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Low Factor VIII Reporting

Hi, Using the ACL 9000, we have seen some low results for the factor VIII activity test, as low as 0.3%. In those instances we were told to dilute the sample 1:2, and retest. On those retests we get the same results, should we report the result as it is or with a less than something value? Miguel A. Vélez MT (ASCP) Immuno Reference Laboratory.

Hello, again, Mr. Vélez, and thank you for your question. Operators typically perform the factor VIII assay on three or four dilutions. They prepare a primary dilution of 1:10 in Veronal or Owren phosphate buffer and further dilute by (for example) 1:2, 1:4, and 1:8, making the final dilutions 1:10, 1:20, 1:40, and 1:80. They assay all four dilutions and multiply the initial assay results by the dilution factors, 1, 2, 4, and 8, and check for parallel results. Most operators consider results that match within ±10% to be parallel. Non-parallel results indicate a plasma inhibitor, which requires follow-up. Most automated coagulometers are programmed to perform factor VIII (and other factor) assays at these or similar dilutions and to provide the necessary computations.

Further, most operators re-assay plasmas that yield results <10% or perhaps <5% for greater sensitivity, using established laboratory protocol. For the re-assay, they typically prepare a 1:5 dilution in place of the standard 1:10, then divide the initial assay result by 2 for a final result. Many instruments are programmed to do this reflexively, providing accurate results in severe hemophilia A; such results help to guide therapy. They could also conceivably perform the assay at 1:2 and divide the initial assay result by 5, though the accuracy suffers as the dilution factor rises.

Clinically, a factor VIII activity level <1% is classified as severe hemophilia A. Levels from 1–5% are moderate, and 5–30% are mild. These categories help define the treatment approach to hemophilia symptoms. There is little clinical reason for providing greater sensitivity when the result is <1%, as the treatment would be unchanged, thus most operators report 1% or <1%, but not fractional results.

For additional information, factor assay methods are provided in greater detail in Audio Module 24, “Factor Assays.” I hope this helps! Geo.

Comments (3)
Factor Assays
Jul 6, 2012 5:54am

Hello George! Thank you for directing me to the Audio Mosul
Hello George! Thank you for directing me to the Audio Mosule 24. It helped! You say there is no clinical reason for providing greter sensitivity when the result is <1%. We currently report <0.25% because our curve reads that low. Is this something we should consider changing? And, a second question if I may. When we run factor assays, we have found that 1:10, 1:20 and 1:40 show parallelism or results which do not agree within 15%. We currently re-dilute those with FVIII DP 1:2 and re-run 1:10, 1:20 and 1:40. Sometimes these results then agree with each other. Other times they agree with one of the original dilutions. We are having a hard time discerning if these are true parallelisms or poor recovery from different dilutions. Any insight???

Jul 6, 2012 3:23am

Hi! I’d like to get some information (if possible a referenc
Hi! I’d like to get some information (if possible a reference) on this clinical situation- If a patient with an uncharacterised bleeding disorder has been transfused plasma or cryoprecipitate in an emergency setting, how soon afterwards can one do a coagulation screen to avoid the results from getting compromised by the transfused factors?

Jun 28, 2012 8:06am

In 2009 we presented a paper at ISTH in Boston where we comp
In 2009 we presented a paper at ISTH in Boston where we compared results of two chromogenic and clottable results for comparing FVIII methodologies using a high curve and low curve for determining levels of FVIII. Please see the following comments:

We compared the Diapharma Group, Inc chromogenic Coamatic FVIII (DP) and the Aniara/Hyphen Biomed chromogenic Biophen FVIII:C (AHB) kits to the Diagnostic Stago, Inc. clot-based FVIII activity assay using a Diagnostica Stago STA-R Evolution coagulation analyzer. Our sample was comprised of 49 normal subjects and 37 with FVIII activity 0.985. Low FVIII activity results generated from the clot-based method and the low chromogenic curves were not significantly different using ANOVA (p=0.19). Likewise, normal or high FVIII activity results generated from the clot-based method and the high chromogenic curves did not differ (p=0.78). We also employed the paired t-test to compare results of chromogenic assays. There were no statistical differences for the low (p=0.07) or high (p=0.12) curve-generated result sets. Results from our sample support the clinical equivalency of the two chromogenic FVIII activity assays to the clot-based assay.

When dealing with very low levels of FVIII using a low range curve might let the facility quantitate low levels with more accuracy. See the following citation:

McGlasson DL, Fritsma GA. Comparison of two chromogenic FVIII activity assays to a standard clot-based FVIII activity assay. Journal of Thrombosis and Haemostasis 2009; Volume 7, Supplement 2: Abstract PP-WE-235

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