Year 1 evaluating fleasibility of cryopreservation and spiking donor blood
In Year 1, only the cryopreservation of D. immitis was evaluated. Two isolates of infected blood were obtained from the NIH/NIAID Filariasis Research Reagent Resource Center, JYD and MO strains, collected into heparized tubes and shipped overnight. One aliquot of fresh blood was removed and designated as a baseline and immediately evaluated using the Modified Knott’s test,  modified to use water to substitute for the 2% formalin. Photomicrographs were taken and the Modified Knott’s’ preparation was permanently mounted onto a slide. The remaining heparinized whole blood was mixed 1:1 with Glycerolyte 57 solution (Baxter, USA), divided into 1 ml aliquots and frozen at − 20 °C. One sample was then immediately thawed (Day 0) while the others remained frozen for later diagnostic test processing on days 7, 21, 70, and 124 post-freezing.
On days 7, 21, and 70 two samples of each isolate were removed from the freezer to perform a Modified Knott’s, again using water instead of the 2% formalin. Total volume of pellets were measured for recovery purposes. New methylene blue (Ricca Chemical Company, USA) was used to stain the microfilaria for all of the Modified Knott’s and carbonate filter tests performed in this study. The detected mf quantity and morphology were recorded for each time point and compared to baseline. Quantity data was collected by evaluating the total number of mf per slide and the average number of mf per 10x objective field. The average number of mf per field was found by taking 10 random views of the slide and averaging the number of mf seen. Morphology of ten individual mf was evaluated using a head scoring of 1–3, cuticle scoring of 1–3, and the measurements of the mf compared to reference values [5, 8]. The head scoring parameters were 3-clear and distinguished; 2-stained, but stained lighter than the body; 1-indistinguished, head and body same coloration. The cuticle scoring parameters were 3-crisp and defined edges of the body; 2-darkly stained; 1-unclear edge/no edge or damaged. Photomicrographs were taken at the different time points for comparison.
The day 124 post-freezing samples were used in the teaching laboratory to run a blind trial on 118 veterinary students. Instructors were also blinded. Pairs of students received a randomly assigned unknown microfilaria-status patient sample, coded by the investigator (SL), in a 3 ml ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) tube. The unknown patient samples consisted of one of the following: non-cryopreserved mf infected blood (nc-mf+), freshly acquired dog donor blood cells spiked with cryopreserved-infected blood (c-mf+), or non-infected normal dog donor blood cells (mf-). Acquisition of the normal dog donor blood cells came from a donor dog in the Ohio State Veterinary Medical Center Blood Bank program. For the dog donor blood, the plasma was removed and remaining blood cells were preserved using Adsol red cell preservative solution (Fenwal, Inc., USA) in the blood bank bag and kept at 4 °C.
The samples and required testing assays were coded to approximately distribute a variety of the samples and assays in each of the six laboratory sessions. The student pairs randomly drew their assigned patient and method, either a Modified Knott’s or carbonate filter assay with the standard 2% formalin fixation component. The student pairs reported if their sample contained detectable mf and if so, measured one or more mf on their slide. If their sample contained no mf they were required to view a positive slide from a different student pair and make written observations about the slides. Students submitted their written results worksheet for this exercise.
Year 2 evaluating the preformanace of semi-purified frozen microfilaria with either 2% acetic acid to 2% formalin
In Year 2, further refinement of the study parameters and a new set of 118 students were evaluated. Changes included purification of mf prior to the addition of cryoprotective medium and the use of 2% acetic acid as a fixative in the techniques. Again the JYD and MO strains of D. immitis were used. For mf purification, heparinized whole blood containing mf was centrifuged for 10 min at 400 G at 25 °C. The separated layers of plasma, buffy coat, and red blood cells (RBC) were removed and a 10 ul aliquot was examined to check for mf. As the RBC layer contained sufficient living mf, further processing of this layer occurred. The 1 part of the RBC-mf layer was mixed with 9 parts of water and gently inverted for 1 min to lyse the host cells. Physiological isomolarity was returned using 9% sodium chloride solution. The mixture underwent centrifugation for 10 min at 400 G to pellet the mf, the resulting pellet was resuspended in an equal volume of Glyerolyte 57, 200 ul aliquoted into individual cryovials, rapidly frozen on dry ice, then stored at − 20 °C until the day before class.
One day prior to the laboratory teaching session, the mf samples were rapidly thawed at 38 °C and added to pre-labeled 3 ml EDTA tubes containing 1 to 2 mls of healthy donor dog blood cells. Similar to Year 1, the 118 blinded veterinary students were randomly assigned a diagnostic technique (Modified Knott’s or carbonate filter), a sample (nc-mf+, c-mf+, or mf-), and fixative (2% acetic acid or 2% formalin) to analyze. Instructors and students were kept blinded from the sample identity and fixative. Fixative bottles were labeled A or B. Students were paired with another student, and each pair randomly drew a patient sample associated with an assigned technique and fixative. Students were required to report if mf were present, if so, measure at least one mf in their slide, observe another student pair’s prepared slide, and make comments about the slides. We did not specifically require students to identify the microfilaria to genus and species. Based on the drawn sample’s patient name, students’ with c-mf + samples using the 2% acetic acid fixative were sent to look at a c-mf + sample processed with 2% formalin fixative and vice versa, so all students compared the two different fixatives relative to their microfilaria sample.
In Year 2 the samples were not evaluated on days 0, 7, 21, and 70 because the feasibility of storing cryopreserved samples was determined in Year 1.
A blind trial was conducted with two experienced laboratory diagnosticians A and B. The Modified Knott’s procedure was used to prepare four slides which consisted of the following: c-mf + in 2% acetic acid fixative, nc-mf + in 2% acetic acid fixative, c-mf + in 2% formalin fixative, and nc-mf + in 2% formalin fixative. The experienced diagnosticians were asked to rank the quality of the four slides based on the morphology of and the ability to detect mf. The best quality was ranked as 1 and the poorest overall as 4. The diagnosticians also measured two mf per slide. Photomicrographs were taken using a Olympus BX41 equipped with a DP74 camera (Olympus America Inc., USA) connected to a Dell Optiplex 7050 (Dell Technologies, USA) with cellSens Standard 1.18 software package (Olympus America Inc) for image acquisition and measurement tools.
Assessment of clinical students during their hospital rotation
A blind trial was conducted on fourth year veterinary students (n = 11) during their clinical rotations. Two different student rotation groups were evaluated. This required the thawing and adding of cryopreserved microfilaria to healthy donor dog blood cells in EDTA tubes either 1 day or 14 days prior to the student assessments. During this 2 week period the spiked blood was kept at 4 °C. Each student was provided with 2% acetic acid fixative and two “unknown labelled blood samples” with one being c-mf + (spiked) and the other mf- (normal dog donor blood cells). The students were instructed to independently perform a Modified Knott’s on the blood samples. Students were required to report on a submission form their results if mf were present, and if so, measure and attempt to identify at least one mf on their slide.
Ethics approval and consent
The Ohio State University Office of Responsible Research Practices Institutional Review Board reviewed and exempted protocol 2019E0658 under 45 CFR 46 (USA). The research was conducted in a commonly accepted educational setting, involving normal educational practices and did not adversely impact students’ opportunity to learn the required educational content. The research compared reagents used for an instructional technique and was therefore exempted from human subject research under 45 CFR 46.