools of newborns. The new procedure is an alternative to the classic qualitative test for adult hemoglobin in meconium based on the differential stability of hemoglobin species in dilute base (Apt test). The HPLC method, based on a commercial device for hemoglobin characterization (Bio-Rad Variant), readily separates fetal and adult hemoglobin from non-hemoglobin components of meconium. To validate the method, blood and meconium were mixed in various proportions and then prepared for analysis with extraction in saline. The HPLC method accurately identified hemoglobin species even when the blood constituted only 5 mL per 100 g of the meconium specimen, and nearly quantitative recovery of hemoglobin was obtained at a blood content of 20 mL per 100 g of the meconium. Analysis time was 6.5 min, and preparation of sample was simple. HPLC detection of fetal blood in stools or other specimens markedly improves detection/characterization of blood in meconium.

Extraction of glyceric and glycolic acids from urine with tetrahydrofuran: utility in detection of primary hyperoxaluria.

Primary hyperoxaluria (PH) is an autosomal recessive metabolic abnormality characterized by excessive oxalate excretion leading to nephrocalcinosis and progressive renal dysfunction. Type I primary hyperoxaluria (PH I) results from a deficiency of alanine:glyoxylate aminotransferase, whereas type II disease has been traced to a deficiency of D-glycerate dehydrogenase. The two syndromes are often distinguished on the basis of organic acids that are coexcreted with oxalate: glycolate and L-glycerate in type I and type II disease, respectively. Routine organic acid analysis with diethyl ether extraction followed by gas chromatographic analysis failed to detect normal and increased concentrations of these diagnostic metabolites. Subsequent extraction of urine with tetrahydrofuran (THF), however, extracted 75% of added glycerate, 42% of added glycolate, and 75% of added ethylphosphonic acid (internal calibrator). THF extraction was analytically sensitive enough to allow determination of normal excretion of glycolate (14-72 micrograms/mg creatinine) and glycerate (0-5 years, 12-177 micrograms/mg creatinine and > 5 years, 19-115 micrograms/mg creatinine). Four of five patients with PH I and both patients with type II disease were correctly identified. Thus, THF extraction is a convenient adjunct to routine organic acid analysis and facilitates the detection of PH.

Accreditation surveys. Coping with the worst-case scenario.

Survey scheduled? Feeling out of control? An administrator from a small Texas hospital shares her worst-case experience-five surveys in 3 months.

Measurement of plasma ibuprofen by gas chromatography-mass spectrometry.

A gas-chromatography-mass spectrometry (GC-MS) method for the determination of plasma ibuprofen was developed. Plasma samples from cystic fibrosis (CF) patients receiving high-dose ibuprofen therapy were analyzed by GC-MS and the result compared to analysis by high-performance liquid chromatography (reference method). Analysis of ibuprofen was sensitive to at least 5 mg/L, and the method was linear to 200 mg/L. Within-run variations of plasma samples were 4.6% (131.7 +/- 6.0 mg/L) and 5.4% (44.4 +/- 2.4 mg/L), respectively. The between-run variation was 9.3% (45.4 +/- 4.2 mg/L) and 7.4% (88.0 +/- 6.5 mg/L). This method is suited for routine clinical use for the monitoring of plasma ibuprofen levels in treatment of CF. It may be particularly applicable in pediatric laboratories, which are likely to possess GC-MS capability.

A new digoxin immunoassay substantially free of interference by digoxin immunoreactive factor.

We have evaluated the new Roche digoxin "On Line" procedure for use in a pediatric population with particular interest in the potential for interference by digoxin-like immunoreactive factor (DLIF). An initial study comparing digoxin values obtained with the new Roche procedure with determinations on an Abbott TDx, American Dade Stratus, and COBAS-FARA using Microgenics Cedia reagents, found good correlations with these established methods. The Roche method was suitably precise and utilized either serum or plasma. Interference by DLIF was assessed by analyzing specimens from patients not receiving digoxin but likely to contain DLIF, with the argument that non-zero values represent cross-reactivity of anti-digoxin antibodies with DLIF endogenous to these specimens. When specimens from neonates, women with second/third trimester pregnancies, and patients with renal and liver failure were assayed with the Roche, Stratus, and TDx methods, all three methods measured DLIF in some specimens, but the Roche method possessed the lowest overall DLIF interference. The modest extent of DLIF interference and the requirement of a small amount of specimen make the Roche method superior in monitoring digoxin in a pediatric population.

Analytical artifacts in hematocrit measurements by whole-blood chemistry analyzers.

Compact analyzers suited to near-patient testing estimate hematocrit by measuring the conductivity of undiluted blood. We evaluated the accuracy of hematocrit determination of one such analyzer (Instrumentation Laboratory BGE analyzer) against an automated cell counter (EPC) and packed cell volume (PCV) microhematocrit. When specimens (n = 34) from outpatient and ward patients were analyzed with all three methods, the BGE analyzer correlated well with both EPC and PCV hematocrit determinations (BGE = 1.00 PCV + 0.3%, S(y)/x = 2.0%), suggesting that all three methods are similar in performance for most patients. However, a patient with increased plasma osmolality showed significant decreases in BGE and PCV hematocrits relative to the EPC method. The differences in hematocrit measurements could be reproduced by adding solutes to blood in vitro or by modifying the plasma osmolality of rats in vivo. Samples from patients undergoing cardiac surgery, whose blood had large changes in protein concentration, showed discrepancies between hematocrits by conductivity and other methods; similar effects could be produced by changes in protein concentration or in vitro addition of polyethylene glycol. We conclude that conductivity measurements provide accurate hematocrit results for physiologically normal subjects but not for some intensive-care and surgical patients.

A new plastic evacuated tube with plasma separator.

A new plastic-walled, evacuated blood collection tube (Terumo Venoject II) with plasma separator was evaluated for characteristics related to the use of plastic in place of glass. Plastic tubes were highly resistant to breakage through handling mishaps or failure during centrifugation. Higher centrifugation speed (13,600g) was well tolerated, and centrifugation times as short as 3 minutes at 13,600g effectively cleared plasma of cellular components, at high plasma yield. Plastic tubes were nearly completely combustible under incineration conditions commonly used for medical waste, and plastic tubes effectively retained vacuum during the typical shelf life of evacuated blood collection tubes. Collection of carbamezepine specimens in plastic tubes decreased levels an average of 6.8% compared to a heparinized glass tube control under conditions approximating routine use; levels of other drugs (phenytoin, phenobarbital, valproic acid, theophylline, and cyclosporine) were less significantly affected. This modest decrease appeared to result from a combination of an immediate interaction with the plastic surface of the tubes and a time-dependent interaction with the olefin-based separator. Modest but clear benefits, including reduction in specimen breakage, reduced centrifugation time and reduced solid waste after incineration derive from the use of plastic in place of glass in evacuated blood collection tubes.

Detection of autoantibodies to amylase by ELISA: comparison of detection of macroamylase and free autoantibody.

New ELISAs for detecting macroamylase or free autoantibodies to amylase were tested with 48 samples that had been characterized by gel chromatography and electrophoresis. The macroamylase ELISA, with anti-IgG or anti-IgA for detection, detected macroamylase in 28 of 33 samples known to contain macroamylase (85% sensitivity), whereas the ELISA for free autoantibody to amylase was positive for only 11 samples. Specificities of both ELISAs were 93%. Among 28 true positives detected with the macroamylase ELISA, 22 contained IgA, 3 contained IgG, and 3 contained both immunoglobulins. Detection of IgM added no true positives. ELISA responses (y) were proportional to log [macroamylase concentration by chromatography (x)] from 0 to 1200 U/L: y = 5.15 x + 1.66; r = 0.72; Sy x = 1.65. As new tools for detecting macroenzymes consisting of enzyme-autoantibody complexes, the ELISAs show that some autoantibodies are detected more sensitively as antibody-antigen complexes than as free antibody.

Automated enzymatic analysis of inulin.

We have developed an automated enzymatic assay for quantitation of inulin in plasma and urine that can be performed on the Cobas FARA II. In the assay, inulinase hydrolyzes inulin to fructose, and sorbitol dehydrogenase converts fructose to sorbitol with consumption of NADH, which is detected by spectrophotometry. The method incorporates a sample blank (inactivated inulinase) for each specimen to subtract contributions of endogenous fructose. Recovery of fructose or inulin was near 100%, with linearity to 300 mg/L. The enzymatic assay (y) agreed well with an anthrone comparison method (x) for analysis of inulin in both urine specimens (y = 1.00x - 138; Sy/x = 714) and plasma specimens (y = 1.00x - 3.5; Sy/x = 5.5). Glucose at 300 mg/L yielded an apparent inulin value of 1.3 mg/L in the enzymatic assay, but reacted at nearly 10% equivalency in the anthrone assay. Interferences from sorbitol, mannitol, and xylitol were negligible. CVs for day-to-day precision studies were 1-4%. The automated enzymatic assay of inulin is faster and avoids the use of caustic reagents required by the classical anthrone method.