Analytical Measurement Range: Examples and Approaches ... Upper LimitNo Upper Limit • Take samples that exceed the upper limit of the analytical rangeupper limit of the analytical

2013 Laboratory Accreditation Program Audioconferences and WebinarsAnalytical Measurement Range: Examples and Approaches

www.cap.org

William Castellani, MD, FCAPMarch 20, 2013

ObjectivesObjectives

• Define and describe the analytical measurement rangey g

• Compare this range to CLIA’88 requirements for calibration and calibration verification

• Discuss strategies for satisfying accreditation requirementsq

© 2013 College of American Pathologists. All rights reserved. 2

ConflictsConflicts

• Nothing to discloseg


It All Begins HereIt All Begins Here

• “Calibration” means a process of testing and adjusting p g j gan instrument or test system to establish a correlation between the measurement response and the concentration or amount of the substance that is being gmeasured by the test procedure.

• “Calibration verification” means the assaying of materials f k t ti i th ti tof known concentration in the same manner as patient

specimens to substantiate the instrument or test system’s calibration throughout the reportable range for

ti t t t ltpatient test results.o Centers for Medicare and Medicaid Services, State Operations Manual,

Appendix C


Reportable Rangep g

• Reportable range means the span of test result values p g pover which the laboratory can establish or verify the accuracy of the instrument or test system measurement responseresponse.

o CLIA ’88, Sec. 493.2, Definitions


Reportable RangeReportable Range

• Two components:po The primary range of measurement

−Analytical measurement range− “Linear” range

o Anything done to the system to expand this rangeDil ti−Dilution

−Concentration– The span of values that can be reliably– The span of values that can be reliably

measured using these modifications is the reportable range in CLIA’88


What Must We Do?What Must We Do?

• Validate or verify yo Reportable range: as part of method

validation/verification− Initial documentation of the analytical

measurement range−Documentation of any dilution or concentrationDocumentation of any dilution or concentration

protocol that can be done to expand the range of values that can be reported by the method

o Analytical measurement range [verification]: every six months thereafter (when necessary)


How Do We Do It?How Do We Do It?

• Set criteria of acceptancep

• Established protocol

M di l l• Medical relevanceo All this should be established by the laboratory

directordirectoro All this should be documented formallyo The actual review may be delegated, though final y g , g

authorization may be reserved for the director



• May decrease the lower limit of the analytical y ymeasurement range by:o Concentrating the sample

−Amicon concentrator−Extraction and resuspension

Increasing the ratio of sample to reagento Increasing the ratio of sample to reagent−Altering the programming of the instrument



• More commonly, may increase the upper limit of the y, y ppanalytical measurement range by:o Decreasing the ratio of sample to reagento Diluting the sample before analysis



• Most often, the manufacturer provides the information or pmechanism for this modificationo Autodilution/autoconcentrationo Dilution protocolo Dilution protocolo Concentration protocol

G d l b t ti ld i l d if i th t• Good laboratory practice would include verifying that these modifications work

• Going beyond the modifications stated in the• Going beyond the modifications stated in the manufacturer’s instructions for use requires laboratory validation


AMR & RR ExamplesAMR & RR Examples

hCG AST

AMR: 3-1000 mIU/ml AMR: 4-900 U/L

RR lower limit: 5 RR lower: 4

RR upper limit: RR upper limit: ppnot definedDilute and reassay until

l i bt i d

pp1,000,000Values above upper RR

t d t thvalue is obtained reported as greater than 1,000,000


General PrinciplesGeneral Principles

• Establish a target valueo May use a patient sample’s result as the “target”o May use a patient sample s result as the targeto May use peer group mean of PT materialo May be established by the provider of the materialy y p

• Establish an acceptable range around the targeto May be an absolute (arbitrary) range [10%]y ( y) g [ ]o May use precision data for control material near the

targeto May be provided by the manufacturero May be provided by the manufacturer

• Document your protocol (approved by director)


Verifying the Reportable RangeVerifying the Reportable Range

• Documenting that the modifications to the sample or g pmethod produce reliable results

• Begin with verifying the dilution protocolo Use the method manufacturer’s recommended diluent

and, if given, dilution factoro Determine an acceptance range for the dilution resulto Determine an acceptance range for the dilution result

• If the manufacturer does not allow for dilution, then the laboratory must validate its own protocoly p


Dilution ProtocolDilution Protocol

• Take samples near the upper limit of the analytical range

• Manually dilute and assay

• Compare against the target using the acceptance rangep g


Dilution ProtocolDilution Protocol

• Dilution Limito If the manufacturer gives either a maximum dilution or

a specific dilution, verify to the limit−Report all results exceeding this diluted range as >

upper limito If the manufacturer gives no guidelines:o If the manufacturer gives no guidelines:

−Laboratory director may specify a maximum diluted value or dilution protocol

−May dilute until you get a value (no upper limit)


No Upper LimitNo Upper Limit

• Take samples that exceed the upper limit of the analytical rangeupper limit of the analytical range.

• Manually dilute and assay × 10 (should get a valid answer at the high end).high end).

• Manually re-dilute × 10.• Compare against the target using

the acceptance range.the acceptance range.• You have verified a × 100 dilution;

at this point, the sample matrix is diluent, not serum/plasma. Any further dilution would not significantly change the matrix.

• Document this claim.


Verify Autodilution MethodVerify Autodilution Method

• Take patient samples that provide results on autodilutionp p p

• Manually dilute the sampleo Do not need to use same dilution factor as theo Do not need to use same dilution factor as the

autodiluter

• Compare the autodilution and manual dilution resultsCompare the autodilution and manual dilution results using the manual dilution result as target value


Verify Autoconcentration Method Part 1Verify Autoconcentration Method, Part 1

• For those methods where the lower limit is extended by yincreasing the ratio of sample to reagent

• Take low samples and serially dilute p yo Suggest x 2 dilution

• Using the neat result of the sample as the target look forUsing the neat result of the sample as the target, look for deviations from linearity


Example: Serum Immunoglobulin AExample: Serum Immunoglobulin A

• Manufacturer’s range: 40 – 800 mg/dL

Dilution Value 1 Value 2 Mean Theoretical

Neat 171 171 171 171

× 2 87 88 88 86

× 4 43 44 44 43

× 8 20 20 20 21

× 16 8 8 8 11× 16 8 8 8 11

× 32 3 3 3 5


Verify Autoconcentration Method Part 2Verify Autoconcentration Method, Part 2

• Once you’ve established the maximum concentration ythat will give reliable results, test samples using the modified concentration protocol programmed into the analyzeranalyzer

• May need to first dilute sample into lower part of the analytical measurement range manually before placinganalytical measurement range manually before placing on analyzer


Example: Serum Immunoglobulin AExample: Serum Immunoglobulin A

Neat result Manual dilution

Result (standard protocol)

Result (autoconcentration)dilution protocol) (autoconcentration)

171 × 8 20 2351 × 2 26 24110 None 110 11288 None 88 89


CLIA Requirement for Calibration/Calibration VerificationCLIA Requirement for Calibration/Calibration Verification

• 493.1255: Calibration and calibration verification d i d t b t ti t thprocedures are required to substantiate the

continued accuracy of the test system throughout the laboratory’s reportable rangey p g


Calibration/Calibration VerificationCalibration/Calibration Verification

• Calibration: Establishes the relationship between l t t t d i t t t i lanalyte content and instrument measurement signal

• Calibration verification: Confirms that the current lib ti tti i lidcalibration settings remain valid


CAP Interpretation of CLIA Calibration VerificationCAP Interpretation of CLIA Calibration Verification

• Validate the set point (CAP Calibration Verification)p ( )

• Prove response over the entire analytical measurement rangeg

• The Laboratory Accreditation Program considers calibration verification to be secondary to calibrationyo If calibration satisfies the CLIA requirements for

calibration verification, no further action is necessary


Calibration Verification RequirementsCalibration Verification Requirements

• Sec. 493.1255(b)(2) [Perform and document calibration ( )( ) [verification procedures] Using the criteria verified or established by the laboratory …

(i) I l di th b t d t ti fo (i) Including the number, type, and concentration of the materials, as well as acceptable limits for calibration verification; and

o (ii) Including at least a minimal (or zero) value, a mid-point value, and a maximum value near the upper limit of the range to verify the laboratory's reportablelimit of the range to verify the laboratory's reportable range of test results for the test system


Frequency of Calibration OR Calibration VerificationFrequency of Calibration OR Calibration Verification

• At least every 6 months

• When recommended by manufacturer

• After major maintenance or serviceAfter major maintenance or service

• When QC indicates

• At complete reagent change unless laboratory• At complete reagent change, unless laboratory has data showing lot uniformity

• At change of critical reagents (laboratoryAt change of critical reagents (laboratory defines)


When is Calibration Sufficient?When is Calibration Sufficient?

Quantitative NoQuantitative Assay?

NoSpecial case: cut-off

Yes

Calibrate at least No Perform calibration every 6 months?

verification at least every 6 months

Yes


Calibrate at Least Every 6 MonthsCalibrate at Least Every 6 Months…

Yes

Use at least 3 l l ?

Verify AMR using

No

levels?

Yes

using materials near low,

id i tMaterials

span AMR

mid-point and high values at

No

Done (low-mid-high)?

least every 6 months

Done


ExamplesExamples

Laboratory calibrates: Verify calibration?

Verify AMR?calibration?

With 1 point dailyWith each new lot change (8-10 months) using 2 calibrators that span the AMR

No Yes

Yes Yesusing 2 calibrators that span the AMREvery 3-4 months with 5 calibrators that span the AMRMonthly with 2 calibrators that span the

NoNo

Monthly with 2 calibrators that span the AMREvery 3 months with 3 points (50, 150,300) but reports 25-600 Yes

No

No

Yes

300) but reports 25 600Every run using kit material to determine a positive/negative cut-off No

Two concentrations around the cut-off


Requirements for [CAP] Calibration VerificationRequirements for [CAP] Calibration Verification

• Target values

• Appropriate matrixo Calibratorso Materials provided by the vendor for the purpose of calibrationo Materials provided by the vendor for the purpose of calibration

verificationo Previously tested unaltered patient/client specimenso Primary or secondary standards or reference materials with matrixo Primary or secondary standards or reference materials with matrix

characteristics and target values appropriate for the methodo Third party reference material if commutable

P fi i t ti t i l ith t i h t i ti d t to Proficiency testing material with matrix characteristics and target values appropriate for the method

• Controls usually do not satisfy this requirement unlessControls usually do not satisfy this requirement unless specifically designed by the manufacturer for this purpose


Requirements for Verification of the Analytical Measurement RangeMeasurement Range

• Target values

• Appropriate matrixo Linearity material of appropriate matrix

Proficiency testing survey materialo Proficiency testing survey materialo Previously tested patient specimens, unalteredo Previously tested patient specimens, altered by admixture with other

i dil ti iki th t h ispecimens, dilution, spiking, or other techniqueo Primary or secondary standards or reference materials with matrix

characteristics and target values appropriate for the methodo Calibrators used to calibrate the analytic measurement systemo Control materials, if they adequately span the AMR


Verification of the Analytical Measurement RangeVerification of the Analytical Measurement Range

• “Guidelines for analyte levels near the low and high y grange of the AMR should be determined by the laboratory director. Factors to consider are the expected analytic imprecision near the limits the clinical impact ofanalytic imprecision near the limits, the clinical impact of errors near the limits, and the availability of test specimens near the limits. It may be difficult to obtain

fspecimens with values near the limits for some analytes (eg, T-uptake, free thyroxine, free phenytoin, prolactin, FSH, troponin, pO2). In such cases, reasonable , p , p 2) ,procedures should be adopted based on available specimen materials.”

Chemistry and Toxicology checklist 9/25/12o Chemistry and Toxicology checklist, 9/25/12


How Do You Determine the Upper and Lower Limit Targets?and Lower Limit Targets?

• Determined by available material:o Define the linear range as going from the low to the

high target sample

• Fixed range:o Within 10% of the top end and 1% of the bottom end

• Clinical use and decision points


Upper Limit Target Based on Clinical Decision PointUpper Limit Target Based on Clinical Decision Point

• Decision point in the lower quarter of the linearof the linear range:o Adult total

bilirubin Use material within 80% of the upper limit of the linear rangelinear range



• Decision point in the mid-half of the linear range:linear range:o Most drugs

Use material within 90% of the upper limit of the linear rangelinear range



• Decision point in the upper quarter of the linearof the linear range:o Neonatal

bilirubino Special case: β hCG

Use material within 95% of the upper limit of the linear rangeβ-hCG linear range


Ongoing Verification of the Upper Limit of the Analytical RangeAnalytical Range

• When a sample exceeding the current verified upper limit p g pp(but within the manufacturer’s range) is assayed, perform two dilutions into the currently verified range (eg × 2 and × 3)(eg, × 2 and × 3)

• If the two dilutions come within defined targets, the upper limit has been extended to the value of this new samplelimit has been extended to the value of this new sample



• Manufacturer’s range:

• Currently verified range:

f f CModified from Martin Kroll, Boston Medical Center



• Manufacturer’s range:

• Newly verified range:

f f CModified from Martin Kroll, Boston Medical Center


Extending the Verification RangeExtending the Verification Range

• Available material with target values may not reach the upper limit ofreach the upper limit of the analytical measurement range

• Example: urine sodiumo manufacturer’s range:

− 0 – 200 mmol/L


Extending the Verification RangeExtending the Verification Range

• Prepare a stock sodium solution of 200 mmol/L

• Do two serial x 2 dilutions (100 and 50 mmol/L target)target)

• Assay each level and plot


Verifying the Lower Limit of the Analytical RangeVerifying the Lower Limit of the Analytical Range

• Negative materialgo Patient sample without the analyte in questiono Surrogate material

−Diluent−Water or aqueous IV fluid

– Matrix of questionable value for most methods

• Diluted low patient samples


Lower Limit Target Based on Clinical Decision PointLower Limit Target Based on Clinical Decision Point

• Relatively straightforward if there is aif there is a lower clinical decision point

Use material < 50% of the lower clinical decision pointpoint


What about the Mid-Point?What about the Mid Point?

• The regulation only states that one mid-point value is g y prequired

• The checklist does not further define how this value should be obtained


Mid-Point MaterialMid Point Material

• The most common approach is 1:1 mixtures of the low ppand high material

• This ratio of 1:1 is strictly “convenient;” for those tests y ;where a critical decision point exists, using a ratio that brings the target of the mid-point material close to this decision point should be considereddecision point should be considered

• The difficulty is in creating an exception to standard laboratory protocollaboratory protocol


Mid Point MaterialMid-Point Material• Most commercial products provide four or more target

samples, though this is not required by regulationsamples, though this is not required by regulation

• A similar distribution of target values can be obtained by sequential mixing

Low2.5 2.52 01 0 1.0 High

Mid2.01.0

1.01.0 1.00.5 0.5 0.5

Mid LoLow Mid Hi HighMid

0.5

Mid-LoLow Mid-Hi HighMid


SummarySummary

• Establish protocols addressing the requirementsEstablish protocols addressing the requirementso material that will be usedo how the target values will be obtainedgo how the acceptance ranges will be determined

• Document the protocols• Document the protocols o Reportable range: Method validation procedureo Calibration verification (CLIA): Qualityo Calibration verification (CLIA): Quality

control/assurance procedure

• Approved by director• Approved by director


Thank you!Thank you!

Questions?


Documents

Analytical Measurement Range: Examples and Approaches ... Upper LimitNo Upper Limit • Take samples that exceed the upper limit of the analytical rangeupper limit of the analytical