201. A Spectrophotometric Method to Accurately Determine Endpoint Titers in Complement Fixation Assays
Session: Poster Abstract Session: Antigen and Antibody Based Diagnostics
Friday, October 21, 2011
Room: Poster Hall B1
Background: In 1899 Jules Bordet discovered that hemolysis was induced by complement which led to the development of the complement fixation (CF) test in 1901.  In 1965 protocols were formally established by the Centers for Disease Control (CDC) to standardize many aspects of CF testing and published as Laboratory Branch Complement Fixation (LBCF).  Since then CF testing has been used to aid in the diagnosis and to monitor treatment for many diseases, including the mycoses histoplasmosis, coccidioidomycosis and aspergillosis.  However, CF testing relies on a subjective determination of cell lysis which can lead to inaccurate results.

Methods: A set of samples was created with known lysis levels (0 to 70% lysis) using the LBCF method with 30% lysis considered the cutoff for positive samples.  Included in the analysis were negative controls, positive controls, negative samples and positive samples for several CF assays currently being performed at ARUP Laboratories.  All samples were then read on a spectrophotometer at 405nm.  Optical densities (OD) were compared to first determine what the OD of 30% lysis was and then how samples compared to the 30% lysis level.

Results: Each positive control showed a clear endpoint that showed a distinct OD difference at the 30% lysis level.  Additionally, titer endpoints for patients samples were able to be accurately determined comparing to the OD at 30% lysis.  Of 29 positive samples compared, 19 endpoints matched, and 9 samples only varied by one titer.  One sample that was negative by the spectrophotometric method had an endpoint difference more than 4 titers from what was determined by a laboratory technologist; however, this patient tested negative by immunodiffusion for the specific fungal antibody being tested for by CF.

Conclusion: Differences between the spectrophotometric method and the technologist’s determinations were to be expected.  The new method utilizes an accurately controlled standard that yields 30% lysis and compares this level using an objective determination with each patient sample.  We feel that by utilizing this method we can accurately endpoint samples and eliminate subjectivity that can yield differences between technologists.


Subject Category: D. Diagnostic microbiology

Ryan Welch, BS, ARUP Laboratories, Salt Lake City, UT, Stephen D. Merrigan, BS, R&D Immunology, Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology, Salt Lake City, UT and Julio Delgado, MD, ARUP Institute for Clinical and Experimental Pathology, Department of Pathology, Salt Lake City, UT

Disclosures:

R. Welch, None

S. D. Merrigan, None

J. Delgado, None

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