Laura J. McCloskey

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.

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.

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.

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.

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 λ 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. J. Clin. Lab. Anal. 24:77–84, 2010.

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

Abstract 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.