Neurotherapeutics (2023) 20:1167–1176 https://doi.org/10.1007/s13311-023-01385-1 ORIGINAL ARTICLE Granulocytes‑Rich Thrombi in Cerebral Large Vessel Occlusion Are Associated with Increased Stiffness and Poorer Revascularization Outcomes Jesús Juega1 · Jiahui Li1 · Carlos Palacio‑Garcia2 · Maite Rodriguez1 · Riccardo Tiberi1 · Carlos Piñana3 · David Rodriguez‑Luna1 · Manuel Requena1 · Álvaro García‑Tornel1 · Noelia Rodriguez‑Villatoro1 · Marta Rubiera1 · Marian Muchada1 · Marta Olivé‑Gadea1 · Federica Rizzo1 · David Hernandez3 · Marta Dios‑Lascuevas3 · Maria Hernandez‑Perez4 · Laura Dorado4 · Helena Quesada5 · Pere Cardona5 · Carolina De La Torre6 · Laura Gallur2 · Jessica Camacho7 · Santiago Ramon‑y‑Cajal7 · Alejandro Tomasello3 · Marc Ribó1 · Carlos A. Molina1 · Jorge Pagola1 on behalf of the ITACAT study group Accepted: 24 April 2023 / Published online: 22 May 2023 © The Author(s) 2023 Abstract We aim to identify a profile of intracranial thrombus resistant to recanalization by mechanical thrombectomy (MT) in acute stroke treatment. The first extracted clot of each MT was analyzed by flow cytometry obtaining the composition of the main leukocyte populations: granulocytes, monocytes, and lymphocytes. Demographics, reperfusion treatment, and grade of reca- nalization were registered. MT failure (MTF) was defined as final thrombolysis in cerebral infarction score IIa or lower and/ or need of permanent intracranial stenting as a rescue therapy. To explore the relationship between stiffness of intracranial clots and cellular composition, unconfined compression tests were performed in other cohorts of cases. Thrombi obtained in 225 patients were analyzed. MTF were observed in 30 cases (13%). MTF was associated with atherosclerosis etiology (33.3% vs. 15.9%; p = 0.021) and higher number of passes (3 vs. 2; p < 0.001). Clot analysis of MTF showed higher percentage of granulocytes [82.46 vs. 68.90% p < 0.001] and lower percentage of monocytes [9.18% vs.17.34%, p < 0.001] in comparison to successful MT cases. The proportion of clot granulocytes (aOR 1.07; 95% CI 1.01–1.14) remained an independent marker of MTF. Among thirty-eight clots mechanically tested, there was a positive correlation between granulocyte proportion and thrombi stiffness (Pearson’s r = 0.35, p = 0.032), with a median clot stiffness of 30.2 (IQR, 18.9–42.7) kPa. Granulocytes- rich thrombi are harder to capture by mechanical thrombectomy due to increased stiffness, so a proportion of intracranial granulocytes might be useful to guide personalized endovascular procedures in acute stroke treatment. Keywords Acute stroke · Mechanical thrombectomy · Flow cytometry · Granulocytes · Recanalization · Thrombi 4 * Marc Ribó Department of Neurology, Germans Trias I Pujol University marcriboj@hotmail.com Hospital, Badalona, Spain 5 1 Department of Neurology, Bellvitge University Hospital, Stroke Unit, Department of Neurology, Vall d’Hebron Hospitalet de Llobregat, Spain University Hospital, Vall d’Hebron Research Institute. 6 Universitat Autonoma de Barcelona, Passeig de la Vall Proteomics Unit, Josep Carreras Leukaemia Research d’Hebron, 119‑129, Barcelona 08035, Spain Institute (IJC), Badalona, Spain 2 7 Hematology Department, Vall d’Hebron University Hospital, Department of Pathology, Vall d’Hebron University Hospital, Barcelona, Spain Barcelona, Spain 3 Department of Neuroradiology, Vall d’Hebron University Hospital, Barcelona, Spain 13 Vol.:(0123456789) 1168 J. Juega et al. Introduction to MT as assessed by the National Institutes of Health Stroke Scale (NIHSS), cerebral infarct characteristics on Achieving complete recanalization in first attempt is the baseline computerized tomography scan, angiographic fea- main goal of mechanical thrombectomy (MT) among tures, and reperfusion treatment modality in each patient patients with an acute stroke due to a large vessel occlusion were collected. The etiology of the stroke was classified (LVO). However, patients who achieve successful reperfu- according to TOAST classification [11]. The study was sion after several passes have better clinical outcomes than approved by the Local Committee of Ethics in Medical those without any reperfusion. Therefore, final revasculari- Research of the Hospital Universitari Vall d’Hebron (PR zation beyond the first pass is still the goal of MT [1–4]. (AG) 234/2017) and was carried out in line with the sec- The rate of MT failure in LVO strokes ranges from 10 to ond Declaration of Helsinki. Written informed consent 30%; these cases are associated with very poor prognosis was obtained from all participants or their relatives. [5]. Main causes of MT failure are difficulty to access to target occlusion or failure to retrieve the clot despite Neuroimaging and Thrombectomy Procedure multiple attempts [6, 7]. The angiographic outcome of MT procedures is highly According to stroke code protocol, every patient underwent influenced by the composition and mechanical properties plain CT at the emergency room or at the angio suite in of the intracranial thrombi. Previous work revealed that order to rule out intracranial hemorrhage [12]. ASPECTS erythrocyte-rich thrombi were associated with shorter scores were recorded for each patient when standard CT arrival-to-recanalization time intervals and more favora- scan was performed. Reperfusion treatment with intravenous ble clinical outcomes [8]. Moreover, recent studies have alteplase (iv-rTPA) and/or MT was administered following shown that neutrophil extracellular traps (NETS) contrib- current guidelines recommendations [13]. Intracranial and/ ute to resistance to reperfusion therapy with more number or extracranial LVO was recorded from the diagnostic angi- of passes and less favorable recanalization [9]. ography. All the images were reviewed by an expert radi- Stent retriever and aspiration techniques are the first-line ologist blinded to clinical data. LVOs occlusion were cat- therapy in endovascular treatment, but complex MT proce- egorized into two groups: proximal large vessel occlusions dures may need to set a permanent intracranial stenting (PIS) (PLVOs) in case of occlusion of intracranial and/or extrac- as a rescue technique after failed standard MT, even though ranial carotid artery; segment M1 of middle cerebral artery PIS requires strong antithrombotic therapy [5, 10]. (M1-MCA) and basilar artery occlusion (BAO); medium- The development of markers to predict failure of stand- vessel occlusions (MeVOs) in case of occlusion of distal ard MT may select patients for early angioplasty and PIS medium arterial arbors (anterior cerebral artery [ACA], in the acute phase of stroke in order to avoid complications M2 middle cerebral artery [M2-MCA] or posterior cerebral associated with multiple attempts. The aim of our study was artery [PCA] [14, 15]. The device employed to perform the to define a profile of thrombectomy-resistant thrombus by first attempt in MT procedure were categorized into: direct mechanical characterization and relationship with composi- aspiration first pass technique (ADAPT), stent retriever, or tion by flow cytometry analysis of retrieved thrombi. combination of both (combined technique) [16]. Additional procedures such as intracranial angioplasty and/or intracranial stenting were registered [5]. Number of passes and modified thrombolysis in cerebral infarction Methods (mTICI) score after MT were documented [17]. MT failure was defined as mTICI score IIa or lower Study Setting and Population (mTICI ≤ IIa) and/or need of intracranial stenting after con- ventional MT procedure. Successful MT was categorized as We conducted a prospective, single-center observational mTICI 2b or higher (final mTICI ≥ 2b). study of patients with acute ischemic stroke and LVO who underwent MT from January 2017 to January 2021 to ana- Clot Analysis by Flow Cytometry Protocol lyze predictors of MT failure and another observational study between March 2021 and June 2021 to perform The thrombi obtained after MT were suspended in a container mechanical characterization with samples from retrieved with RPMI 1640 medium 1640 (Gibco™) and stored at 4 to clots. These studies were included in the multi-center 6 °C for no longer than 24 h until analysis was started in order to ongoing registry ITACAT, “Impact of Thrombus Analy- avoid decrease in detection of cell surface markers from selected sis in Stroke Patients in Catalonia” (355/C/2017). Demo- cell populations with different half-life time [18, 19]. Leuko- graphic data, vascular risk factors, severity of stroke prior cytes were obtained and analyzed as previously published [20]. 13 Granulocytes‑Rich Thrombi in Cerebral Large Vessel Occlusion Are Associated with Increased… 1169 Briefly, the clots were finely minced and disaggregated to obtain in the total leukocyte population. P-value below 0.05 was set- a single cell suspension in 2 to 3 mL of RPMI 1640 using a tled for determining significance in two-tailed test result. For scalpel. Then, cell suspensions were filtered and washed with categorical variables, statistical significance was assessed by phosphate-buffered saline (PBS). Leukocytes were pelleted by chi-square r or Fisher’s exact test. For continuous variables, centrifugation, resuspended in 100 μL of PBS, and labeled by Mann–Whitney U test was employed. Univariate analysis of direct immunofluorescence using CD45 monoclonal antibody demographics, imaging data, MT procedure, and clot analysis conjugated with Krome Orange (Beckman Coulter™). Labeled were performed to define predictors associated with MT fail- cells were acquired in a Navios EX™ flow cytometer and were ure. Among MT with multiple passes, receiver operating char- analyzed using Kaluza software (Beckman Coulter™). Finally, acteristic (ROC) curve was drawn to estimate the best cut-off leukocytes were classified as granulocytes, monocytes, or lym- point based on Youden index for proportion of granulocytes phocytes according to their side scatter complexity and CD45 to predict MT failure. To estimate adjusted odds ratio (aOR) level of expression. A threshold of at least 100 leukocytes was of MTF, predictive models were created using binary logistic set for viable clots. In case there were several thrombi extracted regression analysis and adjusted analysis, adjusted by age, from the same or different vessels, clot obtained in the first pass sex, distal intracranial occlusions (MeVOs), and significant was selected for analysis from each MT. variables obtained in the univariate model. The relationship between thrombi composition and thrombus stiffness was sta- Mechanical Characterization tistically assessed with 95% confidence intervals. Bivariate correlation tests were conducted to find the Pearson’s coef- Retrieved clot fragments were stored in Gibco RPMI 1640 ficient between continuous variables. Statistical analysis was medium (Thermo Fisher, Waltham, MA) at 4–6 °C. Mechan- performed by SPSS 21.0® software. ical experiments were performed within 48 h from the end of the MT procedure. Unconfined compression tests were performed to explore the stiffness of the clots [21–23]. Briefly, test specimens are Results submitted under gradual compression between two parallel rigid plates, and the force loads applied to cause a specific From three hundred acute stroke treatments performed from deformation are registered in a stress–strain curve. The January 2017 to January 2021, two hundred twenty-five experiments were conducted with a Zwick-Line Z0.5TN retrieved clots were obtained. Thirty-nine percent (88/225) of testing machine equipped with a 5N load cell (Zwick-Roell, cases achieved first-pass successful MT and MTF was observed Ulm, Germany) (Online Resource 1). In preparation for the in 30 cases (13% of total MT). Therefore, among 137 endo- mechanical characterization, clot fragments were trimmed vascular treatments (EVT) that required several passes, 78% to a height between 1 and 2 mm, and test specimens’ diam- (107/137) achieved final mTICI ≥ 2b with successful MT, and eters (> 1 mm) were measured with a caliper with a resolu- 21.89% (30/137) were categorized as MT failure (Fig. 1). tion of 0.1 mm. Test parameters were configured to conduct Global data from comparison between MTF versus suc- non-destructive experiments, so the clot samples can be pre- cessful MT (final mTICI ≥ 2b) are stated at Table 1. So served for the subsequent analysis by flow cytometry. Test therefore, patients with MT failure were more frequently samples were pre-conditioned at 37 °C before conducting observed in large arterial atherosclerosis stroke [33.3% (10) the experiments. Unconfined compression tests were config- vs. 15.9% (31); p = 0.021] and in MT that required a higher ured with a preload force of 0.001 N, followed by a gradual number of passes (3 passes vs. 2; p < 0.001). Clot analysis compression up to 50% strain at a rate of 0.1 mm/s. The of MT failure showed higher percentage of granulocytes secant modulus, defined as the slope of a line drawn from [82.46 (69.19–89.02) vs. 68.90% (58.60–79.07); p < 0.001] the origin of the stress–strain diagram and intersecting the and lower percentage of monocytes [9.18% (5.29–20.23) curve at the point of interest (0–45% strain), was used to vs.17.34% (11.90–27.06); p < 0.001]. Neither the use of determine the thrombus stiffness. previous iv-rTPA nor type of device used in first pass was associated with MT failure, as stated in Table 1. Conversely, Statistical Analysis MT recanalization was associated with high-risk cardioem- bolic etiology (HRCE) [60% (117) vs. 36.7% (11); p = 0.016] Categorical variables were presented as absolute number and and dyslipidemia [52.3% (102) vs. 26.7% (8); p = 0.009]. percentage in each group (n, %). The distribution of continu- ous variables was assessed by Kolmogorov–Smirnov test. Comparison Among EVT with Multiple Passes Continuous variables were expressed as medians with inter- quartile range [IQR]. Relative proportion of each leukocyte In cases that required more than one pass, “successful population was expressed as percentage (%) of the type of cell MT” remained associated with dyslipidemia [47.7% (51) 13 1170 J. Juega et al. Fig. 1  Flow chart. EVT is endo- vascular treatment; FC is flow cytometry; MT is mechanical thrombectomy; Mtici is modi- fied thrombolysis in cerebral infarction; PIS is permanent intracranial stenting vs. 26.7% (8); p = 0.040] and HRCE [63.6% (68) vs. Mechanical Characterization and Clot Composition 36.7% (11); p = 0.008]. MT failure was associated with higher proportion of granulocytes [82.46% (69.19–89.02) In another cohort of patients who underwent MT between vs. 69.90% (59.0–76.84); p < 0.001] and lower propor- March and June 2021, thirty-eight clot fragments were ana- tion of monocytes [17.11% (12.18–27.10) vs. 9.18 lyzed from samples obtained from twenty-one MTs. In this (5.29–20.23); p < 0.001] in clot analysis (Table 2). Dif- sub-analysis, unconfined compression tests were conducted ferences in clot composition between groups are repre- before flow cytometry. Clinical characteristics of all patients sented in Fig. 2. and experimental data from mechanical testing and flow In a multivariable model analysis including signifi- cytometry analysis are summarized in Online Resource 2. cant variables obtained from the univariate analysis Median age was 80 [72–88] and median clot stiffness was (dyslipidemia, HRCE, granulocytes %, and monocytes 30.2 (IQR, 18.9–42.7) kPa. Although no significant relation- %), the proportion of granulocytes (aOR 1.07; 95% CI ship was found between monocytes (r = − 0.23, p = 0.177), 1.01–1.14) remained as an independent marker of MT lymphocytes (r = − 0.187, p = 0.274), and clot stiffness, there failure (Table 3). Youden index’s best cut-off point for was a positive correlation between granulocyte proportion clot granulocytes to predict MT failure was 74.40% (AUC and thrombi stiffness (Pearson’s r = 0.35, p = 0.032), as of 0.735; sensitivity of 73.33%, specificity of 69.16%, shown in Fig. 3. positive predictive value of 40%, negative predictive value of 90.24%, and positive likelihood ratio of 2.38) (Fig. 3). In another multivariate analysis, cut-off point Discussion for granulocytes > 74.40% in clot analysis (aOR: 6.99, CI 95%: 2.63–18.59, p < 0.001) was independently asso- Based on our results, high proportion of granulocytes of ciated with higher risk of MT failure after adjustment intracranial clots is a marker of MT failure during stand- for age, sex dyslipemia, HRCE, and occlusion location ard acute EVT of LVO strokes. Most published studies (MeVOs). Conversely HRCE decreased the risk of MT have employed anatomo-pathology techniques to evalu- failure (aOR 0.234, CI 0.082–0.665, p = 0.06), as stated ate the influence of thrombi composition and MT out- in Table 3. come [24]. It has been reported that platelet-rich clots 13 Granulocytes‑Rich Thrombi in Cerebral Large Vessel Occlusion Are Associated with Increased… 1171 Table 1  Global comparison between successful recanalization versus MT failure All EVT (n = 225) Successful MT (n = 195) MT failure (n = 30) P-value Age, years [IQR] 75 [63–82] 75 [63–82] 77 [63–83] 0.762 Sex (female) n, % 105 (46.7) 88 (45.1) 17 (56.7) 0.238 History of atrial fibrillation n, % 65 (28.9) 56 (28.7) 9 (30) 0.885 Active smoker n, % 44 (19.6) 37 (19.0) 7 (23.3) 0.575 High blood pressure n, % 149 (66.2) 129 (66.2) 20 (66.7) 0.956 Diabetes mellitus n, % 43 (19.1) 40 (20.5) 3 (10) 0.173 History of coronary artery disease n, % 43 (19.1) 40 (10.6) 3 (10) 0.173 Dyslipidemia n, % 110 (48,9) 102 (5.3) 8 (26.7) 0.009 Antiplatelets n, (%) 67 (29.8) 59 (30.3) 8 (26.7) 0.689 Anticoagulation n, (%) 35 (15.6) 28 (14.4) 7 (23.3) 0.207 Glycemia (mg/dL) 119 [101–137] 120 [102–141] 112 [97–126] 0.206 NIHSS 16 [10–21] 16 [10–21] 16 [8–20] 0.642 HRCE n, (%) 128 (56.9) 117 (60) 11 (36.7) 0.016 LAA n, (%) 41 (18.2) 31 (15.9) 10 (33.3) 0.021 Other cause n, (%) 9 (4.0) 8 (4.1) 1 (3.3) 0.841 ASPECTS score [IQR] 9 [8–10] 10 [8–10] 9 [7–10] 0.254 PLVOs 169 (75.1) 146 (74.8) 23 (76.6) 0.833 TICA occlusion n, (%) 52 (23.1) 43 (22) 9 (30) 0.337 Medial artery 1 occlusion n, (%) 85 (37.7) 72 (36.9) 13 (43.3) 0.501 Medial artery 1 tandem occlusion n, (%) 20 (8.8) 20 (10.3) 0 0.067 BAO occlusion n, (%) 12 (5.3) 11 (5.6) 1 (3.3) 0.601 MeVOs 56 (24.8) 49 (25.1) 7 (23.3) 0.833 Anterior artery occlusion n, (%) 3 (1.3) 2 (1.05) 1 (3.3) 0.306 Medial artery 2 occlusion n, (%) 44 (19.5) 40 (20.5) 4 (13.3) 0.357 Posterior artery occlusion n, (%) 9 (0.04) 7 (3.6) 2 (6.7) 0.424 iv–rTPA n (%) 78 (34.8) 71 (36.6) 7 (23.3) 0.156 Stent retriever first pass, n (%) 63 (28.1) 57 (29.4) 6 (20.0) 0.288 Combined, first pass n (%) 130 (0.303) 110 (56.7) 20 (66.7) 0.303 ADAPT first pass n (%) 30 (13.4) 27 (13.9) 3 (10.0) 0.558 Number of passes, [IQR] 2 [1–3] 2 [1–3] 3 [2–5] < 0.001 Clot total leukocytes analyzed [IQR] 3394 [1382–11,593] 3374 [1410–11,641] 3679 [1126–11,188] 0.756 Clot granulocytes % [IQR] 70.31 [59.57–80.68] 68.90 [58.60–79.07] 82.46 [69.19–89.02] < 0.001 Clot monocytes % [IQR] 16.35 [10.84–25.94] 17.34 [11.90–27.06] 9.18 [5.29–20.23] < 0.001 Clot lymphocytes % [IQR] 6.65 [2.82–12.90] 8.86 [2.75–13.0] 5.08 [3.0–12.33] 0.340 ADAPT a direct aspiration first pass technique, EVT endovascular treatment, HRCE high risk cardioembolic etiology, IQR interquartile range, Iv-rTPA intravenous fibrinolysis, LAA large arterial atherosclerosis, MT mechanical thrombectomy, NIHSS National Institutes of Health Stroke Scale, PLVOs proximal large vessel occlusions, MeVOs medium vessel occlusions are difficult to retrieve with poorer revascularization neutrophils are involved in neuronal cell death [26]. Flow outcomes; there is a positive correlation between plate- cytometry studies showed that neutrophils are triggered lets and white blood cells, suggesting a possible inflam- by ischemia in experimental models of direct occlusion matory pathway for a pro-coagulant mechanism [25]. in the absence of reperfusion [27], and a recent study in Intracranial clots with high proportion of leukocytes humans observed leukocyte invasion distal to intracranial promote thrombi formation by extracellular DNA traps LVO primarily attributable to strong elevation of the neu- [9]. Web-like structures called NETs are released from trophil subpopulation [28]. activated neutrophils, which create a scaffold for throm- Global analysis of stroke etiology and EVT outcomes bus formation. The amount of leukocytes and NETs may showed a significant association between LAA strokes and increase the adherence to the vessel wall, making the MT failure. However, this was not confirmed in the sub- removal of thrombus more difficult. At the same time, analysis in EVT with multiple passes. 13 1172 J. Juega et al. Table 2  EVT with multiple passes: successful MT vs. MT failure Total (n = 137) Successful MT(n = 107) MT failure (n = 30) P-value Age, years [IQR] 76 [65–82] 76 [65–82] 77 [63–83] 0.938 Sex (female) n, % 73 (53.3) 56 (52.3) 17 (56.7) 0.674 History of atrial fibrillation n, % 42 (30.7) 33 (30.8) 9 (30) 0.930 Active smoker n, % 28 (20.4) 21 (19.6) 7 (23.3) 0.656 High blood pressure n, % 92 (67.2) 72 (67.3) 20 (66.7) 0.949 Diabetes mellitus n, % 21 (15.3) 18 (16.8) 3 (10) 0.359 History of coronary artery disease n, % 25 (18.2) 22 (20.6) 3 (10) 0.186 Dyslipidemia n, % 59 (43.1) 51 (47.7) 8 (26.7) 0.040 Antiplatelets n (%) 33 (24.1) 25 (23.4) 8 (26.7) 0.709 Anticoagulation n (%) 21 (15.3) 14 (13.1) 7 (23.3) 0.168 Glycemia (mg/dL) 116 [102–133] 118 [102–133] 112 [97–126] 0.329 NIHSS 16 [10–21] 16 [10–21] 16 [8–20] 0.750 HRCE n (%) 79 (57.7) 68 (63.6) 11 (36.7) 0.008 LAA n (%) 28 (20.4) 18 (16.8) 10 (33.3) 0.071 Other cause n (%) 2 (1.5) 1 (0.9) 1 (3.3) 0.333 ASPECTS score [IQR] 9 [8–10] 10 [8–10] 9 [7–10] 0.208 PLVOs 103 (75.1) 80 (74.7) 23 (76.6) 0.832 TICA occlusion n (%) 37 (27.0) 28 (26.1) 9 (30) 0.677 Medial artery 1 occlusion n (%) 51 (37.2) 38 (35.5) 13 (43.3) 0.435 Medial artery 1 tandem occlusion n (%) 10 ( 7.2) 10 (9.3) 0 0.083 BAO occlusion n (%) 5 (3.6) 4 (3.7) 1 (3.3) 0.917 MeVOs 34 27 (25.2) 7 (23.3) 0.832 Anterior artery occlusion n (%) 3 (2.2) 2 (1.9) 1 (3.3) 0.629 Medial artery 2 occlusion n (%) 24 (17.5) 20 (18.6) 4 (13.3) 0.497 Posterior artery occlusion n (%) 7 (5.1) 5 (4.7) 2 (6.7) 0.662 iv–rTPA n (%) 45 (32.8) 7 (23.3) 7 (23.3) 0.209 Stent retriever first pass, n (%) 42 (30.7) 36 (33.6) 6 (20.0) 0.152 Combined, first pass n (%) 74 (54.0) 54 (50.5) 20 (66.7) 0.116 ADAPT first pass n (%) 20 (14.6) 17 (15.9) 3 (10.0) 0.420 Number of passes [IQR] 3 [2–4] 3 [2–4] 3 [2–5] 0.073 Clot total leukocytes [IQR] 3092 [1283–9765] 3016 [1265–9636] 3679 [1126–11,188] 0.954 Clot granulocytes % [IQR] 71.03 [60.60–81.06] 69.90 [59.0–76.84] 82.46 [69.19–89.02] < 0.001 Clot monocytes % [IQR] 15.61[10.23–24.40] 17.11 [12.18–27.10] 9.18 [5.29–20.23] < 0.001 Clot lymphocytes % [IQR] 6.42 [3.01–12.69] 6.63 [3.0–12.80] 5.08 [3.0–12.33] 0.886 ADAPT a direct aspiration first pass technique, EVT endovascular treatment, HRCE high risk cardioembolic etiology, IQR interquartile range, Iv-rTPA intravenous fibrinolysis, LAA large arterial atherosclerosis, MT mechanical thrombectomy, NIHSS National Institutes of Health Stroke Scale, PLVOs proximal large vessel occlusions, MeVOs medium vessel occlusions Our study evidences the relationship between [0–45%] = 26 ± 2 kPa) and fibrin-rich (secant modulus granulocyte-rich clots and increased thrombus stiffness. [0–45%] = 170 ± 39 kPa) thrombi harvested from clinical Previous studies have shown that fibrin/platelet-rich thrombi cases [22]. Similar clot stiffness values using the uncon- are stiffer than erythrocyte-rich clots and present higher fined compression experiments (tangent modulus at 40% frictional properties that might contribute to resistance strain = 59 ± 63 kPa) has been published and found no asso- to MT. On another note, erythrocyte-rich clots might ciation between total leukocytes and thrombus stiffness [21]. be more prone to fracture and present higher risk of per However, our study focused on specific leukocyte subpopu- procedural embolization since the primary mechanism of lations achieving a positive correlation between granulocyte clot deformation is fibrin rupture [21, 22, 29, 30]. proportion and thrombi stiffness, which may explain why Clot stiffness quantified in our study matched previ- high granulocyte content within the clot of LVO leads to ously reported values on erythrocyte-rich (secant modulus resistance to recanalization with standard MT procedures. 13 Granulocytes‑Rich Thrombi in Cerebral Large Vessel Occlusion Are Associated with Increased… 1173 Fig. 2  Differences in clot analysis among EVT with multiple passes. lower proportion of monocytes [17.11% (12.18–27.10) vs. 9.18 MT failure was associated with higher proportion of granulocytes (5.29–20.23); p < 0.001] in clot analysis [82.46% (69.19–89.02) vs. 69.90% (59.0–76.84); p < 0.001] and Our protocol of FC that analyzed intracranial clots might higher number of passes are associated with worse outcome help to identify thrombectomy-resistant thrombus in acute probably due to vessel harm during mechanical thrombec- stroke treatment. FC technique allowed to perform a fast path- tomy, clot fragmentations and higher risk of clinical complica- way of clot analysis, avoiding time-consuming steps of standard tions [7]. Obtaining a marker after first pass that predict high anatomo pathologic techniques. To our knowledge, no previous risk of mechanical thrombectomy failure with conventional studies have employed FC to study clot composition in cases MT techniques may help to select patients for advanced pro- of MT failure. cedures such us permanent intracranial stenting (PIS) as a First goal of MT is to achieve first pass recanalization (TICI rescue reperfusion technique in order to avoid complications 3 after first pass) with conventional techniques (stent retriever, and procedure delay [31]. direct aspiration, or combination of these techniques) within On the other hand, because neutrophils and NETs are impor- fast procedures [1] but there are no evidence of which should tant constituents of cerebral thrombi, there are currently some be the best strategy after first attempt failure. Besides sudden groups trying to develop new thrombolytic agents for targeting recanalization strongly predicts favorable outcome in patients NETs with DNase 1 that might have potential acute stroke treat- undergoing EVT even after previous unsuccessful attempt, ment in combination with endovascular treatment [32]. Table 3  Models of multivariate Univariate analysis Multivariate analysis analysis for predictors of MT failure among EVT with Unadjusted OR 95% CI Adjusted OR 95% CI P-value multiple passes Dyslipidemia 0.39 0.16–0.97 0.02 0.19–0.85 0.022 HRCE 0.33 0.14–0.76 0.29 0.11–0.76 0.012 Granulocytes % 1.07 1.03–1.11 1.07 1.01–1.14 0.028 Monocytes % 0.925 0.878–0.975 0.99 0.92–1.07 0.914 Univariate analysis Multivariate analysis Unadjusted OR 95% CI Adjusted OR 95% CI P-value Age 0.99 0.96–1.02 1.01 0.98–1.04 0.341 Sex (female) 1.19 0.52–2.69 1.49 0.560–4.012 0.420 Dyslipidemia 0.39 0.16–0.97 0.36 0.133–0.988 0.047 HRCE 0.33 0.14–0.76 0.24 0.092–0.670 0.006 MeVOS 0.90 0.348–2.336 0.97 0.326–2.921 0.965 Granulocytes > 74.40% 6.16 2.48–15.27 6.99 2.635–18.59 < 0.001 CI confidence interval, HRCE high risk cardioembolic etiology, MeVO medium vessel occlusion, OR odds ratio 13 1174 J. Juega et al. Samples stored for 24–48 h may be used for immunophenotyp- ing, but time spent in mechanical characterization of clots may provoke losses in absolute cell numbers in FC analysis; these lim- itations should be taken into account for future analysis [19]. We did not employ a specific marker of neutrophils in our analysis, and we assumed that higher percentage of granulocytes was due to higher neutrophils and potentially higher NETS. Dyslipidemia was associated with successful recanalization in our study, but our data was only based on medical records and not on choles- terol levels in blood tests. Future studies to explore the interaction between granulocytes and platelets in intracranial thrombi and their impact on endovascular treatment are warranted. Despite registering the type of device employed, we did not analyze its influence on clot composition variation, so future work should evaluate the physical properties of the clot according to FC com- position to design next-generation EVT devices [21, 34, 35]. Conclusions There is a positive correlation between granulocyte propor- tion and thrombi stiffness that may explain endovascular resistance to recanalization. Influence of granulocytes within thrombus may be a target for future reperfusion treatments. Supplementary Information The online version contains supplemen- tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 13311-0​ 23-0​ 1385-1. Required Author Forms Disclosure forms provided by the authors are available with the online version of this article. Author Contribution All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by Jesus Juega, Jiahui Li, and Carlos Palacio. The first draft of the manuscript was written by Jesus Juega, and all authors commented on previous versions Fig. 3  Correlation between clot stiffness and clot composition by flow of the manuscript. All authors read and approved the final manuscript. cytometry Funding Open Access Funding provided by Universitat Autonoma de Barcelona. This work was supported by “Project 355/C/2017, Fundació There are new sensors designed to discriminate different La Marató de TV3 in Strokes and Traumatic Spinal Cord and Brain Injury, 2017 Call of Projects.” types of thrombus during EVT, but no device has been dem- onstrated yet to improve the decision-making process during Availability of Data and Material The data that support the findings EVT of acute stroke treatment [33]. Future works might focus of this study are available on request from the corresponding author. on the development of a simplified FC analysis that might Code Availability Not applicable. analyze part of the intracranial clot during MT by a point- of-care test might be worthy as a guide for patient-specific Declarations treatment strategy. Moreover, intraarterial blood testing [28] around the clot might potentially offer promising results Ethics Approval The study was approved by the Ethical Committee in without the need for the analysis of the clot itself, but this line with the last Helsinki guidelines. hypothesis should be tested in further studies. Consent to Participate Written informed consent was obtained from This study has several limitations. Clots were not obtained in all participants. every procedure after catheter removal. In fact, in some MT fail- ure cases, no thrombus is collected after many passes. The small Consent for Publication The authors seek consent from individuals to publish their codified data. cohort of patients included in the second study makes the results less robust compared to the global results of the first analysis. 13 Granulocytes‑Rich Thrombi in Cerebral Large Vessel Occlusion Are Associated with Increased… 1175 Conflict of Interest The authors declare no competing interests. consensus statement on present knowledge and promising direc- tions. Stroke. 2020:STROKEAHA120028956. Open Access This article is licensed under a Creative Commons Attri- 15. Ospel J, Goyal M. A review of endovascular treatment for medium bution 4.0 International License, which permits use, sharing, adapta- vessel occlusion stroke. J Neurointerv Surg. 2021;13(7):623–30. tion, distribution and reproduction in any medium or format, as long https://​doi.​org/​10.​1136/​neuri​ntsurg-​2021-​017321. as you give appropriate credit to the original author(s) and the source, 16. 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