James D. Landmark

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.

Transfusion-Associated Graft Versus Host Disease in the Immunocompetent Patient: An Ongoing Problem

Transfusion associated-graft versus host disease (TA-GVHD) is a rare complication of blood transfusion. It carries a very high mortality rate. Although the phenomenon has been well described in immunocompromised patients, this review focuses on the immunocompetent host. Cases of TA-GVHD continue to be reported following a variety of surgical procedures, especially cardiac procedures requiring cardiopulmonary bypass. Additional risk factors for TA-GVHD include blood component transfusion in populations with limited genetic diversity, the use of directed donations from family members, and the transfusion of fresh blood. As there is no effective treatment, the focus is on prevention.

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.

Autoantibodies to red blood cell antigens occur frequently with hemolysis among pediatric small bowel transplant recipients: clinical implications and management.

Reports have linked pediatric solid organ transplant recipients with the development of hemolytic autoimmune antibodies, especially in the setting of the immunosuppressant tacrolimus. This study aims to identify whether these observations also occurred at an institution that frequently performs pediatric multivisceral transplants and to characterize the treatment and outcome. Chart review was performed on all patients with RBC autoantibodies. Laboratory and clinical data were used to identify hemolysis. For transplant recipients with RBC autoantibodies, the type of transplant and outcome of the AIHA were profiled. One hundred twenty‐eight patients were identified with RBC autoantibodies, of which 22 patients were solid organ transplant recipients, including 18 SB graft recipients. Sixteen of the 18 had evidence of hemolysis. The incidence rate of AIHA in this population is estimated to be 10%, resulting in significant cost. Treatment included immunosuppressant modulation, steroids, IVIG, and plasma exchange, with 12 of the 16 patients responding. RBC autoantibodies occur in up to 10% in pediatric SB transplant recipients, with high cost of obtaining compatible blood. Neither tacrolimus nor receipts of a donor spleen were associated with the development of AIHA. Treatment using steroids and IVIG appears to be effective.

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.

Predicted influence of sample hematocrit on injected mass of internal standard in mass spectrometry assays utilizing simple protein precipitation for sample preparation

Abstract Background: The injected mass of internal standard is often monitored in mass spectrometry assays to flag aberrant specimen processing. When simple protein precipitation is used for sample preparation, the protein volume (solids fraction) of the specimen is in principle a variable affecting the final concentration of the internal standard in the liquid phase. We examined the predicted extent of this variation for an example of protein precipitation for sample preparation used in a mass spectrometry assay for immunosuppressants in whole blood. Methods: Liquid and solid mass fractions of samples with hematocrit ranging from 15% to 60% were measured after protein precipitation of samples using a whole blood/precipitating reagent ratio of 1:4. Relative supernatant volumes as a function of hematocrit were determined. Results: Liquid volume variation was consistent with a predicted variation in the internal standard concentration of only approximately ±1% across this hematocrit range. Data were in close agreement with calculations based simply on protein density and concentration. Conclusions: Hematocrit affects the injected mass of internal standard in assays that utilize protein precipitation for sample preparation, due to predictable variation in solids and liquid fractions. The effect is small, however, compared to the typical variation observed for injected mass of internal standard. Clin Chem Lab Med 2007;45:215–9.