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.

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.

Predicted effects of hemoglobin A1c assay precision on a patient population distribution of serial hemoglobin A1c difference values.

BACKGROUND: Interpretation of serial measurements of % hemoglobin A1c includes an assessment of differences from preceding values (DHbA1c). We examined predicted effects of different assay precisions on an observed population distribution for DHbA1c. METHODS: Primary data were 5260 DHbA1c values from sequential HbA1c measurement pairs obtained within 1 calendar year. Each DHbA1c was replaced by a distribution obtained from sampling each component HbA1c value according to a normal distribution characterized by a fixed coefficient of variation (CV) of either 1%, 3% or 5% (forming data sets A, B and C, respectively). Data sets B and C, with inferior precision, were compared with the reference data set A (highest precision). RESULTS: Using DHbA1c bin widths of 0.5% HbA1c, differences in assay precision caused significant redistribution of numbers within bins. For instance, for CV=5%, there was a 7.2% decrease in the number of results within the DHbA1c bin=(-0.5 to0.5. CONCLUSION: Different HbA1c assay CVs can significantly affect the fraction of patients within different clinical categorizations for DHbA1c and consequently may differently influence patient care recommendations.

Validation of use of annular once-punched filter paper bloodspot samples for repeat lead testing.

BACKGROUND: Lack of a second sample often precludes the ability to perform repeat lead measurements on filter paper bloodspot samples. We investigated whether annular specimens remaining from once-punched filter paper bloodspot specimens could provide accurate Pb measurements when measurements were scaled for the remainder area relative to the original punch area. METHODS: 50 microl bloodspot specimens were prepared using Pb-spiked EDTA whole blood. After removal of 6 mm punches, bitmap images of the remainder specimens were obtained using a scanner. Image analysis was used to determine the bloodspot area of the remainder sample relative to the area of the original punch. Measurement of Pb for punches and for remainder specimens was performed by ICP-MS. RESULTS: Area-corrected Pb measurements for remainder samples were significantly higher than for the punches, by an average factor of 1.52+/-0.12 (p<0.05, n=28). The difference was due to a discontinuity (an increase) in the per-area Pb at the bloodspot perimeter. Area-corrected results for annular specimens that excluded the perimeter were identical to those of the punch. CONCLUSION: Area-corrected Pb measurement using annular once-punched bloodspot remainder specimens can accurately reproduce lead measurements obtained from the original punch when the bloodspot perimeter area is excluded.

Vincristine-laden platelet transfusion for patients with refractory thrombocytopenia.

BACKGROUND: The aim of the present study was to assess the efficacy of vincristine-laden platelet transfusion for patients with refractory thrombocytopenia. PATIENTS AND METHODS: Twenty evaluable patients who received vincristine-laden platelets for refractory thrombocytopenia were included in this retrospective study. Vincristine (1 mg) was added to the platelets and incubated for one hour prior to transfusion. Serial platelet counts following vincristine-laden platelet transfusion and units of platelets transfused in the week prior to and the week after transfusion of vincristine-laden platelets were evaluated. RESULTS: The underlying diseases of the patients were lung cancer (n =4), breast cancer following autologous hematopoietic stem cell transplantation and acute myeloid leukemia (n=3 each), myelodysplastic syndrome (n=2), acute lymphoid leukemia, chronic lymphoid leukemia, chronic myeloid leukemia, multiple myeloma, ovarian cancer, aspergillosis, cytomegalovirus infection and systemic lupus erythematosus (n = 1 each). The median rate of change of platelet count after transfusion of vincristine-laden platelets was 550/microL/day (range, -1,000 to 12,8001/microL/day; p=0.003). The median change in the number of units of platelets transfused in the week following vincristine-laden platelet transfusion was -1.5 as compared to the week prior to the transfusion (p=0.031). Patients with a primary marrow disorder exhibited no difference in either the rate of change in platelet count or in the difference in the units of platelets transfused compared to those without a primary bone marrow disorder. CONCLUSION: Vincristine-laden platelet transfusion was associated with significantly increased platelet counts and a subsequent decrease in platelet transfusion.

Effects of sterilizing gamma irradiation on bloodspot newborn screening tests and whole blood cyclosporine and tacrolimus measurements.

Sterilizing irradiation of the US mail has been proposed as a method to prevent delivery of viable anthrax spores. Because newborn screening samples (bloodspots) and cyclosporine and tacrolimus specimens (whole blood) are delivered routinely through the mail, we studied whether sterilizing gamma irradiation could affect these test results. Specimens were exposed to 18 kGy gamma irradiation (100 hours x 18,000 rad/h), a "kill dose" for Bacillus pumilus spore strips. Irradiation had no significant effect on whole blood cyclosporine or tacrolimus results, but it had a degradative effect on bloodspot phenylalanine, hemoglobins, biotinidase, galactose-1-phosphate uridyltransferase, thyroxine, and thyrotropin. Such irradiation potentially could cause false-negative results for the detection of phenylketonuria and likely would lead to an increase in secondary testing for hemoglobin variants, but it is unlikely to lead to false-negative or false-positive results for the remaining newborn screening tests. These experiments cannot rule out possible greater effects by larger doses or other types of irradiation.

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.

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.

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.

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

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.