Douglas F. Stickle

1,5-anhydroglucitol monitoring in diabetes: a mass balance perspective.

1,5-anhydroglucitol (AG) is a nonmetabolizable glucose analogue found in plasma due to ingestion. The normal steady-state concentration can be dramatically decreased by inhibition of tubular reabsorption during periods of hyperglycemia. For this reason, monitoring of AG has been plausibly advocated for detection of periodic glucosuric hyperglycemia. In this review, we examine the influence of variation in factors affecting both steady-state and transient changes in plasma AG. Among normals, the lower and upper limits of the plasma AG reference range vary by a factor of 5. Using a simplified mass balance model (a single compartment model with 3-6x larger-than-plasma volume of distribution), reasonable inter-individual variations of ingestion rate, glomerular filtration rate and fractional post-filtration reabsorption are each able to account for the wide range of normal, steady-state AG concentrations. In monitoring of changes in AG, inter-individual variations in the threshold for glucose excretion, volume of distribution and glomerular filtration rate are all likely to significantly affect correspondence of integral changes in AG to integral glucosuria/hyperglycemia. This combination of variables, affecting both steady-state and transient changes, is significantly confounding with respect to interpretation of serial plasma AG concentrations. Resolution of information content of AG monitoring is thus largely that of crossing simple characterization of deltas [+,0,-] for changes in AG concentration against the information content of hemoglobin A1c monitoring. Despite this limitation, AG monitoring can in principle provide information about glycemic control in the short term that is not apparent through monitoring of hemoglobin A1c alone. However, whether AG monitoring can lead to improved outcomes in diabetes management remains to be established.

New onset of heterophilic antibody interference in prostate-specific antigen measurement occurring during the period of post-prostatectomy prostate-specific antigen monitoring

Laboratories evaluated whether an interference was causing a false-positive PSA for the Immulite 2000 immunoassay after a time course of increasing prostate-specific antigen (PSA) in a post-prostatectomy patient led to salvage therapy, which had no effect on the elevated PSA. Serial dilutions of PSA for the patient sample (6.1 ng/mL; post-prostatectomy reference range: <0.1 ng/mL [undetectable]) were linear (r > 0.99). However, the PSA measurement was reduced to 0.1 ng/mL after pretreatment of the sample with heterophilic antibody blocking reagent. PSA was undetectable (<0.1 ng/mL) when measured using two alternative immunoassays. These results were consistent with the presence of heterophilic antibody interference for the Immulite 2000 assay. In this case, heterophilic antibody interference with PSA measurement must have originated during the period of post-prostatectomy monitoring, and the apparent progressive increases in PSA may have been due solely to the progressive increase of this heterophilic antibody assay interference. In the absence of clinical correlation, positive PSA monitoring results should always be assessed for heterophilic antibody interference for at least one time point.

A survey of apparent blood volumes and sample geometries among filter paper bloodspot samples submitted for lead screening

BACKGROUND Sample collection instructions for the bloodspot lead screening program conducted by the Nebraska Medical Center recommend continuous application of a single finger-stick blood drop per printed filter paper circle (a volume of approximately 50 microl). In this study, we assessed whether apparent blood volumes and geometries of finger-stick bloodspot samples submitted for lead testing were consistent with collection recommendations. METHODS Samples were 422 extra bloodspots from 138 patients that were submitted for lead analysis. Using image analysis, apparent blood volumes were computed by comparison of bloodspot areas to bloodspot areas for standards of known volume. Circularity of samples was also assessed by image analysis. RESULTS Mean blood volume (25+/-13 microl) was approximately 50% of that needed to fill a printed circle. The distribution of volumes had three local maxima, consistent with bloodspot formation by multiple discrete applications of blood drops of small volumes (17+/-6 microl) rather than by continuous application of blood. Multi-drop samples were also apparent from non-circular geometries. CONCLUSIONS Bloodspots submitted for lead analysis showed an apparently inherent drop volume of less than 20 microl per drop and the application of multiple drops. Non-ideality of such specimens indicates the need for continuing education of bloodspot collectors.

Increased perimeter red cell concentration in filter paper bloodspot samples is consistent with constant-load size exclusion chromatography occurring during application

BACKGROUND Filter paper bloodspot samples exhibit increased red cell concentration near the bloodspot perimeter compared to the interior. We examined whether simulation of size exclusion chromatography separating cell and liquid components during bloodspot formation was consistent with the observed red cell distribution. METHODS Whole blood was defined as a mixture of 60% liquid and 40% solids (hematocrit). Bloodspot formation was simulated by step-wise center additions of 1 microl blood up to a total volume of 50 microl. Partitioning of the liquid component of the liquid volume into space not available to the solid component was calculated using a partitioning coefficient K=Vi/Vm, where Vi is the immobile (stationary) liquid volume and Vm is the mobile liquid volume. After each volume addition step, relative red cell concentration was calculated as the solids volume fraction of total volume as a function of radial distance from the center. RESULTS Simulation for K=0.3 resulted in final red cell distribution that was quantitatively consistent with bloodspot data. CONCLUSIONS Increased perimeter red cell concentration in bloodspots is consistent with partitioning of the liquid fraction into mobile and immobile components during bloodspot formation according to the principles of constant-load size exclusion chromatography.

Dynamic changes in plasma proinsulin/insulin ratio during insulin secretion influence correlation between radioimmunoassay (RIA) and IMX measurements of insulin.

Because proinsulin and insulin have different circulatory half-lives, the ratio of proinsulin to insulin in plasma depends on the dynamics of insulin secretion. This variation can potentially influence comparison of IMX assays and radioimmunoassays (RIAs) for [insulin], given that the antibody used in the IMX assay has negligible cross-reactivity with proinsulin compared to the 40% cross-reactivity with proinsulin of the antibody used in the RIA. Simulation of a simple mass balance model for insulin and proinsulin concentrations during an oral glucose tolerance test predicts that the ratio (R) of RIA to IMX insulin measurements of [insulin] should transiently decrease, pass through a minimum, increase past the initial value, pass through a maximum and eventually return to the initial value. Using time course specimens from patients, this pattern of variation in R was observed in the majority (12/16) of cases studied. The variation in R for time course specimens (CV = 26%) was significantly greater than for other specimens (fasting, random or undesignated; P < 0.05). Thus, when comparing IMX and RIA measurements of [insulin], variation in R for samples from differing states of dynamic insulin secretion contains a component that is attributable to dynamic changes in the ratio of [proinsulin]/[insulin] in plasma.

Average glucose from hemoglobin A1c for altered red blood cell lifetimes: Predictions based on a model for hemoglobin A1c formation

BACKGROUND A model for hemoglobin A1c (HbA1c) formation was used to predict the relationship between average glucose (AG) and %HbA1c under conditions of altered red blood cell lifetime (RCL). METHODS Using a kinetic mass balance model for formation of HbA1c in red blood cells as a function of age (time in circulation), whole blood %HbA1c vs. glucose was calculated based on the nonlinear distribution of red blood cells as a function of age across RCL. RESULTS Model calculations provided a close fit to the standard relationship of estimated average glucose to %HbA1c for normal RCL (r>0.999). Results for altered RCL were calculated assuming simple time-scale compression or expansion of the distribution of red blood cells as a function of RCL. For a given %HbA1c, the operative average glucose needed to have achieved a given %HbA1c was predicted to be altered by RCL according to average glucose×RCL=constant. CONCLUSIONS Model calculations estimate the extent to which standard reporting of AG vs. HbA1c underestimates or overestimates AG under conditions of altered RCL. Conditions of altered RCL may often be operative in patients with certain hemoglobin variants.

Interpatient distributions of bloodspot area per fixed volume of application: Comparison between filter paper and non-cellulose dried matrix spotting cards

BACKGROUND Non-cellulose dried matrix spotting (DMS) cards are an alternative to filter paper (FP) for bloodspots. We compared the interpatient distributions of bloodspot areas between DMS and FP for a fixed volume of application of whole blood, and examined correlations of areas with hematocrit. METHODS EDTA-whole blood adult patient samples (n=49; 25 males, 24 females) were utilized after routine measurement of hemoglobin and hematocrit. Replicate (4×) bloodspots were produced by bolus drop application of 50μL whole blood via a fixed-volume pipettor to either FP or DMS. Dried bloodspot areas were determined by image analysis. RESULTS Hematocrits (HCT) were normally distributed (HCT=30.9±5.3%). For both FP and DMS, bloodspot areas (a, cm(2)) across patients were normally distributed: for FP, a=1.11±0.056cm(2) (±5.0%); for DMS, a=0.378±0.037cm(2) (±9.9%). Relative bloodspot area differences across the population range were >20% for both DMS and FP. Correlation of bloodspot areas to hematocrit was negative for FP (r=-0.80) but positive for DMS (r=+0.78). CONCLUSIONS Interpatient variation in blood volume per area is a preanalytical variable for both DMS and FP bloodspots. Hematocrit is but one interpatient variable, as correlations of fixed-volume bloodspot areas with hematocrit across patients were substantially inexact (r(2)<0.65).

Decreasing the Cutoff for Elevated Blood Lead (EBL) Can Decrease the Screening Sensitivity for EBL

Change in the definition of elevated blood lead (EBL) from greater than or equal to 10 μg/dL (cutoff A) to greater than or equal to 5 μg/dL (cutoff B) was recently endorsed in the United States. A potential effect of this change is to decrease the screening sensitivity for EBL detection. We demonstrate this effect by simulated sampling of an example patient distribution for lead. Using lead-dependent assay imprecision, simulated sampling of the patient distribution tracked individual misclassifications relative to the EBL cutoff. Decreasing the EBL cutoff from A to B reduced screening sensitivity for EBL detection in this population to less than 90%, a decrease of 4%. The result was due to the fact that, for B, a greater fraction of the EBL population was near the EBL cutoff and therefore subject to misclassification due to assay imprecision. The effect of the decreased EBL cutoff to reduce EBL screening sensitivity is likely to apply to EBL screening programs generally.

Distribution of serum concentrations reported for macroenzyme aspartate aminotransferase (macro-AST)

Background The presence of macroenzyme (M) is often the explanation of an isolated elevation of aspartate aminotransferase (AST). Where M is identified, it is reasonable for the clinician to ask where an individual patients result fits in with known concentrations of M. In this context, we conducted a survey of literature to examine the distribution of reported serum concentrations of macro-AST. We also analyzed the distribution data to examine whether elevations were consistent with simple alteration of circulatory half-life (t1/2) of M relative to normal AST. Methods Distributions of M were compiled from the literature. These distributions were compared to predictions based on fixed changes in t1/2 applied to the reference interval for AST. Results There was a bimodal distribution of literature values for M (n =51), comprised roughly of populations A (M <200 U/L; 60% of total) and B (M >200 U/L; 40% of total). The two distributions were reasonably well characterized by a simple projection to the right of the reference interval for AST according to increased t1/2 (A: t1/2 =3.3 days; B: t1/2 =19.8 days) relative to AST (t1/2 =0.7 days). Conclusions Knowledge of distributions for M may be useful in discussion with clinicians regarding significance of M for individual patients. Distributions for M were consistent with the simplest explanation for elevated AST due strictly to an extended circulatory lifetime for M. Caveats to analysis, however, include selection within literature data mainly for patients with various co-morbidities.

Validation, quality control, and compliance practice for mass spectrometry assays in the clinical laboratory

Abstract Clinical laboratory assays using mass spectrometry are invariably developed in-house. Careful attention must therefore be given to both regulatory requirements and standards of professional practice regarding the validation, quality control, and competency/compliance aspects of offering, such assays. Here we review multiple guidelines and sources of information regarding this process: from federal requirements of the Clinical Laboratory Improvement Act (CLIA), accreditation requirements of the College of American Pathologists (CAP), Food and Drug Administration (FDA) guidance related to the pharmaceutical industry, and recommendations/reviews from the literature. We also discuss proposals for new FDA regulations regarding laboratory-developed tests (LDTs) that may have dramatic impact on clinical laboratories that utilize mass spectrometry. Only liquid and gas-chromatography mass spectrometry are discussed in this chapter.