Purpose: Red blood cell (RBC) disorders are ranging from different types of anemia over inherited membrane disorders to parasitic infections. Other diseases like neurological disorders also impair normal RBC function leading to loss of cell elasticity and impaired oxygen transport. Thus RBCs represent a very interesting cell population as biomarker carrier in several diseases. Therefore, tools to evaluate the morphology of RBCs as well as expression and staining patterns of biomarkers on RBCs are highly anticipated. Our newly designed RBC chips for Chipcytometry only need a few drops of blood to analyze these arameters on a high amount of RBCs in parallel. In addition, storage of RBCs is possible for later analysis of interesting markers.
Methods: ZellSafe microfluidic chips were modified to contain a cell-adhesive surface suitable for RBC immobilization and storage. Heparinized blood was diluted and RBCs loaded on the chip. After adhesion to the chips, RBCs were stored in erythrocyte fixation buffer up to 15 weeks. Surface biomarkers and RBC shape were re-analyzed at different time points. Several biomarkers can be analyzed on the same cell due to Chipcytometry characteristics based on an iterative process of staining, imaging and bleaching.
Results: Biconcavity - a hallmark of RBCs- was well retained after immobilization on the chip. Expression of several RBC surface markers including CD35, CD44, CD47, CD71, CD235a and CD238 was analyzed on fresh RBCs and after storage at different time points. Surface expression of markers was retained after storage (e.g., for CD235a storage is possible for even 15 weeks). As some of these markers are altered in diseased persons, our method provides an excellent tool to analyze changes in surface expression and even after storage of cells. In addition, by mixing RBCs with white blood cells markers on both cell types can be analyzed at the same time. Differentiation of populations can be easily performed using lineage markers like CD45, CD3, CD14 or CD19. Therefore also leukemias which can alter both, morphology of RBCs and surface makers on PBMCs, can be examined.
Conclusion: With analyzing and storage of RBCs on a chip, we offer an outstanding new tool for red blood cell research. RBC disorders are marked by changes in expression of different markers. RBC Chipcytometry provides a new technique to directly compare RBC morphology and biomarker expression. As image generation and analysis of the results can be performed automatically on a high number of cells there is nearly no limit of throughput to reach statistical significance in a study. We are currently expanding the panel of RBC markers including blood cell antigens for blood typing.
Anke Hofmeister-Brix– Zellkraftwerk Diagnostics