The dating of thrombus organization in cases of pulmonary embolism: an autopsy study

Abstract

Background: Pulmonary embolism (PE) is associated to high mortality rate worldwide. However, the diagnosis of PE often results inaccurate. Many cases of PE are incorrectly diagnosed or missed and they are often associated to sudden unexpected death (SUD). In forensic practice, it is important to establish the time of thrombus formation in order to determine the precise moment of death. The autopsy remains the gold standard method for the identification of death cause allowing the determination of discrepancies between clinical and autopsy diagnoses. The aim of our study was to verify the morphological and histological criteria of fatal cases of PE and evaluate the dating of thrombus formation considering 5 ranges of time.

Methods: Pulmonary vessels sections were collected from January 2010 to December 2017. Sections of thrombus sampling were stained with hematoxylin and eosin. The content of infiltrated cells, fibroblasts and collagen fibers were scored using a semi-quantitative three-point scale of range values.

Results: The 30 autopsies included 19 males (63.3%) and 11 females (36.7%) with an average age of 64.5 ± 12.3 years. The time intervals were as follows: early (≤1 h), recent (> 1 h to 24 h), recent-medium (> 24 h to 48 h), medium (> 48 h to 72 h) and old (> 72 h). In the first hour, we histologically observed the presence of platelet aggregation by immunofluorescence method for factor VIII and fibrinogen. The presence of lymphocytes has been identified from recent thrombus (> 1 h to 24 h) and the fibroblast cells were peripherally located in vascular tissue between 48 and 72 h, whereas they resulted central and copious after 72 h.

Conclusions: After a macroscopic observation and a good sampling traditional histology, it is important to identify the time of thrombus formation. We identified histologically a range of time in the physiopathology of the thrombus (early, recent, recent-medium, medium, old), allowing to determine the dating of thrombus formation and the exact time of death.

Clinical trial number: NCT03887819.

Trial registration: The trial registry is Cliniclatrials.gov, with the unique identifying number NCT03887819. The date of registration was 03/23/2019 and it was "Retrospectively registered".

Keywords: Forensic autopsy; Histology; Pulmonary embolism; Sudden unexpected death; Thrombus dating.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Thrombus formation in the first hour in SUD. a) H&E staining showing the thrombus overview of platelet aggregation below the vascular wall (5x magnification); b) H&E staining showing the thrombus overview of platelet aggregation below the vascular wall (10x magnification); c) Immunofluorescence indicating the accumulation of factor VIII in the vascular wall (white arrow); d-e) Immunofluorescence indicated fibrinogen amount in the vascular wall (white arrows)

Fig. 2

Immunohistochemistry for anti-CD3 antibody at different times. a) Negative immunohistochemistry for anti-CD3 in a case of SUD (10x magnification). The white arrow indicates the T lymphocytes at different time b) > 1 h to 24 h; c) > 24 h to 48 h; d) > 48 h to 72 h in different cases of PE death

Fig. 3

Fibrosis score atdifferent times and revascularization. a) In the first hour and b) up to 24 h’ absence of fibrosis with absence of red signal. In the following hours c) > 24 h to 48 h, d) > 48 h to 72 h, e) > 72 h there is an increase in the signal (PricoSirius Red/Fast Green stain; 10x magnification). f) The black arrow indicates revascularization in the old thrombus after 72 h (Immunohistochemistry for anti-CD31 antibody; × 10 magnification)