Addition of serum-containing medium to cerebrospinal fluid prevents cellular loss over time

Abstract

Immediately after sampling, leukocyte counts in native cerebrospinal fluid (CSF) start to decrease rapidly. As the time lapse between CSF collection to analysis is not routinely registered, the clinical significance of decreasing cell counts in native CSF is not known. Earlier data suggest that addition of serum-containing medium to CSF directly after sampling prevents this rapid decrease in leukocyte counts and, thus, may improve the accuracy of CSF cell counting and cell characterization. Here, we prospectively examined the effect of storage time after lumbar puncture on counts of leukocytes and their major subsets in both native CSF and after immediate addition of serum-containing medium, measured by flow cytometry and microscopy. We collected CSF samples of 69 patients in tubes with and tubes without serum-containing medium and determined counts of leukocytes and subsets at 30 minutes, 1 hour, and 5 hours after sampling. Compared to cell counts at 30 minutes, no significant decrease in cell number was observed in CSF with serum-containing medium 1 and 5 hours after sampling, except for the granulocytes at 1 hour. In native CSF, approximately 50% of leukocytes and all their subsets were lost after 1 hour, both in flow cytometric and microscopic counting. In 6/7 (86%) samples with mild pleocytosis (5–15 × 106 leukocytes/l), native CSF at 1 hour was incorrectly diagnosed as normocellular. In conclusion, addition of serum-containing medium to CSF directly after sampling prevents cell loss and allows longer preservation of CSF cells prior to analysis, both for microscopic and flow cytometric enumeration. We suggest that this protocol results in more accurate CSF cell counts and may prevent incorrect conclusions based on underestimated CSF cell counts.Electronic supplementary materialThe online version of this article (doi:10.1007/s00415-011-5970-8) contains supplementary material, which is available to authorized users.

Flow cytometry for the microscopy of body fluids in patients with suspected infection

“…Conversely some deterioration was seen, even with refrigeration, with samples collected in glass tubes without an anticoagulant 33. For CSFs it has been shown that adding serum could stabilise the cells for 5–24 h,57 58 but this approach is not usually adopted for practical reasons.…”

Flow cytometric characterization of cerebrospinal fluid cells

“…Within 30 minutes after sampling, the CSF cell number decreases significantly (9,14,34,(84)(85)(86). Also, differences in survival rate between different leukocyte subsets were observed: monocytes and granulocytes showed a more rapid cell loss than lymphocytes (9,34,85; Table 3).…”

“…In an earlier study, we showed that addition of serum-containing medium (RPMI-1640 with HEPES, L-Glutamine, Penicillin/Streptomycin, heat-inactivated fetal bovine serum, and heparin) preserves CSF cells until at least five hours after sampling (9). Another study showed that immediate addition of Earle's balanced salt solution with human serum albumin to CSF prevents total WBC loss until at least 24 hour after sampling (86).…”

The addition of RPMI significantly improves the cellularity of cerebrospinal fluid cytology specimens over time

“…This result is consistent with previous studies that used media to preserve cell markers for flow cytometry on CSF specimens. [9][10] Based on these results, we concluded that laboratories such as ours who are not open 24 hours a day, 7 days a week should routinely be adding media to all CSF specimens that cannot be processed immediately. Alternatively, if the cytology laboratory is in a hospital with a hematology laboratory that is open 24 hours, they might consider having the hematology laboratory make the specimen for cytology at the time of receipt.…”

“…8 Studies using CSF and flow cytometry have shown significant cell marker loss (30%) in as little as 4 hours, which can be prevented by the addition of media. 9,10 Media is also used routinely in blood storage and preservation. 11 To clarify the possible role of media in the preservation of CSF cytology specimens, we examined the effect that the addition of media to cytology CSF specimens had on the cellularity of specimens processed 24 hours and 48 hours after receipt.…”