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.
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.
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).
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.