Storage of blood clots for histological analysis: How long is too long in saline and paraformaldehyde?

To investigate the composition of blood clots following mechanical thrombectomy, it is essential to ensure optimum storage for highest quality histological and immunofluorescence analysis. We investigated for how long clots can be stored in paraformaldehyde (PFA), saline and heparinised saline before the tissue integrity is compromised. Whole blood and fibrin-rich clot analogues were made under dynamic flow conditions. Clots were stored in 4% PFA, saline or heparinised saline for timepoints ranging from 1 hour to two months. Five µm sections were stained with Martius Scarlet Blue to visualise red blood cells (RBCs), white blood cells (WBCs) and fibrin. Semi-quantitative analysis of the integrity of clot components used a scoring system (0: Poor; 1: Sub-par; 2: High). Quantitative analysis used Orbit Image Analysis software. Autofluorescence was assessed using a relative scale. Clots stored in PFA for up to two months were qualitatively similar to those stored for all shorter periods (median score: 2 per component). Clots stored in saline/heparinised saline for one week showed degradation of RBCs and WBCs, but fibrin remained intact (median score: 1, 1, 2 respectively). Degradation of the samples stored in saline/heparinised saline made accurate quantification using Image Analysis software difficult from 24h. Samples stored in PFA for up to two weeks showed an edging autofluorescence effect, which became more evident with prolonged storage. For optimum histology, ideally clots should not be stored in saline before fixation and should ideally be stored in formalin for less than one month to minimise the impact of autofluorescence on immunofluorescence.