Industry Division Newsletter

Winter 2013
2013 Division Officers Division Goals
Chair: Jennifer Huff, D.V.M., Ph.D.
Chair-Elect: Open • To provide a forum for AACC members to meet, identify
Secretary: Agim Beshiri, M.D. common interests and concerns, and develop programs that assist them in
Treasurer: Suzette Chance, Ph.D. the workplace. Although the focus of the Division is on issues relevant to
Past Chair: Doug Clark industry, or issues from the industry perspective, membership in the
Division is open to any member of the association.

Nominating Committee:
Karl De Vore • To identify and develop synergies between industry and
John Ellison clinical laboratory members which contribute to the health care system and
Mihaela Necula, Ph.D. enhance patient care.
Jim Pierson-Perry

Contents
Message from Past Division Chair.......................................................................... 1 - 2
Industry Division Awardees of recent years……………………………………... 2
Election Update……………………………………………………………………..2-3
Assay Interference: A Need for Increased Understanding and Ease of Testing…..4 - 11

Message from Past Division Chair

Doug Clark

We welcome our new Chair of the Industry Division, Jennifer Huff, along with all members of the
Industry Division. Please encourage your colleagues to join the Industry Division, if they have not, and
to actively participate if they already are members.

Our Division’s annual business meeting was held during this year’s AACC National Meeting in Los
Angeles, CA, on Tuesday, July 17th from 4-6 pm. We had two guest speakers at the meeting. Dr. Herbert
Vesper from the CDC spoke about the progress and future plans for steroid standardization projects.
Standardization programs are available for Testosterone and Estradiol and the development of a program
for Vitamin D is well underway.

Dr. Michael Bennett from the Children’s Hospital of Philadelphia spoke about the need for Pediatric
Reference ranges for methods provided by Industry. Availability of patient samples that cover all of the
age and gender groups needed to develop pediatric ranges for children is a problem for Industry.

At the AACC annual meeting in Los Angeles our Division sponsored the following seminars and
workshops:

• OEM Lecture Series

• Challenges and Clinical Impacts of Standardization of Immunoassays
• JCTLM/IVD Industry meeting

Page 1 of 11
 • Testing for Chronic Kidney Disease (CKD): Creatinine, GFR, UAlb and More: Industry
Responses to Clinical Needs
For 2013, Jennifer Huff will lead the Division’s annual business meeting and technical discussions. The
Industrial Division meeting in Houston will be held on Monday, July 29, 2013 from 2-4 pm. Jennifer will
provide more details in the near future so save the time slot to attend the meeting. Refreshments will be
provided.

Keep an eye out for two upcoming webinars sponsored by the Industry Division on our changing
regulatory landscape.

See you in Huston!

Industry Division Awardees of Recent Years

Best Abstract Award
This award is made to the author(s) of an abstract for the AACC national meeting which is deemed to
present a significant contribution to the IVD industry in one of the following areas: management,
regulatory affairs, or improved patient care through a new or improved medical device. The selection is
done through a vote of the Division’s officers and Nominating Committee members.
Awardees for the past three years were:

2010 A Panel of Cervicovaginal Fluid Biomarkers Efficiently Predicts Gestational Age at Delivery by
Alison Woodworth, Ph.D., DABCC, FACB, Assistant Professor of Pathology, Associate Director of
Clinical Chemistry, Vanderbilt University Medical Center, Nashville, TN
2011 Development of Real-time PCR assay for the Detection of Five Major Pneumonia Pathogen
Panels by Yu-Jeong Kim, Ph.D., Daejeon, Korea
2012 Direct Identification of Microbes in Urine Specimens Using Mass Spectrometry by Mari DeMarco
P.h.D, Clinical Chemistry Fellow Washington University School of Medicine Laboratory and Genomic
Medicine

Louis J. Dunka, Jr. Award

The Louis J. Dunka, Jr. Memorial Service Award is presented annually by the AACC Industry Division to
an individual for outstanding accomplishment that is evidenced by the initiation or support of a single
event or continuous effort that has served the AACC Industry Division Membership and/or the IVD
industry.

Awardees for the past three years were:

2009 Ronald H. Laessig, Ph.D. (posthumously) Emeritus Director, Wisconsin State Laboratory of
Hygiene, Madison, WI
2010 Jack Levine, M.B.A Siemens Healthcare Diagnostics, Tarrytown, NY
2011 James Pierson-Perry Ph.D. Siemens Healthcare Diagnostics, Glasgow, De

Election Update

After reviewing the by-laws for the Industrial Division, we found that we are behind in our elections of
some positions for 2013. The terms for Karl Devore and Mihaela Necula on the Nominating Committee
were up for renewal at the end of 2012. Also the term of Suzette Chance, our Treasurer was up for
renewal at the end of 2012. We need a Chair Elect for 2013 who will serve as the Chair for 2014.

Page 2 of 11
Please let Doug Clark or Jennifer Huff know if you are interested in any of these positions or if you
currently hold one of the positions it you would like to serve another term. We can hold a special election
to catch up with our by-laws if we can get nominees. It is not a huge time commitment so become
involved in the Division. With more members involved we can work on becoming more active Division
and provide useful articles, webinars, and information to all members of the Industrial Division.

The follow is an informative article from John H. Contois, PhD at Sun Diagnostics about Interference
Testing for Industry.

Page 3 of 11
Assay Interference: A Need for Increased Understanding and Ease of Testing

John H. Contois and Rae-Anne Nguyen

Sun Diagnostics, LLC
New Gloucester, Maine

Laboratory Directors are responsible for the quality of data that is reported from their
laboratories, but they rely on the manufacturer for a great deal of information. The quality of
interference testing data provided by the manufacturer is seldom questioned, and, in-house
interference testing is not normally performed. Only 8% of laboratories performed in-house
interference studies (2). Therefore, the burden is on the manufacturer to provide the highest
quality interference data possible. While relying on the manufacturer’s interference claims is
considered sufficient to meet regulatory requirements, this information may not translate to
performance in a specific clinical laboratory, and individual laboratories should be encouraged
to verify manufacturer’s data by performing in-house interference testing.

In addition, the laboratory must implement an effective detection system to identify specimens
with clinically important interferents and a specific policy to prevent reporting of inaccurate
results. One study reported that 9.7% of all specimens contained at least one visible
interferent; 76% were lipemic, 16% were hemolyzed, and 6% were icteric (3), yet visual
inspection of samples alone is not effective. The use of automated, spectrophotometric
measurement of bilirubin, hemoglobin, and lipemia (serum indices), along with clearly defined
decision rules, is effective (4), but must include an understanding of the relationship between
index scale and the impact on each test. Interestingly, recent abstract reports that only 38% of
laboratories were using automated serum indices for assessment of interference (2).
Manufacturers should encourage their customers to use serum indices and provide training to
help end users better understand how indices can better detect problem specimens.

It is also important for the laboratory to understand how the indices were determined by the
manufacturer, and for the manufacturer to describe their interference testing protocols in
detail. It is important to correlate icterus and hemolysis indices with bilirubin and hemoglobin
concentrations. Lipemia index is typically based on the spectrophotometric response of
Intralipid®, a synthetic lipid emulsion, and cannot be correlated with the response of human
triglyceride-rich lipoproteins. The manufacturer should assess lipemia using intact human
lipoproteins, primarily chylomicrons, instead of Intralipid.

Information provided by the manufacturer may not be adequate for the laboratory to gauge
the impact that interference will have on their results. Often, the data is reported without
defining a “significant” change or what analyte concentration was tested along with the
interferent, and interpretation is often based on an arbitrary 10% rule (5). The experimental

Page 4 of 11
design may be vague, such that the user does not have adequate information to make an
informed decision about the adequacy of interference testing (5).

There are several reasons for encouraging a laboratory to perform in-house interference testing
experiments, such as: verifying the commutability of interference data from the manufacturer
to their specific instrument/reagent system; to provide additional information that may not be
provided by the manufacturer, such as assessing interference with at least two levels of
analyte, as recommended by CLSI due to potential interdependence of interferent and analyte
concentrations; and determining the applicability of data generated from testing that used
artificial materials, such as Intralipid, instead of human lipoproteins to test lipemia interference.

Common Interferents

Interference from hemolysis (hemoglobin and /or red blood cell contents), lipemia
(triglyceride-rich lipoproteins or turbidity), icterus (unconjugated or conjugated bilirubin), and
proteins (albumin and gamma-globulins, paraproteins) are so common with routine chemistry
assays, that validation by manufacturers always includes interference testing for these
substances. Newer instruments and reagent formulations are better able to limit the effect of
common interferents; sample blanking, bichromatic measurements, and additives to inhibit
interferents have all been effective to a certain degree, but problems have not been eliminated.
Unfortunately, we cannot develop universal rules regarding interference, as instruments and
reagent systems can vary widely in the response to specific interferents (6, 7). Some
investigators have developed a mathematic formula for the correction of interference, but this
is generally not a good idea. The best approach is for laboratorians to understand the
limitations of the assay in their laboratory, and not report results that are inaccurate to a
degree that they will impact clinical interpretation.

Hemolysis

Hemolysis occurs in about 3% of all specimens received in the laboratory and accounts for 40%
to 70% of all unsuitable specimens (8). Dimeski describes four mechanisms for interference
from hemolysis: additive, spectral, chemical, and dilutional (9). Interference may be due to
hemoglobin or to other red cell constituents that are released into plasma or serum. These
substances can directly or indirectly interfere with a number of assays, including ALT, AST,
creatinine, CK, iron, LDH, lipase, magnesium, phosphorus, potassium, urea, albumin, ALP,
chloride, GGT and sodium (10-12).

Differentiating between in vivo and in vitro hemolysis is vitally important to patient

management. It is sometimes possible to differentiate between the two biochemically. Free
hemoglobin in the circulation binds to haptoglobin and the complex is rapidly cleared.
Therefore, a decrease in serum haptoglobin is consistent with in vivo hemolysis. Increased

Page 5 of 11
bilirubin and reticulocyte counts are also characteristic of in vivo hemolysis. The bilirubin is
derived from the breakdown of heme, while the presence of reticulocytes indicates the
physiological response to anemia. Another clue differentiating in vitro and in vivo hemolysis is
the concentration of intracellular contents: in vitro hemolysis is typically characterized by an
increase in cellular components such as K, LDH, and AST, which would be quickly normalized
during in vivo hemolysis. Given the clinical significance of hemolysis, the cause of hemolysis
should be investigated and documented. Procedures for handling and reporting hemolyzed
specimens should be available, including appropriate rejection criteria. As a rule of thumb,
when most or all specimens from a patient are hemolyzed, in vivo hemolysis should be
suspected and reported to the physician. When intermittent specimens are hemolyzed, it is
likely to be preanalytical, in vitro hemolysis. In vivo hemolysis is relatively rare, accounting for
only about 3% of all hemolyzed specimens (10), which is all the more reason to be vigilant.

Hemoglobin, which absorbs light strongly at around 550 nm, can cause an apparent increased
analyte concentration when monitored near this wavelength. Bichromatic readings and sample
blanking will minimize interference; however, the high background absorbance may still be
problematic (13). Hemoglobin may also have “pseudo” peroxidase activity which may interfere
with bilirubin measurement by diazonium methods (3). Artifactual release of red cell
constituents will have obvious effects on the accuracy of serum concentrations, especially K,
Mg, P, LDH, AST and ALT (10); AST activity is approximately 40-fold higher in erythrocytes than
in plasma, so that even slight hemolysis can alter results (10). Potassium is typically 25 times
more concentrated in red blood cells than in plasma. Adenylate kinase released from red cells
can increase creatine kinase (CK) activity (10), although the addition of AMP or other analogs
can inhibit adenylate kinase (11). Although most phosphate in red blood cells is organic, it can
stimulate the release of inorganic phosphate in serum (6). It is inappropriate to use
hemoglobin to assess interference from hemolysis because only the direct effect of hemoglobin
is evaluated. The effect of dilution and other red cell components should be considered, as well.
CLSI therefore recommends interference testing by spiking hemolysate into a non-hemolyzed
serum pool (14).

Various techniques have been used to reduce or correct for hemolysis interference.
Deproteinization by ultrafiltration or precipitation can remove hemoglobin but cannot correct
for the release of intracellular contents. Sample blanking and bichromatic measurement will
decrease the absorbance effect of hemoglobin but, again, will not correct for the release of
intracellular contents. It is critically important that the laboratory have a clear understanding of
the effect of hemolysis on each laboratory test and clearly know when a test result cannot be
accurately reported.

Lipemia

Page 6 of 11
Lipemia is best defined as turbidity in a sample caused by elevated triglycerides, mostly as
chylomicrons and very low density lipoproteins (VLDL), visible to the naked eye and caused by
recent dietary fat intake, abnormal lipoprotein metabolism, the infusion of lipids (parenteral or
enteral feeding), and heparin therapy. Interference from lipemia is due to light scattering or
absorbance or, with severe lipemia, volume displacement (9). Turbidity is visually evident with
triglycerides above about 300 mg/dL. Chylomicrons, because they are larger and more
triglyceride-rich, are the more common source of turbidity, while VLDL contributes much less
(15). Other causes of turbidity include elevated proteins (monoclonal gammopathies) and cold
agglutinins.

Lipemia interferes with almost all spectrophotometric measurements by absorbing and

scattering light. Sample blanking and bichromatic measurements help but do not eliminate this
interference. Triglyceride-rich lipoproteins in large concentrations also have a volume depletion
effect, whereby the apparent concentration of analyte is decreased because lipoproteins
replace the volume of available water. In other words, the volume taken up by lipoproteins is
included in the analyte concentration. With immunoassays, the analyte may not be accessible
to antibody, resulting in falsely low results.

For interference testing, lipemia is often simulated using Intralipid®, an emulsion containing
20% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water. Unfortunately, the
photometric response to this synthetic “fat” differs from physiological lipemia, making it
inappropriate to use Intralipid® for interference studies (16). It is far more accurate to assess
lipemia using intact human triglyceride-rich lipoproteins (15) as lipids are far more complex
than Intralipid®. CLSI recommends use of a high-triglyceride serum sample to evaluate lipemia
interference (14).

To prevent lipemia it is advisable to request fasting specimens (8-12 hour fast) and to halt
parenteral feedings for 8 hours prior to specimen collection. If a turbid sample is received in the
laboratory and the requested test(s) are affected, it is possible to remove lipoproteins by
centrifugation. Other approaches such as extraction of lipids with organic solvents,
precipitation of lipoproteins with polyanions, cyclodextrin, or polyethylene glycol, and
delipidation with detergents, lipase, and deoxycholate, have all been suggested. It is important
to document the process used to remove the turbidity from the sample and to remember to
measure cholesterol, triglycerides, and apolipoproteins before centrifugation or other means of
lipid removal. Laboratories will also want to ensure that their process to eliminate lipid
interference does not introduce additional errors. Although equations can be used to correct
for volume depletion, the equations do not consider other interfering effects, such as light
scattering, that may also introduce error.

Icterus

Page 7 of 11
Icterus is another name for jaundice, but it also specifically refers to serum or plasma with
elevated levels of conjugated or unconjugated bilirubin. Bilirubin occurs in serum as relatively
insoluble free bilirubin, as water soluble conjugate (mono- and di-glucuronides), and covalently
bound to albumin. Conjugated bilirubin is often seen in urine when present in high
concentrations in serum. Unlike hemolysis and lipemia, icterus is not easy to detect visually.
Icterus has been reported to interfere with many assays, including creatinine (Jaffe reaction),
phosphate (molybdate), albumin (dye-binding), and assays based on oxidase or peroxidase
reactions (glucose, cholesterol, triglycerides, and uric acid). Hyperbilirubinemia is relatively
common in hospitalized patients (ICU, gastroenterology, surgical, pediatrics), making
assessment of bilirubin interference, and choice of methods, especially important.

Bilirubin causes spectrophotometric interference because of its strong absorbance between

340 and 500 nm (3). Spectrophotometric interference is due to the fact that the absorption of
bilirubin and the chromophores typically used for reaction with hydrogen peroxide overlap (17).
It is important to evaluate interference from both conjugated and unconjugated bilirubin.

Possible solutions to the problem of icterus interference have been proposed, including use of
different chromogens, longer wavelengths, and additives such as ferrocyanide (17, 18) or
bilirubin oxidase (18). Unfortunately, none of the methods fully resolve the problem. The best
guidance is for laboratorians to evaluate or understand the effect of icterus on their test
systems and ensure that inaccurate results are not reported.

Proteins

Most protein interference is cause by paraproteins (monoclonal immunoglobulins) that may

affect many different assays, including bilirubin, phosphate, HDL cholesterol, GGT, CRP, and
glucose (19). The interference may be due to physical or chemical alteration in the sample or it
may be due to a volume displacement effect. A very high protein concentration will, like very
high concentrations of chylomicrons and VLDL, reduce the available water (9). The practical
effect of these “solids” is volume depletion; the aspirated sample is diluted with protein so that
the analyte concentration is artifactually low. As with lipemia interference, the use of
equations to calculate for volume depletion is discouraged. The equations do not consider
other interfering effects, such as light scattering, that may also introduce error and the mean
specific volumes are estimates that can vary widely between individuals.

Viscosity is a common problem with specimens containing monoclonal immunoglobulins,

especially IgM, and this may affect sample delivery.

Serum Indices

Page 8 of 11
Visual inspection alone is not an effective method for screening for interferents that may be
present in a sample. Serum indices are semi-quantitative estimates of hemoglobin, bilirubin,
and lipemia (turbidity) in a sample using spectrophotometry. These indices are typically
expressed in arbitrary units (+1, +2, etc) but can also be correlated to actual concentrations of
hemoglobin, bilirubin, and triglycerides. Absorbances are recorded at several wavelengths to
allow the calculation of the indices, and although they are similar, each manufacturer has a
unique procedure. In general, we can assume that absorbance for turbidity is first monitored at
wavelengths >600 nm. Then absorbance at ~450-575 nm is measured to assess bilirubin
(icterus), and absorbance at 400-600 nm is used to assess hemoglobin interference. Algorithms
are used to resolve overlapping areas and calculate indices. Jay reported clinically meaningful
changes in AST, chloride, LDH, potassium, and sodium in specimens without visually apparent
hemolysis (11). Although carotenoids and other pigments may interfere with this assessment,
automated measurement of serum indices is necessary for an effective program (3).
Manufacturers should encourage laboratories to use serum indices as part of their program to
detect abnormal specimens.

Increase Understanding and Evaluation

The responsibilities of ensuring assay results are accurate falls on both the instrument/reagent
manufacturer and the laboratory.

Manufacturers should provide specific details of their experiments including the materials used,
the protocols followed and the levels of interferent and analyte tested. Manufacturers must
provide a full description of the experimental design and statistical methods used to assess
interference with each assay (5). When serum indicies are listed, the procedure used to
determine the indicies should be clearly outlined.

Laboratories should verify how their instruments and assays perform in their environment, with
their patient population, and compare that to the claims of their manufacturer. They must
assess the quality and completeness of the data supplied by the manufacturer. There should be
clear methods for identifying samples with interferents and procedures for dealing with
affected samples. Labs must understand assay limitations and interference thresholds so that
inaccurate results are not reported.

CLSI guidance document EP-7A outlines procedures for evaluating assays for interference and is
intended for both manufacturers and laboratories. The recommendation includes an initial
screening for interferents and then, if necessary, a dose response experiment to determine
affect at different interferent concentrations.

Using the guidance outlined in the CLSI document, EP-7A (14), Sun Diagnostics has developed
an interference test kit to aide manufacturers and laboratories in evaluating interference from

Page 9 of 11
the substances described in this paper. ASSURANCE™ Interference Test Kit contains human
triglyceride-rich lipoproteins for evaluation of lipemia interference; human hemolysate for
evaluation of hemolysis interference; a mixture of human serum albumin and gamma-globulins
to evaluate protein interference; and conjugated and unconjugated bilirubin to evaluate
interference from icterus. The samples are highly concentrated (up to 20x; the CLSI
recommendation) and contain no added stabilizers or preservatives so that the sample matrix is
largely intact. Additionally, Sun Diagnostics has developed two very useful tools to accompany
the interference test kit: a recommended testing protocol that clearly outlines the steps for
both screening and dose response interference experiments and a data analysis spreadsheet
that performs the calculations necessary to help determine if a substance is interfering with an
assay.

With the availability of a kit, interference testing becomes more practical. Manufacturers and
laboratories no longer need to find patient samples that fit their needs, store untested patient
samples, or obtain artificial interferent substitutes, such as IntraLipid®. ASSURANCE™ contains
stable, highly concentrated, purified and/or human-sourced materials that are representative
of actual interferents encountered during testing. The human-sourced material is tested and
found negative/non-reactive for infectious disease. The recommended testing protocols and
data analysis tools can be worked into the routine procedure for interference testing.
ASSURANCE™ Interference Test Kit from Sun Diagnostics will simplify interference testing.

References

1. Ji JZ, Meng QH. Evaluation of the interference of hemoglobin, bilirubin, and lipids on Roche
Cobas 6000 assays. Clin Chem Acta 2011; 412:1550-1553.
2. Aslan B et al. Abstract A71: Clinical chemistry sample interferences reporting patterns in
Ontario laboratories. Clin Chem Supplement 2012; 58(10):A23-A24.
3. Roche Diagnostics. Cobas analyzer (c501) handbook: Serum Indicies: Reduction of clinical
errors in laboratory medicine. http://www.roche-
diagnostics.cz/download/program/Serum%20Indices_maly.pdf
4. Vermeer HJ, Thomassen E, de Jonge N. Automated processing of serum indicies used for
interference detection by the laboratory information system. Clin Chem 2005; 51:244-247.
5. Kazmierczak SC, Catrou PG. Analytical interference: more than just a laboratory problem. Am J
Clin Pathol 2000; 113:9-11.
6. Glick MR, Ryder KW, Jackson SA. Graphical comparisons of interferences in clinical chemistry
instrumentation. Clin Chem 1986; 32(3):470-475.
7. Glick MR, Ryder KW. Analytical systems ranked by freedom from interferences. Clin Chem 187;
33(8):1453-1458.
8. Lippi G e. al. Abstract: Haemolysis: An overview of the leading cause of unsuitable specimens
in clinical laboratories. Clin Chem & Lab Med. 2008 46(6):764-772.
9. Dimeski G. Interference testing. Clin Biochem Rev 2008; 29 Suppl (i): S43-S48.
10. Thomas L. Haemolysis as influence and interference factor. eJIFCC vol 13 no 4:
http://www.ifcc.org/ejifcc/vol13no4/130401002.htm.

Page 10 of 11
11. Thomas L. Haemolysis as influence and interference factor. eJIFCC vol 13 no 4:
http://www.ifcc.org/ejifcc/vol13no4/130401002.htm.
12. Lippi G et. al. Abstract: Influence of hemolysis on routine clinical chemistry testing. Clin Chem
& Lab Med. 2006 44(3):311-316.
13. Cook SL, Bruns DE. Persistent hemolysis in a patient with pancreatitis. Clin Chem 2012; 58:974-
978.
14. CLSI, INTERFERENCE TESTING IN CLINICAL CHEMISTRY; APPROVED GUIDELINE, 2ND ED, CLSI
DOCUMENT EP07-A2, WAYNE, PA, 2005.
15. Bornhorst JA, Roberts RF, Roberts WL. Assay-specific differences in lipemic interference in native
and intralipid-supplemented samples. Clin Chem 2004; 50:2197-2201.
16. Nanji AA, Poon R, Hinberg I. Lipaemic interference: effects of lipaemic serum and intralipid. J Clin
Pathol 1988; 41:1026-1027.
17. Spain MA, Wu AHB. Bilirubin interference with determination of uric acid, cholesterol, and
triglycerides in commercial peroxidase-coupled assays, and the effect of ferrocyanide. Clin
Chem 1986; 32(3):518-521.
18. Artiss JD, McEnroe RJ, Zak B. Bilirubin interference in a peroxidase-coupled procedure for
creatinine eliminated by bilirubin oxidase. Clin Chem 1984; 30(8):1389-1392.
19. Artiss JD, Zak B. Problems with measurements caused by high concentrations of serum solids.
Crit Rev Clin Lab Sci 1987; 25: 19-41.
20. www.westgard.com/basic-method-validation-3rd-edition-faqs.htm
21. Sun Diagnostics, LLC: www.sundiagnostics.us and www.sundiagnostics.us/assurance

Page 11 of 11