High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA

<p>Abstract</p> <p>Background</p> <p>Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome.</p> <p>Methods</p> <p>High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene.</p> <p>Results</p> <p>High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of <it>P. falciparum </it>parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection.</p> <p>Conclusions</p> <p>High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots.</p

Delayed Appearance of High Altitude Retinal Hemorrhages

When closely examined, a very large amount of climbers exhibit retinal hemorrhages during exposure to high altitudes. The incidence of retinal hemorrhages may be greater than previously appreciated as a definite time lag was observed between highest altitude reached and development of retinal bleeding. Retinal hemorrhages should not be considered warning signs of impending severe altitude illness due to their delayed appearance

Donor-recipient killer immunoglobulin like receptor (KIR) genotype matching has a protective effect on chronic graft versus host disease and relapse incidence following HLA-identical sibling hematopoietic stem cell transplantation

Impact of donor-recipient killer immunoglobulin-like receptor (KIR) gene-gene matching on transplant outcomes is still inconclusive. Recent data suggest that killer cell immunoglobulin-like receptor (KIR) regulated natural killer cell (NK cell) activity may contribute to graft versus leukemia (GvL) effects and graft versus host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This case-control study aims to evaluate the effects of both aKIR and iKIR donor-recipient genotype matching on the outcomes of T cell replete HLA-identical sibling allo-HSCTs in a homogenous young patient population with myeloid leukemias. Five transplant outcomes including relapse rate (RR), disease-free survival (DFS), overall survival (OS), cumulative incidences of acute GvHD (aGvHD), and chronic GvHD (cGvHD) are investigated. Out of 96 HLA-identical sibling donor-recipient pairs, 34 were matched for activating KIR (aKIR), 38 for inhibitory KIR (iKIR), and 20 for both aKIR and iKIR. Fourty-four pairs were mismatched for both iKIR and aKIR. In univariate analysis, aKIR-matching resulted with a decrease in relapse rate (RR) (hazard ratio [HR]: 0.4; p = 0.04) and an increase in disease-free survival (DFS) (HR: 0.5; p = 0.03). In addition, cGvHD ocurred less frequently in the aKIR-matched (odds ratio [OR]: 0.4; p = 0.04) or iKIR-matched (OR: 0.3; p = 0.009) cohorts. Matching for both aKIR and iKIR was also associated with a decrease in cGvHD incidence (OR: 0.3; p = 0.02). iKIR-matching had no effects on RR, OS, or DFS. Analysis of donor haplotype effects showed haplotype-BB to have a tendency towards reduced relapse rate (HR: 0.4; p = 0.08) and better OS (HR: 0.4; p = 0.04); haplotype-Bx to increase the incidence of cGvHD (OR: 4.1; p = 0.03). In multivariate analysis, DFS advantage remained significant for aKIR-matching (HR: 0.5; p = 0.04); cGvHD incidence was reduced in the presence of iKIR-match (OR: 0.3; p = 0.02) and increased in the presence of haplotype-AB and -BB donors (OR: 7.9; p = 0.02; OR: 5.1; p = 0.03, respectively). In an attempt to investigate the pathogenesis underlying KIR-matching, we searched for residual NK/T cells on day 0 peripheral blood samples of six additional recipients and noted the presence of CD3(+) (7.0-91.4 x 10(6)/L) and CD56(+)57(+) (0.8-12.7 x 10(6)/L) cells. In conclusion, conditioning regimen surviving recipient NK/T cells potentially influenced by KIR-matching may contribute to GvL/GvH reactions