Acquired immune hemolytic anemia associated with IgA erythrocyte coating: investigation of hemolytic mechanisms

We have investigated the hemolytic mechanisms in a patient with acquired immune hemolytic anemia whose red cells appeared to be coated with IgA alone. The clinical course was similar to that of patients with hemolytic anemia mediated by warm-reacting IgG antibody. Splenic sequestration of red cells was demonstrated, and marked reduction of hemolysis occurred after corticosteroid therapy. Antibody was eluted from the patient's red cells and used to sensitize normal red cells in vitro. These sensitized red cells were not lysed by fresh autologous serum, nor did they fix detectable amounts of C3. However, red cells sensitized by eluted antibody were lysed by normal human peripheral blood monocytes in a system designed to demonstrate antibody-dependent cell-mediated cytotoxicity. Monocyte-mediated hemolysis of sensitized red cells was inhibited by the addition of low concentrations of normal serum IgA to the system, but not by IgG. The ability of the eluate to induce monocyte-mediated hemolysis was abolished by its adsorption on Sepharose-bound anti-IgA, but not by preincubation with Sepharose-bound anti-IgG. In addition, normal human monocytes were demonstrated to ingest eluate-sensitized red cells. These data demonstrate an in vitro interaction of IgA-sensitized red cells with leukocytes and suggest a possible mechanism for the patient's hemolysis.     

Evaluating HIV prevention effectiveness: the perfect as the enemy of the good

There is a need to better understand the effectiveness of HIV-prevention programs. Cluster randomized designs have major limitations to evaluate such complex large-scale combination programs. To close the prevention evaluation gap, alternative evaluation designs are needed, but also better articulation of the program impact pathways and proper documentation of program implementation. Building a plausible case using mixed methods and modeling can provide a valid alternative to probability evidence. HIV prevention policies should not be limited to evidences from randomized designs only.     

Manual planimetric measurement of carotid plaque volume using three-dimensional ultrasound imaging

We investigated the utility of three manual planimetric methods to quantify carotid plaque volume. A single observer measured 15 individual plaques from 15 three-dimensional (3D) ultrasound (3D US) images of patients ten times each using three different planimetric approaches. Individual plaque volumes were measured (range: 32.6-597.1 mm3) using a standard planimetric approach (M1) whereby a plaque end was identified and sequential contours were measured. The same plaques were measured using a second approach (M2), whereby plaque ends were first identified and the 3D US image of the plaque was then subdivided into equal intervals. A third method (M3) was used to measure total plaque burden (range: 165.1-1080.0 mm3) in a region (+/- 1.5 cm) relative to the carotid bifurcation. M1 systematically underestimated individual plaque volume compared to M2 (V2 = V1 + 14.0 mm3, r = 0.99, p = 0.006) due to a difference in the mean plaque length measured. Coefficients of variance (CV) for M1 and M2 decrease with increasing plaque volume, with M2 results less than M1. Root mean square difference between experimental and theoretical CV for M2 was 3.2%. The standard deviation in the identification of the transverse location of the carotid bifurcation was 0.56 mm. CVs for plaque burden measured using M3 ranged from 1.2% to 7.6% and were less than CVs determined for individual plaque volumes of the same volume. The utility of M3 was demonstrated by measuring carotid plaque burden and volume change over a period of 3 months in three patients. In conclusion, M2 was determined to be a more superior measurement technique than M1 to measure individual plaque volume. Furthermore, we demonstrated the utility of M3 to quantify regional plaque burden and to quantify change in plaque volume.     

Atypical Femoral Fractures: Transverse Morphology at Lateral Cortex Is a Critical Feature

In 2010, the American Society for Bone and Mineral Research (ASBMR) task force defined major and minor features to assist in the case finding and reporting of atypical femoral fractures (AFFs). One major feature that was proposed was a “transverse or short oblique configuration.” Our primary aim was to compare the conventional overall fracture morphology (OFM) with its associated angle (OFMA) and our proposed lateral cortical fracture angle (LCFA) in the assessment of fracture configuration in suspected AFFs and non‐AFFs. The radiographs of 79 patients with AFFs and 39 patients with non‐AFFs were each analyzed by two blinded reviewers to obtain the OFM, OFMA, and LCFA. Using the overall fracture morphology to assess the suspected AFFs resulted in discordance between reviewers in 18 cases (22.8%), of which 5 (6.3%) were discordant between short oblique (>30° to 60°) and long oblique (>60° to 90°) configurations, therefore affecting their classifications as AFFs. By assessing only the critical component within the lateral cortex, all the suspected AFFs fell well within the classification as transverse fractures with a mean LCFA of 4.8° (range 0.3 to 18.0, SD = 4.23). The inter‐reader variability was also lower for LCFA versus OFMA (4.1° versus 6.9°, p = 0.001) when used to assess AFFs. Fracture angles were significantly different in AFFs versus non‐AFFs regardless of whether the OFMA or LCFA methodology was employed, but the greater difference associated with LCFA suggests its greater discriminating power. When LCFA was used in conjunction with 0° to 30° as the criteria for transverse morphology, all the AFFs and non‐AFFs were correctly classified. By using a standardized and precise method in measuring the fracture angle, specifically using only the component of the lateral cortex and limiting to truly transverse fractures, ie, between 0° and 30°, the LCFA is a robust and accurate method to assess the fracture morphology in suspected AFFs. © 2014 American Society for Bone and Mineral Research.     

Stem cell factor retards differentiation of normal human erythroid progenitor cells while stimulating proliferation

Stem cell factor (SCF), the ligand for the c-kit tyrosine kinase receptor, markedly stimulates the accumulation of erythroid progenitor cells in vitro. We now report that SCF delays erythroid differentiation among the progeny of individual erythroid progenitors while greatly increasing the proliferation of these progeny. These effects appear to be independent of an effect on maintenance of cell viability. Highly purified day-6 erythroid colony-forming cells (ECFC), consisting mainly of colony-forming units-erythroid (CFU-E), were generated from human peripheral blood burst-forming units-erythroid (BFU-E). Addition of SCF to the ECFC in serum-free liquid culture, together with erythropoietin (EP) and insulin-like growth factor 1 (IGF-1), resulted in a marked increase in DNA synthesis, associated with a delayed peak in cellular benzidine positivity and a delayed incorporation of 59Fe into hemoglobin compared with cultures without SCF. In the presence of SCF, the number of ECFC was greatly expanded during this culture period, and total production of benzidine-positive cells plus hemoglobin synthesis were ultimately increased. To determine the effect of SCF on individual ECFC, single-cell cultures were performed in both semisolid and liquid media. These cultures demonstrated that SCF, in the presence of EP and IGF-1, acted on single cells and their descendants to delay erythroid differentiation while substantially stimulating cellular proliferation, without an enhancement of viability of the initial cells. This was also evident when the effect of SCF was determined using clones of ECFC derived from single BFU-E. Our experiments demonstrate that SCF acts on individual day-6 ECFC to retard erythroid differentiation while simultaneously providing enhanced proliferation by a process apparently independent of an effect on cell viability or programmed cell death.     

Inaccuracies associated with the automated measurement of mean cell hemoglobin concentration in dehydrated cells

Because of discrepancies between electronically and manually measured values of mean cell hemoglobin concentration (MCHC) encountered in studies of pathologic red cells, we studied the effect of cell water content on MCHC measurements by both methods. A series of red cell samples with varying water contents (54%-164% normal) were prepared from normal cells using the antibiotic nystatin. MCHC was then measured, using the microhematocrit centrifuge and three different electronic cell counters in common laboratory use. For MCHC values above 36 g/dl as measured by the spun hematocrit method, all three electronic counters under estimmated the MCHC, with increasing error as the true MCHC increased. For MCHC values below 30 g/dl, the values from two conductivity based instruments agreed with those from the spun hematocrit method, whereas one instrument based on light scattering overestimated the MCHC. These results indicate that inaccuracies in the measured mean cell volume (MCV) of dehydrated or otherwise undeformable cells may lead to spurious values for MCHC when electronic cell counters are used.