Introduction

Cerebrospinal fluid is a hypotonic fluid with a specific gravity of 1.007. Due to this fact the membrane of the cells within it quickly rupture (Steele et al. 1986). This phenomenon means that accurate counts on CSF have to be done rapidly to ensure that the cells are still intact. This effect can be delayed by refrigerating specimens, but within a matter of days the cells will succumb to the harsh conditions. Different types of cells degenerate at different rates, so not only will cell counts decrease, but the results will bias certain cell types over time. White blood cells degenerate faster than red blood cells, and among white blood cells granulocytes degenerate faster than agranulocytes.

Spinal fluid cell counts are used to aid in the diagnosis of malignancies, meningitis, and hemorrhages, so it is essential that counts are sufficiently accurate to avoid a false diagnosis.

CLSI does not state any specific time limit for processing. But it does state that cellular degeneration can begin within an hour, so testing should be done as soon as possible (“CLSI Guideline, Jan. 2006, Doc. H56-a, Vol. 26, No. 26.” 2006).

In “Application of Fluorescence In Situ Hybridization on Cerebrospinal Fluid Cytospins for the Detection of Residual Leukemic Cells in Patients With Childhood Acute Lymphoblastic Leukemia” by Hwang et al. an image is presented to demonstrate the effects of delayed processing on the cells with in a cerebrospinal fluid specimen.

Papers

We will be looking at two papers that analyze the effects of leaving CSF at room temperature and at 4 °C.

Chow et al.

In the paper “Lysis of Erythrocytes and Leukocytes in Traumatic Lumbar Punctures” whole blood was put into acellular CSF to simulate a traumatic tap. These samples were analyzed in a hemacytometer at various time intervals to find the rate of cell lysis.

It was found that red blood cells had survival of 90% after 24 hours at room temperature and at 4 °C. For white blood cells, at 120 minutes survival was less than 60% at room temperature and around 85% at 4 °C (Chow and Schmidley 1984).

Steele et al.

In the paper “Leukocyte Survival in Cerebrospinal Fluid” by Steele et al. white blood cells of different types were separated and survival was compared after samples were held at room temperature.

Neutrophils were found to have 50% survival after 2 hours, while lymphocytes and monocytes had a 88% and 80% survival rate respectively.

Discussion

Both papers on the topic had similar conclusions on the topic. The low survival rate seen by Chow et al. was likely due to a high percentage of neutrophils in their sample because the cells were sourced from peripheral blood.

How can cell degeneration be reconciled with delays that are frequently present in spinal fluid testing?

In a large hospital settings a 30 minute delay can be expected before the specimen is available to be processed, with longer delays occurring frequently. Once a specimen is received, if processing can not begin immediately consider a method of preserving a portion of the specimen for the cell count. If multiple tubes are sent then save atube used for cell counts in the refrigerator. If only one tube or a syringe is sent then a portion should aliquoted from the original specimen to be used for the cell count. If processing was delayed a comment on the results can be used to qualify the nucleated cell count and differential results.

Storage procedures must be considered holistically, as refrigeration of the entire specimen can eliminate the viability of fastidious organisms in the culture.

For samples that may be used for flow cytometry similar criteria apply as above. If the cells are lysed for the cell count, they will be equally useless for flow cytometric analysis. Further research is required into the survival rate of blast cells. As these samples are often transported to another facility for analysis it is essential that they are refrigerated during transport and analysed within hours of collection as degeneration is rapid even while at 4 °C.

Implementation of such policies is simple on paper, but requires continuous monitoring and proper staffing to ensure that all specimens can be processed immediately.

Bibliography

Chow, George, and James W Schmidley. 1984. “Lysis of Erythrocytes and Leukocytes in Traumatic Lumbar Punctures.” Archives of Neurology 41 (10): 1084–85.

“CLSI Guideline, Jan. 2006, Doc. H56-a, Vol. 26, No. 26.” 2006.

Steele, Russell W, Daniel J Marmer, Marcus D O’brien, Samuel T Tyson, and Christopher R Steele. 1986. “Leukocyte Survival in Cerebrospinal Fluid.” Journal of Clinical Microbiology 23 (5): 965–66.

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