Comparisons of plasma aldosterone and renin data between an automated chemiluminescent immunoanalyzer and conventional radioimmunoassays in the screening and diagnosis of primary aldosteronism.

Determining values of plasma renin activity (PRA) or plasma active renin concentration (ARC), plasma aldosterone concentration (PAC), and aldosterone-to-renin ratio (ARR) is essential to diagnose primary aldosteronism (PA), but it takes several days with conventional radioimmunoassays (RIAs). Chemiluminescent enzyme immunoassays for PAC and ARC using the Accuraseed® immunoanalyzer facilitated the determination, but relations between Accuraseed® immunoanalyzer-based and RIA-based values in samples of PA confirmatory tests and adrenal venous sampling remained to be elucidated. We addressed this issue in the present study. This is a prospective, cross-sectional study. ARC and PAC values were measured by the Accuraseed® immunoanalyzer in samples, in which PRA and PAC values had been measured by the PRA-FR® RIA and SPAC®-S Aldosterone kits, respectively. The relations between Accuraseed® immunoanalyzer-based and RIA-based values were investigated with regression analyses. The optimal cutoff of Accuraseed® immunoanalyzer-based ARR for PA screening was determined by the receiver operating characteristic analysis. After log-log transformations, linear relations with high coefficients of determination were observed between Accuraseed® immunoanalyzer-based and RIA-based data of renin and aldosterone. Following the PA guidelines of Japan Endocrine Society, Accuraseed® immunoanalyzer-based cutoffs were calculated from the regression equations: the basal PAC for PA screening >12 ng/dL, PAC for the saline infusion test >8.2 ng/dL, ARC for the furosemide-upright test <15 pg/mL, and ARR for the captopril challenge test >3.09 ng/dL per pg/mL. The optimal cutoff of Accuraseed® immunoanalyzer-based ARR for PA screening was >2.43 ng/dL over pg/mL not to overlook bilateral PA patients. The present study provided conversion formulas between Accuraseed® immunoanalyzer-based and RIA-based values of renin, aldosterone, and ARR, not only in basal samples but also in samples of PA confirmatory tests and adrenal venous sampling. Although validation studies are awaited, the present study will become priming water of harmonization of renin and aldosterone immunoassays.

Thyroglobulin Antibody Screen Prior to Mass Spectrometry Provides Measurable Cost Savings and Optimal Laboratory Utilization.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods that allow accurate quantitation of thyroglobulin (Tg) in the presence of Tg antibodies (TgAbs) have recently become available. Due to cost differences between LC-MS/MS and immunoassay, some laboratories now offer a reflex test strategy that uses LC-MS/MS only for TgAb-positive samples. The goal of this study was to examine utilization of Tg testing strategies and cost savings. METHODS: Test ordering patterns were examined for over 150,000 orders for TgAb and Tg in our laboratory. The average list price was determined from three separate commercial laboratories offering this testing. RESULTS: Data showed that 89% of orders for Tg used the reflex test option, resulting in a savings of over $3 million compared with testing all samples by LC-MS/MS. Of the Tg by LC-MS/MS orders not using the reflex option, 1,663 also included a separate order for TgAb on the same patient sample, representing approximately $170,000 in potentially unnecessary costs from TgAb-negative samples. CONCLUSIONS: Identifying situations to use more expensive testing methods (eg, LC-MS/MS) only when necessary, such as for TgAb-positive patients, leads to considerable cost savings and a more economical use of valuable health care resources.

CA 19-9: handle with care.

Since its inception in the mid-1980s of the 20th century testing for carbohydrate antigen 19-9 (CA 19-9) has raised expectation for an earlier diagnosis and accurate monitoring of several malignant diseases. After almost 30 years, the available evidences have confirmed the appropriateness and usefulness of determining CA 19-9 levels as a prognostic indicator and as a reliable tool for monitoring pancreatic and gastrointestinal cancer, but concerns have been raised about its applications in screening, which is actually not recommended, and in the diagnosis of malignancies, due to several interferences that limit the specificity and to the insufficient sensitivity of this marker. In this paper we aimed to review the basic concepts of CA 19-9 testing and its current applications, with a major focus on the most recent evidences dealing with assay interference, methods comparison and monitoring of malignant diseases. The prognostic value and monitoring recommendations for pancreatic, gastric and colorectal cancers are described in depth.

[Changes in serum vitamin D assay usage and the need for evidence-based recommendations]. / Évolution des dosages sériques de vitamine D dans un hôpital d'adultes Plaidoyer pour l'élaboration de recommandations en vue d'une juste prescription.

Result of renewed interest due to the large amount of literature that reported numerous epidemiological data demonstrating the high prevalence of vitamin D deficiency, the number of prescriptions of serum vitamin D assays has grown exponentially in recent years with a cost for health insurance that increased almost fivefold in four years. The quantitative and qualitative analysis of assays carried out from 2007 to 2011 in a French university adult short-stay hospital shows changes in practices not only quantitatively but also qualitatively resulting in an overtime increase in the frequency of prescriptions in patients younger, less vitamin D deficient and more frequently male. In the absence of French guidelines, this development cannot be qualified as deviant but justifies the urgent need to establish evidence-based recommendations for good prescriptions and adequate assays of blood vitamin D.

A novel triple mix radiobinding assay for the three ZnT8 (ZnT8-RWQ) autoantibody variants in children with newly diagnosed diabetes.

BACKGROUND AND AIMS: Autoantibodies against the zinc transporter 8 (ZnT8A) are common in type 1 diabetes (T1D). ZnT8A analyses are complicated by the fact that there are three variants of the autoantigen at amino acid position 325 representing ZnT8-R (Arginine), ZnT8-W (Tryptophan) and ZnT8-Q (Glutamin). The aims of the study were: 1) to develop an autoantigen triple mix Radio-Binding Assay (RBA) for ZnT8A; 2) to identify the individual ZnT8-R,-W,-QA reactivity and 3) to validate the triple mix ZnT8A RBA in children with newly diagnosed T1D. METHODS: Serum samples were obtained from 2664 (56% males, n=1436) patients in the Swedish nationwide Better Diabetes Diagnosis (BDD) study representing patients with T1D (97%, n=2582), T2D (1.7%, n=46), MODY (1.0%, n=28) and secondary diabetes (0.3%, n=8). cDNA coding for the C-terminal end of each variant was prepared by site-directed mutagenesis and subcloned into a high efficiency in vitro transcription translation vector. The ZnT8 variants were labeled with 35S-methionine and used in a standard RBA separating free from autoantibody-bound autoantigen with Protein A-Sepharose. RESULTS: ZnT8-TripleA was detected in 1678 (65%) patients with T1D, 4 (9%) T2D, 3 (11%) MODY and in none (0%) of the patients with secondary diabetes. Among the T1D patients ZnT8-RA was detected in 1351 (52%) patients, ZnT8-WA in 1209 (47%) and ZnT8-QA in 790 (31%) demonstrating that 1661 (64%) had one or several ZnT8A. The ZnT8-TripleA assay showed a false positive rate of 1.9% (n=49). Only 1.2% (n=32) of the T1D patients were false negative for ZnT8-TripleA compared to 0/46 (0%) of the T2D patients. The precision (intra assay CV) and reproducibility (inter assay CV) of the ZnT8-TripleA assay did not differ from the RBA of the individual ZnT8 variants. CONCLUSION: We conclude that the ZnT8-TripleA assay had low false positive and false negative rates. The ZnT8-TripleA assay would therefore be highly suitable not only to analyze patient with newly diagnosed diabetes but also for screening the general population since this assay demonstrated high sensitivity and very high specificity.

Modern laboratory evaluation of peptide and amines: a continuing role for radioimmunoassay?

Modern medicine, and specifically clinical diagnosis, relies, among other diagnostic procedures, on the measurements of the biogenic analytes for elucidation and correlation of specific neuroendocrine markers. Tremendous advances have been made in imaging and radioactive uptake procedures to elucidate tumor presence and characterization. However, such advances only partially provide the fundamental degree of tumor activity and clinical confirmational validity. The author points out in some detail the problems that may arise when the methodological differences presented by each investigational study and investigators are not standardized. This variation causes a concern with the specific objectives of the investigator and the specific aims of the research project at hand, and ultimately for the validity of the published results.

A Bayesian precision profile for measuring the quality of immunoassay experiments.

A precision profile, the relationship between the concentration of a substance and its measured precision, is a convenient way of conveying the ability of an immunoassay to accurately measure the concentration of a substance in blood serum. A precision profile is characterized by the definition of precision. Historically, precision has been evaluated as the standard error of an estimator of the concentration in a sample conditional on the true concentration. In this paper, Bayesian predictive inference is used to develop a new measure of precision based on the accuracy with which an assay could infer the concentration in a hypothetical new sample. This leads to a natural procedure for evaluating a precision profile that avoids using approximations such as those inherent in traditional methods.

An optimised, highly sensitive radioimmunoassay for the simultaneous measurement of estrone, estradiol and estrone sulfate in the ultra-low range in human plasma samples.

Following the introduction of potent aromatase inhibitors for the treatment of breast cancer patients, highly sensitive methods have become mandatory to evaluate the influence of these drugs on plasma estrogen levels. Commercially available kits for estrogen measurements are not suitable for these kinds of evaluations due to their detection limits that are close to baseline estrogen levels in postmenopausal women. We describe here an optimised radioimmunoassay suitable for the simultaneous measurement of plasma estrone (E1), estradiol (E2) and estrone sulfate (E1S) levels in the ultra-low range. Following incubation with [3H]-labelled estrogens as internal standards, crude estrogen fractions were separated by ether extraction. The E1S fraction was hydrolysed with sulfatase followed by eluation on a Sephadex column. Free estrogens (E1, E2) were separated by chromatography (LH-20). Estrone and E1S (following hydrolysis) were converted into E2, and each estrogen fraction was measured by the same highly sensitive and specific radioimmunoassay using estradiol-6-(O-carboxymethyl)-oximino-2-(2-[125 I]-iodo-histamine) as ligand. Although several purification steps were involved, the internal recovery values for tritiated estrogens were found to be 88%, 90%, and 49% for E1, E2 and E1S, respectively. The intra-assay coefficient of variation was <5% for all recovery measurements. The detection limits were calculated following repeated blank measurements and found to be 1.14 pmol/L for E1, 0.67 pmol/L for E2, and 0.55 pmol/L for E1S, respectively. The intra-assay coefficient of variation (CV) was found to be 3.4% for E1, 5.1% for E2 and 6.1% for E1S, while the inter-assay CV was 13.6%, 7.6% and 7.5% for E1, E2, and E1S, respectively. Considering normal plasma levels for E2 (15 pmol/L), E1 (80 pmol/L) and E1S (400 pmol/L) in postmenopausal women, the method allows theoretically to detect suppression of plasma E2, E1 and E1S levels by 95.5%, 98.6% and 99.9% when starting from average, normal postmenopausal levels. Thus, the method presented here is to our knowledge the currently most sensitive assay available for plasma estrogen measurements in the ultra-low range and, as such, a reliable tool for a proper evaluation of potent aromatase inhibitors and other potential drugs influencing on plasma estrogen levels.

Quantification of dexamethasone and corticosterone in rat biofluids and fetal tissue using highly sensitive analytical methods: assay validation and application to a pharmacokinetic study.

A sensitive, specific, accurate and precise LC/MS/MS method was developed for the simultaneous measurement of dexamethasone and corticosterone in rat plasma. The method was extended to dexamethasone analysis in rat plasma ultrafiltrate and fetal tissues. Samples were processed using SPE involving Oasis HLB cartridges, which offered complete extraction recovery for the analytes. Samples were subsequently analyzed using LC/MS/MS. A structurally related corticosteroid, prednisolone, was used as the internal standard. Using a 500 microL plasma sample, limits of quantification of 0.2 and 2.0 ng/mL were achievable for dexamethasone and corticosterone. This level of sensitivity allowed characterization of maternal/fetal dexamethasone profiles after administration of multiple doses of dexamethasone sodium phosphate to rats. However, this sensitivity was not satisfactory for corticosterone during pharmacokinetic studies involving dexamethasone due to its strong adrenosuppressive effect. This led us to investigate the suitability of a commercially available radioimmunoassay kit, which through extensive testing and minor modifications was found to offer extremely sensitive, specific, accurate and precise analysis of corticosterone. Knowledge of the steroid profiles captured using these highly sensitive analytical tools may potentially help in the optimization of corticosteroid therapy during pregnancy.

Evaluation of immunoassays for the measurement of insulin-like growth factor-I and procollagen type III peptide, indirect biomarkers of recombinant human growth hormone misuse in sport.

Insulin-like growth factor-I (IGF-I) and procollagen type III peptide (P-III-P) have been proposed as indirect biomarkers for the detection of the misuse of recombinant human growth hormone in sport. An extended intra- and inter-laboratory validation of commercially available immunoassays was carried out. For total IGF-I, two radioimmunoassay (RIA) kits (IGF-I/RIA1, Nichols Institute Diagnostics and IGF-I/RIA2, Mediagnost) and one enzyme-linked immunosorbent assay (ELISA) (R&D) were evaluated. For P-III-P, two RIA kits (P-III-P/RIA3, Cis-bioInternational and P-III-P/RIA4, Orion Diagnostica) were studied. The intra-laboratory precision and accuracy values for all IGF-I assays were better than 15%. The IGF-I/ELISA showed the lowest limit of quantification (LOQ) and its calibration curve covered the range of concentrations found in human serum samples. Higher agreement between laboratory results was obtained for IGF-I/ELISA and IGF-I/RIA1. Low inter-technique correlation was obtained for the three assays; the only comparable results were obtained between IGF-I/ELISA and IGF-I/RIA1. For P-III-P, intra-laboratory precision and accuracy values better than 15% were obtained for both assays in almost all cases. The calibration curve for P-III-P/RIA4 covered the range of concentrations of serum samples, while 30% of the values for P-III-P/RIA3 were below the calibration sample with the lowest concentration. Inter-laboratory correlation was also higher for P-III-P/RIA4. In summary, ELISA and RIA4 were the most suitable assays for measurement of IGF-I and P-III-P, respectively, in serum samples. However, the validation studies carried out show the need for harmonization of immunoassay parameters to improve the reproducibility and comparability of results between different laboratories and in different studies.

Evaluation of a new automated electrochemiluminescent sex hormone-binding globulin (SHBG) immunoassay.

Serum sex hormone-binding globulin (SHBG) regulates the cellular bioavailability of SHBG-bound steroid hormones. Since variations in SHBG levels may affect the concentration of free, i.e., biologically active testosterone in serum, SHBG levels are commonly measured as a supplement to total testosterone determination. The recently developed electrochemiluminescence Elecsys SHBG immunoassay was evaluated analytically on a Modular E170 (Roche Diagnostics, Mannheim, Germany) immunoanalyzer. Major differences in SHBG concentrations have been described among the commercially available methods; we therefore compared the new method with an established SHBG immunoradiometric assay (IRMA) in 99 routine serum samples. To provide reference values to clinicians, SHBG concentration was measured by Elecsys in 304 serum samples from healthy volunteers and several relevant clinical subgroups. The within-run and total imprecision coefficients of variation were

Estimation of lot-to-lot relative potency in the preparation of radioimmunoassay calibrators.

The aim of this study was to determine guidelines for estimating lot-to-lot differences in the potency of calibrator materials or batches of standards for radioimmunoassays. Thirty one lots of standards for thirteen different analytes were compared to the previous lot for that analyte with the relative potency computed by nine different methods. Assays were performed manually. The nine different calculation methods included non-simultaneous fitting of pairs of standard curves, full or partial simultaneous fitting, and least squares or robust minimisation. The simultaneous methods were found superior to the non-simultaneous in minimising the variance of the relative potency estimates, while robust fitting procedures did not result in a lower variance than least-squares minimisation. The root mean square coefficient of variation for the simultaneous estimation of the relative potency by least squares was 6.1%. On this basis, it is recommended that relative potency estimations in radioimmunoassay be based on at least eight independent pair-wise standard curve comparisons. Additional guidelines for preparing and comparing batches of standards are also given.

Proenzyme psa for the early detection of prostate cancer in the 2.5-4.0 ng/ml total psa range: preliminary analysis.

OBJECTIVES: To determine the clinical utility of using proenzyme prostate-specific antigen (pPSA) for early detection of prostate cancer in the 2.5 to 4.0 ng/mL total PSA range. pPSA, the precursor form of PSA that contains a 7 amino acid leader peptide, and truncated forms such as [-2]pPSA and [-4]pPSA can be measured in serum by research immunoassay. METHODS: Archival serum from 119 men (noncancer, 88; cancer, 31), obtained before biopsy and in the total PSA range of 2.5 to 4.0 ng/mL, were assayed for total PSA, free PSA (fPSA), and pPSA. pPSA was defined as the sum of the [-2], [-4], and [-7] forms, and the percent pPSA (%pPSA) was defined as pPSA/fPSA. RESULTS: pPSA averaged 4.6% +/- 0.4% (SEM) of total PSA and 39.3% +/- 3.5% of fPSA. PSA and %fPSA values were similar between the noncancer and cancer groups, and %pPSA tended to be higher in the cancer group (50.1% +/- 4.4%) compared with the noncancer group (35.5% +/- 6.7%; P = 0.07). Using receiver operating characteristic analysis to assess clinical utility, the area under the curve for %pPSA was 0.688 compared with 0.567 for %fPSA. At a fixed sensitivity of 75%, the specificity was significantly greater for %pPSA at 59% compared with %fPSA at 33% (P <0.0001). CONCLUSIONS: In the 2.5 to 4.0 ng/mL total PSA range, 75% of cancers can potentially be detected with 59% of unnecessary biopsies being spared using %pPSA; use of %fPSA would result in sparing only 33% of unnecessary biopsies. A large prospective clinical trial is needed to confirm these preliminary findings.

Development of radioimmunoassay for measurement of serum digoxin in digitalized patients using novel anti-digoxin antiserum.

There is an antiserum elicited by digoxin 3'-hemisuccinate-bovine serum albumin (BSA) conjugate possessing high specificity for digoxin. Our study focused on development of RIA using this novel antiserum for measurement of digoxin in serum from digitalized patients. The property of the new antiserum was investigated by RIA with digoxin 3'-hemisuccinyl-[3H]leucine. The separation of bound and free fractions was performed using a dextran-coated charcoal suspension. The new antiserum bound approximately 50% of digoxin 3'-hemisuccinyl-[3H]leucine with a final dilution of 1:30000. The intra- and inter-assay coefficients of variation were <9% in the range of 0.52-4.17 ng/ml. The mean digoxin concentration in serum samples (n=35) from digitalized patients was estimated to be 0.68 ng/ml, which was lower than its measurement of digoxin with the commercial antidigoxin BSA serum and monoclonal anti-digoxin. It is apparent that the RIA described here has sufficient precision. The RIA system was available for the measurement of digoxin in serum from digitalized patients.

Evaluation and comparison of therapeutic monitoring of whole-blood levels of cyclosporin A and its metabolites in renal transplantation by HPLC and RIA methods.

BACKGROUND: The aim of the work was to evaluate the possibility to estimate the level of cyclosporin A (CyA) metabolites as the difference of radioimmunoassay (RIA) non-specific and RIA specific methods. METHODS: Blood samples of renal transplant patients were analyzed by three different methods: RIA specific method (CYCLO-Trac, DiaSorin, USA) (RIA(SP)), RIA non-specific method (Immunotech, Czech Republic) (RIA(NS)), and high performance liquid chromatography (HPLC) method. RESULTS: Although values obtained by RIA(SP) correlated well those obtained by HPLC (RIA(SP)=0.995.HPLC+9.68; r(2)=0.962, n=448), the results of HPLC methods were lower by 8%. The values obtained by RIA(NS) were 2.57 times higher than the values obtained by RIA(SP) (RIA(SP)=0.356RIA(NS); r(2)=0.713, n=448). The ratio (CyA+CyA metabolites)/(CyA) calculated as the ratio RIA(NS)/RIA(SP) values for 42 renal transplant patients was relatively stable for each particular patient. The sum of selected CyA metabolites (M1+M17+M21) measured by HPLC correlated well with that estimated from the difference of RIA(NS)-RIA(SP): HPLC(metab)=0.921.(RIA(NS)-RIA(SP))+21.3; (r(2)=0.746, n=448). CONCLUSION: The combination of both the specific and non-specific methods for the determination of CyA presents an improved means for the TDM of CyA and CyA metabolites in renal transplant patients. Moreover, a combination of both methods can help to elucidate some unexpected events, such as the persistence of high cyclosporin blood levels.

Description of a generally applicable model for the evaluation of uncertainty of measurement in clinical chemistry.

There is a growing pressure on clinical chemistry laboratories to conform to quality standards that require the evaluation and expression of the uncertainty of results of measurement. Nevertheless, there is some reluctance to accept the uncertainty concept in the analytical community due to difficulty in evaluating uncertainty in practice. For example, often the uncertainty of some uncertainty components is not known very well in clinical chemistry measurements, such as those associated with matrix effects or with the values of the calibrators. Moreover, it is not clear how to interpret uncertainty in relation to diagnostic criteria, reference ranges and other decision limits in clinical chemistry practice. Hence, the value of reporting the uncertainty of the measurement result is not obvious. In this paper it is suggested a relatively simple, logical procedure for evaluating measurement uncertainty based on the principles in the Guide for the Expression of Uncertainty of Measurement (GUM). The measurement process is partitioned into elements that are well known to the analyst, namely sampling, calibration, and analysis. The corresponding model function expresses the result of a measurement as the value obtained by the analytical procedure multiplied by the correction factors for sampling bias, for bias caused by the calibrators, and for other types of bias. Under normal conditions, when the measurement procedure is validated and corrected for all known bias, the expected value of each correction factor is one. The uncertainty that remains with regard to sampling, manufacturing of calibrators and other types of bias is combined with the analytical imprecision to yield a combined uncertainty of a result of measurement. The advantages of this approach are: (i) Data from the method validation, internal quality control and from participation in external quality control schemes can be used as input in the uncertainty evaluation process. (ii) The partition of the measurement into well-defined tasks highlights the different responsibilities of the clinical chemistry laboratory and of the manufacturer of reagents and calibrators. (iii) The approach can be used to harmonize the uncertainty evaluation process, which is particularly relevant for laboratories seeking accreditation under ISO 17025. The application of the proposed model is demonstrated by evaluating the uncertainty of a result of a measurement of prolactin in human serum. In the example it is shown how to treat the uncertainty associated with a calibrator supplied with a commercial analytical kit, and how to evaluate the uncertainty associated with matrix effects.

Quantitative genetic analysis of circulating levels of biochemical markers of bone formation.

Carboxyterminal propeptide of type 1 collagen (PICP) and bone Gla-protein-osteocalcin (BGP) are the most important components of the organic bone matrix and play a key role in bone formation. To investigate whether and to what extent variation of the plasma levels of these indices of bone turnover depends on genetic factors, we studied 355 adults belonging to nuclear pedigrees. Genetic analysis was carried out in 2 steps: 1) variance decomposition analysis was performed using the FISHER statistical package; and 2) complex segregation analysis implemented in the program package MAN. The effect of age and gender differences, gender hormones, as well as PTH and vitamin-D (calcidiol) plasma levels were evaluated simultaneously with the parameters of variance analysis. The results showed that about 50% of PICP variation is attributable to genetic factors. The effect of age was significant among men and postmenopausal women, whereas calcidiol influenced variation of PICP in premenopausal women. The results of variance analysis showed that some 40% of BGP, adjusted for confounding variables, can be explained in genetic factors. Age and PTH were important covariates for osteocalcin in men and premenopausal women. Exploration of the maximum likelihood estimates of the various hypotheses concerning the mode of intergenerational transmission of PICP and BGP demonstrated a good correspondence to the Mendelian mode of inheritance (i.e., major gene effect).

Effects of physostigmine and calcium on acetylcholine efflux from the hippocampus of freely moving rats as determined by in vivo microdialysis and a radioimmunoassay.

The effects varying the concentration of Ca2+ in perfused artificial cerebrospinal fluid ([Ca2+]csf) on basal acetylcholine (ACh) efflux from the hippocampus of freely moving rats, in the presence and absence of the cholinesterase (ChE) inhibitor physostigmine, were investigated using in vivo microdialysis and a highly specific radioimmunoassay for ACh. In the absence of physostigmine, basal ACh efflux was 3.4+/-0.7 pg/30 min (mean +/- SEM) at [Ca2+]csf = 1.26 mM. Stepwise increases in [Ca2+]csf elicited a gradual increase in ACh efflux that was significant at [Ca2+]csf = 5.04 mM. Inhibition of ChE by addition of 10 microM physostigmine to the perfusate increased the efflux of ACh to 103.2+/-21.1 pg/30 min ([Ca2+]csf = 1.26 mM), and the efflux was augmented still further by increasing [Ca2+]csf, a change that became significant at [Ca2+]csf = 3.78. These results illustrate the sensitivity of basal ACh efflux from the hippocampus to changes in the extracellular Ca2+ concentration, and suggest that a more accurate picture of hippocampal cholinergic activity is obtained by microdialysis using normal artificial cerebrospinal fluid, under physiological conditions, rather than in the presence of a ChE inhibitor.