Daily blood sampling in septic mice: an optimal and effective monitoring tool

Background

Daily assessment of circulating immune/inflammatory and organ function parameters is strongly advocated in septic patients. In models of murine sepsis, such monitoring becomes challenging given the limited blood volume available for analysis. We studied the influence of daily versus single sampling in acutely (days 1 to 5) septic mice upon their short/long-term survival, organ function and complete blood count (CBC). We additionally tested the reliability of CBC differential in resuspended cell pellet versus whole blood analysis.

Methods

Seventy-four female OF-1 mice (18 to 21 g body weight) were subjected to cecal ligation and puncture (CLP). Blood sampling volumes (by facial vein puncture) of 35 μl (n = 40) and 20 μl (n = 34) were tested. The samples were immediately diluted 1:10 to a final volume of either 350 μl (group 1) or 200 μl (group 2). Half of each group was sampled either daily for 5 days or only on day 5 post CLP. For comparison of resuspended versus regular CBCs, 150 μl (of the original 350 μl) was analyzed immediately after sampling. The remaining 200 μl was then spun, plasma removed (180 μl), the cell pellet re-suspended with an equal volume of the diluent and CBC performed.

Results

Repetitive daily bleeding, regardless of the volume, did not affect either short-term (5 days) or long-term (28 days) CLP mortality. By day 5, changes between groups in the level of circulating IL-6, IL-1 receptor antagonist and organ function/metabolic parameters (ALT, LDH, glucose and urea) were identical. In group 1 (35 μl), the red blood cell (RBC) count was reduced by 22% while the hemoglobin (Hb) concentration decreased by 23% (both P < 0.05). However, only a minimal decrease of RBC and Hb by 10% and 11%, respectively (both P < 0.05), was observed in group 2 (20 μl). In neither group were platelet or white blood cell counts affected by repetitive bleeding. Except for lymphocytes, the comparison of regular and resuspended CBCs displayed a high correlation for all cell types (r > 0.9, slope > 0.9). On each post-CLP day, the lymphocytes correlation remained moderate, reaching r = 0.6 (slope = 0.6) on average (days 1 to 5). This effect was reproduced when tested in non-CLP OF-1 mice (n = 12) at 1:2 dilution (r = 0.5, slope = 0.7).

Conclusion

Although we noted a statistically significant (and inversely proportional) decrease in RBC and Hb after repetitive daily bleeding, its biological impact was probably marginal. Differential blood analysis in resuspended pellet was highly reliable for all (except lymphocytes) cell populations tested. The results indicate that low-volume daily blood sampling allows a multi-directional and minimally invasive monitoring of various immunoinflammatory parameters in acutely septic mice.