Body Fluid Validation

Body Fluid Validation

I was once asked by a peer from another hospital if I had formed a plan to a new checklist question on the CAP All Common checklist.  She said we now have to validate body fluids and a disclaimer such as “This test has not been validated on this fluid type” was not going to be enough.

According to the CAP question COM.40620; Methods for body fluid analysis have been validated or verified.  In the note section it gave me a clue for how to accomplish this… “by performing an appropriate study (e.g. a dilution study using admixtures of samples, spiking samples, further dilution)”.

As my serum assays are validated, by plan was to take a representative number of assays and make a 1:1 dilution of a body fluid and serum.  Knowing the initial body fluid result, the serum result, and the expected recovery, I would be able to calculated the expected recovery and compare expected to realized and see if that was with-in with-in a TAE set by my medical director.  

I set up 10 specimens and tested for ALB, GLU, LDH, and TP.  The chart below provides an explanation for the needed results and a place for pass/fail criteria.

All passed and with this study tucked away I can show an inspector we validated Body Fluids.

Hope this works for you!

Validating your Analytical Measuring Range by Alternate Methods

If you are CAP certified you are used to the idea of validating your AMR, or the range the analyzer is able to measure without manipulation (dilution or spiking).  This is not to be confused with other ranges such as Clinical Reportable Ranges (CRR) that I will discuss in another post.

Alternate, in this context, means another way than by buying commercial available AMR samples.

Likely, most of your analytes are covered by linearity surveys from CAP...but not all!

LDL, Urine Protein, CSF Protein, Lactic Acid and the list goes on....

So what are some "Alternate Methods".  My favorite is using a calibrator of a different lot used to calibrate the assay in question.  But sometimes a calibrator does not come close enough to the high end of AMR.  I like to use the "rule" of 10% as being "close enough".

Secondly I like to use a high patient sample diluted to cover the AMR.  Using calculated target values the points covering the low, mid, and high can plotted to validate the analyte over the AMR of the analyzer.

How?  Read on and feel free to copy and modify the following your your procedures :)
Note: I got the jist of this from the folks at Binding Site who make our Kappa and Lambda Free Light assay reagents (used on the Roche 8000).

Making a Serial Dilution or Preparing Samples for AMR Validation

When you don’t have linearity samples to validate your Analytical Measuring Range then you can prepare dilutions of a high sample that covers the range, all the way down to 2% of the upper value.  Using this method avoids making repeatedly higher dilutions from the high sample and the use of too many different pipettes…both likely sources of error.

Needed:

·         Class A pipettes or NIST certified pipettes (an adjustable pipette delivering 250 and 750uL works best. 

·         Sample of at least 2.25mL containing the analyte being tested. 

·         Appropriate diluent.

If the sample you have is (ideally) above the upper measuring range of your analyzer you will need to spike it to be under the upper limit AND with-in 10%.  Spike and retest until this is achieved.  Remember you will need at least 2.25 mLs of this sample.

Once this “100%” solution is achieved, measure 3x and take the average.  From this sample and value you will make dilutions and calculate the expected recovery.  Note that not all levels are necessary for the AMR study but the lowest, mid -range, and highest at a minimum.

Arrange 7 tubes in a rack and label with the final dilution as stated in the chart below, and prepare the indicated dilutions.  Use a fresh tip between samples and good pipetting practices.  Fill in the expected recovery.

Final Dilution	Expected Recovery	Volume of 100% solution	Volume DIluent
75%		750uL of 100% dilution	250uL
50%		750uL of 100% dilution	750uL
25%		750uL of 50% dilution	750uL
12.5%		750uL of 50% dilution	750uL
6.25%		750uL of 12.5% dilution	750uL
3.13%		750uL of 6.25% dilution	750uL
1.56%		750uL of 3.13% dilution	750uL

Run samples in duplicate, if may be beneficial to extend the analyzer’s reportable range in the event the lower samples exceed the AMR.

Crunch the data in a statistical program such as EP Evaluator from Data Innovations.  In this case use the statistical module Accuracy & Linearity- Linearity and Calibration Verification, choose Confirm Linearity and Confirm Accuracy.  It is ideal to set limits for error based on CLIA, CAP, API, or other sources and this should be approved by the laboratory director.

For example: The total allowable error (TAE) for ALT is 20% (CLIA).  IF using EP Evaluator enter also a budget for systematic error.  Hopefully, no more than 50% for systematic error budget is used.

The statistical software will indicate if the experiment passes or fails.  Alternatively, a graph can be plotted and each point can be assessed if it is with-in the TAE.  Just add a column to the above chart next to expected recovery for the result and another column for the acceptable range (ALT: up to +/-20% from the Expected Recovery).

Comments? Suggestions?

email: joe@abetterlabnow.com

This blog is to introduce myself as a PRN laboratory technical consultant and provide free content to my peers. Working in a laboratory is more exciting than ever and the changes our profession faces makes for an opportunity for those with my experience to work in multiple settings.
Contact me if you need help with a project, prepare for an inspection, write procedures or train your staff.

Yours,
Joe Noffsinger MT (ASCP), MBA