Effective and cheap removal of leukocytes and platelets from Plasmodium vivax infected blood

A 10 ml syringe (with the plunger removed) was tipped with two 1 cm² pieces of Grade 105 lens cleaning tissue (Whatman^®). The tissue was placed in the syringe so as to cover the outlet lumen. Only syringes with a centred rather than an offset outlet should be used. Ten ml of loosely packed of CF11 cellulose powder (Whatman^®) were added to the syringe and then packed down to ~5.5 ml of packed cellulose. The tip and bottom of the syringe were covered with aluminium foil, and then autoclaved. When ready to use, the CF11 column was wetted with ~5 ml of isotonic phosphate buffered saline (PBS) solution (without Ca²⁺ and Mg²⁺, pH 7.3).

Due to the limited quantity of P. vivax IRBC healthy donor blood was used for the initial side-by-side comparisons of leukocytes and platelet removal methods. Ten ml of whole blood were collected onto Lithium heparin from five healthy donors by venepuncture. The whole blood was centrifuged at 500 g for 5 min at room temperature. The plasma supernatant was removed, but the buffy coat fraction containing white blood cells (WBC) was added back to the red blood cell (RBC) pellet. The RBC+WBC mix from each donor was divided into five two ml portions to which an equal volume of RPMI was added with gentle mixing. The first tube was used as the control for this study. The 2^nd tube was centrifuged again as above and the PBS supernatant and buffy coat carefully removed from the packed RBC, this tube was the 'Buffy Coat Removal' treatment. The 3^rd tube was loaded into a five ml syringe (the plunger removed) and mounted onto a Plasmodipur™ filter (Euro-Diagnostica^®) that was pre-rinsed with a sterile PBS solution. Then gentle pressure was applied to the syringe attached to the Plamodipur unit and the filtered RBC/PBS (50% haematocrit) mix was collected as the 'Plasmodipur' treatment. The 4^th and 5^th tubes were added to PBS-wetted CF11 columns. The filtered 4^th sample was then collected as the 'CF11' treatment. The filtered 5^th sample was then added to another unused CF11 column (pre-wetted with 5 ml of PBS). The double filtered 5^th sample was the 'CF11x2' treatment. In addition to the above samples, three more healthy volunteers were recruited, and 8 ml of whole blood were collected onto Lithium heparin. This blood was processed as above and divided into four 2 ml samples. The first and 2^nd tubes were processed as described above for the control and the 'CF11' treatments, respectively. The contents of the 3^rd tube were layered over 2 ml of Lymphoprep™ (Greiner Bio-One^®) and centrifuged at 600 g for 20 min at 20°C, the leukocytes that banded at the interface were removed using a pipette, and the supernatant RBC fraction was the 'Lymphoprep' treatment. The 4^th tube was processed as above for the 'Lymphoprep' treatment, but then subsequently processed using the 'Plasmodipur' treatment protocol described above. This final sample was called the 'Lymphoprep and Plasmodipur' treatment.

Please note; unless specifically stated, the term 'CF11 filtration' involved only a single CF11 filter, as opposed to 'CF11x2' which involved processing the sample using two CF11 columns.

Complete blood counts of the control samples and the suspensions obtained from the six treatments were conducted using an Automated Hematology Analyzer (Model pocH-100i, Sysmex Company) and by microscopic examination (x100 oil immersion) of Giemsa-stained thick (250 fields) and thin smears (450 fields). The data from the thin smears were used for differential lymphocyte counts.

Thick and thin smears were routinely made for the pre- and post-CF11 filtration from each of the P. vivax isolates collected, prior to ex vivo drug sensitivity testing at the Shoklo Malaria Research Unit (SMRU), Mae Sod, Thailand. The pre- and post-CF11 filtration, thick and thin smears from 30 randomly selected isolates collected during 2008 were examined as follows. Parasitaemias were determined from the number of IRBC per ten 100× oil immersion fields (200 RBC per field) on the thin film. The percentage of early (ring-like parasites with a single chromatin dot) and mature (amoeboid-like cytoplasm or presence of haemozoin) asexual stages was determined from examining 200 parasites in the thick smears under 100× oil immersion. Due to their very low numbers in the pre- and post-filtration smears schizonts (parasites with 3 or more chromatin dots and haemozoin) were combined with the mature stage count. Gametocyte counts were too low for statistical comparison.

Parametric analysis of non-paired data was calculated using one-way analysis of variance. Non-parametric analysis of the paired data was performed using Wilcoxon or Friedman's tests and post-hoc analysis using Dunn's test (GraphPad Prism 5.01).

The clinical IRBC samples examined in this study were collected under the following ethical guidelines in the approved protocol OXTREC 027-05 (University of Oxford, Centre for Clinical Vaccinology and Tropical Medicine, UK)

Plasmodipur filters were the most rapid method for the filtration of donor blood with ~10 min per isolate as compared to the ~20 min per isolate required for CF11 filtration. A Plasmodipur filtering unit costs ~50–55 USD while a single CF11 column could be made at a total cost of ~1 USD. The effectiveness of the two types of columns to remove leukocytes and platelets from clinical blood samples was then assessed (Figure 1A and 1B). Filtration on CF11 and Plasmodipur reduced the mean initial leukocyte counts from 9.4 × 10³ per μl [95%CI 5.2–13.5] to 0.01 × 10³ [95%CI 0.01–0.03] and 0.7 × 10³ per μl [95%CI 0.4–1.8] respectively (Figure 1A). Filtration on CF11 was particularly effective at removing platelets, reducing the platelet count from 211.6 × 10³ per μl [95%CI 107.5–315.7] to 0.8 × 10³ per μl [95%CI -0.7–2.2], a significantly greater platelet reduction when compared to the mean platelet count of the Plasmodipur filtrate which was 14.0 × 10³ per μl [95%CI -2.3–30.5]) (p = 0.02). Only by pre-processing the blood sample with Lymphoprep prior to Plasmodipur filtration was it possible to reduce the mean leukocyte (0.3 × 10³ per μl [95%CI -1.1–1.8]) and platelet (0.0 × 10³ per μl) contamination to the same levels as that obtained by CF11 filtration. However, pre-treatment with Lymphoprep adds another 50 mins and a further ~8–9 USD to procedure, thus negating the benefits of the relatively speedier Plasmodipur filtration. It was interesting to note that the composition of the contaminating leukocytes in the suspension obtained after Lymphoprep/Plasmodipur or CF11 filtration differed greatly. Leukocyte contamination in the Lymphoprep/Plamodipur filtrate was predominantly neutrophils (98%) with the remainder made up of lymphocytes and basophils/monocytes (Figure 2). By contrast, no neutrophils were found in the CF11 filtrate where the small number of contaminating leukocytes was either lymphocytes (59%) or basophils/eosinophils (41%). The specific removal of neutrophils by CF11 is of particular advantage in ex vivo culture, where neutrophils readily phagocytise maturing IRBCs [6].

Filtration of P. vivax infected isolates resulted in median parasitaemia change of -0.1% [IQR -0.3–0.03] (Figure 3A). This corresponds to a 7.14% reduction in IRBCs from the initial median parasitaemia of 0.7% [IQR 0.38–1.12] to a post filtration median parasitaemia of 0.65% [IQR 0.30–1.0] (p = 0.01). The magnitude of IRBC loss was much less than the major losses previously reported for CF11 filtration, namely from a starting parasitaemia of 0.37% to 0.18% in the filtrate for one sample and 0.43% to 0.01% for the other, representing a 52% and 98% reduction in parasitaemia respectively [11]. It was further reported that CF11 selectively retained mature trophozoites and schizonts, with only ring stages passing through the CF11 unimpeded[11]. In this study we failed to observe any selective retention of mature P. vivax stages, the median pre- and post-filtration composition of late stages in the P. vivax samples was 17% and 19% respectively. The median change in the composition of late stages pre- and post-filtration was an increase of 1.0% [IQR -4.2 to 4.2] (p = 0.06) (Figure 3B). This is consistent with our experience with routine use of CF11 filtration of P. vivax isolate where the loss of mature forms were not noted[12, 13, 15], and where CF11 filtration did not effect P. vivax maturation of early or late stage trophozoites.

It should be noted that equally satisfactory removal of leukocytes and platelets, with little loss of parasites, were obtained when the PBS solution was used to wash and resuspend the blood and the CF11 columns was replaced by RPMI or McCoy's 5A medium (GIBCO^®)