Histological Analysis of in vitro Thrombus Formation in a Backward Facing Step Flow Domain Using Carstairs' Stain

Open Access
Kinney, Joanna E
Area of Honors:
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Dr. Keefe B Manning, Thesis Supervisor
  • William O Hancock, Honors Advisor
  • Peter J Butler, Faculty Reader
  • thrombosis
  • histology
  • bioengineering
  • heart
  • blood
Prosthetic cardiac devices can alter a patient’s natural hemodynamics by introducing stagnant areas and recirculation regions within blood vessels. These flow conditions are known to induce thrombosis, which is consequently one of the leading obstacles in the development of such implantable devices. The present in vitro study uses a backward-facing step (BFS) model to mimic the sudden expansion often associated with prosthetic cardiac devices. The BFS is integrated into a closed flow loop that circulates bovine blood at 0.76 L/min (Re = 490). Bovine blood is taken from a donor and used in the BFS loop to form clots in two groups: (1) reconstituted blood with 45% hematocrit and 2.14 x 108 platelets/mL and (2) whole blood. Thrombi are formed by circulating the bovine blood through the loop for 15 minutes, 30 minutes, 45 minutes, 60 minutes, 6 hours, and 12 hours, where n=2 for each blood group and time. These thrombi are then fixed in 4% paraformaldehyde, sectioned in histology, and stained using Carstairs’ staining procedure to observe platelets, red blood cells, and fibrin within each thrombus. Quantification of thrombus constituency is performed using ImageJ to examine thrombus compositional trends with respect to distance from the backward-facing step, spatial height with the thrombus, formation time, and blood properties. The results of the present study showed that platelets and RBCs are more populous on the model-side of the thrombus while fibrin is more concentrated on the flow-side of the thrombus. Analysis demonstrates that fibrin concentration may increase with time until fibrin becomes the main component within each thrombus. Evidence is also provided that whole blood forms longer, more stable thrombi than reconstituted blood (Wilcoxon Paired T-Test, P < 0.05); this finding is likely due to leukocytes and other buffy coat components exclusively present within the whole blood. No clear trends are observed over distance from the step; however, future studies should increase sample size to further investigate potential correlations.