Spectrophotometry for Cerebrospinal Fluid Pigment Analysis

Abstract

The use of spectrophotometry for the analysis of the cerebrospinal fluid (CSF) is reviewed. The clinically relevant CSF pigments -oxyhemoglobin and bilirubin -are introduced and discussed with regard to clinical differential diagnosis and potentially confounding variables (the four "T"s or: traumatic tap, timing, total protein and total bilirubin). The practical laboratory aspects of spectrophotometry and automated techniques are presented in the context of analytical and clinical specificity and sensitivity. The perceptual limitations of human color vision are highlighted and the use of visual assessment of the CSF is discouraged in light of recent evidence from a national audit in the United Kingdom. Finally, future perspectives including the need for longitudinal CSF profiling and routine spectrophotometric calibration are outlined.

Cerebrospinal fluid analyses for the diagnosis of subarachnoid haemorrhage and experience from a Swedish study. What method is preferable when diagnosing a subarachnoid haemorrhage?

“…This does not occur in vitro in tapped CSF (except in samples with very high white blood cell count), which strongly reduces the risk for false-positive results caused by a traumatic tapping. Bilirubin has a broad maximum absorption peak in the blue region of the optical spectrum at 450-460 nm [28]. On spectrophotometry of an acute bleeding, where oxyhaemoglobin is still present in high concentrations, bilirubin may be visible as a gentle slope to the right of the sharp oxyhaemoglobin peak ( Figure 1B).…”

“…It is a rapid ( < 1 min) technique that requires a small sample amount (50-500 µL) [28]. Because sensitivity and specificity vary for different markers that may be elevated depending on the duration since symptom onset, the laboratory technician must know the time point of symptom debut, sampling, and centrifugation to perform a sound interpretation (Table 1).…”

“…This process starts shortly (2-4 h) after the bleeding, and oxyhaemoglobin can be detected in CSF from approximately 2-12 h after ictus [34]. Oxyhaemoglobin, the oxygenated form of haemoglobin, has an absorbance maximum around 415 nm, in the violet region of the optical spectrum ( Figure 1A) [28]. Minor secondary absorbance peaks, sometimes referred to as Soret bands, may be detected at 540 and 580 nm, in the green region of the optical spectrum.…”

“…Bilirubin may be false positive due to hyperbilirubinemia (a serum sample should therefore be taken together with CSF to allow adjustment for this), significant bloodbrain barrier damage, malignancies, or treatment with rifampicin [28,64].…”

Pneumatic tube transport of blood-stained cerebrospinal fluid specimens has no clinically relevant effect on rates of haemolysis compared to manual transport

“…This process takes at least 6 h and up to 15 h to form post bleed. 2,[5][6][7][8][9] Hence, current advice is to wait at least 6 h and preferably 12 h after the onset of headache before performing lumbar puncture to investigate suspected SAH. 8 Mechanical haemolysis of red blood cells in collected CSF specimens may also release oxyhaemoglobin but bilirubin is not produced due to the absence of in vivo enzyme systems.…”