Estimation of hematocrit in filter paper dried bloodspots by potassium measurement: advantage of use of perimeter ring samples over circular center sub-punch samples.

BACKGROUND: Quantitative assays using dried filter paper bloodspots (DBS) may be adversely affected by hematocrit (HCT) as an unknown variable. Studies have demonstrated the utility of the measurement of potassium (K) from DBS punches to estimate HCT. Because there is significant accrual of RBCs at the DBS perimeter, we investigated whether K measurement from ring-shaped specimens inclusive of the perimeter might provide an advantage over conventional interior circular sub-punch samples to estimate HCT. METHODS: Primary samples were Li-heparin whole blood, with HCT as measured on concurrently-drawn K-EDTA specimens. DBS were formed by bolus addition of 40 µL whole blood to filter paper cards. Total bloodspot area was determined by image analysis. Removal of center sub-punch (P) samples of fixed area produced remainder ring (R) samples inclusive of the perimeter. Samples were extracted in K-EDTA (2.5 mmol/L) and measured for diluent-corrected K per area (α, µmol K/cm2). RESULTS: Forty-three patient samples were utilized. α was normally distributed: α(P)=1.23±0.26 µmol K/cm2; α(R)=1.86±0.41 µmol K/cm2; α(R)/α(P)=1.51±0.15. α was correlated with HCT: α(P)=0.030 HCT(%)+0.015 µmol K/cm2 (r2=0.795); α(R)=0.052 HCT(%)+0.010 µmol K/cm2 (r2 = 0.912), but with higher resolution and lesser error for α(R). CONCLUSIONS: K per area (α) was significantly higher in R samples vs. P samples, with higher resolution for α(R) vs. HCT. Use of ring samples inclusive of the perimeter to estimate HCT for DBS via K measurement can provide an advantage over use of center sub-punch samples.

Myth and reality: practical test system for the measurement of anti-DNA antibodies in the diagnosis of systemic lupus erythematosus (SLE).

The myth persists that only the labor intensive Farr radioimmunoassay and Crithidia luciliae immunofluorescence (CL-IFA) are systemic lupus erythematosus (SLE)-specific tests. We compared them to ELISA with bacteriophage lambda DNA (EL-dsDNA) and denatured calf thymus DNA (EL-ssDNA). By percentile ranking, the specificity cut-off level was set both out of clinical context (SOCC) on 100 blood bank donors, and in clinical context (SICC) on 100 patients with either rheumatoid arthritis or scleroderma (50/50). Clinical sensitivity was calculated on 100 random SLE patients. At 95% SICC, the sensitivity of Farr, CL-IFA, EL-dsDNA, and EL-ssDNA was similar (95%CI): 76% (66-84), 76% (66-84), 63% (53-72), and 75% (65-83), respectively; 87% of the patients were positive by at least one method and 55%by all methods. At 99% SICC, the sensitivity was also similar (95% CI): 57% (47-67), 47% (37-57), 58% (47-67), and 43% (33-53), respectively. The areas under ROC curve were similar (95% CI) when patients were used as controls for specificity. At 99% SOCC, EL-ssDNA identified 89% positive, 2 negative but positive by another method at 95% SICC, and 9 negative (i.e. 89/2/9), followed by CL-IFA (80/6/14), Farr (76/12/12), and EL-dsDNA (64/23/13). Thus, at relatively low cost and easy automation, under the same conditions of specificity, the two ELISA tests combined were at least as good, if not superior, to CL-IFA or Farr: they showed similar clinical sensitivity and also identified more patients with anti-DNA antibodies.

Model analysis of effect of canagliflozin (Invokana), a sodium-glucose cotransporter 2 inhibitor, to alter plasma 1,5-anhydroglucitol.

BACKGROUND: Renal reabsorption of 1,5-anhydroglucitol (AG) is competitively inhibited by elevated glucose and leads to depleted plasma AG in diabetes. Plasma AG recovery in diabetes normally correlates with improved glycemic control. However, use of sodium-glucose co-transporter 2 (SGLT2) inhibitors (e.g., canagliflozin) to treat diabetes by inhibition of renal glucose reabsorption can negate this correlation, via an indirect effect (increase of renal filtrate glucose concentration) to inhibit AG reabsorption by sodium-glucose co-transporter 4 (SGLT4). Conversely, then, AG measurement might be useful as an independent marker for SGLT2 inhibitor activity. METHODS: Using an AG mass balance model, we analyzed literature data on plasma AG before and after initiation of canagliflozin therapy (CT) to quantitatively characterize the effect of CT on AG reabsorption. RESULTS: According to model calculations, modest decreases (<5%) in fractional reabsorption of AG account for the drastic decrease in [AG] observed during CT. Decreases are predicted to be rapid (t1/2<3days) after CT initiation. CONCLUSION: CT negates the usual premise of AG measurement (that [AG] should increase with improved glycemic control). However, according to model calculations, a substantial and likely rapid effect of CT on [AG] means that AG measurement might provide an early marker for CT activity.

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).

Receiver-operating characteristics of adjusted urine measurements of oxycodone plus metabolites to distinguish between three different rates of oxycodone administration.

OBJECTIVES: In a study by Couto et al. (Use of an algorithm applied to urine drug screening to assess adherence to an oxycontin regimen. J Opioid Manag 2009;5:359-64), adjusted urine measurements of oxycodone plus metabolites noroxycodone and oxymorphone were determined among volunteer subjects in three groups according to oxycodone administration rates (A: 80 mg/day; B: 160 mg/day; C: 240 mg/day). We performed receiver-operating characteristic (ROC) analyses of the distribution data from this study to determine the ability to correctly categorize individual measurements with respect to each group. DESIGN AND METHODS: For groups A-C, assumed reference ranges were defined as median-centered intervals encompassing a designated central percentage of the group's distribution. By varying assumed reference ranges across all possible reference ranges, ROC analyses of the ability of each group's reference ranges to appropriately include or exclude members of all groups were calculated. This generated six ROC curves (sensitivity vs. specificity): A vs. (B or C); B vs. (A or C); C vs. (A or B). RESULTS: Overlaps of distributions A, B, and C were large, such that none of the ROC curves exceeded areas-under-curves of 0.8. The greatest sensitivity-specificity combination had a sensitivity of 74% for C with specificity of 75% for A, for which oxycodone administration rates were different by a factor of 3. CONCLUSIONS: ROC analyses of data from a previous study demonstrated that, even under experimentally controlled conditions, adjusted urine drug measurements could not be used reliably to correctly categorize individual subjects' results according to their known oxycodone administration rates in the range of 80-240 mg/day. Misclassifications of results were 25% or greater.

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.

Predicted effects of proposed tightened acceptance criterion for Pb proficiency testing (PT) on PT sample pass rates for Pb point-of-care testing.

BACKGROUND: There is current advocacy for change in Pb proficiency testing (PT) acceptance criterion from ± 4 µg/dl ([Pb] <40 µg/dl; criterion a) to ± 2 µg/dl ([Pb] <20 µg/dl, criterion b). We examined the effect of this proposed change on PT sample pass rates for point-of-care testing (POCT) as predicted by imprecision of POCT PT sample results. METHODS: Inter-site standard deviations (s) of POCT PT results were tabulated as a function of [Pb] and characterized as a linear function of [Pb] (r(2)>0.8). Given s, predicted minimum, random-error-only PT failure rates (Fp) as a function of [Pb] were computed as the fraction of a normal distribution of results ([Pb]± s) that would fall outside of boundaries of acceptance criterion a or b. RESULTS: For [Pb]=2-20 µg/dl, current observed PT sample failure rates using criterion a range from 3 to 6%, which are greater than the predicted minimum failure rates based on s alone (Fp(a)=0-6%). In contrast, predicted minimum failure rates based on s using criterion b are greatly increased (Fp(b)=5-35%). CONCLUSIONS: Given the degree of inter-site imprecision among POCT Pb PT results, adoption of criterion b for PT acceptance will dramatically increase Pb PT sample failure rates for POCT due to random-error alone.

Demonstration of autoimmunity in the tight skin-2 mouse: a model for scleroderma.

The tight skin-2 (Tsk2/+) mouse has been proposed as an animal model of systemic sclerosis (SSc) because this animal exhibits increased collagen synthesis and accumulation in the dermis. The Tsk2/+ mouse also has been reported to have a mononuclear cell infiltrate in the dermis; however, to date no evidence of autoimmunity has been described in this animal model. We report here that Tsk2/+ mice harbor numerous autoantibodies in their plasma including some, which are similar to those, present in SSc patients. Immunofluorescence with HEp-2 cells revealed the presence of anti-nuclear Abs (ANAs) in the plasma of 92% of the Tsk2/+ mice. In contrast, <5% of cage-mated CAST/ei mice had a positive ANA and none of the C3H/HeJ age-matched controls were positive. Homogenous, speckled, rim, nucleolar, centromere as well as combinations of these patterns were observed. The proportion of Tsk2/+ animals with a positive ANA increased slightly with age. ELISAs showed that 93% of the Tsk2/+ animals were positive for anti-Scl70, 82% for anti-centromere, 5% for anti-RNP/Sm, and none were positive for anti-RNA-polymerase II Abs. Indirect immunofluorescence with Crithidia luciliae and ELISA for anti-dsDNA Abs showed that 76% of Tsk2/+ mice were positive for this autoantibody. The high frequency of anti-Scl70 and anti-centromere autoantibodies indicates that Tsk2/+ mice display some humoral immune alterations which are similar to those found in patients with SSc. However, the Tsk2/+ mice also develop autoantibodies to dsDNA and a majority of the mice develop multiple autoantibody specificities (anti-Scl70, anti-CENP-B, and anti-dsDNA) indicating that the mouse may be a useful model to study autoimmunity in a wider spectrum of connective tissue diseases.

Pregnancy outcomes in female renal recipients: a comparison of systemic lupus erythematosus with other diagnoses.

This study compares pregnancy outcomes in systemic lupus erythematosus (SLE) patients post renal transplant with recipients with other primary diagnoses, utilizing data from the National Transplantation Pregnancy Registry, Philadelphia, PA. Recipients were referred from transplant centers nationwide. A retrospective analysis was performed using data from questionnaires, hospital records and telephone interviews. Outcomes of pregnancies post renal transplant secondary to lupus nephritis (SLE: n = 38; 60 pregnancies) were compared with the pregnancy outcomes of renal recipients with other diagnoses (non-SLE: n = 247; 374 pregnancies). Drug-treated hypertension during pregnancy was less common in the SLE group than in the non-SLE group (45.0% vs. 62.5%, p = 0.015). There were fewer cesarean sections in the SLE group (30.2 vs. 53.2%, p = 0.008). There was no primary or gestational diabetes in the SLE group. There were no other statistical differences in maternal conditions or pregnancy outcomes between the SLE and non-SLE groups, or in the incidence of post pregnancy graft loss. Female recipients transplanted for renal failure secondary to lupus nephritis can successfully maintain pregnancy. Outcomes are comparable to renal recipients with other diagnoses. Newborns in both groups were often premature and had low birthweight. Overall childhood health was reported to be good; there were no apparent predominant structural malformations among the children.