Platelet-rich clots as identified by Martius Scarlett Blue staining are isodense on NCCT

Abstract

Background

Current studies on clot characterization in acute ischemic stroke focus on fibrin and red blood cell (RBC) composition. Few studies have examined platelet composition in acute ischemic stroke clots. We characterize clot composition using the Martius Scarlet Blue stain and assess associations between platelet density and CT-density.

Materials and Method

Histopathological analysis of the clots collected as part of the multi-institutional STRIP registry was performed using Martius Scarlett Blue stain and the composition of the clots was quantified using Orbit Image Analysis (Idorsia Ltd.) machine learning software. Prior to endovascular treatment, each patient underwent non-contrast Computed Tomography (NCCT) and the CT density of each clot was measured. Correlations between clot components and clinical information were assessed using the Chi-Squared test.

Results

Eighty-five patients were included in the study. The mean platelet density of the clots was 15.7% (2.5–72.5%). There was a significant correlation between platelet-rich clots and the absence of hyperdensity on NCCT, (ρ) =0.321, p=0.003*, n=85). Similarly, there was a significant inverse correlation between the percentage of platelets and the mean HU on NCCT (ρ=−0.243, p=0.025*, n=85).

Conclusion

Martius Scarlett Blue stain can identify patients who have platelet-rich clots. Platelet-rich clots are isodense on NCCT.

INTRODUCTION

Mechanical thrombectomy is standard of care for treatment of acute ischemic stroke secondary to large vessel occlusion [1]. The composition of the occlusive clot has been shown to significantly influence the outcome for patients treated with both rtPA and mechanical thrombectomy devices [2–7]. Retrieval of occlusive clots has afforded researchers the opportunity to study their histological composition, thereby increasing our understanding of their inherent characteristics and in the future potentially helping the interventional community to individualize patient care based on the suspected composition of the occlusive clot. In order for this to have an impact in the clinical setting, accurate characterization of all clot components as well as correlations between clot composition and diagnostic imaging findings must first be identified.

Initial studies assessing correlations between clot histological composition and diagnostic imaging have typically used the standard Hematoxylin and Eosin (H&E) histopathological stain. Red blood cell-rich clots have been shown to be associated with a Hyperdense Artery Sign (HAS) on NCCT and a positive Susceptibility Vessel Sign (SVS) on Magnetic Resonance Imaging (MRI), both of which are associated with improved outcome after treatment [8, 9]. However, the H&E stain fails to accurately distinguish between Fibrin and Platelets and thus studies using H&E have tended to refer to clots in terms of Fibrin/Platelet or Fibrin/Other content [10, 11]. The quantity of platelets can vary greatly between clots and it is well established that certain AIS patients benefit significantly from treatment with anti-platelet therapy [12]. We present a histological stain, Martius Scarlett Blue, capable of identifying platelet-rich regions of acute ischemic stroke clots and assess associations between platelet content and diagnostic imaging findings.

MATERIALS AND METHODS

Patient Selection and Clinical Data

This investigational study was performed as part of the multi-institutional Stroke Thromboembolism Registry of Imaging and Pathology (STRIP) registry. The study was institutional review board approved and HIPAA compliant. Patients were included in the study if they were >18 years, had undergone mechanical thrombectomy treatment for acute ischemic stroke and had a non-contrast computed tomography (NCCT) scan prior to endovascular treatment. Patients treated with recombinant tissue plasminogen activator (rt-PA) alone, patients without an available NCCT scan prior to endovascular treatment and patients with incomplete data were excluded from the study (Figure 1). An example of the data abstraction form is provided in the supplementary material (Supplementary File 1).

Figure 1: Flowchart describing the patient cohort selection process.

Computed Tomography Imaging

Prior to endovascular treatment, each patient had a NCCT scan performed and expert readers at each site evaluated the mean and maximum clot attenuation on NCCT as measured by the placement of regions of interest (ROIs) along the clot. A positive HAS was defined as ≥50 Hounsfield Units (HU). Because this is a multi-institutional study there was variability in the CT scanners and techniques used. All imaging findings were reported at the site where the thrombectomy was performed. There was no core lab for imaging.

Clot Collection, Processing and Histology

On retrieval, each clot was immediately fixed in 10% phosphate-buffered formalin. Clots were shipped to the histology core facility. Upon arrival at the core facility, gross photos were taken of each clot. All clots were then processed using a standard tissue processing protocol and embedded in paraffin. The formalin-fixed paraffin-embedded clot material was cut into 3–5μm sections. Representative slides from each clot were stained with Hematoxylin and Eosin (H&E) and Martius Scarlett Blue (MSB) stains as per their protocols (Supplementary Files 2&3). Following staining a representative MSB stained slide was sent for whole slide scanning (Aperio ScansScope AT Turbo, Leica Biosystems). Histologic quantification was performed on the digital slide using Orbit Image Analysis Software (Orbit Image Analysis, Idorsia Ltd.) as described previously [13].

Immunofluorescence

The slides were dried by heating to 56˚C for 2 hours in an oven, deparaffinized in Xylene (2× 10mins) followed by rehydration through alcohols and in distilled water. Sections were pretreated with 0.1 mol/L citric acid buffer in a microwave for 15 minutes. Slides were left to cool at room temperature for 30 minutes before being rinsed in Tris-buffered saline (TBS). The slides were incubated with 4% normal donkey serum in TBS buffer for 30 minutes at 37°C, followed by incubation with primary antibodies CD42b (mouse monoclonal antibody, pre-diluted; Abcam) and Fibrinogen (rabbit polyclonal antibody, 1:200; Dako) in TBS buffer for 1 hour at 37°C, then 4°C overnight. After incubation, the sections were rinsed in TBS buffer, followed by incubation with secondary antibodies (Cy3 conjugated donkey anti-mouse IgG (1:200); Alexa-Flour 488–conjugated donkey anti-rabbit IgG, (1:100); Jackson ImmunoResearch Inc.) for 2 hours at room temperature. The sections were rinsed in TBS (4× 5mins) and counterstained with DAPI (1:250). Finally, the sections were rinsed in TBS and then dehydrated through alcohols, cleared in xylene, and mounted with EZ-Mount. Negative controls were performed with non-immune normal serum used instead of the primary antibody. The sections were viewed and imaged with a fluorescence confocal microscope.

Statistical analysis

All statistical correlations were assessed using IBM SPSS Statistics 22. Correlations between clot composition and Hounsfield Units (HU) density on NCCT imaging were assessed using the Chi-Squared test. The Mann-Whitney U test for non-parametric data was used to assess correlations between continuous and categorical variables.

RESULTS

Patient Cohort

In total 85 patients with a diagnosis of AIS and treated with mechanical thrombectomy met the inclusion criteria and were included in the study. Table 1 shows the clinical demographics of the patient cohort. The median age of the patients was 66 years (range 20–91 years). Fifty-one percent of patients had been treated with rt-PA prior to mechanical intervention. The majority of cases had an Internal Carotid Artery (ICA) or Middle Cerebral Artery (MCA) occlusion (40% and 84% respectively) and twenty-five cases (30%) had occlusions that spanned two or more locations (data not shown). Stentriever devices were used in 61% of patients, whilst aspiration alone was used to treat the remaining 39% of patients. TICI 2b/3 was achieved in 96% of patients treated, with a mean number of passes of 2.1 ± 1.4.

Table 1.

Clinical details of patient cohort.

		Number of Patients (n=85)	(%)
Age (Years):
Mean	66
Range	20–91
Site:
ICA		34	40%
M1		56	66%
M2		15	18%
A1		2	2%
Basilar		5	6%
Vertebral		2	2%
P1		2	2%
rt-PA:
Yes		43	51%
No		42	49%
No of Passes Required:
Mean	2.1
1		39	46%
2		19	22%
3		15	18%
4		6	7%
5+		6	7%
Final TICI Score:
1		0	0%
2a		3	4%
2b		46	54%
3		36	42%
Imaging Characteristics	Mean HU:
All Clots	54.0	85	100%
Cardioembolic	53.4	44	52%
Large Artery	49.7	19	22%
Unknown	56.9	13	15%
Other	59.6	9	11%
Technique:
Stentriever		33	39%
Aspiration		33	39%
Both		19	22%

ICA: Internal Carotid Artery; M1 and M2 segment of the Middle Cerebral Artery; A1: Anterior Cerebral Artery A1 segment; P1: Posterior Cerebral Artery P1 segment; t-PA: tissue-plasminogen activator; TICI Score: Thrombolysis in Cerebral Infarction (TICI) Score; HU: Hounsfield Units.

MSB Stain Identifies platelet-rich regions in clots

The MSB-stain allows for a significantly better differentiation of the major components of clots than the H&E stain as can be seen in Figure 2. The MSB stain can accurately identify the presence of platelets as distinct from fibrin strands (Figure 2 A&C). Platelets cannot be accurately distinguished from fibrin in the H&E stained image, even at high magnification (Figure 2 B&D). The ability of the MSB stain to identify platelet-rich regions is confirmed by immunofluorescence staining that demonstrates the presence of platelets, as detected using an anti-platelet (CD42b) antibody, in the areas identified as being platelet-rich by the MSB stain (Figure 3).

Figure 2: Comparison of MSB and H&E stains in an acute ischemic stroke clot.

A&C are examples of an MSB-stained slide from a clot demonstrating the presence of Red Blood Cells (Yellow), White Blood Cells (Blue), Fibrin strands (Red) and Platelets (Grey, identified by Black arrows). B&D are examples of a slide H&E stained slide from the same clot demonstrating the presence of Red Blood Cells (Red), White Blood Cells (Blue) and Fibrin/ Platelets (Purple).

Figure 3: Immunofluorescence staining confirms the presence of platelet-rich regions in clots.

(A) A low magnification image (4x) of an MSB stained slide demonstrating the presence of Fibrin strands (Red), Red Blood Cells (Yellow), White Blood Cells (Blue) and Platelets (Grey) in an acute ischemic stroke clot stained with Martius Scarlett Blue Staining. (B-D) Immunofluorescence images demonstrating the presence of; (B) Nucleated cells (DAPI, Blue), (C) Platelets (CD42b, Red) and (D) Fibrinogen (Green). (E) is a merged immunofluorescence image demonstrating the co-localization of platelets with Fibrinogen (Yellow).

AIS Clot Compositions

The composition of the major clot components was heterogeneous amongst the patient cohort as can be seen in Figure 4. Red Blood cells and fibrin were typically the dominant components of AIS clots with their mean compositions being 39.4% and 41.8% respectively, while the average White Blood Cell composition was 3.9%. The composition of platelets/other components varied from 2.5 to 72.5% of the total area, with a mean value of 15.7%.

Figure 4: Clot composition of the patient cohort.

This is a graphical representation of the histological clot composition of each patient in the cohort as determined by Martius Scarlett Blue staining represented as the percentage of Red Blood Cells (Yellow), White Blood Cells (Blue), Fibrin strands (Red) and Platelets (Grey).

Treatment with rt-PA prior to mechanical thrombectomy resulted in significantly reduced Platelets (12.2% vs 19.4%, p=0.018) and WBCs (2.6% vs 3.5%, p=0.024), but did not significantly affect Red Blood Cells (42.9% vs 35.8%, p=0.173) or Fibrin (42.3% vs 41.3%, p=0.937). There was no significant difference in clot composition between cases treated with Stentriever devices and aspiration. Clot composition also did not significantly affect the Final TICI score nor the number of procedural passes.

Platelet Content versus clot attenuation on NCCT

There was a significant correlation between platelet-rich clots (≥15.7%) and the absence of a HAS (<50 HU) on NCCT, (ρ=0.321, p=0.003*, n=85). In addition, there was also a significant inverse correlation between the percentage of platelets and the mean HU on NCCT as shown in Figure 5 (ρ=−0.243, p=0.025*, n=85). An example of a patient with a isodense vessel on NCCT prior to treatment that was subsequently found to have a had platelet-rich thrombi is shown in Figure 6. No significant correlations between the other major clot components and a HAS were observed.

Figure 5: Correlation between % Platelet/Other composition and HU density on NCCT.

A significant inverse correlation between the percentage of platelets and the mean HU on NCCT was observed (ρ=−0.243, p=0.025*, n=85).

Figure 6: Example of a platelet-rich clot that is isodense on NCCT.

(A) An NCCT image of the patient prior to mechanical thrombectomy. There is an absence of a Hyperdense Artery Sign in the left ICA Terminus and M1 branch of the MCA. (B) A digital subtraction angiography (DSA) image of the patient prior to mechanical thrombectomy demonstrating occlusion of the ICA Terminus and MCA. (C) A DSA image of the patient after treatment with mechanical thrombectomy to remove the occlusive clot demonstrating full revascularization (TICI3) in the MCA. (D) is the gross photograph of the retrieved clot after fixation in PFA. (E&F) Low (0.5x) and High (40) magnification image of the MSB stained slide from the clot demonstrating the presence of platelet-rich region (34.6% Platelets, Grey) within the clot.

DISCUSSION

In this study we demonstrate that the Martius Scarlett Blue stain can identify platelet-rich regions in acute ischemic stroke clots. These findings are important as we are now able to characterize the histological composition of acute ischemic stroke clots more comprehensively than ever before, by including one of the most essential components to thrombosis-platelets. We also demonstrate that platelet-rich clots, as determined by the MSB stain, are isodense on NCCT scans. These findings are important as they 1) provide additional insight into the composition of clot in large vessel occlusion, 2) allow for more accurate radiological-pathological correlation between clot histology and density on CT and 3) could potentially provide insights into treatment strategies for secondary stroke prevention.

Previous studies investigating the composition of acute ischemic stroke clots using basic histological stains have failed to accurately distinguish between fibrin and platelets [2, 10, 11, 14–16]. Activation of platelets has long been known to initiate the coagulation cascade and logically, platelets and platelet-related factors are key components of clots [17]. The ability to identify platelet-rich regions as distinct from fibrin, now allows us to investigate correlations between platelet-content and clinical and procedural parameters, in addition to more accurately representing the ‘true’ fibrin content of the clots.

In order for this to have an impact on the acute treatment of stroke, correlations between histological and mechanical clot characteristics and diagnostic imaging modalities must first be identified [18, 19]. Accurate identification of clot composition on diagnostic imaging would provide valuable information on the degree and ease of revascularization using mechanical thrombectomy techniques as the mechanical properties of clots are known to change significantly depending on composition [20]. We demonstrate for the first time, that platelet-rich clots are isodense on CT. This suggests that patients whose clots appear isodense on NCCT are more likely to be platelet-rich. Platelet-rich clots have long been known to be more resistant to standard thrombolytic therapy [21, 22] and therefore those patients might benefit significantly from treatment with novel anti-platelet therapy that has previously been shown to improve outcomes in certain patients [12].

The ability to quantify platelet composition might also play a significant role in the medical management of the acute ischemic stroke patients in order to prevent a recurrent stroke, as the cause of a secondary stroke is typically directly related to the original stroke. Accurate knowledge of the platelet-content of the occlusive clot would allow clinicians to better select patients that might derive a clinical benefit from dual anti-platelet therapy which would be of particular importance in cryptogenic stroke patients.

Our study has limitations. First, TICI scores were self-reported at each site and not measured using a central core lab which may have resulted in some site-to-site variability. Second, whilst the MSB stain is more accurate than the H&E stain in identifying major clot components, it still does not specifically identify other potentially key clot components such as von Willebrand factor and calcification. Therefore, the authors represent this subgroup as Platelet and Other components as we acknowledge that there are potentially other components in addition to platelets in these regions. Immunohistochemical analysis using specific antibodies is the only way to accurately distinguish between platelets and platelet related factors such as von Willebrand factor. An additional histological stain such as Von Kossa staining should be used if calcification is suspected. There was variability in the make and model of CT scanners used between different sites and all imaging findings were reported at the site where the thrombectomy was performed, not at a core imaging lab, which might result in some slight intra-site variability when measuring HU density.

CONCLUSIONS

Martius Scarlett Slue stain can identify patients who have platelet-rich clots. These platelet-rich clots are isodense on NCCT. This correlation may inform acute treatment approach and thereby lead to better patient outcomes.