Session 7102 You Want to Measure ”? This ” in “ That › ascpannualmeeting › 2015 › AM15FCA › ... · 2015-10-26 · Session 7102 You Want to Measure ... Body Fluid Testing

Session 7102

You Want to Measure

“That” in “This”? Body Fluid Testing in the Clinical Laboratory

Academy of Clinical Laboratory Physicians

and Scientists (ACLPS)

Jonathan Genzen, MD, PhDUniversity of Utah / ARUP Laboratories

Richard Torres, MDYale University / VA Connecticut Healthcare

In the past 12 months, neither presenters have had any significant

financial interest or other relationship with the manufacturer(s) of

the product(s) or provider(s) of the service(s) that will be discussed

in our presentations.

Speaker Disclosures

Background & Regulatory Introduction

SPEAKERS AND OVERVIEW

Body Fluid Testing in Clinical Chemistry

Body Fluid Testing in Hematology

Jonathan Genzen, MD, PhDMedical Director, ARUP Automated Core Laboratory

Richard Torres, MDDirector, Yale Flow Cytometry Laboratory

(focus on validations)

PERSPECTIVE

Presenters are NOT:• On the CAP Checklist Committee

• On the CLSI C49 Body Fluid Committee

• Employees of, or speaking on behalf of, CAP, COLA,

CMS, or the FDA

Presenters ARE:• Presenting opinions citing existing regulations and practice

• Presenting ideas for possible improvements

• Providing references to the literature where available

For clarification of regulatory issues,

please contact your accreditation agency

BODY FLUIDSDefinition: “Body fluid, bodily fluids or biofluids are liquids originating

from inside the bodies of living people. They include fluids that are excreted or

secreted from the body as well as body water that normally is not.” (WIKIPEDIA)

Amniotic fluid

Aqueous (vitreous) humour

Aspiration fluid

Bile

Breast milk

Cerebrospinal fluid

Cerumen

Chyle

Chyme

Dialysate

Drain fluids

Exudates

Feces (stool & diarrhea)

Gastric fluid

Mucus / nasal drainage

Pericardial Fluid

Peritoneal (Ascites) fluid

Plasma

Pleural fluid

Pus

Saliva

Serous fluid

Semen

Serum

Sputum

Synovial fluid

Sweat

Tears

Urine

Vaginal secretion

Vomit

Whole blood

Table modified from: https://en.wikipedia.org/wiki/Body_fluid

FOCUS: Fluid types / matrices not specifically evaluated

in FDA-cleared package inserts

● Differentiation of transudates versus exudates

Transudate* any fluid that has passed through a presumably normal membrane

as a result of imbalanced hydrostatic and osmotic forces

(characteristically low in protein)

Exudate* any fluid that has exuded out of a tissue or its capillaries, more

specifically because of injury or inflammation (characteristically high

in protein and WBCs)

● Evaluation of other cause(s) of fluid accumulation

● Identification of fluid source(s) and leakage(s)

● Supportive evidence for malignancy / other conditions

WHY ANALYZE BODY FLUIDS

* Source: Stedman’s Medical Dictionary, 23rd Ed.

● Light’s criteria for pleural effusions (pleural fluid) [1,2]

• Pleural fluid-to-serum protein ratio >0.5

• Pleural fluid-to-serum LDH > 0.6

• Pleural fluid LDH >2/3 upper limit of normal serum LDH

EXAMPLES

REFERENCES

[1] Light RW et al. 1972. Pleural effusions: the diagnostic separation of transudates and exudates. Annals of Internal Medicine. 77:507-513.

[2] Porcel JM, Light RW. 2006. Diagnostic approach to pleural fluids in adults. American Family Physician. 73(7):1211-1220.

[3] Koch M et al. 2011. Bile leakage after hepatobiliary and pancreatic surgery: a definition and grading of severity by the International Study

Group of Liver Surgery. Surgery. 149(5):680-688.

[4] ARUP Lab Test Directory

● Bile leakage (drain fluid) [3]

The International Study Group of Liver Surgery defines bile leakage as:

“bilirubin concentration in the drain fluid at least 3 times the serum bilirubin

concentration on or after postoperative day 3 or as the need for radiologic or

operative intervention resulting from biliary collections or bile peritonitis”

● Total protein (CSF) [4]

• 1 month through adulthood: 14-45 mg/dL

ADDITIONAL RESOURCES

Kjeldsberg et al.

2015

ASCP Press

Block and Franke

2015

AACC Press

Clinical ContextReference Ranges

Validation Advice

Reference Ranges

Update Pending

CLSI C49-A

2007

Henry’s

2011

Chapter 29

Clinical ContextReference Ranges

http://www.mayomedicallaboratories.com/articles/communique/2013/03-bodyfluid-testing/index.html

Practical Guide to the Analytical Validation of Body Fluid Chemistry Testing

http://www.aruplab.com/bodyfluids

Body Fluid Reference Intervals and/or Interpretative Information for Select Analytes

FREE WEB RESOURCES

• Body fluid testing is NOT inherently bad.

• Body fluid testing can help to SUPPORT THE

PRACTICE OF MEDICINE.

• Inappropriate use or erroneous interpretation

of body fluid results IS inherently bad.

• U.S. laboratories MUST adhere to CLIA and

applicable regulatory/accreditation

requirements regarding validation of assays

(including validation of matrices not included in

package inserts).

BODY FLUID TESTING

CLIA Clinical Laboratory Improvement Amendments

COLA Commission on Office Laboratory Accreditation

TJC The Joint Commission

NYDOH New York Department of Health

ISO International Standards Organization

FDA Food and Drug Administration

CLIACLIA

COLA

CAP

TJC

NYDOH

COLA

CAP

TJC

NYDOH

ISOISO FDAFDA

Awaiting C49 CLSI

Update (2015)

SPECTRUM OF REGULATIONS

Body Fluids

as LDTs

CLIA(Clinical Laboratory Improvement Amendments)

(2) Establishment of performance specifications. Each laboratory that modifies

an FDA-cleared or approved test system, or introduces a test system not subject

to FDA clearance or approval (including methods developed in-house and

standardized methods such as text book procedures), or uses a test system in

which performance specifications are not provided by the manufacturer must,

before reporting patient test results, establish for each test system the

performance specifications for the following performance characteristics, as

applicable:

(i) Accuracy.

(ii) Precision.

(iii) Analytical sensitivity.

(iv) Analytical specificity to include interfering substances.

(v) Reportable range of test results for the test system.

(vi) Reference intervals (normal values).

(vii) Any other performance characteristic required for test performance.

§ 493.1253 Standard: Establishment and verification of performance specifications.

Source - https://www.law.cornell.edu/cfr/text/42/493.1253

COLA(formerly the Commission on Office Laboratory Accreditation)

“Using a different sample matrix” is a Modification of Manufacturer Instructions.

When the laboratory alters or fails to follow the manufacturer’s instructions…the

laboratory must comply with…requirements for Performance Specifications for non-

FDA approved tests (VER 5-11)

Item QC 10 R

Source - https://clients.cola.org/files/pdf/COLA%20Laboratory%20Accreditation%20Manual.pdf

B) FDA Approved Methods Modified by the Lab…

VERIFICATION OF PERFORMANCE SPECIFICATIONS

Items V5-11• Accuracy

• Precision

• Reportable Range

• Reference Range

• Analytical Sensitivity

• Analytical Specificity (including interfering substances)

• Any Other Performance Characteristic Required for Test Performance

Including Linearity

“QUALITY CONTROL”

COM.40000 Method Validation/Verification ApprovalThere is a summary statement, signed by the laboratory director (or

designee who meets CAP director qualifications) prior to use in patient

testing, documenting evaluation of validation/verification studies and

approval of each test for clinical use.

**NEW/REVISED** 04/21/2014 Phase II

NOTES :

The summary statement must include a written assessment of the validation/verification study,

including the acceptability of the data. The summary must also include a statement approving

the test for clinical use with the approval signature such as, "This validation study has been

reviewed, and the performance of the method is considered acceptable for patient testing."

For an FDA-cleared/approved test, a summary of the verification data must address analytic

performance specifications, including analytic accuracy, precision, interferences, and reportable

range, as applicable.

In addition, for modified FDA-cleared/approved tests or LDTs, the summary must address

analytical sensitivity, analytical specificity and any other parameter that is considered

important to assure that the analytical performance of a test (e.g. specimen stability, reagent

stability, linearity, carryover, and cross-contamination, etc.), as appropriate and applicable.

CAP Laboratory Accreditation Program, All Common Checklist

CAP

Source - http://www.cap.org/web/home/lab/accreditation

COM.40620 Body Fluid Validation

Testing of body fluid specimens using methods intended for

other specimen types (eg blood or other fluid) have been

validated by the laboratory for accuracy, precision, analytic

sensitivity, analytic interferences, and reportable range.

**NEW/REVISED** 04/21/2014 Phase II

NOTES : Elaborates on procedures, extent of performance specifications,

reference intervals, alternative performance assessment, rare/unusual specimens

Method performance specifications for blood specimens may be used

for body fluids if the laboratory can reasonably exclude the existence

of matrix interferences affecting the latter either by reference in the

procedure manual to published literature or by evaluation for

interferences due to matrix effects by performing an appropriate study

(e.g. a dilution study using admixtures of samples, spiking samples,

further dilution).


CAP


COM.40620 Body Fluid Validation - CONTINUED

MORE NOTES :

“The reference range must be defined and reported with results, unless the

value is reported in comparison to its concentration in blood. Reference range

citations from the manufacturer’s insert or published literature citations may

be used to determine the range (COM.50000)…”

“Alternative performance assessment is required (COM.01500) and may

be performed using clinical assessment by chart review.”

“For clinically unique specimens…submitted with a unique request…it may

not be possible to establish test performance characteristics…In such cases

result must be accompanied by a comment such as, “The reference range and

other method performance specifications have not been established for this

body fluid. The test result must be integrated into the clinical context for

interpretation.”


CAP


“Method performance specifications for blood specimens may be used

for body fluids if the laboratory can reasonably exclude the existence of

matrix interferences affecting the latter…”

OPINION (JRG)

would be included not just on

the CAP checklist, but also

incorporated into CLIA law

In an ideal world…

CLIA is “our law”…and laws can be changed

BODY FLUID CHALLENGES

• Pre-Analytic Issues

• Analytic Issues

• Post-Analytic Issues

How many of these do you

really have control over?

PRE-ANALYTIC ISSUES

Modified from: Table 2.1, Kjeldsberg et al. Body Fluid Analysis, 2015, ASCP Press

Pre-Analytic

Collection container

Collection technique

Specimen handling

Specimen labeling

Presence of additives,

anticoagulants, preservatives

Transport time and temp

Specimen accessioning

General vs specific orderables

Centrifugation

Pre-treatment

types, inventory, cost

lack of standardization, clinic and operating room (OR), not phlebotomists

multiple locations involved (office, OR, accessioning, lab)

patient ID, site description

may be incompatible with orders / multiple orders

specimen degradation

general (“Sodium, Body Fluid”) versus specific (“Sodium, Pleural Fluid”)

interpretation of fluid types by lab staff

elimination of cells / particulates

hyaluronidase for viscous specimens

ANALYTIC ISSUES


Analytic

Specimen integrity

Cellularity

Viscosity

Instrument failure

(clot detection?)

Insufficient volume and

test prioritization

Indices

pH

QC

bad specimen = bad result (ex. “just tell me what the concentration is”)

are you measuring concentration in fluid or cells (e.g. frozen specimens)?

is the specimen aspirated appropriately by the instrument?

will the instrument tell you if it is not aspirated appropriately?

how do you handle / prioritize multiple orders on limited volume specimens?

hemolysis, lipemia, icterus

are assays affected by pH extremes?

availability of QC for routine body fluid testing (ex. Amniotic fluid AFP)

POST-ANALYTIC ISSUES


Post-Analytic

Result reporting

Reference intervals /

interpretive guidance

Specimen storage

Tracking source type

Results in comparison to

corresponding

serum/plasma

CAP requirements

can be very challenging

stability, container size

display in chart

obtaining specimens, “linking” results

BODY FLUID VALIDATION STUDIES

(i) Accuracy.

(ii) Precision.

(iii) Analytical sensitivity.

(iv) Analytical specificity to include interfering substances.

(v) Reportable range of test results for the test system.

(vi) Reference intervals (normal values).

(vii) Any other performance characteristic required for test performance.

● Set validation objectives (i.e. CLIA)

● Decide what fluids to validate

● Consider accreditation requirements

● Identify available resources

● Staff, time, AND specimens

Assay %

LDH 20.0

Total Protein 17.5

Glucose 16.2

Cholesterol 14.1

Triglycerides 6.8

Top 5 Pleural Requests

(43.6%)

~75%~1/3 of all your body fluid requests!

(and that’s just from pleural estimates)

Thatcher, Crabtree, Straseski, Genzen

Unpublished data.

BODY FLUID REQUESTS

BODY FLUID VALIDATION STUDIES

● Mixed Recovery

● Spiked Recovery

● Dilution Recovery

● Stability

● Precision

● Analytical Sensitivity

● Interferences

● Reference Intervals

Ma

trix

Acc

ura

cy

Lin

ea

rity

/ R

an

ge

For more

information

Examples on

the following

slides!

Block and Franke, 2015.

900 μl BODY FLUID

~ 1 ng/mL analyte

100 μl Serum

+ + + + +

GOALS: 1) Evaluate ACCURACY

2) Exclude MATRIX EFFECT

0 ng/mL 10000 ng/mL5000 ng/mL 7500 ng/mL2500 ng/mL

1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL

= = = = =

All body fluid specimens / spiking sera measured BEFORE mixing (need baseline values)

Measured SPIKED CONC. 1 243 480 760 998

EXPECTED FINAL CONC. 0.9 250.9 500.9 750.9 1000.9([X]fluid x 0.9) + ([X]spike x 0.1)

Calculate PERCENT RECOVERY[X]spiked conc.

expected final conc.X 100

111% 96.9% 95.8% 101.2% 99.7%

[X]fluid

[X]spike

SPIKED RECOVERY

Based on Block and Franke, “Quick Guide to Body Fluid Testing”, AACC Press, 2015.


2) Exclude MATRIX EFFECTSPIKED RECOVERY

MIXED RECOVERY

Low Conc High Conc(low end AMR) (high end AMR)

1) Obtain 2 body fluid specimens (low and high)

2) Create a 3rd (50:50 mix)

3) Create 4th and 5th 50:50 mixes (intermediates)

A E

Mid ConcC

Mid-Low ConcB

Mid-High ConcD

100% 75% 50% 25% 0%% of Low0% 25% 50% 75% 100%% of High

Expected = (1.00 x A) + (0 x E) = (.75 x A) + (.25 x E) = (. 5 x A) + (.5 x E) = (. 25 x A) + (.75 x E) = (0 x A) + (1.0 x E)

Measured 1 247 504 734 998

= (1.00 x 1) + (0 x 998) = (.75 x 1) + (.25 x 998) = (.5 x 1) + (.5 x 998) = (.25 x 1) + (.75 x 998) = (0 x 1) + (1.0 x 998)= 1 = 250.25 = 499.5 = 748.75 = 998

PERCENT

RECOVERYMeasured

ExpectedX 100

100% 98.7% 101% 98% 100%



3) Assess LINEARITY / RANGE


MIXED RECOVERYGOALS: 1) Evaluate ACCURACY


3) Assess LINEARITY / RANGE

DILUTION RECOVERY

High Conc(high end AMR)

A

100% 50% 25% 12.5% 6.25%% of A

Expected

PERCENT

RECOVERYMeasured

ExpectedX 100

100% 95.2% 97.8% 102.6% 99.4%

B C D E1:2 1:4 1:8 1:16

Serial Dilutions

Measured 998 475 244 128 59

Choose high conc. fluid specimen and appropriate diluent

Absolute 998 499 249.5 124.8 62.4



3) Assess LINEARITY/RANGE


DILUTION AND MIXED RECOVERYLinearity / Reportable Range

Plot data from Mixing Recovery and/or Dilution Recovery experiments

PRECISIONGOALS: 1) Evaluate IMPRECISION

High Conc. Low Conc.

Run Multiple Times(Within and/or Between Day)

Obtain high and low specimens (or pools)

Analytic Sensitivity• Replicates of specimens near lower limit of quantification

Interferences• Spiking of relevant endogenous / exogenous substances

Stability• Relevant to your laboratory processes

OTHER STUDIES

Reference Intervals• Can be very challenging

• Many fluids do not exist “normally”

• Chart review!

• Literature!


Tumor Markers (Beckman DXI)

EXAMPLE REFERENCES

Body Fluid Validations (Beckman AU)

Matrix Study (Roche cobas 8000)

Owen et al., Clin Biochem. 2015 Sep;48(13-14):911-4.

RECOVERY STUDIES – PLEURAL FLUID

Roche Cobas 8000


RECOVERY STUDIES – PLEURAL FLUID

Roche Cobas 8000

METHODS AND KITS


Part 1 – The End

Questions?

[email protected]

Documents

Session 7102 You Want to Measure ”? This ” in “ That › ascpannualmeeting › 2015 › AM15FCA › ... · 2015-10-26 · Session 7102 You Want to Measure ... Body Fluid Testing