Strong interference of hemoglobin concentration on CSF total protein measurement using the trichloroacetic acid precipitation method.
ABSTRACT Among other methods, trichloroacetic acid precipitation is used to quantify total protein in cerebrospinal fluid (CSF).
We analyzed the influence of hemoglobin on total protein concentration assayed by the trichloroacetic acid method and compared the results to the benzethonium chloride method.
Four CSF samples were spiked with different amounts of hemoglobin, leading to overestimation of protein concentration when assayed by the trichloroacetic acid method. Using the benzethonium chloride method, measurement of protein concentration was minimally disturbed. In addition, albumin and total protein concentrations were measured in 135 clinical samples. The total protein/albumin ratio remained constant when protein was measured with the benzethonium chloride method, while ratios increased when protein was assayed by the trichloroacetic acid method.
Strong interference by hemoglobin leads to overestimation of the total protein concentration in CSF when assayed by the trichloroacetic acid method and may lead to false conclusions when evaluating the blood-brain barrier.
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ABSTRACT: During the past two decades, the comet-based in vitro DNA repair assay has been used regularly to measure base excision repair (BER)-related DNA incision activity. Most studies focus on the assessment of BER in human lymphocytes or cultured cells by estimating the activity of a cell extract on substrate DNA containing specific lesions such as 8-oxoguanine. However, for many 'real-life' studies, it would be preferable to measure BER in the tissues of interest instead of using in vitro models or surrogate 'tissues' such as lymphocytes. Various attempts have been made to use the comet-based repair assay for BER with extracts from rodent tissues, but high non-specific nuclease activity in such tissues were a significant impediment to robust estimates of BER. Our aim in this study was to optimise the in vitro repair assay for BER for use with rodent tissues using extracts from liver and brain from C57/BL mice. Because the DNA incision activity of an extract is dependent on its protein concentration, the first optimisation step in preventing interference by non-specific nuclease activity was to determine the protein concentration at which there is a maximal difference between the total and non-specific damage recognition. This protein concentration was 5 mg/ml for mouse liver extracts and 1 mg/ml for brain extracts. Next, we tested addition of proteinase inhibitors during the preparation of the tissue extracts, but this did not improve the sensitivity of the assay. However, addition of 1.5 μM aphidicolin to the tissue extracts improved the detection of DNA repair incision activity by reducing non-specific nuclease activity and possibly by blocking residual DNA polymerase activity. Finally, the assay was tested on tissue samples from an ageing mouse colony and in mice undergoing dietary restriction and proved capable of detecting significant inter-animal differences and nutritional effects on BER-related DNA incision activity.Mutagenesis 02/2011; 26(3):461-71. · 3.50 Impact Factor
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ABSTRACT: Blood contamination is commonly observed in ventricular cerebrospinal fluid (CSF) samples from patients with extraventricular drainage systems. Because the introduction of blood may interfere with the white blood cell count as a useful marker for the diagnosis of an infection, correction for blood content would be desirable. In a retrospective study, we analysed the use of correction formulas in 724 blood-contaminated ventricular CSF samples. Using a standard correction method the white blood cell count was not normalised in most CSF samples, with pleocytosis indicating an inflammatory stimulus set by the blood itself or by the foreign body. When correcting white blood cell counts in the CSF of culture-positive patients, some samples were normalised or overcorrected. In addition, correction of the CSF white blood cell count did not increase sensitivity and specificity for the detection of culture-positive CSF samples. Correction is not necessary when using the white blood cell count as a parameter to predict CSF infection in ventricular CSF samples.Clinical Chemistry and Laboratory Medicine 02/2008; 46(6):842-8. · 3.01 Impact Factor