session 7102 you want to measure ”? this ” in “ that › ascpannualmeeting › 2015 ›...
TRANSCRIPT
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 Laboratory Accreditation Program, All Common Checklist
CAP
Source - http://www.cap.org/web/home/lab/accreditation
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 Laboratory Accreditation Program, All Common Checklist
CAP
Source - http://www.cap.org/web/home/lab/accreditation
“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
Modified from: Table 2.1, Kjeldsberg et al. Body Fluid Analysis, 2015, ASCP Press
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
Modified from: Table 2.1, Kjeldsberg et al. Body Fluid Analysis, 2015, ASCP Press
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.
GOALS: 1) Evaluate ACCURACY
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%
GOALS: 1) Evaluate ACCURACY
2) Exclude MATRIX EFFECT
3) Assess LINEARITY / RANGE
Based on Block and Franke, “Quick Guide to Body Fluid Testing”, AACC Press, 2015.
MIXED RECOVERYGOALS: 1) Evaluate ACCURACY
2) Exclude MATRIX EFFECT
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
GOALS: 1) Evaluate ACCURACY
2) Exclude MATRIX EFFECT
3) Assess LINEARITY/RANGE
Based on Block and Franke, “Quick Guide to Body Fluid Testing”, AACC Press, 2015.
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!
Based on Block and Franke, “Quick Guide to Body Fluid Testing”, AACC Press, 2015.
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
Owen et al., Clin Biochem. 2015 Sep;48(13-14):911-4.
RECOVERY STUDIES – PLEURAL FLUID
Roche Cobas 8000
METHODS AND KITS
Owen et al., Clin Biochem. 2015 Sep;48(13-14):911-4.