Between-assay CVs Research Articles

Analytical performances of the D-100TM hemoglobin testing system (Bio-Rad) for HbA1c assay.

Glycated hemoglobin (HbA1c) is widely used for the monitoring of glycemic balance in diabetic patients and has also been proposed as a tool for the diagnostic of diabetes mellitus. Accordingly, HbA1c quantification must be performed using robust, reliable and efficient methods. Here are reported the results of the evaluation of a new high performance liquid chromatography (HPLC) system for HbA1c quantification, the D-100TM system from Bio-Rad Laboratories. The analytical performances of the method as well as the influence of the most frequent interferences regarding HbA1c assays (e.g., labile HbA1c, carbamylated hemoglobin, high HbF) have been tested. Intra- and between-assay CVs were respectively lower than 0.93% and 1.46% (HbA1c results expressed in NGSP units) and lower than 1.67% and 2.27% (HbA1c results expressed in IFCC units). The linearity proved to be excellent from 15 mmol/mol (3.5%) to 184 mmol/mol (19.0%) (r=0.999). The results were well correlated with those obtained by another HPLC method (VARIANTTM II Hemoglobin A1c Program reorder pack 270-2101NU-Bio-Rad): HbA1c[VARIANTTM II, mmol/mol]=1.013×HbA1c[D-100TM, mmol/mol]+0.637 (r=0.993, n=2000). The D-100TM system provided results consistent with IFCC-assigned external quality control samples and the presence of labile HbA1c, carbamylated hemoglobin and HbF did not interfere with HbA1c measurement. The D-100 TM system proved to be a robust and reliable method for HbA1c measurement suitable for routine practice in clinical chemistry laboratories.

Rapid detection of aneuploidy (trisomy 21) by allele quantification combined with melting curves analysis of single-nucleotide polymorphism loci.

Molecular approaches for the detection of chromosomal abnormalities will allow the development of rapid, cost-effective screening strategies. We present here a molecular alternative for the detection of aneuploidies and, more specifically, trisomy 21. We used the quantitative value of melting curve analysis of heterozygous genetic loci to establish a relative allelic count. The two alleles of a given single-nucleotide polymorphism (SNP) were differentiated by thermodynamic stability with a fluorescently labeled hybridization probe and were quantified by relative areas of derivative melting curves detected after fluorescence resonance energy transfer. Heterozygous SNPs provided internal controls for the assay. We selected six SNPs, heterozygous in at least 30% of a random population, to form a panel of informative loci in the majority of a random population. After normalization to a heterozygous control, samples segregated into three categories; nontrisomic samples had mean allele ratios of 0.96-1.09, whereas trisomic samples had mean ratios of 1.84-2.09 or 0.46-0.61, depending on which allele was duplicated. Within-run mean CVs of ratios were 6.5-27%, and between-assay mean CVs were 13-24%. The use of melting curve analysis of multiple SNPs is an alternative to the use of small tandem repeats for the detection of trisomies. Because of the high density of SNPs, the approach may be specifically useful for very fine mapping of the regions of chromosome 21 that are critical for Down syndrome; it is also applicable to aneuploidies other than trisomy 21 and to specimens that are not amenable to cytogenetic analysis.

Measurement of Immunoreactive Angiotensin-(1–7) Heptapeptide in Human Blood

The renal enzyme renin cleaves from the hepatic alpha(2)-globulin angiotensinogen angiotensin-(1-10) decapeptide [Ang-(1-10)], which is further metabolized to smaller peptides that help maintain cardiovascular homeostasis. The Ang-(1-7) heptapeptide has been reported to have several physiological effects, including natriuresis, diuresis, vasodilation, and release of vasopressin and prostaglandins. To investigate Ang-(1-7) in clinical settings, we developed a method to measure immunoreactive (ir-) Ang-(1-7) in 2 mL of human blood and to estimate plasma concentrations by correcting for the hematocrit. A sensitive and specific antiserum against Ang-(1-7) was raised in a rabbit. Human blood was collected in the presence of an inhibitor mixture including a renin inhibitor to prevent peptide generation in vitro. Ang-(1-7) was extracted into ethanol and purified on phenylsilylsilica. The peptide was quantified by radioimmunoassay. Increasing doses of Ang-(1-7) were infused into volunteers, and plasma concentrations of the peptide were measured. The detection limit for plasma ir-Ang-(1-7) was 1 pmol/L. CVs for high and low blood concentrations were 4% and 20%, respectively, and between-assay CVs were 8% and 13%, respectively. Reference values for human plasma concentrations of ir-Ang-(1-7) were 1.0-9.5 pmol/L (median, 4.7 pmol/L) and increased linearly during infusion of increasing doses of Ang-(1-7). Reliable measurement of plasma ir-Ang-(1-7) is achieved with efficient inhibition of enzymes that generate or metabolize Ang-(1-7) after blood sampling, extraction in ethanol, and purification on phenylsilylsilica, and by use of a specific antiserum.

Clinical and Analytical Evaluation of an Enzyme Immunoassay for Myelin Basic Protein in Cerebrospinal Fluid

RIA of myelin basic protein (MBP) in cerebrospinal fluid (CSF) is commonly used as a biochemical marker of demyelination in patients with multiple sclerosis (MS). Our aim was to develop a sufficiently sensitive ELISA for MBP and evaluate it clinically in patients with MS. The ELISA used anti-bovine MBP antibody coated on plates and biotinylated anti-MBP antibody. The bound antibody complex was quantified with streptavidin-horseradish peroxidase. MBP was determined in CSF from 84 MS patients and 55 patients with other neurological diseases. The respective within- and between-assay CVs were 4.7% and 7.2% at 200 ng/L, and 6. 3% and 8.8% at 2000 ng/L. The detection limit was 30 ng/L. Most of the MS patients with acute exacerbations had markedly increased MBP in the CSF. Longitudinal studies of six MS patients with recurrent exacerbation confirmed this observation. MBP concentrations from 78 MS patients, as tested with our ELISA, correlated well with those obtained by RIA (r = 0.9; P: <0.01), but the detection limit of the ELISA was much lower than that of the RIA. This convenient ELISA with higher sensitivity than the existing assays is a suitable routine assay that provides a diagnostic indicator of myelin breakdown in the central nervous system; moreover, it is an excellent indicator of MS disease activity.

ELISA for Urinary Trehalase with Monoclonal Antibodies: A Technique for Assessment of Renal Tubular Damage

alpha,alpha-Trehalase, located on renal proximal tubules, is a glycoprotein that hydrolyses alpha,alpha-trehalose to two glucose molecules. Urinary trehalase reflects damage to renal proximal tubules, but its activity has not been measured routinely because measurement of catalytic activity is rather complicated and because conventional assays for enzyme activity might not reflect all of the trehalase protein because of enzyme inactivation in urinary samples. We established novel monoclonal antibodies for human trehalase and a sandwich ELISA for quantification of urinary trehalase. We determined the urinary trehalase protein concentration with this ELISA and trehalase catalytic activity, and the results of these two methods were compared. The ELISA system was more sensitive than the detection of enzyme activity and could detect a subtle difference in the amount of trehalase present in renal diseases. The within- and between-assay CVs in the ELISA were 6.7-7.6% and 6.2-8.2%, respectively. Highly significant increases in both the quantity and activity were seen in patients with nephrotic syndrome (acute phase), Lowe syndrome, and Dent disease. The quantities were 70- to 200-fold greater, whereas enzyme activities were, at most, 10-fold higher than those of control subjects. In the detection of small amounts of trehalase in patients with chronic glomerulonephritis and renal anomalies, quantities were better than enzyme activities. We have established an ELISA system for quantification of urinary trehalase that uses novel monoclonal antibodies. Our ELISA system is simpler and more sensitive than a conventional activity assay and reflects trehalase protein. This ELISA can be a useful as a common tool for clinical assessment of renal proximal tubular damage.

Modified and improved anti-acetylcholine receptor (AChR) antibody assay: comparison of analytical and clinical performance with conventional anti-AChR antibody assay

We developed a modified anti-acetylcholine receptor (AChR) antibody (Ab) assay based on a radioreceptor assay and a calibration curve. We compared the analytical and clinical performances of this modified assay with those of the conventional anti-AChR Ab radioreceptor assay. Serum specimens were from patients with myasthenia gravis (MG) (n = 156) and from control subjects (n = 106). The modified assay demonstrated lower within-assay (4.0-6.6%) and between-assay (5.3-7.8%) CVs, greater linearity, lower cost, and shorter assay time than the conventional method. ROC curve analysis indicated almost identical specificity and sensitivity (> 0.92) for these two anti-AChR Ab assays. The modified and conventional assays were also equivalent for blocking anti-AChR Ab assay. Moreover, the modified anti-AChR Ab assay, differently from the conventional assay, allowed us to reveal anti-AChR Ab concentration differences among different clinical grades of MG.

Clinical and technical evaluation of ACS™BR serum assay of MUC1 gene-derived glycoprotein in breast cancer, and comparison with CA 15-3 assays

The mucin glycoprotein-detecting assay CA 15-3 is a valuable tool for monitoring the course of disease in breast cancer patients. Assays of CA 15-3 are based on the use of two MAbs to polymorphic epithelial mucin (PEM). We evaluated the technical and clinical performance of the Chiron ACS BR, an automated competitive chemiluminescence assay using a single MAb, B27.29, and compared the assay's results with those of the Centocor CA 15-3 RIA, the Abbott IMx CA 15-3, and the Boehringer Mannheim Enzymun-Test CA 15-3. The study population consisted of 253 healthy women, 66 patients with benign breast disease, 168 breast cancer patients, and 76 patients with other carcinomas. In the technical evaluation, we assessed the precision and linearity on dilution of the ACS BR assay. Cutoff values (upper limits of values seen in healthy subjects) were determined for all four assays. Agreement between the assays was studied by linear regression analysis. The ACS BR assay gave within- and between-assay CVs of 2.2% and 3.9%, respectively. Three samples from healthy women gave discordant values by ACS BR and were not included in the calculations. All four assays exhibit a highly similar pattern when monitoring breast cancer disease; the closest agreement of values was obtained between ACS BR and Centocor CA 15-3. We conclude that the ACS BR assay is a fast and reliable immunoassay for measuring PEM in serum. Although it detects a slightly different epitope on the PEM molecule than is targeted in other assays, for cancer serum samples it agreed better with the original Centocor CA 15-3 assay than did the other two CA 15-3 assays tested.

Analytical and clinical performance of improved Abbott IMx CA 125 assay: comparison with Abbott CA 125 RIA

We compared the improved Abbott IMx cancer antigen (CA) 125 assay (cat. no. 7A89) with the Abbott CA 125 RIA. Serum specimens were from healthy perimenopausal women (n = 124) and from patients with benign gynecologic and nongynecologic diseases (n = 124), ovarian carcinoma (n = 104), or other malignancies (n = 193). The IMx assay detected as little as 0.193 kAU/L CA 125 (AU = arbitrary Abbott unit), demonstrated up to 29% overestimation upon serum dilution, low within-assay (2.7-5.6%) and between-assay (4.8-8.2%) CVs, and no high-dose hook effect < or = 46,000 kAU/L nor influence from human anti-mouse antibodies in serum of women injected with OC 125 F(ab')2. Values by IMx were 20% lower than by RIA for healthy perimenopausal women (n = 100; IMx = 0.80 RIA - 2.5 kAU/L), and at least 50% higher for those with benign or malignant ovarian disorders at concentrations < 100 kAU/L. Receiver-operating characteristic (ROC) curve analysis of ovarian neoplasma vs perimenopausal controls indicated a gain of specificity and sensitivity with the improved IMx assay over the RIA, but ROC performance was the same with either assay if patients with benign ovarian disorders were used as controls.

Neonatal concentrations of apolipoproteins B and A-1 measured by radioimmunoassay in dried blood spots

Apolipoproteins B and A-1 concentrations have been measured by radioimmunoassay in eluates of whole blood spotted on filter paper. The stability of apolipoproteins has been studied during a period of 2 months. Apolipoprotein levels are determined within 15 days after collection in order to avoid the significant decrease of B/A-1 ratio observed afterwards. The within-assay and between-assay CVs for middle concentrations are 6.4% and 6.8%, respectively, for apo B, and 8.2% and 13.5% for apo A-1. The coefficient of correlation between dried blood and plasma B/A-1 ratio is r 2 = 0.907 ( n=30). The apolipoprotein concentrations have been measured in 994 3-day-old newborns; the mean ± SD is 0.214±0.073 g/l (apo B), 0.495±0.147 g/l (apo A-1) and 0.453±0.164 (B/A-1 ratio). The apo B concentration is unaffected by birth weight or gestational age, but it is correlated with sex. Linear multivariate regression has been found for the apo A-1 concentration which is influenced by gestational age and by sex. Girls have higher levels than boys for both parameters ( P<0.0001 for apo B; P=0.0037 for apo A-1). This radioimmunoassay technique for apolipoproteins meets the practical requirements of a neonatal screening program for familial hypercholesterolemia.

Improved rapid procedure for simultaneous determinations of hemoglobins A1a, A1b, A1c, F, C, and S, with indication for acetylation or carbamylation by cation-exchange liquid chromatography

Although the clinical utility of glycohemoglobin (HbA1c) as an indicator of blood glucose control is generally accepted, the use of cation-exchange liquid chromatography for its quantification is controversial, given numerous studies that show no correlation between HbA1c and other indicators of blood glucose control in uremic subjects. We report a specific cation-exchange liquid-chromatographic method that measures simultaneously HbA1a, HbA1b, HbA1c, HbF, HbA0, HbC, and HbS quickly and reproducibly. Carbamylation or acetylation is indicated by extra peaks. Carbamylated hemoglobin increased the result for HbA1c by as much as 1% above the nonuremic value. The method is a fast (approximately 15 min per sample), easy to perform, reproducible, and sensitive screening method for abnormal hemoglobins. Within-assay CVs were < 0.7% in all cases and between-assay CVs were < 1.4%.

Evaluation of Glycaemic Control Limits Using the Ames DCA 2000 HbA1c Analyser

The Ames DCA 2000 is a benchtop analyser that measures HbA1c by an agglutination inhibition immunoassay using a monoclonal antibody. Laboratory and nursing staff measured HbA1c on-site in 78 patients with Type 1 diabetes at the outpatient clinic. Significant correlations were noted with both the Corning Glytrac total HbA1 assay (r = 0.89) and the Novoclone assay for HbA1c (r = 0.95). Mean within-assay CV was 1.6% and 3.0% at HbA1c of 5.4% and 13.0%, respectively, while between-assay CVs were 4.2% and 3.8%. These results are as good as our routine laboratory method based on the Corning HbA1 assay. Locally derived reference population data for HbA1c were produced and patients were assigned to categories of good, acceptable, and poor glycaemic control using conventional recommendations for Type 2 diabetes. There was poor agreement between the methods, with only 22% of patients achieving good/acceptable control using the DCA 2000, while 46% of patients had an HbA1 in this range. It appears that the convention for derivation of control limits for HbA1 does not hold for this HbA1c assay.

Competitive solid-phase enzyme immunoassay for melatonin in human and rat serum and rat pineal gland

We describe a solid-phase competitive enzyme immunoassay for determination of melatonin in serum. The detection limit of the assay is 1.0 fmol/well. Low cross-reactivity of the antiserum with other indoles, parallel serum extract dilution and melatonin standard curves, good analytical recovery, and within- and between-assay CVs of 6.4-14.4% provide validation of the assay. Linear regression analysis of melatonin concentrations measured with this assay (y) and with a commercial 3H RIA (x) in 88 sera yielded the relation y = 0.62 x - 0.76, Sy/x = 0.03. Values for melatonin in serum samples from healthy subjects are lower during the day than during the night. Melatonin response in rat serum and pineal gland to isoproterenol injection is similar to published RIA data. The analytical procedure is also simple. Thus, the assay should have practical applications in investigation of pineal function in both clinical and basic studies.

Direct Enzyme Immunoassay of Estradiol in Serum of Women Enrolled in an in Vitro Fertilization and Embryo Transfer Program

We present a fast and simple direct enzyme immunoassay for the sexual steroid hormone estradiol-17 beta in serum of women enrolled in an in vitro fertilization and embryo transfer program. We added 25 microL of standards or samples and 125 microL of a mixture of monoclonal antibody and displacing agents to the wells of a microtiter plate previously coated with a second antibody. After 30 min 50 microL estradiol-horseradish peroxidase conjugate is added. Total incubation time is 75 min. The immunoassay is stopped by washing the microtiter plate. The amount of bound conjugate is then measured at 450 nm with hydrogen peroxide as a substrate and 3,3',5,5'-tetramethylbenzidine as chromogen. The detection limit is 0.24 nmol/L. The assay is thus limited in its application. Analytical recovery is 90.8% (range 80.4-102.9%); within- and between-assay CVs range between 1.3% and 15.4%. Total assay time, including preparation of reagents and data reduction, is only 2.5 h. Results of this enzyme immunoassay compare well with those obtained with four different commercial radioimmunoassays (r = 0.88-0.93).

Determination of fetal hemoglobin by cation exchange liquid chromatography

A sensitive liquid Chromatographic method for the analysis of the major component of fetal hemoglobin (HbF 0) in blood using high-resolution strong cation exchange column is described. The imprecision of the method was assessed in 20 repeated assays of blood samples containing 0.67%, 0.97%, 2.2% and 3.3% of HbF 0. Within-assay CVs were 5.0%, 2.8%, 1.8% and 1.5% and between-assay CVs 5.8%, 3.7%, 1.9% and 2.7%, respectively. The HbF 0 concentration was stable in EDTA blood for at least 20 days at +8°C and 3 days at +22°C. Blood HbF 0 levels (mean ± S.D. were 0.41 ± 0.20% for 21 healthy males, and 0.48 ± 0.19% for 22 healthy non-pregnant females. The method is simple and reproducible, and allows the sensitive determination of blood HbF 0.

Free thyroxin measured in dried blood spots from normal, low-birth-weight, and hypothyroid neonates

We have adapted a new radioimmunoassay for free thyroxin (FT4) measurement in dried blood spots for use in neonatal screening for hypothyroidism. The method is easy, fast, and cheap. Within-assay and between-assay CVs are respectively 9.6% and 13.2%. In 997 neonates three days postpartum with normal thyrotropin concentrations, the mean FT4 concentration was 27.2 pmol/L (SD 7.3 pmol/L). There was no significant difference in mean FT4 concentration between boys and girls. FT4 concentrations increased linearly with birth weight or with gestational age, as expressed by multiple linear regression: FT4 (pmol/L) = 0.0016 birth weight (g) + 0.6931 gestational age (weeks) - 4.8772. Only gestational age significantly affected the FT4 value. For five hypothyroid infants tested on day three postpartum, FT4 values were all below the 1st percentile of values from healthy neonates. Thus, when the neonatal concentration of thyrotropin is above normal, FT4 measured in the same sample can provide a reliable earlier diagnosis of hypothyroidism.

A sensitive immunochemiluminescence assay for human serum amyloid a protein

We describe an immunochemiluminescence assay for human plasma serum amyloid A protein (SAA) in which specific rabbit polyclonal antibodies against synthetic peptides are used. The detection of the antigen-antibody reaction at 425 nm is based on a brief emission of light by a luminophor component (signal) in response to chemical energy. The working range of the assay covers plasma SAA concentrations from 5 to 100 micrograms/L. The lower detection limit is 5 micrograms/L, the within- and between-assay CVs are less than 12%. Bilirubin, cholesterol and triglyceride in final concentrations of up to 220 mumol/L, 8.1 mmol/L and 2.68 mmol/L, respectively, do not interfere with the assay. Results were correlated with those obtained by the enzyme-linked immunosorbent assay using the same antibodies (r = 0.95; p less than 0.001; n = 50). This method is inexpensive, simple and easily automated.

Direct chemiluminescence immunoassay of estradiol in saliva

A sensitive and simple direct solid-phase chemiluminescence immunoassay is described for estradiol in saliva. In this assay, a second antibody is bound to the wells of microtiter plates. Either buffer with standards or saliva (100 microL) is incubated in these wells with monoclonal anti-estradiol antibody and with estradiol-isoluminol conjugate. Incubation time is 2 h. Chemiluminescence of the bound fraction is measured in a manually operated luminometer (Biocounter). The assay has a detection limit of 3.8 pmol/L; analytical recovery of added estradiol is 96.8% (SD 7.0%); within- and between-assay CVs range between 2.5% and 12.7%. Forty unknown saliva samples can be assayed and results calculated within 4.5 h. Results of a slightly modified procedure-with black microtiter plates and a prototype of an automated plate reader (Luminoskan)--compare well with those of the described method (r = 0.97). Because steroid-binding globulins have been found in saliva, the effect of displacing agents on the results of the direct chemiluminescence assay is described.

Measurement of pepsinogen group I in endoscopic gastroduodenal biopsies

A technique for determining pepsinogen group I (PGI) concentrations in endoscopic gastroduodenal biopsies is described and validated. This method has a good precision (intra-assay CVs 4.1% to 8.4%; between-assay CVs 4.6% to 9.3%), and analytical recovery is satisfactory (94% to 102%). Results were uninfluenced by the storage interval of the samples. At PGI-producing sites (i.e., fundus, corpus) peptic activity was greater than in the antrum and duodenum. PGI concentrations in serum and in gastroduodenal biopsies were not correlated. This simple, reliable method can quantify better than other assays peptic activity in humans, without being influenced by gastric secretory volumes. It will also facilitate prospective studies on the effect of various secretagogues in vivo and evaluations of the influence of anti-ulcer drugs on peptic secretion.

Automated quantification of creatine kinase MB isoenzyme in serum by radial partition immunoassay, with use of the Stratus analyzer

We evaluated the analytical and clinical performances of a new radial partition immunoassay for measuring the mass concentration of creatine kinase (CK)-MB in serum. All pipetting, washes, incubations and data reduction were performed in 8 min by the Stratus (Dade) fluorometric analyzer. Within-assay and between-assay CVs were respectively 5.5% and 8.4% at 21 micrograms/L, and 4.2% and 3.4% at 48 micrograms/L. Assaying serial dilutions of serum samples with high CK-MB concentrations demonstrated excellent linearity. Results of the Stratus technique correlated well (n = 115, r = 0.98) with those of the Tandem-E CKMB II assay. There was no interference from hemolysis, bilirubin, rheumatoid factor, or added CK-MM (up to 3500 U/L); consequently, CK-MB can be determined in undiluted serum, even in the presence of high total CK activity. The mean CK-MB concentration in 105 blood donors was 1.9 (SD 1.3) micrograms/L. For seven myocardial infarction patients who received prompt fibrinolytic therapy, the mean CK-MB concentration was 4.5 (SD 1.8) micrograms/L at admission, and maximum concentrations, 119 (SD 94) micrograms/L, were recorded 16 h later. CK-MB returned to concentrations less than 10 micrograms/L within 72 h.

Simultaneous liquid-chromatographic determination of vitamin K1 and vitamin E in serum.

We describe a high-performance liquid chromatographic procedure for the simultaneous measurement of vitamins K1 and E in human serum. Delipidated human serum (free of vitamins K1 and E) was used to make standard solutions of these vitamins, and cetyl naphthoate and alpha-tocopheryl acetate were the internal standards for vitamin K1 and vitamin E, respectively. A simple, novel separation method utilizing liquid-liquid partition chromatography was used as a preparative "clean-up" procedure. Cetyl naphthoate and vitamin K1 (after post-column reduction) were detected by fluorescence, alpha-tocopheryl acetate and vitamin E by ultraviolet absorption. Sensitivity (detection limit) of the assay was 30 pg for vitamin K1 and 5 ng for vitamin E per injection. The method is specific, precise, and more rapid than previously described procedures. Within- and between-assay CVs were 8.1% and 12.9%, respectively, for vitamin K1; 3.5% and 6.0%, respectively, for vitamin E. Analytical recoveries of vitamins K1 and E were 80% and 93%, respectively, from serum and from delipidated serum (standards). The average neonatal serum concentration of vitamin K1 was 83 ng/L, 2.5 mg/L for vitamin E; for normolipidemic adults, the values were 343 ng/L and 7.9 mg/L, respectively, and for hyperlipidemic adults, 541 ng/L and 11.1 mg/L, respectively.