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Paraprotein interference with turbidimetric gentamicin assay.

Dimeski G, Bassett K, Brown N - Biochem Med (Zagreb) (2015)

Bottom Line: A second sample was received and analysed on a Roche Cobas system to obtain a result.To be able to provide more rapid results it was shown ethanol could be used as a precipitant of proteins in both calibrators and patient samples with acceptable recovery.IgM paraprotein was identified as the cause of interference with the gentamicin, vancomicin and valproate assays.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Pathology, Pathology Queensland, Princess Alexandra Hospital, Brisbane, Australia.

ABSTRACT

Introduction: Gentamicin due to its low level of resistance and rapid bactericidal activity is commonly used to treat gram-negative bacteria. However, due to its toxic effects it needs to be monitored. To date, no interference has been reported with gentamicin assays.

Materials and methods: A patient with leg cellulitis and sepsis received a single dose of gentamicin and a sample was sent for gentamicin analysis. The sample showed high blank absorbance readings on Beckman DxC800 and DC800 analysers with various dilutions. A second sample was received and analysed on a Roche Cobas system to obtain a result. A third sample was received 107 hours later with the same results and this sample was then analysed neat and post ethanol precipitation on all the turbidimetric assays available on the DxC800 analyser.

Results: The high blank absorbance was observed upon addition of the reactive reagents due to protein precipitation. Although not obvious from the patient protein results, it was shown the presence of high IgM paraprotein, 18.9 g/L (reference range 0.4-2.3 g/L) was the cause of precipitation, giving high blank readings. Of all the other turbidimetric assays, only vancomicin and valproate showed similar high blank absorbance readings. To be able to provide more rapid results it was shown ethanol could be used as a precipitant of proteins in both calibrators and patient samples with acceptable recovery.

Conclusion: IgM paraprotein was identified as the cause of interference with the gentamicin, vancomicin and valproate assays. Protein interference in these assays can be overcome by precipitation with ethanol.

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Related in: MedlinePlus

Reaction absorbance curves for 1. Gentamicin: A- normal sample, B -case study neat sample, C-case study 1/20 diluted sample; 2. Vancomicin: D- normal sample, E- case study neat sample; 3. Valproate: F- normal sample, G- case study neat sample.
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f1: Reaction absorbance curves for 1. Gentamicin: A- normal sample, B -case study neat sample, C-case study 1/20 diluted sample; 2. Vancomicin: D- normal sample, E- case study neat sample; 3. Valproate: F- normal sample, G- case study neat sample.

Mentions: A 93 year old female with severe dementia presented with leg cellulitis and sepsis and was administered 320 mg gentamicin (Pfizer, Perth, Australia) at one of our smaller hospitals. No further gentamicin was administered during her hospital stay. On presentation the patient’s sample was analysed on a Beckman DxC600 general chemistry analyser (Beckman Coulter, Brea, CA, USA) as per Beckman Coulter recommendations and the results were: creatinine 177 µmol/L (reference range (RR) 46-108), urea 12.0 mmol/L (RR 2.9-8.2), total protein 63 g/L (RR 60-83), albumin 22 g/L (RR 35-50), and globulins 41 g/L (RR 25-45). A blood sample was collected ~40 hours post gentamicin administration and the laboratory could not obtain a result due to persistent high blank absorbance errors on the Beckman DxC600 general chemistry analyzer (Beckman Coulter, Brea, CA, USA). The sample was diluted with normal saline with ratios starting from 1/3 and going as high as 1/20. The sample was then referred to our laboratory and dilutions were repeated on a Beckman DxC800 general chemistry analyzer using the exact same method. The absorbance error could not be eliminated to obtain a result as shown by the absorbance curves in Figure 1. A second sample was then collected at ~50 hours post gentamicin administration and this was dispatched for analysis on a Roche Cobas system (Roche Diagnostics, Mannheim, Germany) and the result was 3.3 mg/L. No further gentamicin was administered. Review of the absorbance curves suggested an interference related problem. Although the total protein level did not suggest presence of paraproteins the globulins level was high enough not to rule out the presence of paraproteins. The sample was then diluted off board with the reagent A (reaction buffer) in the ratio used in the method and no precipitation was observed. Unfortunately by this stage due to limited volume no further tests could be conducted.


Paraprotein interference with turbidimetric gentamicin assay.

Dimeski G, Bassett K, Brown N - Biochem Med (Zagreb) (2015)

Reaction absorbance curves for 1. Gentamicin: A- normal sample, B -case study neat sample, C-case study 1/20 diluted sample; 2. Vancomicin: D- normal sample, E- case study neat sample; 3. Valproate: F- normal sample, G- case study neat sample.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401306&req=5

f1: Reaction absorbance curves for 1. Gentamicin: A- normal sample, B -case study neat sample, C-case study 1/20 diluted sample; 2. Vancomicin: D- normal sample, E- case study neat sample; 3. Valproate: F- normal sample, G- case study neat sample.
Mentions: A 93 year old female with severe dementia presented with leg cellulitis and sepsis and was administered 320 mg gentamicin (Pfizer, Perth, Australia) at one of our smaller hospitals. No further gentamicin was administered during her hospital stay. On presentation the patient’s sample was analysed on a Beckman DxC600 general chemistry analyser (Beckman Coulter, Brea, CA, USA) as per Beckman Coulter recommendations and the results were: creatinine 177 µmol/L (reference range (RR) 46-108), urea 12.0 mmol/L (RR 2.9-8.2), total protein 63 g/L (RR 60-83), albumin 22 g/L (RR 35-50), and globulins 41 g/L (RR 25-45). A blood sample was collected ~40 hours post gentamicin administration and the laboratory could not obtain a result due to persistent high blank absorbance errors on the Beckman DxC600 general chemistry analyzer (Beckman Coulter, Brea, CA, USA). The sample was diluted with normal saline with ratios starting from 1/3 and going as high as 1/20. The sample was then referred to our laboratory and dilutions were repeated on a Beckman DxC800 general chemistry analyzer using the exact same method. The absorbance error could not be eliminated to obtain a result as shown by the absorbance curves in Figure 1. A second sample was then collected at ~50 hours post gentamicin administration and this was dispatched for analysis on a Roche Cobas system (Roche Diagnostics, Mannheim, Germany) and the result was 3.3 mg/L. No further gentamicin was administered. Review of the absorbance curves suggested an interference related problem. Although the total protein level did not suggest presence of paraproteins the globulins level was high enough not to rule out the presence of paraproteins. The sample was then diluted off board with the reagent A (reaction buffer) in the ratio used in the method and no precipitation was observed. Unfortunately by this stage due to limited volume no further tests could be conducted.

Bottom Line: A second sample was received and analysed on a Roche Cobas system to obtain a result.To be able to provide more rapid results it was shown ethanol could be used as a precipitant of proteins in both calibrators and patient samples with acceptable recovery.IgM paraprotein was identified as the cause of interference with the gentamicin, vancomicin and valproate assays.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Pathology, Pathology Queensland, Princess Alexandra Hospital, Brisbane, Australia.

ABSTRACT

Introduction: Gentamicin due to its low level of resistance and rapid bactericidal activity is commonly used to treat gram-negative bacteria. However, due to its toxic effects it needs to be monitored. To date, no interference has been reported with gentamicin assays.

Materials and methods: A patient with leg cellulitis and sepsis received a single dose of gentamicin and a sample was sent for gentamicin analysis. The sample showed high blank absorbance readings on Beckman DxC800 and DC800 analysers with various dilutions. A second sample was received and analysed on a Roche Cobas system to obtain a result. A third sample was received 107 hours later with the same results and this sample was then analysed neat and post ethanol precipitation on all the turbidimetric assays available on the DxC800 analyser.

Results: The high blank absorbance was observed upon addition of the reactive reagents due to protein precipitation. Although not obvious from the patient protein results, it was shown the presence of high IgM paraprotein, 18.9 g/L (reference range 0.4-2.3 g/L) was the cause of precipitation, giving high blank readings. Of all the other turbidimetric assays, only vancomicin and valproate showed similar high blank absorbance readings. To be able to provide more rapid results it was shown ethanol could be used as a precipitant of proteins in both calibrators and patient samples with acceptable recovery.

Conclusion: IgM paraprotein was identified as the cause of interference with the gentamicin, vancomicin and valproate assays. Protein interference in these assays can be overcome by precipitation with ethanol.

Show MeSH
Related in: MedlinePlus