Enrichment of leukocytes in peripheral blood using 3D printed tubes

Abstract

Leukocytes have an essential role in patient clinical trajectories and progression. Traditional methods of leukocyte enrichment have many significant limitations for current applications. It is demonstrated a novel 3D printing leukocyte sorting accumulator that combines with centrifugation to ensure label-free initial leukocyte enrichment based on cell density and size. The internal structure of leukocyte sorting accumulator (revealed here in a new design, leukocyte sorting accumulator-3, upgraded from earlier models), optimizes localization of the buffy coat fraction and the length of the period allocated for a second centrifugation step to deliver a higher recovery of buffy coats than earlier models. Established methodological parameters were evaluated for reliability by calculating leukocyte recovery rates and erythrocyte depletion rates by both pushing and pulling methods of cell displacement. Results indicate that leukocyte sorting accumulator-3 achieves a mean leukocytes recovery fraction of 96.2 ± 2.38% by the pushing method of layer displacement. By the pulling method, the leukocyte sorting accumulator-3 yield a mean leukocytes recovery fraction of 94.4 ± 0.8%. New procedures for preliminary enrichment of leukocytes from peripheral blood that avoid cellular damage, as well as avert metabolic and phase cycle intervention, are required as the first step in many modern clinical and basic research assays.

Fig 1. Schematic diagram of the leukocyte sorting accumulators.

(A) Drawings of the LSAs models (from left to right: LSA-1, LSA-2 and LSA-3). (B) 3D modeling of the LSAs. (C) Photographs of the LSAs. LSA models consisting of the threaded-caps, the main body, and the threaded-booster.

Fig 2. Processing of leukocyte extraction by LSA-3 the pushing style method.

(A) 2–3 ml of whole blood was being prepared. (B, C) The sample is filling in the lower funnel chamber. (D) The buffy coat (red rectangle) was formed by centrifuging at 2500g for 10min. (E) with anticlockwise rotating the threaded-booster of the main body, the buffy coat layer (red rectangle) responds by moving up to the upper from the lower funnel.

Fig 3. Processing of leukocytes by LSA-3 designs pulling style method.

(A) 2–3 ml of whole blood was being prepared. (B) The LSA-3 is assembled. (C) the blood is introduced in the upper funnel chamber. (D) The buffy coat (red rectangle) is formed at 2500g for 10min centrifugation conditions. (E) with clockwise rotate the threaded-booster cause the buffy coat (red rectangle) moving down to a specific location within the upper funnel.

Fig 4. Schematic illustration of the working principles of the LSAs.

(A) Flow-process diagram for enrichment of leukocytes by the LSAs models. The position of buffy coat in the upper funnel is identified by the red rectangle. (B) 3D printed LSA-3 including the upper funnel, the central channel and the lower funnel. (C) Whole blood separated by the cell density in three fractions: Rich platelets, the buffy coat and rich erythrocytes.

Fig 5. Enrichment of leukocytes and depletion of erythrocytes by the LSAs.

(A) The mean leukocytes recovery rate of the LSAs and traditional centrifugal blood cell. (B) The mean erythrocyte depletion rate of the LSAs and traditional centrifugal blood cell.

Fig 6. Morphology analysis of leukocytes respective after cell lysis and the LSA-3 pushing style method.

(A) Wright-Giemsa staining of whole blood samples obtained using a 20x microscope objective. (B) The LSA-3 pushing style method of cell displacement. Leukocytes concentration has been significantly enhanced (20x objective). (C) Nucleated cells with clear cytoplasm after Wright-Giemsa staining are considered viable leukocytes (magnification, x40; representative leukocytes identified by red ellipse). (D) After cell lysis buffer treatment of whole blood cells, the leukocytes clear cytoplasm (indicating viability and homeostasis) was coated with rich erythrocyte debris (magnification, x40, identified by green ellipse).