A refined subclavian cardiac puncture method for longitudinal blood collection in mice

Murine blood sampling is essential for pharmacokinetic research and chronic disease modeling. However, conventional techniques such as tail vein or retro-orbital blood collection have low sample volumes and limited frequency, rendering these approaches inadequate for long-term, volume-intensive studies. This study aimed to establish subclavian cardiac puncture (SCP) as a technique for repeated, high-volume blood collection in mice, and to define its maximum safe volume and frequency without compromising systemic physiology. Thirty-six healthy adult C57BL/6 mice (20-30 g, with equal male and female numbers) were randomized into six groups: control, sham, and four experimental cohorts receiving weekly blood removal at 7.5%, 15%, 22.5%, or 30% of total blood volume (TBV) for four weeks (n = 6 per group). Under isoflurane anesthesia, blood collection was performed on mice via SCP. Mice were secured in supine position before puncture, with needle inserted 1-1.5 cm horizontally at the midpoint of left clavicle; slow blood aspiration upon pulsatile blood backflow effectively prevented pneumothorax and cardiac trauma. All syringes and collection tubes were pre-rinsed with EDTA-2K anticoagulant, and TBV was calculated by the standard 72 mL/kg body weight formula to guarantee accurate blood withdrawal proportion. Monitoring indicators included weight, systolic blood pressure, water intake, fur condition, and hematological indices, such as red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Morphological observations of blood smears were also conducted. The results showed that the 30% TBV group tolerated blood collection for two weeks; the 22.5% TBV group for three weeks; and the 7.5% and 15% TBV groups for all four weeks without distress. The 30% TBV group showed the smallest increase in body weight and developed mild anisocytosis at the later stage of the experiment; in this stage, blood pressure decreased compared to the baseline level on day 0 of blood collection, and RBC, HGB, HCT, and MCHC values were below the normal reference ranges on day 21 of blood collection. The 22.5% TBV group also showed a later-stage decrease in blood pressure, with RBC, HGB, and HCT values below the normal reference ranges on day 21 of blood collection. These results suggest that the refined SCP enables safe, repeated, high-volume sampling in mice (with a safe blood collection volume of 15% TBV), supports pharmacokinetic and longitudinal biomarker studies, enhances data reliability, and reduces the number of animals used and stress.