Comparative Analysis of Cardiac Puncture and Perfusate Blood Collection for Murine Extracellular Vesicle Isolation

Abstract

Reliable characterization of circulating extracellular vesicles (EVs) in rodents may be significantly influenced by how blood is collected, yet systematic comparisons of commonly used sampling methods remain limited. Here, we directly evaluate the effects of cardiac puncture and perfusate blood collection on EV yield and surface-marker profiles in naïve mice, as well as in mice subjected to neurotrauma using a contusion spinal cord injury (SCI) model. Using matched isolation procedures and MACSPlex immunophenotyping, we analyzed newly generated cardiac puncture plasma alongside previously published perfusate-derived datasets, with both cohorts matched for age, sex, weight, injury severity, and post-injury timepoint. Cardiac puncture produced substantially higher particle concentrations due to access to undiluted blood, whereas perfusate samples exhibited modest increases in select markers, such as CD9, consistent with method-associated influences on platelet-derived vesicles. Despite these quantitative differences, both approaches yielded broadly similar EV phenotypes, and SCI-associated marker patterns remained stable across sampling methods. The consistency between cardiac puncture and perfusate datasets validates the robustness of our earlier perfusate-based findings and demonstrates that key biological signatures are preserved regardless of collection technique. These results provide practical guidance for optimizing murine EV studies and underscore the importance of methodological transparency and standardization in preclinical EV research.

1. Introduction

Extracellular vesicles (EVs) are critical mediators of intercellular communication and promising therapeutic agents as well as biomarkers across a wide range of physiological and pathological conditions [1,2,3,4,5,6,7]. Murine models provide a genetically tractable and physiologically relevant system for studying EV biology in vivo, enabling controlled investigation of EV dynamics in health and disease [8,9,10,11]. However, the limited blood volume obtainable from individual mice poses a significant technical constraint, restricting the amount of plasma available for EV isolation as well as for parallel biochemical, molecular, or pharmacological analyses [12,13]. This necessitates careful optimization of blood collection and sample preparation procedures to maximize yield, reproducibility, and analytical sensitivity [14,15,16].

Several techniques are commonly employed for mouse blood collection, each with distinct advantages and limitations depending on the study context. Cardiac puncture is a terminal blood collection method where a needle is inserted through the chest wall directly into the heart to quickly withdraw a large volume of undiluted blood. The total circulating blood volume in mice is generally about 6–8% of the animal’s body weight, and cardiac puncture typically yields approximately 50–75% of this total volume [17,18,19]. Perfusate blood collection, obtained via vascular perfusion, is a specialized technique designed to maximize total fluid recovery by washing the vasculature with buffer solution, thereby providing a larger volume sample [20]. Other common non-terminal blood collection methods in mice, such as submandibular, saphenous, and tail vein bleeding, typically yield smaller volumes of blood compared to terminal procedures [21].

Following collection, whole blood samples undergo further processing to isolate plasma, which serves as the starting material for EV isolation and analysis. Blood is typically anticoagulated immediately upon collection to prevent clotting, then centrifuged to separate plasma from cellular components. The isolated plasma fraction contains circulating EVs along with other soluble factors, making the initial volume and quality of whole blood critical for maximizing plasma yield. Consequently, the choice of blood collection method directly influences the amount and integrity of plasma obtained and thus impacts downstream EV recovery and characterization [22].

In this study, we leverage previously published perfusate-derived plasma EV data following spinal cord injury (SCI) in mice alongside newly generated cardiac puncture samples to systematically compare the two blood collection methodologies under matched experimental conditions (Figure 1) [9]. Using MACSPlex immunophenotyping as our primary analytical platform, we assessed how the choice of sampling method influences detectable EV surface marker profiles within the context of SCI-associated systemic inflammation. This work aims to provide evidence-based recommendations to support methodological standardization and improve the quality of murine EV research.

2. Materials and Methods

2.1. Animals

Adult female C57BL/6J mice (the Jackson Laboratory, Bar Harbor, ME, USA) were used. All housing conditions, husbandry procedures, and SCI methods are described in our previous publication [9]. For the present study, cardiac puncture samples were collected from mice euthanized at approximately 180 days of age. Perfusate-derived plasma EV data were obtained from our previously published dataset, which was generated from age-, sex-, weight-, and strain-matched female C57BL/6J mice euthanized at the same approximate age. Both cohorts underwent an identical injury paradigm consisting of a 50 kdyne thoracic contusion at the thoracic T8 level and were analyzed at the same post-injury timepoint corresponding to the subacute phase (7 days post-injury). All procedures were approved by the University of Miami Institutional Animal Care and Use Committee (Protocol #22-099).

2.2. Thoracic Contusion Injury

A moderate thoracic contusion SCI was generated using a 50 kdyne impact force. Adult female C57BL/6J mice were anesthetized, and a midline incision was made to expose the dorsal vertebral column. The paraspinal muscles were gently separated to reveal the T8 vertebra, where a laminectomy was performed to expose the dorsal surface of the spinal cord without disrupting the underlying dura. The vertebral column was stabilized using clamps positioned rostral and caudal to the laminectomy site, and a 50 kdyne impact was delivered using the Infinite Horizon impactor under automated force control. All impact parameters were recorded by the device to ensure consistency across animals [23]. Following injury, musculature and skin were closed in layers, and mice received standard postoperative care including hydration support and manual bladder expression. Animals survived 7 days post-injury, representing the subacute phase of SCI in this model. At the 7-day endpoint, mice were euthanized, and blood was collected for downstream EV isolation.

2.3. Blood Collection

Perfusate-derived plasma samples were obtained from our previously published dataset [9]. Briefly, animals were euthanized via CO₂ inhalation followed by thoracotomy. A blunt-tipped needle was inserted into the left ventricle and advanced toward the ascending aorta to enable transcardial perfusion with sterile 0.9% saline. The perfusion protocol was standardized across all animals, delivering a total volume of 4 mL at a constant flow rate of 8 mL/min to ensure consistent vascular clearance. Perfusate blood was collected into 0.5 M EDTA-coated collection dishes using S-Monovette^® 1.2 mL blood collection syringes (22G; Sarstedt, Nümbrecht, Germany) and immediately placed on ice, typically yielding approximately 3 mL of blood after centrifugation. No new perfusate samples were generated for the present study. For cardiac puncture samples, whole blood was collected immediately post-euthanasia by direct cardiac puncture without perfusion. A 1.2 mL K₃EDTA-coated syringe fitted with a 22G blunt needle was inserted into the left ventricle, and blood was aspirated slowly to avoid ventricular collapse, typically yielding approximately 0.5 mL of blood after centrifugation.

2.4. Platelet-Free Plasma Preparation

Blood samples were centrifuged at 1000× g for 15 min at 4 °C to separate plasma from cellular components. The upper plasma layer was carefully transferred to a clean microcentrifuge tube and centrifuged again at 1500× g for 15 min at 4 °C to obtain platelet-free plasma, which was subsequently stored at −80 °C until EV isolation.

2.5. EV Isolation

EV isolation was performed according to previously described protocols [9]. Thawed platelet-free plasma was centrifuged at 14,000× g for 35 min at 4 °C to remove residual debris. Plasma was loaded onto qEVoriginal 70 nm size-exclusion chromatography columns (Izon Science, Christchurch, New Zealand) pre-equilibrated with sterile PBS. Fractions were collected in pre-chilled microcentrifuge tubes. EV-rich fractions, typically eluting between fractions 7 and 10 based on nanoparticle tracking analysis (NTA) and concentrated using Vivaspin^® 2 centrifugal filters (5 kDa molecular weight cut-off; Sartorius, Göttingen, Germany).

2.6. Nanoparticle Tracking Analysis

EV size and concentration were determined using a NanoSight NS300 instrument (Malvern Panalytical, Malvern, UK). Samples were diluted in sterile 0.22 μm-filtered PBS to achieve an optimal particle concentration of 100–300 particles per frame. Five technical replicates per sample were acquired at room temperature, with 30 frames captured per replicate. Data were processed using NanoSight NTA software (v3.4), and results were reported as mean particle concentration (particles/mL) and modal particle diameter (nm). Total particle yield per animal was calculated by multiplying the particle concentration (particles/mL) by the total plasma volume obtained from each collection method (cardiac puncture: ~0.5 mL; perfusate blood: ~3 mL).

2.7. Multiplex Bead-Based Flow Cytometric Analysis of EV Surface Proteins by MACSPlex Exosome Kit Mouse

EV surface proteins were analyzed using the MACSPlex Exosome Kit, Mouse (Miltenyi Biotec, Bergisch Gladbach, Germany) according to previously described methods [9]. SEC-isolated EVs were adjusted to 1 × 10⁹ particles/mL and incubated with antibody-coated capture beads under gentle agitation at room temperature for one hour. Fluorochrome-conjugated detection antibodies against the tetraspanins CD9, CD63, and CD81 were used to confirm successful EV binding. Following washes, bead populations were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) and gated based on their fluorescence profiles in the PE and FITC channels. Data were processed using FlowJo v10.10.0. Background fluorescence from buffer-only controls was subtracted, and CD9 expression was used for normalization.

2.8. Statistical Analysis

All statistical analyses were performed using GraphPad Prism v10. Normality was assessed using the Shapiro–Wilk test. Comparisons between blood collection methods were performed using unpaired t-tests with Welch’s correction for parametric data or Mann–Whitney U tests for non-parametric data. For comparisons involving two independent variables, two-way ANOVA followed by Tukey’s post hoc test was applied. Cardiac puncture groups comprise n = 4 biological replicates per group. Perfusate-derived data were obtained from a previously published dataset comprising n = 4 biological replicates for the naïve group and n = 3 for the 50 kdyne SCI group. For MACSPlex flow cytometric analyses, all biological replicates were acquired in technical duplicate on the flow cytometer. All statistical tests are specified in the figure legends. Significance levels are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

3. Results

3.1. Cardiac Puncture Yields Higher Particle Concentration but Comparable Total Particle Recovery

To evaluate the impact of blood collection methodology on plasma-derived EV yield, we compared cardiac puncture and perfusate blood collection using NTA. Particle concentration analysis revealed that cardiac puncture consistently produced significantly higher concentrations than perfusate blood collection in both naïve and injured animals. In naïve mice, cardiac puncture yielded a mean concentration of 6.47 × 10⁹ particles/mL compared to 2.09 × 10⁹ particles/mL for perfusate blood, representing approximately a 3.1-fold increase (p < 0.01) (Figure 2a). Similarly, in 50 kdyne SCI animals, cardiac puncture demonstrated a mean concentration of 6.93 × 10⁹ particles/mL compared to 1.80 × 10⁹ particles/mL for perfusate blood, a 3.9-fold increase in particle concentration (p < 0.05) (Figure 2b). No significant differences in particle size distribution were observed between collection methods in either group (Supplementary Figure S1). A subset of perfusate samples in the previously published dataset yielded particle concentrations below the minimum threshold required for reliable MACSPlex quantification (1 × 10⁹ particles/mL) and were therefore excluded from surface marker analysis as previously described. All cardiac puncture samples produced measurable EV concentrations. However, when accounting for the total plasma volume obtained by each method (cardiac puncture: ~0.5 mL; perfusate: ~3 mL), total particle yield per animal showed a trend toward higher recovery with perfusate blood collection in both naïve and 50 kdyne SCI groups, though these differences did not reach statistical significance (Figure 2c,d).

3.2. Perfusate Blood Collection Enriches CD9 Expression in Naïve EVs

To characterize the tetraspanin composition of EVs isolated by each method, we performed MACSPlex analysis targeting the canonical EV markers CD81, CD63, and CD9. In both naïve and 50 kdyne SCI groups, CD9 was the most abundantly detected tetraspanin, consistent across both collection methods (p < 0.0001). Notably, perfusate blood-derived EVs exhibited significantly higher CD9 expression compared to cardiac puncture samples in the naïve group (p < 0.001) (Figure 3a). However, no significant difference in CD9 expression was observed between collection methods in the 50 kdyne SCI group (Figure 3b). CD81 and CD63 were detected at substantially lower levels across all samples, with no significant differences between collection methods. Comprehensive MACSPlex profiling across all 39 surface markers revealed that both collection methods produced qualitatively similar binding profiles, with the same subset of markers consistently detected across naïve and 50 kdyne SCI groups (Figure 3c,d).

3.3. Cardiac Puncture-Derived EVs Exhibit Higher Prominin-1 Expression in Naïve Mice

To enable direct comparison of EV surface marker profiles between cardiac puncture and perfusate blood collection methods, MACSPlex intensities were normalized to CD9, the most abundant tetraspanin detected across all samples. This approach accounted for differences in particle concentration and baseline tetraspanin levels, facilitating accurate assessment of relative marker expression between collection methods (Figure 4a,b). Comparative surface marker profiling revealed largely consistent expression patterns between the two collection methods, with the majority of markers showing no significant differences. Volcano plot analysis identified Prominin-1, CD41, and CD11b as markers surpassing the differential expression threshold (Figure 4c). CD11b displayed low mean expression intensity (<0.2) and was excluded from interpretation to avoid overrepresenting background noise (Supplementary Figure S2a). Prominin-1 remained a key marker of differential expression, with quantitative analysis revealing 2.6-fold higher levels in cardiac puncture-derived EVs compared to perfusate blood-derived EVs from naïve mice (p < 0.01), a finding reproduced and validated upon inclusion of the fourth biological replicate collected on a separate experimental day (Figure 4d). Conversely, CD41 was significantly elevated in perfusate-derived naïve EVs compared to cardiac puncture samples, with quantitative analysis revealing 2.3-fold higher levels in perfusate-derived EVs (p < 0.05) (Figure 4d).

3.4. Surface Marker Profiles Remain Consistent Between Collection Methods in 50 Kdyne Subacute SCI Mice

To assess whether the collection method-dependent differences observed in naïve animals were maintained following SCI, MACSPlex analysis was performed on plasma-derived EVs from 50 kdyne SCI mice (thoracic T8 contusion injury using the Infinite Horizons impactor) collected via both cardiac puncture and perfusate blood methods. Surface marker intensities were normalized to CD9 to control for variations in particle number and tetraspanin content, enabling direct comparison between collection approaches (Figure 5a,b). In contrast to naïve animals, comparative profiling of 50 kdyne SCI samples revealed no significant differences in surface marker expression between the two collection methods. Volcano plot analysis identified CD2 as the only marker exceeding the differential expression (Figure 5c). However, CD2 displayed minimal baseline signal (<0.2) and was therefore excluded to prevent misinterpretation of low-abundance events (Supplementary Figure S2b). Additionally, direct comparison between naïve and 50 kdyne SCI groups within cardiac-puncture-derived samples revealed no significant injury-induced changes in surface marker profiles (Supplementary Figure S3).

4. Discussion

This study addresses a critical methodological challenge in murine EV research regarding how blood collection techniques influence EV yield and characterization. Specifically, we provide a systematic evaluation comparing cardiac puncture and perfusate blood collection methods and their impact on particle yield, tetraspanin abundance, and EV surface-marker profiles under both homeostatic and SCI conditions. By integrating newly generated cardiac puncture data with previously published perfusate-derived EV datasets, we directly assess methodological differences and validate the robustness of prior findings under matched experimental conditions [9].

Although this study integrates newly collected cardiac puncture samples with previously generated perfusate-derived datasets, several steps were implemented to minimize batch-related variability and ensure biological comparability. Both datasets were produced under closely matched experimental conditions, including identical mouse strain, sex, age, injury severity (50 kdyne), and post-injury survival period (7 days). EV isolation, NTA, and MACSPlex analyses were performed by the same personnel using the same protocols, instrument settings, and reagent lots whenever possible [14,24]. All instruments were calibrated using identical standards, and data processing followed an unchanged analytical workflow. A uniform QC threshold of 1 × 10⁹ particles/mL for MACSPlex analysis, as recommended by the manufacturer, was applied consistently across both datasets prior to downstream analysis, and CD9 normalization was applied uniformly to control for differences in particle input and tetraspanin abundance between datasets. These measures substantially reduce the likelihood of technical drift between datasets and support the interpretation that observed differences between blood collection methods primarily reflect methodological or biological effects rather than batch artifacts. Nevertheless, the cross-dataset design represents an inherent limitation of this study, as samples were not collected simultaneously, and the possibility of residual batch-related variation cannot be completely excluded.

Consistent with prior studies, our data underscore the pronounced effects of blood collection technique on EV particle concentration and total yield, emphasizing the need for methodological standardization [25]. Cardiac puncture yielded substantially higher particle concentrations compared to perfusate blood collection, likely reflecting the recovery of undiluted blood and the avoidance of dilution effects inherent to perfusion-based sampling. Notably, several samples in the previously published perfusate-derived dataset fell below the minimal particle threshold required for reliable MACSPlex analysis, resulting in the exclusion of seven samples from downstream quantification [9], whereas all cardiac puncture samples exceeded this threshold and were successfully analyzed. These observations highlight a critical distinction between particle concentration, absolute recovery, and practical assay utility, while cardiac puncture produces more concentrated samples and improves assay success rates, perfusion compensates through larger volumes, potentially mobilizing additional EV populations from the vasculature. Other studies have similarly reported that inferior vena cava puncture under continuous cardiac perfusion yielded approximately 2.1 times more undiluted blood than standard cardiac puncture without altering cellular composition or viability [20]. It should be noted, however, that this blood was not processed into plasma or plasma-enriched EVs, and downstream applicability may therefore differ.

Our analysis further revealed that CD9, a tetraspanin widely recognized as a key platelet EV marker, was the most abundantly detected marker across both collection methods and animal groups. Because MACSPlex assays were performed using an equal particle input for all samples, the higher CD9 signal detected in perfusate-derived EVs from naïve mice indicates a methodological enrichment of CD9⁺ vesicles relative to total particle number, rather than differences in overall EV abundance. This pattern suggests that perfusion may selectively increase the proportion of platelet-derived vesicles potentially through mechanical shear, altered vascular flow, or dislodgement of platelet aggregates during the flushing process mechanisms, that are well documented to promote platelet activation and EV shedding [26,27]. It should also be noted that the anticoagulant type and concentration differed between methods, with 0.5 M EDTA in collection dishes for perfusate and K₃EDTA in syringes for cardiac puncture, which could influence platelet activation or vesicle release and may contribute to the observed differences in CD9 signal [28].

Beyond platelet-associated markers, Prominin-1 (CD133), a pentaspan membrane glycoprotein and stem/progenitor cell marker, was significantly elevated in EVs isolated from naïve cardiac blood plasma compared to naïve perfusate plasma EVs [29]. Prominin-1 has been reported to actively induce EV formation by binding cholesterol-enriched regions of the plasma membrane and remodeling membrane curvature at specialized protrusions such as microvilli [30]. Its interaction with cholesterol is critical for EV biogenesis, influencing vesicle morphology and stability, and mutations affecting this interaction substantially reduce EV production. While blood collection techniques influence our measurements, biological variables independent of collection method, such as systemic lipid metabolism, dietary cholesterol intake, or cellular membrane composition, may also modulate Prominin-1-mediated EV formation and stability [31,32,33].

For comparisons in the subacute SCI model at a severity of 50 kdyne, surface marker expression profiles of plasma-derived EVs were consistent between cardiac puncture and perfusate collection methods. Additionally, when comparing naïve and 50 kdyne SCI samples within each collection method independently, neither approach detected significant injury-induced alterations in surface marker profiles, demonstrating consistency across methodologies. These observations align with our prior work characterizing circulating EV dynamics after different severities of SCI, which identified prominent platelet-associated vesicle signatures and a characteristic tetraspanin profile marked by abundant CD9 and low CD63 and CD81 detection. In this study, those same features were preserved regardless of plasma collection method, suggesting that SCI-driven biological changes, rather than blood collection technique, are the primary determinants of the observed EV phenotypes.

While cardiac puncture provided significantly higher particle concentration, total particle yield per animal was comparable between both collection methods. This stability in total particle recovery, combined with consistent surface-marker profiles across methods, reinforces the distinction between EV concentration and EV phenotype, supporting the robustness of previously reported SCI-associated EV signatures. Consequently, our data affirm that perfusate collection remains a reliable and robust method within this pathological context, preserving confidence in prior and future biomarker investigations using this technique. While both methods yield biologically consistent EV profiles in the context of SCI, validating the robustness of our previously published perfusate-derived findings, several practical considerations favor cardiac puncture for future studies. The higher particle concentration achieved through cardiac puncture reduces concerns about dilution-related artifacts that could influence downstream assays, particularly when analyzing low-abundance EV cargo or detecting subtle biological differences between experimental groups. An additional practical advantage of cardiac puncture relates to assay success rate. In our prior perfusate-derived datasets, several samples fell below the minimum particle concentration required for reliable MACSPlex quantification, resulting in the exclusion of seven samples from downstream analysis [9]. In contrast, all cardiac puncture samples in the current dataset exceeded this concentration threshold and were successfully analyzed. This difference highlights a key limitation of perfusate blood collection, dilution during perfusion increases the likelihood of samples dropping below assay detection limits, thereby reducing usable sample numbers and potentially introducing bias. The consistently higher concentration obtained via cardiac puncture mitigates this issue, improving assay reliability and overall data retention.

Beyond phenotypic characterization, circulating plasma-derived EVs from SCI animals have been shown to exert measurable functional effects in vitro and in vivo, underscoring the biological relevance of the EV populations characterized in the present study. Plasma EVs isolated from SCI mice induced pro-inflammatory cytokine secretion and neuronal apoptosis in vitro, and systemic administration of these EVs was sufficient to impair neurological function in intact recipient animals, implicating circulating EVs as active mediators of secondary injury cascades following neurotrauma [34]. Consistent with this, circulating plasma EVs derived from septic mice have similarly been shown to selectively activate microglia in vitro in a dose-dependent manner, with conditioned media from EV-treated microglial cultures subsequently inducing neuronal apoptosis, and intracerebroventricular administration of these EVs provoking a marked innate immune response in the brain in vivo [35]. Together, these findings highlight the broader importance of circulating EV phenotype and subpopulation composition in determining downstream biological activity. While functional characterization of plasma EVs was beyond the scope of the present methodological study, future investigations should examine whether the collection method-dependent differences in EV subpopulation composition observed here, particularly the enrichment of platelet-derived vesicles in perfusate samples, translate into measurable differences in biological potency. Such studies would likely require pooling of samples across animals to achieve sufficient EV quantities for replicated functional assays, as has been demonstrated in prior plasma EV functional studies [34].

Overall, our findings demonstrate that although cardiac puncture consistently provides higher particle concentration, both cardiac puncture and perfusate blood collection produce broadly similar EV populations, with only modest quantitative differences whose magnitude depends on physiological state. The consistency between these newly generated cardiac puncture datasets and our previously published perfusate-derived EV profiles further validates the robustness of the earlier findings and highlights the reproducibility of core biological signatures across sampling methods. These results underscore the importance of methodological transparency and standardization in murine EV research, as differences in blood collection can influence specific readouts without altering global EV phenotypes [13,16]. Nevertheless, several limitations should be considered when interpreting the broader applicability of these findings. This study was conducted under a single biological context, specifically female C57BL/6J mice at 7 days post-SCI and should therefore be interpreted as a controlled methodological comparison rather than a comprehensive evaluation across sex, strain, or injury stage. Furthermore, our analyses focused on EV recovery and surface-marker phenotyping and did not assess biological function, meaning the present data do not establish functional equivalence between EV preparations obtained by the two collection methods. This is particularly relevant given that plasma EV isolates contain vesicles from multiple cellular sources, and assay-dependent outcomes such as cellular uptake or inflammatory signaling may vary by recipient cell type and experimental conditions. Future studies should extend this comparison across sexes and post-injury timepoints and incorporate appropriately powered functional testing to determine how collection method influences downstream biological readouts.

5. Conclusions

In summary, this study systematically evaluates how blood collection techniques influence the quantitative and phenotypic properties of plasma-derived EVs in both naïve and SCI mice. Although cardiac puncture consistently yielded higher particle concentrations, both cardiac puncture and perfusate methods achieved comparable total particle recovery per animal and elicited similar surface-marker profiles, indicating that global EV phenotypes remain stable across sampling approaches. Several perfusate-derived samples in the previously published dataset fell below the minimal particle threshold required for reliable MACSPlex analysis and were excluded from downstream quantification, whereas all cardiac puncture samples were successfully analyzed, highlighting a practical advantage of cardiac puncture in retaining usable samples. The cross-validation performed in this study, comparing newly generated cardiac puncture data with previously published perfusate-derived EV datasets, confirms the reproducibility and robustness of the earlier findings. This demonstrates that the biological signatures identified previously are valid, even across different blood collection methods, and reinforces confidence in the interpretability of prior perfusate-based studies. While methodological choices, particularly regarding blood acquisition, can introduce measurable differences in EV concentration, yield, and specific marker abundance, these did not obscure biologically driven injury effects, underscoring the reliability of perfusate-based sampling within SCI studies. At the same time, cardiac puncture offers several practical advantages for future murine EV research. Higher particle concentration supports assays requiring high particle density, and the shorter procedure time and reduced technical complexity facilitate implementation across research settings. Together, these findings provide a framework for selecting appropriate blood collection techniques in murine EV research. While our findings are specific to female C57BL/6J mice at the 7-day subacute SCI timepoint, they demonstrate that, despite the practical advantages of cardiac puncture, prior perfusate-based studies remain valid and biologically informative. Additional studies across different experimental contexts would further benefit this area of method development. Clarifying how sampling strategies shape EV readouts will strengthen the interpretability of preclinical biomarker studies and support reproducible pipelines for translational EV research.