A method for reducing the sloughing of thick blood films for malaria diagnosis

Abstract

BackgroundThe gold standard for malaria diagnosis is the examination of thick and thin blood films. Thick films contain 10 to 20 times more blood than thin films, correspondingly providing increased sensitivity for malaria screening. A potential complication of thick film preparations is sloughing of the blood droplet from the slide during staining or rinsing, resulting in the loss of sample. In this work, two methods for improving thick film slide adherence (‘scratch’ (SCM) and ‘acetone dip’ (ADM) methods) were compared to the ‘standard method’ (SM) of thick film preparation.MethodsStandardized blood droplets from 26 previously examined EDTA whole blood specimens (22 positive and four negative) were concurrently spread on glass slides using the SM, ADM, and SCM. For the SM and ADM prepared slides, the droplet was gently spread to an approximate 22 millimeters in diameter spot on the slide using the edge of a second glass slide. For the SCM, the droplet was spread by carefully grinding (or scratching) it into the slide with the point of a second glass slide. Slides were dried for one hour in a laminar flow hood. For the ADM, slides were dipped once in an acetone filled Coplin jar and allowed to air dry. All slides were then Giemsa-stained and examined in a blinded manner. Adherence was assessed by blinded reviewers.ResultsNo significant or severe defects were observed for slides prepared with the SCM. In contrast, 8 slides prepared by the ADM and 3 prepared using the SM displayed significant or severe defects. Thick films prepared by the three methods were microscopically indistinguishable and concordant results (positive or negative) were obtained for the three methods. Estimated parasitaemia of the blood samples ranged from 25 to 429,169 parasites/μL of blood.ConclusionsThe SCM is an inexpensive, rapid, and simple method that improves the adherence of thick blood films to standard glass slides without altering general slide preparation, microscopic appearance or interpretability. Using the SCM, thick films can be reliably examined less than two hours after sample receipt. This represents a significant diagnostic improvement over protocols requiring extended drying periods.

Evaluación del desempeño mediante paneles de láminas, una herramienta para la clasificación de los microscopistas senior del Programa de Control de Malaria en Colombia

“…Para la elaboración de los paneles es necesario contar con el material suficiente en las salidas de campo, el cual se debe calcular estimando un porcentaje de pérdida de láminas por errores. Estos errores pueden presentarse en cualquiera de las etapas, por ejemplo, al extender las muestras o en la coloración y el montaje permanente de la muestra; también, pueden ocurrir accidentes mecánicos y presentarse problemas inherentes a la técnica, como el desprendimiento de la gota gruesa debido a la humedad, la elevada temperatura ambiental, la limpieza inadecuada del portaobjetos o por el fácil desprendimiento de la sangre cuando procede de un paciente anémico (17). La calidad y la estandarización de los paneles de láminas son factores relevantes que permiten controlar variables y obtener paneles más homogéneos (13,14).…”

<i>Schistosoma mansoni</i> Infection: A Major Contributor of Reduced Effective T Helper Responses against <i>Plasmodium falciparum</i> and <i>Schistosoma mansoni</i> Co-Infection in <i>ex vivo</i>: A Cross-Sectional Study to Assess Th1, Th2 & Th17 Immune Responses

“…Slides were examined under a compound microscope with ×100 oil immersion objective lens. P. falciparum was identified by seeing the infected red blood cell (RBC) with the Plasmodium not changing shape, multiple infection of a RBC with Plasmodium and presence of double nuclear (chromatin dot) on Plasmodium as described previously [47] These enhanced the activation of protein kinase C to induce T helper cells to produce cytokines. Hence cytokines produced by T helper cells were prevented from leaving the golgi apparatus resulting in a build up [6].…”

Update on Malaria Diagnostics and Test Utilization

“…All rights reserved.) making thick films that allows for improved adherence and faster turnaround times (13). The process is similar to that of making a normal thick film, but the edge of a glass microscope slide is used for spreading the blood, while simultaneously firm pressure is applied to create small scratches in the underlying slide.…”

“…The scratches allow for improved adherence of the blood film to the slide without affecting the smear morphology. The smear can then be stained as soon as it is dry, generally within 20 to 30 min of smear preparation (13).…”