Automated Bethesda Titre

Automated Bethesda Titre
Nov 16, 2017 7:23am

From Jasmina, Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh India

Hello George. This is regarding running the factor VIII assay for the Bethesda assay on automated  analysers. When performing the Bethesda assay on the coagulation analysers like the ACL TOP (IL ), we make the doubling dilutions of sample and add them to the normal plasma manually on the bench and then transfer each sample after the incubations to the analyser for readings on the standard FVIII , one stage assay programme. Is this appropriate or is it necessary to have a separate FVIII assay protocol for the Bethesda assay on the analyser? The analyser will further dilute each of the tubes by another 1: 10.Will that affect the results predictably? Would be glad to hear from anyone using this assay on the ACL TOP. Thanks.


Hello, Dr. Ahluwalia, it is good to hear from you. I've forwarded your question to a colleague, Heather DeVries, who is familiar with the ACL TOP and who manages Bethesda assays regularly.


Heather writes, Greetings! This procedure is exactly what we do, and was validated by Larry Smith at Cedars-Sinai, if I remember correctly (Geo: actually, I think Larry was at Memorial Sloan Kettering then, now travels for Abbott). The additional 1:10 that the instrument is making would have been part of that validation. We use NASCOLA proficiency for this, and have not had any problems, so I trust the assay. If they were to build a test from scratch, the entire procedure would need re-validation…not fun for the Bethesda!

2 Comments

From Jasmina, Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh India

Hello George. This is regarding running the factor VIII assay for the Bethesda assay on automated  analysers. When performing the Bethesda assay on the coagulation analysers like the ACL TOP (IL ), we make the doubling dilutions of sample and add them to the normal plasma manually on the bench and then transfer each sample after the incubations to the analyser for readings on the standard FVIII , one stage assay programme. Is this appropriate or is it necessary to have a separate FVIII assay protocol for the Bethesda assay on the analyser? The analyser will further dilute each of the tubes by another 1: 10.Will that affect the results predictably? Would be glad to hear from anyone using this assay on the ACL TOP. Thanks.


Hello, Dr. Ahluwalia, it is good to hear from you. I've forwarded your question to a colleague, Heather DeVries, who is familiar with the ACL TOP and who manages Bethesda assays regularly.


Heather writes, Greetings! This procedure is exactly what we do, and was validated by Larry Smith at Cedars-Sinai, if I remember correctly (Geo: actually, I think Larry was at Memorial Sloan Kettering then, now travels for Abbott). The additional 1:10 that the instrument is making would have been part of that validation. We use NASCOLA proficiency for this, and have not had any problems, so I trust the assay. If they were to build a test from scratch, the entire procedure would need re-validation…not fun for the Bethesda!

By Dr Jasmina Ahluwalia
Nov 17, 2017 10:49pm
Thank you, George and Heather. This should be good to get us going.
By LINDA
Nov 20, 2017 10:35am
Hi Dr Ahluwalia. Speaking from the technical side, the 1:10 dilution that the ACL TOP makes with the factor diluent for the FVIII is the standard sample pre-dilution. It’s done for all FVIII samples including QC and each calibration dilution starting with the 100% dilution down though the last dilution it makes with the calibrator. When you run a sample, it is treated exactly as the 100% calibration dilution. The 1:10 sample pre-dilution is required for the test and will not affect the results from your Bethesda samples. It would not be necessary to have a separate FVIII assay protocol.

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