Effects of blood contamination of cerebrospinal fluid on western blot analysis for detection of antibodies against Sarcocystis neurona and on albumin quotient and immunoglobulin G index in horses

Margaret M. Miller From the Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348-1692 (Miller, Sweeney, Russell), and Equine Biodiagnostics Inc, A165 ASTeCC Building, University of Kentucky, Lexington, Kentucky 40506-0286 (Sheetz, Morrow).

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Corinne R. Sweeney From the Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348-1692 (Miller, Sweeney, Russell), and Equine Biodiagnostics Inc, A165 ASTeCC Building, University of Kentucky, Lexington, Kentucky 40506-0286 (Sheetz, Morrow).

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Gail E. Russell From the Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348-1692 (Miller, Sweeney, Russell), and Equine Biodiagnostics Inc, A165 ASTeCC Building, University of Kentucky, Lexington, Kentucky 40506-0286 (Sheetz, Morrow).

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R. Michael Sheetz From the Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348-1692 (Miller, Sweeney, Russell), and Equine Biodiagnostics Inc, A165 ASTeCC Building, University of Kentucky, Lexington, Kentucky 40506-0286 (Sheetz, Morrow).

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Jennifer K. Morrow From the Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348-1692 (Miller, Sweeney, Russell), and Equine Biodiagnostics Inc, A165 ASTeCC Building, University of Kentucky, Lexington, Kentucky 40506-0286 (Sheetz, Morrow).

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Objective

To determine effects of blood contamination on western blot (WB) analysis of CSF samples for detection of anti-Sarcocystis neurona antibodies, and on CSF albumin and IgG concentrations, albumin quotient (AQ), and IgG index in horses.

Design

Prospective in vitro study.

Samples

Blood with various degrees of immunoreactivity against S neurona was collected from 12 healthy horses. Cerebrospinal fluid without immunoreactivity against S neurona was harvested from 4 recently euthanatized horses.

Procedure

Blood was serially diluted with pooled nonimmunoreactive CSF so that final dilutions corresponded to 10-3 to 100 μl of blood/ml CSF, and WB analysis was performed on contaminated CSF samples. Number of RBC, albumin and IgG concentrations, AQ, and IgG index were also determined.

Results

Antibodies against S neurona were detected in CSF contaminated with 10-3 μl of strongly immunoreactive blood/ml. In CSF samples contaminated with 10 μl of blood/ml, AQ remained within reference range. Volume of blood required to increase IgG index varied among blood samples and was primarily influenced by serum IgG concentrations. Number of RBC in contaminated samples was correlated with volume of blood added, but not with degree of immunoreactivity detected in contaminated CSF samples.

Conclusions and Clinical Relevance

During collection of CSF from horses, contamination with blood may introduce serum antibodies against S neurona at concentrations sufficient for detection by WB analysis, thus yielding false-positive results. When blood is moderately or strongly immunoreactive, the amount of contaminating albumin may be small enough as to not increase AQ above reference range. In these cases, AQ and IgG index should be interpreted with caution. (J Am Vet Med Assoc 1999;215:67—71)

Objective

To determine effects of blood contamination on western blot (WB) analysis of CSF samples for detection of anti-Sarcocystis neurona antibodies, and on CSF albumin and IgG concentrations, albumin quotient (AQ), and IgG index in horses.

Design

Prospective in vitro study.

Samples

Blood with various degrees of immunoreactivity against S neurona was collected from 12 healthy horses. Cerebrospinal fluid without immunoreactivity against S neurona was harvested from 4 recently euthanatized horses.

Procedure

Blood was serially diluted with pooled nonimmunoreactive CSF so that final dilutions corresponded to 10-3 to 100 μl of blood/ml CSF, and WB analysis was performed on contaminated CSF samples. Number of RBC, albumin and IgG concentrations, AQ, and IgG index were also determined.

Results

Antibodies against S neurona were detected in CSF contaminated with 10-3 μl of strongly immunoreactive blood/ml. In CSF samples contaminated with 10 μl of blood/ml, AQ remained within reference range. Volume of blood required to increase IgG index varied among blood samples and was primarily influenced by serum IgG concentrations. Number of RBC in contaminated samples was correlated with volume of blood added, but not with degree of immunoreactivity detected in contaminated CSF samples.

Conclusions and Clinical Relevance

During collection of CSF from horses, contamination with blood may introduce serum antibodies against S neurona at concentrations sufficient for detection by WB analysis, thus yielding false-positive results. When blood is moderately or strongly immunoreactive, the amount of contaminating albumin may be small enough as to not increase AQ above reference range. In these cases, AQ and IgG index should be interpreted with caution. (J Am Vet Med Assoc 1999;215:67—71)

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