Characterization of the ‘White’ Appearing Clots that Cause Acute Ischemic Stroke

Background: Most clots retrieved from patients with acute ischemic stroke are ‘red’ in color and are predominantly composed of red blood cells and brin. ‘White’ clots represent a less common entity and their histological composition is largely unknown. The aim of this study was to investigate the composition, imaging and procedural characteristics of ‘white’ clots retrieved by mechanical thrombectomy. Nineteen ‘white’ thrombi selected by visual inspection from 293 were as of the multi-institutional Non-contrast computed tomography (NCCT), histological and immunohistochemical analyses were performed. Components were quantied using Orbit Image Analysis. clots presented signicantly platelets/other and red blood compared to the clots which showed a mean of 23% and 44%, respectively. The mean platelet (CD42b) content in ‘white’ clots was 43%; von Willebrand Factor (vWF) mean expression was 38%.


Introduction
Several studies have highlighted the variations in the histopathological features of acute ischemic stroke clots (AIS) related to etiology and the impact of thrombus composition on the effectiveness of thrombolysis and mechanical thrombectomy (MT). [1][2][3][4][5][6][7] However, only a few papers have analyzed the homogeneous/heterogeneous aspect of color ('red', 'white', mixed), size and shape of the retrieved clots; most AIS clots are mainly 'red' and histologically show a red blood cells and brin-dominant pattern. [4][5][6][7] 'White' clots account for a small subset of thrombi and their exact composition has been less characterized. It has been shown that 'white' coronary thrombi are smaller, predominantly composed of brin compared to 'red' ones and are associated with lower mortality while the AIS 'white' clots are signi cantly correlated with atypical etiologies. [8,9] Therefore, we investigated 'white' clots retrieved by MT focusing on their composition, imaging and procedural parameters.

Clot collection
This study was approved by the regional hospital ethics committees and National University of Ireland Galway research ethics committee (16-SEPT-08) in accordance with the ethical standards of the Declaration of Helsinki.
A total of 293 patients with AIS underwent MT between March and November 2018 at two centers within the multi-institutional RESTORE registry: Beaumont Hospital (Dublin, Ireland) and Sahlgrenska University Hospital (Gothenburg, Sweden).
Clot length and mean Houns eld Units (HU) density were evaluated on NCCT prior to mechanical thrombectomy.

Clot analysis
Clots were immediately placed in formalin, shipped to core laboratory and upon arrival, gross photos of each clot were taken. Clots retrieved from 19 patients out of 293 cases were described as 'white' based on their gross appearance (Fig. 1). The clots were para n-embedded, sectioned and stained with Martius Scarlett Blue (MSB) to identify main components [10], von Kossa and Alizarin Red stainings to con rm calci cation and mineralization when suspected and immunostained for platelets (rabbit monoclonal anti-CD42b, Abcam ab227669), von Willebrand Factor (monoclonal mouse anti-human vWF, Dako M0616) and fatty-acid binding protein 4 (anti-FABP4 rabbit polyclonal, Abcam ab13979) for adipocytes.
Stained slides underwent whole slide scanning (Olympus VS120) at 20x magni cation. Histological and immunohistochemical quanti cation was performed using Orbit Image Analysis software (www.orbit.bio). [11] Statistical analysis Data were analyzed using IBM SPSS-25 software. Nonparametric Kruskal-Wallis H-test was applied to compare basic characteristics between 'white' clots and other cases. Results were expressed as mean ± SD, median [IQ1-IQ3] or number and % of cases, as appropriate.
Study cohort 'White' clots represented 6% of our cohort and were selected based on the macroscopic appearance ( Fig. 1). Clinical and procedural information for the entire cohort are summarized in Table 1. Table 1 Clinical and intervention characteristics of study population.
Calci cation, collagen and adipocytes are present in 'white' clots Calci cation suspected on gross examination in one case (Fig. 4A) was con rmed by von Kossa and Alizarin Red (Fig. 4C). Collagen was identi ed on MSB staining in the same case (Fig. 4B).
Adipocyte-like structures were noticed in two 'white' clots ( Fig. 4B). Since the lipid content is lost during processing of clots through xylene, the FABP4 antibody was used as a marker for adipocytes. FABP4 was expressed by adipocytes in Case 16 while in Case 15, it was detected only in the nuclei in proximity to adipocytes (Fig. 4D).

Discussion
We carried out histopathological analysis of 19 'white' clots selected by their macroscopic aspect. We used the MSB stain which allows for a signi cantly better differentiation of the major components of clots than the traditional hematoxylin & eosin stain. [10] 'White' clots were platelet-rich compared to 'red' thrombi. We con rmed the presence of calci cation in one case using von Kossa stain. Mineralization was demonstrated by Alizarin Red suggesting that arterial mineralization and calci cation are governed by morphogenetic signals involved in skeletal mineralization. [12] It has been shown that emboli with large artery atherosclerosis etiology as well as a high proportion of cryptogenic stoke cases are platelet-rich. [13] In our cohort, two 'white' clots had a large artery atherosclerosis source while six cases were cryptogenic. The high platelet content of 'white' clots may support the use of antiplatelet therapy for secondary prevention in cryptogenic stroke.
We acknowledge that there are also other components in the platelets/other-rich areas identi ed by MSB so we performed immunohistochemistry to distinguish between platelets and platelet-related factors such as vWF. [14] The high expression of CD42b and vWF in our 'white' clots highlights their critical role in 'white' clots formation. Recent studies have described important histological features that may explain the recombinant tissue plasminogen activator (rtPA)-resistance of AIS thrombi: the presence of a dense outer shell containing mainly platelets, vWF and extracellular DNA as well as the presence in the plateletrich thrombi of dense brin structures lined with vWF, extracellular DNA and lled with platelets. [15,16] Given these observations, we suggest that platelet and vWF-rich composition of 'white' clots may impair thrombolysis and therefore, antiplatelet treatment could be used in these cases to target non-brin components and improve rtPA e cacy.
Increased levels of platelets and calcium may also render 'white' clots stiffer and impact the MT outcome. [14,17] However, despite the platelet-rich composition of 'white' clots compared to 'red' clots in our cohort, there was no signi cant difference in terms of number of passes during MT and nal modi ed Thrombolysis in Cerebral Infarction (mTICI) score.
We identi ed also the presence of adipocyte-like structures in two 'white' clots. Originally described as an adipocyte marker, FABP4 plays an important role in atherogenesis. In particular, FABP4 expression within the carotid atherosclerotic plaque is associated with its vulnerability and adverse outcome. [18] FABP4 immuno uorescence con rmed the presence of adipocytes in one case which may represent a histological marker of fat embolism or a vulnerable atherosclerotic plaque.
Imaging may hint to clot composition. [5,6,19] A recent study has demonstrated that platelet-rich thrombi identi ed by MSB staining are isodense on NCCT. [10] We found that 'white' thrombi are smaller and less hyperdense compared to the other clots.
Our study has limitations. The low number of 'white' clots may lead to under or overrepresentation of some characteristics presented. Formalin xation may change the macroscopic appearance and processing through xylene causes the loss of lipid content.

Conclusions
'White' clots represent a distinct subset of clots extracted from AIS patients. They are characterized by platelet and vWF-rich composition, are smaller and less hyperdense on NCCT than the other clots. The platelet-rich structure of 'white' clots may support the potential use of antiplatelet treatment to prevent stroke recurrence in these cases. Authors' contributions OMM, and KD were involved in all stages of the manuscript from concept design to drafting the manuscript. AO'H, SP, PB, JA, BM, MD, AB, AA, TG, TT, AR and JT were responsible for collecting and recording the clinical and procedural information from patients. All authors reviewed, edited, and approved the nal manuscript prior to submission.