Flow cytometry based suspended microarray assays are susceptible to error in the assays and instrument. I present the Intraplexing method for compensating for these sources of error, evidence for the major sources of variance, and straightforward models which suggest other minor contributing factors of error. Intraplexes require little sample, are inexpensive, sensitive, and quantify within statistical confidence limits.
The Intraplex method uses ratios of sensitivity in multiplexed assay sets to the same analyte within a sample. The intraplex method compensates for the sources of variance that have been identified in suspended microarray assays. It requires no changes to instruments to construct precision assays.
The major sources of error are: The suspended microarray system generates statistical distributions of counts. The number of microbeads picked up for different bead regions (sets) will vary in a classic bell curve. This problem can be ignored with small numbers of multiplexed assays, but it is significant when the number of assays multiplexed together rises.
The microparticles on which assays are conducted vary significantly in size.
Random carryover of microbeads between wells is an intractable source of error. I show that instruments carry over microbeads in sufficient numbers to be considered a statistically valid sample. This source of error is the probable primary reason why it has been so difficult for suspended microarray assays to get through approval as diagnostics.
Opto-electronics may not respond similarly on different instruments. The opto-electronic system does not necessarily respond identically between different instruments at differing signal intensity. Inter-instrument ratio can be unity for calibration point and two logs for lower intensity signal.
There are other sources of error. Aside from microsphere agglutination, they appear to be minor contributors compensated for by the Intraplex method; however, these sources of error deserve further study.
Intraplexing can enable reliable, precise, time course studies in small animals for virtually any assay of interest. If Intraplexing is used, then the assay should be suitable for use in human diagnostics, as well as making for better science. I believe the Intraplexing method should become standard practice for assay development and analysis.
|Advisor:||Baldwin, Enoch P.|
|Commitee:||Calvert, Christopher C., Stahlberg, Henning|
|School:||University of California, Davis|
|School Location:||United States -- California|
|Source:||DAI-B 70/08, Dissertation Abstracts International|
|Subjects:||Biostatistics, Microbiology, Biophysics|
|Keywords:||Intraplex, Luminex biophysics, Luminex sources of error, Multiplexing, Suspended microarrays|
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