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7 Interferences from Bilirubin, Hemolysis, and High Lipid Content

7 Interferences from Bilirubin, Hemolysis, and High Lipid Content

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2.8 Interferences from Endogenous and Exogenous Components

Hemoglobin is mainly released by hemolysis of red blood cells (RBC).

Hemolysis can occur in vivo, during venipuncture and blood collection, or

during sample processing. Hemoglobin interference depends on its concentration in the sample. Serum appears hemolyzed when the hemoglobin concentration exceeds 20 mg/dL. The absorbance maxima of the heme moiety in

hemoglobin are at 540 to 580 nm wavelengths. However, hemoglobin

begins to absorb around 340 nm and then absorbance increases at

400À430 nm as well. Interference of hemoglobin (if the specimen is grossly

hemolyzed) is due to interference with the optical detection system of

the assay.

All lipids in plasma exist as complexed with proteins that are called lipoproteins, and particle size varies from 10 nm to 1000 nm (the higher the percentage of the lipid, the lower the density of the resulting lipoprotein and

the larger the particle size). The lipoprotein particles with high lipid content

are micellar and are the main source of assay interference. Unlike bilirubin

and hemoglobin, lipids normally do not participate in chemical reactions

and mostly cause interference in assays due to their turbidity and capability

of scattering light, as in nephelometric assays.



Immunoassays are affected by a variety of endogenous and exogenous

compounds, including heterophilic antibodies. The key points regarding

immunoassay interferences include:

Endogenous factors such as digoxin-like immunoreactive factors only

affect digoxin immunoassays. Please see Chapter 15 for a more detailed


Structurally similar molecules are capable of cross-reacting with the

antibody to cause falsely elevated (positive interference) or falsely

lowered results (negative interference). Negative interference occurs less

frequently than positive interference, but may be clinically more

dangerous. For example, if the result of a therapeutic drug is falsely

elevated compared to the previous measurement, the clinician may

question the result, but if the value is falsely lower, the clinician may

simply increase the dose without realizing that the value was falsely

lower due to interference. That can cause drug toxicity in the patient.

Interference from drug metabolites is the most common form of

interference, although other structurally similar drugs may also be the

cause of interference. See also Chapter 15.




I m m un o a ss a y P l a t f o r m an d D e s i g ns



Heterophilic antibodies are human antibodies that interact with assay antibody

interferences. Features of heterophilic antibody interference in immunoassays


Heterophilic antibodies may arise in a patient in response to exposure to

certain animals or animal products or due to infection by bacterial or

viral agents, or non-specifically.

Among heterophilic antibodies, the most common are human antimouse antibodies (HAMA) because of wide use of murine monoclonal

antibody products in therapy or imaging. However, other anti-animal

antibodies in humans have also been described that can interfere with an


If a patient is exposed to animals or animal products, or suffers from an

autoimmune disease, the patient may have heterophilic antibodies in


Heterophilic antibodies interfere most commonly with sandwich assays

that are used for measuring large molecules, but rarely interfere with

competitive assays. Most common interferences of heterophilic

antibodies are observed with the measurement of various tumor markers.

In the sandwich-type immunoassays, heterophilic antibodies can form

the “sandwich complex” even in the absence of the target antigen; this

generates mostly false positive results. False negative results due to the

interference of heterophilic antibodies are rarely observed.

Heterophilic antibodies are absent in urine. Therefore, if a serum

specimen is positive for an analyte, for example, human chorionic

gonadotropin (hCG), but beta-hCG cannot be detected in the urine

specimen, it indicates interference from heterophilic antibodies in the

serum hCG measurement.

Another way to investigate heterophilic antibody interference is serial

dilution of a specimen. If serial dilution produces a non-linear result, it

indicates interference in the assay.

Interference from heterophilic antibodies may also be blocked by adding

any commercially available heterophilic antibody blocking agent in the

specimen prior to analysis.

For analytes that are also present in the protein-free ultrafiltrate

(relatively small molecules), analysis of the analyte in the protein-free

ultrafiltrate can eliminate interference from heterophilic antibodies

because, due to large molecular weights, heterophilic antibodies are

absent in protein-free ultrafiltrates.

Heterophilic antibodies are more commonly found in sick and hospitalized

patients with reported prevalences of 0.2%À15%. In addition, rheumatoid

2.10 Interferences from Autoantibodies and Macro-Analytes

factors that are IgM type antibodies may be present in the serum of patients

suffering from rheumatoid arthritis and certain autoimmune diseases.

Rheumatoid factors may interfere with sandwich assays and the mechanism

of interference is similar to the interference caused by heterophilic antibodies. Commercially available rheumatoid factor blocking agent may be used

to eliminate such interferences.


A 58-year-old man without any familial risk for prostate cancer

visited his primary care physician and his prostate-specific

antigen (PSA) level was 83 ng/mL (0À4 ng/mL is normal). He

was referred to a urologist and his digital rectal examination

was normal. In addition, a prostate biopsy, abdominal tomodensitometry, whole body scan, and prostatic MRI were performed, but no significant abnormality was observed.

However, due to his very high PSA level (indicative of advance

stage prostate cancer) he was treated with androgen deprivation therapy with goserelin acetate and bicalutamide. After 3

months he still had no symptoms, his prostate was atrophic on

digital rectal examination, and he had suppressed testosterone

levels as expected. However, his PSA level was still highly

elevated (122 ng/mL) despite no radiographic evidence of

advanced cancer. At that point his serum PSA was analyzed

by a different assay (Immulite PSA, Cirrus Diagnostics, Los

Angeles) and the PSA level was , 0.3 ng/mL. The treating

physician therefore suspected a false positive PSA by the original Access Hybritech PSA assay (Hybritech, San Diego, CA),

and interference of heterophilic antibodies was established by

treating specimens with heterophilic antibody blocking agent.

Re-analysis of the high PSA specimen showed a level below

the detection limit. This patient received unnecessary therapy

for his falsely elevated PSA level due to the interference of heterophilic antibody [9].


A 64-year-old male during a routine visit to his physician was

diagnosed with hypothyroidism based on elevated TSH (thyroid stimulating hormone) levels, and his clinician initiated

therapy with levothyroxine (250 microgram per day). Despite

therapy, there were still increased levels of TSH (33 mIU/L)

and his FT4 level was also elevated. The endocrinologist at

that point suspected that TSH levels measured by the Unicel

Dxi analyzer (Beckman Coulter) were falsely elevated due to

interference. Serial dilution of the specimen showed non-

linearity, an indication of interference. When the specimen

was analyzed using a different TSH assay (immunoradiometric assay (IRMA), also available from Beckman Coulter),

the TSH value was 1.22 mIU/L, further confirming the interference with the initial TSH measurement. The patient had a

high concentration of rheumatoid factor (2700 U/mL) and the

authors speculated that his falsely elevated TSH was due to

interference from rheumatoid factors [10].



Autoantibodies (immunoglobulin molecules) are formed by the immune

system of an individual capable of recognizing an antigen on that person’s




I m m un o a ss a y P l a t f o r m an d D e s i g ns

own tissues. Several mechanisms may trigger the production of autoantibodies, for example, an antigen formed during fetal development and then

sequestered may be released as a result of infection, chemical exposure, or

trauma, as occurs in autoimmune thyroiditis. The autoantibody may bind to

the analyte-label conjugate in a competition-type immunoassay to produce a

false positive or false negative result. Circulating cardiac troponin I autoantibodies may be present in patients suffering from acute cardiac myocardial

infarction where troponin I elevation is an indication of such an episode.

Unfortunately, the presence of circulating cardiac troponin I autoantibodies

may falsely lower cardiac troponin I concentration (negative interference)

using commercial immunoassays, thus complicating the diagnosis of acute

myocardial infarction [11]. However, falsely elevated results due to the presence of autoantibodies are more common than false negative results.

Verhoye et al. found three patients with false positive thyrotropin results

that were caused by interference from an autoantibody against thyrotropin.

The interfering substance in the affected specimens was identified as an

autoantibody by gel-filtration chromatography and polyethylene glycol

precipitation [12].

Often the analyte can conjugate with immunoglobin or other antibodies to

generate macro-analytes, which can falsely elevate the true value of the analyte. For example, macroamylasemia and macro-prolactinemia can produce

falsely elevated results in amylase and prolactin assays, respectively. In

macro-prolactinemia, the hormone prolactin conjugates with itself and/or

with its autoantibody to create macro-prolactin in the patient’s circulation.

The macro-analyte is physiologically inactive, but often interferes with many

prolactin immunoassays to generate false positive prolactin results [13]. Such

interference can be removed by polyethylene glycol precipitation.


A 17-year-old girl was referred to a University hospital for

having a persistent elevated level of aspartate aminotransferase (AST). One year earlier, her AST level was 88 U/L as

detected during her annual school health check, but she had

no medical complaints. She was not on any medication and

had a regular menstrual cycle. Her physical examination at

the University hospital was unremarkable. All laboratory test

results were normal, but her AST level was further elevated

to 152 U/L. All serological tests for hepatitis were negative.

On further follow-up her AST level was found to have

increased to 259 U/L. At that point it was speculated that her

elevated AST was due to interference, and further study by

gel-filtration showed a species with a molecular weight of

250 kilodaltons. This was further characterized by immunoelectrophoresis and immunoprecipitation to be an immunoglobulin (IgG kappa-lambda globulin) complexed AST that

was causing the elevated AST level in this girl. These complexes are benign [14].

Key Points


The Prozone or hook effect is observed when a very high amount of an analyte is present in the sample but the observed value is falsely lowered. This

type of interference is observed more commonly in sandwich assays. The

mechanism of this significant negative interference is the capability of a high

level of an analyte (antigen) to reduce the concentrations of “sandwich”

(antibody 1:antigen:antibody 2) complexes that are responsible for generating the signal by forming mostly single antibody:antigen complexes. The

hook effect has been reported with assays of a variety of analytes, such as

β-hCG, prolactin, calcitonin, aldosterone, cancer markers (CA 125, PSA), etc.

The best way to eliminate the hook effect is serial dilution. For example, if

the hook effect is present and the original value of an analyte (e.g. prolactin)

was 120 ng/mL, then 1:1 dilution of the specimen should produce a value of

60 ng/mL; but if the observed value was 90 ng/mL (which was significantly

higher than the expected value), the hook effect should be suspected. In

order to eliminate the hook effect, a 1:10, 1:100, or even a 1:1000 dilution

may be necessary so that the true analyte concentration will fall within the

analytical measurement range (AMR) of the assay..


A 16-year-old girl presented to the emergency department

with a 2-week history of nausea, vomiting, vaginal spotting,

and lower leg edema. On physical examination, a lower abdomen palpable mass was found. The patient admitted sexual

activity, but denied having any sexually transmitted disease.

Molar pregnancy was suspected, and the quantitative β-subunit of human chorionic gonadotropin (β-hCG) concentration

was 746.2 IU/L; however, the urine qualitative level was negative. Repeat of the urinalysis by a senior technologist also

produced a negative result. At that point the authors

suspected the hook effect and dilution of the serum specimen

(1:1) produced a non-linear value (455.2 IU/L), which further

confirmed the hook effect. After a 1:10 dilution, the urine test

for β-hCG became positive, and finally, by using a 1:10,000

dilution of the specimen, the original serum β-hCG concentration was determined to be 3,835,000 IU/L. Usually the hook

effect is observed with a molar β-hCG level in serum because

high amounts of β-hCG are produced by molar pregnancy



Immunoassays can be competitive or immunometric (non-competitive, also known

as sandwich). In competitive immunoassays only one antibody is used. This

format is common for assays of small molecules such as a therapeutic drugs or




I m m un o a ss a y P l a t f o r m an d D e s i g ns

drugs of abuse. In the sandwich format two antibodies are used and this format is

more commonly used for assays of relative large molecules.

Homogenous immunoassay format: After incubation, no separation between

bound and free label is necessary.

Heterogenous immunoassay format: The bound label must be separated from the

free label before measuring the signal.

Commercially available immunoassays use various formats, including FPIA, EMIT,

CEDIA, KIMS, and LOCI. In the fluorescent polarization immunoassay (FPIA), the

free label (a relatively small molecule) attached to the analyte (antigen) molecule

has different Brownian motion than when the label is complexed to a large

antibody (140,000 or more Daltons). FPIA is a homogenous competitive assay

where after incubation the fluorescence polarization signal is measured; this signal

is only produced if the labeled antigen is bound to the antibody molecule.

Therefore, intensity of the signal is inversely proportional to the analyte


EMIT (enzyme multiplied immunoassay technique) is a homogenous competitive

immunoassay where the antigen is labeled with glucose 6-phosphate

dehydrogenase, an enzyme that reduces nicotinamide adenine dinucleotide (NAD,

no signal at 340 nm) to NADH (absorbs at 340 nm), and the absorbance is

monitored at 340 nm. When a labeled antigen binds with the antibody molecule,

the enzyme label becomes inactive and no signal is generated. Therefore, signal

intensity is proportional to analyte concentration.

The Cloned Enzyme Donor Immunoassay (CEDIA) method is based on

recombinant DNA technology where bacterial enzyme beta-galactosidase is

genetically engineered into two inactive fragments. When both fragments

combine, a signal is produced that is proportional to the analyte concentration.

Kinetic interaction of microparticle in solution (KIMS): In the absence of antigen

molecules free antibodies bind to drug microparticle conjugates to form particle

aggregates that result in an increase in absorption that is optically measured at

various visible wavelengths (500À650 nm).

Luminescent oxygen channeling immunoassays (LOCI): The immunoassay

reaction is irradiated with light to generate singlet oxygen molecules in

microbeads (“Sensibead”) coupled to the analyte. When bound to the respective

antibody molecule, also coupled to another type of bead, it reacts with singlet

oxygen and chemiluminescence signals are generated that are proportional to the

concentration of the analyteÀantibody complex.

Usually total bilirubin concentration below 20 mg/dL does not cause interferences,

but concentrations over 20 mg/dL may cause problems. The interference of

bilirubin is mainly caused by its absorbance at 454 or 461 nm.

Various structurally related drugs or drug metabolites can interfere with



Heterophilic antibodies may arise in a patient in response to exposure to certain

animals or animal products, due to infection by bacterial or viral agents, or use of

murine monoclonal antibody products in therapy or imaging. Heterophilic

antibodies interfere most commonly with sandwich assays used for measuring

large molecules, but rarely with competitive assays, causing mostly false positive


Heterophilic antibodies are absent in urine. Therefore, if a serum specimen is

positive for an analyte (e.g. human chorionic gonadotropin, hCG), but beta-hCG

cannot be detected in the urine specimen, it indicates interference from a

heterophilic antibody in the serum hCG measurement. Another way to investigate

heterophilic antibody interference is serial dilution of a specimen. If serial dilution

produces a non-linear result, it indicates interference in the assay. Interference

from heterophilic antibodies can also be blocked by adding commercially available

heterophilic antibody blocking agents to the specimen prior to analysis.

Autoantibodies are formed by the immune system of a person that recognizes an

antigen on that person’s own tissues, and may interfere with an immunoassay to

produce false positive results (and less frequently, false negative results). Often the

endogenous analyte of interest will conjugate with immunoglobin or other

antibodies to generate macro-analytes, which can falsely elevate a result. For

example, macroamylasemia and macro-prolactinemia can produce falsely elevated

results in amylase and prolactin assays, respectively. Such interference can be

removed by polyethylene glycol precipitation.

Prozone (“hook”) effect: Very high levels of antigen can reduce the concentrations

of “sandwich” (antibody 1:antigen:antibody 2) complexes responsible for

generating the signal by forming mostly single antibody:antigen complexes. This

effect, known as the prozone or hook effect (excess antigen), mostly causes

negative interference (falsely lower results). The best way to eliminate the hook

effect is serial dilution.


[1] Jolley ME, Stroupe SD, Schwenzer KS, Wang CJ, et al. Fluorescence polarization immunoassay

III. An automated system for therapeutic drug determination. Clin Chem 1981;27:1575À9.

[2] Jeon SI, Yang X, Andrade JD. Modeling of homogeneous cloned enzyme donor immunoassay. Anal Biochem 2004;333:136À47.

[3] Snyder JT, Benson CM, Briggs C, et al. Development of NT-proBNP, Troponin, TSH, and FT4

LOCI(R) assays on the new Dimension (R) EXL with LM clinical chemistry system. Clin

Chem 2008;54:A92 [Abstract #B135].

[4] Dai JL, Sokoll LJ, Chan DW. Automated chemiluminescent immunoassay analyzers. J Clin

Ligand Assay 1998;21:377À85.

[5] Forest J-C, Masse J, Lane A. Evaluation of the analytical performance of the Boehringer

Mannheim Elecsyss 2010 Immunoanalyzer. Clin Biochem 1998;31:81À8.

[6] Babson AL, Olsen DR, Palmieri T, Ross AF, et al. The IMMULITE assay tube: a new approach

to heterogeneous ligand assay. Clin Chem 1991;37:1521À2.




I m m un o a ss a y P l a t f o r m an d D e s i g ns

[7] Christenson RH, Apple FS, Morgan DL. Cardiac troponin I measurement with the

ACCESSs immunoassay system: analytical and clinical performance characteristics. Clin

Chem 1998;44:52À60.

[8] Montagne P, Varcin P, Cuilliere ML, Duheille J. Microparticle-enhanced nephelometric

immunoassay with microsphere-antigen conjugate. Bioconjugate Chem 1992;3:187À93.

[9] Henry N, Sebe P, Cussenot O. Inappropriate treatment of prostate cancer caused by heterophilic antibody interference. Nat Clin Pract Urol 2009;6:164À7.

[10] Georges A, Charrie A, Raynaud S, Lombard C, et al. Thyroxin overdose due to rheumatoid

factor interferences in thyroid-stimulating hormone assays. Clin Chem Lab Med


[11] Tang G, Wu Y, Zhao W, Shen Q. Multiple immunoassays systems are negatively interfered

by circulating cardiac troponin I autoantibodies. Clin Exp Med 2012;12:47À53.

[12] Verhoye E, Bruel A, Delanghe JR, Debruyne E, et al. Spuriously high thyrotropin values due

to anti-thyrotropin antibody in adult patients. Clin Chem Lab Med 2009;47:604À6.

[13] Kavanagh L, McKenna TJ, Fahie-Wilson MN, et al. Specificity and clinical utility of methods

for determination of macro-prolactin. Clin Chem 2006;52:1366À72.

[14] Matama S, Ito H, Tanabe S, Shibuya A, et al. Immunoglobulin complexed aspartate aminotransferase. Intern Med 1993;32:156À9.

[15] Er TK, Jong YJ, Tsai EM, Huang CL, et al. False positive pregnancy in hydatidiform mole.

Clin Chem 2006;52:1616À8.


Pre-Analytical Variables



Accurate clinical laboratory test results are important for proper diagnosis

and treatment of patients. Factors that are important to obtaining accurate

laboratory test results include:

Patient Identification: The right patient is identified prior to specimen

collection by matching at least two criteria.

Collection Protocol: The correct technique and blood collection tube

have been used for sample collection to avoid tissue damage, prolonged

venous stasis, or hemolysis.

Labeling: After collection, the specimen was labeled properly with correct

patient information; specimen misidentification is a major source of preanalytical error.

Specimen Handling: Proper centrifugation (in the case of serum or

plasma specimen analysis) and proper transportation of specimens to the


Storage Protocol: Maintaining proper storage of specimens prior to

analysis in order to avoid artifactual changes in analyte; for example,

storing blood gas specimens in ice if the analysis cannot be completed

within 30 min of specimen collection.

Interference Avoidance: Proper analytical steps to obtain the correct result

and avoid interferences.

LIS Reports: Correctly reporting the result to the laboratory information

system (LIS) if the analyzer is not interfaced with the LIS.

Clinician Reports: The report reaching the clinician must contain the right

result, together with interpretative information, such as a reference range

and other comments that aid clinicians in the decision-making process.


3.1 Laboratory

Errors in PreAnalytical,

Analytical, and


Stages .................... 35

3.2 Order of Draw of

Blood Collection

Tubes..................... 37

3.3 Errors with


Preparation ........... 38

3.4 Errors with

Patient Identification

and Related

Errors ..................... 38

3.5 Error of

Collecting Blood in

Wrong Tubes: Effect

of Anticoagulants. 40

3.6 Issues with

Urine Specimen

Collection .............. 42

3.7 Issues with

Specimen Processing


Transportation...... 42

3.8 Special Issues:

Blood Gas and


A. Dasgupta and A. Wahed: Clinical Chemistry, Immunology and Laboratory Quality Control

DOI: http://dx.doi.org/10.1016/B978-0-12-407821-5.00003-6

© 2014 Elsevier Inc. All rights reserved.



P r e -A na ly t ica l V ar i ab le s

Ionized Calcium

Analysis................. 43

Table 3.1 Common Laboratory Errors

Key Points ............. 44

Type of Error

References ............ 45

Pre-Analytical Errors

Tube filling error

Patient identification error

Inappropriate container

Empty tube

Order not entered in laboratory information system

Specimen collected wrongly from an infusion line

Specimen stored improperly

Contamination of culture tube

Analytical Errors

Inaccurate result due to interference

Random error caused by the instrument

Post-Analytical Errors

Result communication error

Excessive turnaround time due to instrument downtime

Failure at any of these steps can result in an erroneous or misleading laboratory result, sometimes with adverse outcomes. The analytical part of the analysis involves measurement of the concentration of the analyte corresponding

to its “true” level (as compared to a “gold standard” measurement) within a

clinically acceptable margin of error (the total acceptable analytical error,

TAAE). Errors can occur at any stage of analysis (pre-analytical, analytical, and

post-analytical). It has been estimated that pre-analytical errors account for

more than two-thirds of all laboratory errors, while errors in the analytical

and post-analytical phases account for only one-third of all laboratory errors.

Carraro and Plebani reported that, among 51,746 clinical laboratory analyses

performed in a three-month period in the author’s laboratory (7,615 laboratory orders, 17,514 blood collection tubes), clinicians contacted the laboratory regarding 393 questionable results out of which 160 results were

confirmed to be due to laboratory errors. Of the 160 confirmed laboratory

errors, 61.9% were determined to be pre-analytical errors, 15% were analytical

errors, while 23.1% were post-analytical errors [1]. Types of laboratory errors

(pre-analytical, analytical, and post-analytical) are summarized in Table 3.1.

In order to avoid pre-analytical errors, several approaches can be taken,


The use of hand-held devices connected to the LIS that can objectively

identify the patient by scanning a patient attached barcode, typically a

wrist band.

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