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36 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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within each chamber communicate through a small aperture
(100 µm) or sensing zone. When a nonconductive particle or cell
passes through the aperture, it displaces an equivalent volume
of conductive uid. is increases the conductance and creates
a voltage pulse for each cell counted, the intensity of which is
proportional to the cell volume.
14
In hematology analyzers, blood is separated into two samples for measurement. One volume is mixed with a diluent and
delivered to a chamber where platelet and erythrocyte counts
are performed. Particles with volumes between 2 and 20 femtoliter (fL) are counted as platelets, and particles with volumes
>36 fL are counted as erythrocytes. e other volume is mixed
with a diluent, and an erythrocyte lysing reagent is used to permit leukocyte (>36 fL) counts to be performed. e number of
cells in this size range may be subtracted from the erythrocyte
count performed in the other chamber.
Modern hematology analyzers employ additional technologies to enhance the resolution of blood cell analysis. e RF
energy is used to assess important information about the internal structure of cells such as nuclear volume. Laser light scatter
is used to obtain information about cell shape and granularity.
e combination of these and other technologies— such as light
absorbance for hemoglobin measurements— provide accurate
blood cell dierentials, counts, and other important blood cell
indices. ese basic principles are common to many hematology analyzers used in clinical laboratories. However, each uses
dierent proprietary detection, measurement and soware systems, and ways of displaying the data.
Flow cytometers incorporate the principles of uorometry
and light scatter to the analysis of particles or cells that pass
within a uid stream. is technology provides multiparametric measurements of intrinsic and extrinsic properties of cells.
Intrinsic properties, including cell size and cytoplasmic complexity, are properties that can be assessed directly by light scatter and do not require the use of any type of probe. Extrinsic
cellular properties, such as cell surface or cytoplasmic antigens,
enzymes or other proteins, and DNA/RNA, require the use of a
uorescent dye or probe to label the components of interest and
a laser to induce the uorescence (older systems used mercury
arc lamps as a light source) to be detected.
e basic ow cytometer consists of four types of components: uidics, optics, electronics, and data analysis. Fluidics
refers to the apparatus that directs the cells in suspension to
the ow cell where they will be interrogated by the laser light.
Fluidics systems use a combination of air pressure and vacuum
to create the conditions that allow the cells to pass through the
ow chamber in single le. e optical components include the
laser (or other light source), ow chamber, monochromatic lters, dichroic mirrors, and lenses. ese are used to direct the
scattered or uorescent light to detectors, which measure the
signals that are subsequently analyzed.
35
e light scattered by the cell when it reaches the ow chamber is used to measure its intrinsic properties. Forward- scattered
light (FSC) is detected by a diode and reects the size of the
passing cell. Side- scattered light (SSC) is detected by a photo-
multiplier tube at an angle approximately 90 degrees to the laser
beam. e SSC is a function of the cytoplasmic complexity of
the cell, including the granularity of the cell. e correlated measurements and analysis of FSC and SSC can allow for dierentiation among cell types (ie, leukocytes) and can be depicted on
a scattergram.
e analysis of extrinsic properties is more complicated. e
measurement of DNA or RNA, for example, requires the use of
intercalating nucleic acid dyes such as propidium iodide. e
detection of antigenic determinants on cells can be performed
with uorescent- labeled moAbs directed at these antigens. In
each case, the principle of detection involves the use of laser
light to excite the uorescent dye and detect its emitted signal.
Fluorescent dyes are characterized by their excitation (absorption) and emission wavelength spectra and by the dierence
between the maxima of these spectra or Stokes shi (discussed
in the spectrophotometry section). ese properties permit the
use of multiple uorescent probes on a single cell.
To illustrate the operation of a ow cytometer, consider a fourcolor, six- parameter (FSC and SSC) conguration ( Figure2-8).36
An argon gas laser with a wavelength of 488 nm is commonly
used because it simultaneously excites several dierent dyes that
possess dierent emission wavelengths. Fluorochromes conjugated with moAbs that may be used include uorescein isothiocyanate, phycoerythrin (PE), energy- coupled dye, and Cy5PE
(tandem dye composed of the carbocyanine derivative Cy5 and
PE) with peak emission wavelengths of approximately 520, 578,
613, and 670 nm, respectively. e emitted light at each of these
wavelengths is detected at an angle of 90 degrees. e array of
optical lters selects light in each wavelength region and directs
it to a dierent photomultiplier tube where it is detected, amplied, and converted into an electronic signal. is measurement
can be made on thousands of cells in a matter of seconds. e
result is a histogram that identies distinct cell populations
based on light scatter and extrinsic properties. In the case of
blood, a histogram will distinguish lymphocytes, monocytes,
and granulocytes by light scatter. e B cell, T cell, T- cell subsets, and natural killer cell populations can all be distinguished.
is important method of cell analysis has found many applications in medicine, making it a relatively common clinical laboratory instrument. Flow cytometry analysis is routinely used to
assist in classifying the type of leukemia and lymphoma, derive
prognostic information in these and other malignancies, monitor immunodeciency disease states such as HIV/AIDS, enumerate stem cells by cluster dierentiation (CD34), and assess
various functional properties of cells.
Image Cytometry
Image cytometry, more commonly known as histology, is a laboratory method that uses instruments and techniques to analyze
tissue specimens. Examining individual cells, rather than the
collection of cells that make up a tissue, is referred to as cytology.
e basic components of an image cytometry system may
include a microscope, camera, computer, and monitor. Variations and complexity of these systems exist, which are beyond
the scope of this chapter. However, the essence of these instruments is the ability to acquire images in two or three (confocal
microscopy) dimensions to study the distribution of various
components within cells or tissues. e high optical resolution
CHAPTER 2 • InTRoduCTIon To Common LAboRAToRy AssAys And TECHnoLogy 37
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FIGURE 2-8. Schematic of a four- color ow cytometry system. The laser beam is focused onto the ow cell
through which the cell suspension is directed. Scattered light is detected by the forward and side scatter detectors.
Emitted light from specic moAb labeled with uorochromes are detected. Appropriate dichroic long pass lters
direct the specic wavelength of light through a narrow band pass lter and then to the appropriate PMT.
(Courtesy of Beckman Coulter.)
of these systems is an important determinant in obtaining
morphometric information and precise data about cell and
tissue constituents through the use of uorescence/absorbancebased probes, as in ow cytometry.37 Specic applications of
image cytometry generally involve unique methods of cell or
tissue preparation and other modications. is lends to the
versatility of this technology, which yields such applications as
the measurement of DNA content in nuclei to assess prognosis
in cancer and the detection of specic nucleic acid sequences to
diagnose genetic disorders.
In Situ Hybridization
Among the methods of image cytometry, in situ hybridization is perhaps the most commonly used in the clinical labo-
ratory, particularly in molecular cytogenetics laboratories. In
situ hybridization is used to localize nucleic acid sequences
(entire chromosomes or parts, including genes) in cells or
tissues through the use of probes, which consist of a nucleic
acid sequence that is complementary to the target sequence
and labeled in some way that makes the hybridized sequence
detectable. ese principles are common to all methods of in
situ hybridization, but they dier in the type of probe that is
used. Fluorescent probes, which provide excellent spatial resolution, have become a preferred method of in situ hybridization
for many applications. (Radioactive probes are also used for this
application. However, because their spatial resolution is limited,
detection and artifacts are oen produced.)
Fluorescent in situ hybridization (FISH) is a powerful molecular cytogenetics technique used for detecting genes and genetic
anomalies and monitoring dierent diseases at the genetic level.
ese assays are more sensitive and can detect chromosomal
abnormalities that cannot be appreciated by routine chromosome analysis (ie, karyotyping). Typically, metaphase chromosomes or interphase nuclei are denatured on a slide along with
a uorescent labeled DNA probe. e probe and chromosomes
are hybridized, and the slide is washed, counterstained, and
analyzed by uorescent microscopy. ere are various types
of FISH probes that can be utilized, such as DNA probes to
detect nick translations or RNA probes that can detect in vitro
transcription. An appropriate arrangement of lters is used to
direct the relevant wavelength of light from the light source to
excite the uorescent molecule on the probe. All but the emission wavelength of light is blocked with a special lter permitting the signal from the probe to be visualized.38 In molecular
cytogenetics, these assays are commonly used to identify gene
fusions or translocations.
MOLECULAR DIAGNOSTICS
Molecular diagnostics (MDx) were initially introduced into the
clinical laboratories as manual, labor- intensive techniques. is
discipline has experienced an overwhelming period of maturation in the past several years. Testing has moved quickly
from highly complex, labor- intensive procedures to more
38 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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user- friendly, semiautomated protocols, and the application
potential of MDx continues to evolve. Nucleic acid amplication technologies are among the procedures that have most revolutionized MDx testing.
Nucleic Acid Amplification
Polymerase chain reaction (PCR) is the most frequently used of
these technologies. Other amplication techniques that are used
in clinical laboratory procedures include ligase chain reaction,
transcription mediated amplication, branched DNA amplication, isothermal amplication, and nucleic acid sequence- based
amplication.
e PCR technology is used principally for detecting
microbiologic organisms and genetic diseases (Table2-2).
Examples of microorganisms identied by this process include
chlamydia, cytomegalovirus, Epstein-Barr virus, HIV, mycobacteria, and herpes simplex virus. As PCR amplies nucleic
acid sequences, if one knows the sequence of interest, then
an assay can be developed and optimized for use in patient
testing. While the list of cleared and approved targets is limited to the most commonly encountered microorganisms,
new assays are being developed by many diagnostic companies. PCR can oen identify organisms with greater speed and
sensitivity than conventional methods. For clinical microbiology laboratories, PCR methods are attractive because they
are rapid, sensitive, and specic. Many laboratories have
moved from culture- based methods to molecular amplication methods for the rapid identication of patients that
may be colonized with multidrug- resistant organisms, such
as Clostridioides dicile, methicillin- resistant Staphylococcus
aureus, or vancomycin- resistant enterococci. Rapid identication of these patients is crucial in healthcare settings that
oen place these patients on contact precautions to try and
reduce the spread of these organisms. e PCR applications
in microbiology can also be used to identify organisms carrying antibiotic resistance genes, such as the Klebsiella pneu-
moniae carbapenemase bla
all β- lactam antibiotics among members of the Enterobacte-
rales and other gram- negative bacilli.
Genetic diseases diagnosed using PCR include α-1 antitrypsin deciency, cystic brosis, sickle cell anemia, fragile X syndrome, Tay-Sachs disease, drug- induced hemolytic anemia, and
Von Willebrand disease. In addition, cancer research has beneted from PCR through the diagnosis of various cancers (eg,
chronic myeloid leukemia and pancreatic and colon cancers)
as well as through the detection of residual disease aer treatment.39 is technique is used to amplify specic DNA and RNA
sequences enzymatically.
In addition, PCR takes advantage of the normal DNA replication process. In vivo, DNA replicates when the double helix
unwinds and the two strands separate. A new strand forms on
each separate strand through the coupling of specic base pairs
(eg, adenosine with thymidine and cytosine with guanosine).
e PCR cycle is similar and consists of three separate steps
(Figure2-9)28:
1.
Denaturation: e reaction tube is heated causing the double
stranded DNA to separate.
gene that confers resistance to
KPC
2. Primer annealing: Sequence- specic primers are allowed to
bind to opposite strands anking the region of interest by
decreasing the temperature.
3.
Primer extension: DNA polymerase then extends the hybridized primers, generating a copy of the original DNA template.
e eciency of the extension step can be increased by rais-
ing the temperature. Typical temperatures for the three steps are
201.2°F (94°C) for denaturation, 122°F to 149°F (50°C to 65°C)
for annealing, and 161.6°F (72°C) for extension. Note that cycle
temperatures are inuenced by the specic enzyme used, the
primer sequence, and the genomic sample. Because one cycle is
typically completed in less than three minutes, many cycles can
occur within a short time, resulting in the exponential production of millions of copies of the target sequence.40 e genetic
material is then identied by agarose gel electrophoresis.
One potential disadvantage of this method is contamina-
tion of the amplication reaction with products of a previous
PCR (carryover), exogenous DNA, or other cellular material.
Contamination can be reduced by prealiquoting reagents, using
dedicated positive- displacement pipettes, and physically separating the reaction preparation from the area where the product
is analyzed. In addition, multiple negative controls are necessary
to monitor for contamination. Also common in clinical laboratories are instrument platforms that can perform real- time (q)
PCR as well as multiplex PCR, which allows amplication of
two or more products in parallel in a single reaction tube.40 In
real- time detection methods, a labeled oligonucleotide probe
containing a uorophore on the 5ʹ end and a quencher on the 3ʹ
end bind to the DNA template. With the probe bound, the
quencher prevents the uor from emitting light. During DNA
synthesis, the extending forward primer causes strand displacement. As the activity of the DNA polymerase enzyme continues
reading along the template, the exonuclease activity of the polymerase enzyme cleaves the probe, resulting in the generation of
light that is detected by the instrument. As each cycle of PCR
continues, more uorescent molecules are released, resulting in
increasing uorescence proportional to the amount of amplicon
present. Several in vitro diagnostic companies such as BD Diagnostics, BioFire, Cepheid, and Nanosphere have U.S. Food and
Drug Administration (FDA)- approved platforms that can allow
for simultaneous detection of multiple microorganism targets.
Use of these multiplex assays is attractive because they require
minimal sample volumes to generate multiple results. ese tests
are typically referred to as syndromic panels— named as such
for the most common samples tested, which include upper and
lower respiratory tract specimens, stool, blood, and cerebrospinal uid to detect CNS infections. Other companies are developing similar panels that can aid in the detection of prosthetic
joint infections, which are routinely diagnosed using culture
methods, but those suer from decreased analytic sensitivity.
GENOMICS, EPIGENETICS,
PROTEOMICS, AND METABOLOMICS
Newly developed techniques capable of examining the DNA,
messenger RNA (mRNA), and proteins of cells have provided a
CHAPTER 2 • InTRoduCTIon To Common LAboRAToRy AssAys And TECHnoLogy 39
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FIGURE 2-9. Stages of a single PCR reaction cycle. Beginning with your DNA template, the sample is added into
a microtube with DNA polymerase, forward and reverse sequence specic primers that will bind to and amplify
the region of interest within the template, and excess amounts of deoxynucleotide triphosphates (dNTP). During
Step
1 (denaturation), the sample is heated between 93°C and 96°C to separate the double- stranded DNA into two
single stranded templates. During Step 2 (annealing), the forward and reverse sequence specic primers will bind to
complementary sequences within the template to facilitate replication. Annealing occurs between 50°C and 70°C.
During Step 3, extension of the template occurs at 68°C and 75°C. During extension, DNA polymerase will catalyze
the addition of complementary dNTP to the primer using the sample DNA as the template. This completes one
cycle of the PCR reaction yielding two copies of the amplied region of interest.
framework for detailed molecular classications and treatments
of diseases. Genetic analysis of cystic brosis, for example, has
shown the disease to be the result of more than 1,500 dierent
mutations in the gene cystic brosis transmembrane conductance regulator.41 e most common mutation accounts for
two- thirds of cystic brosis cases. Several related developments, especially in the areas of tumor classications, are based
on the elds of genomics, epigenetics, and proteomics. e
most important laboratory procedures are array- based comparative genomic hybridization and the data derived from these
studies— bioinformatics.
Genomics
e study of all the genes of a cell, its DNA sequences, and the
ne- scale mapping of genes is the science of genomics. A genome
is the sum total of all genes of an individual organism. Knowledge of full genomes has created multiple possibilities, mainly
concerned with patterns of gene expression associated with
various diseases.
42,43
Epigenetics
Epigenetics refers to modications of the genome that are functionally relevant but do not involve a change in the nucleotide
sequence. Histone deacetylation and DNA methylation are
examples of such changes, both of which serve to suppress gene
expression without altering the sequence of the silenced genes.
Such changes may continue to exist for many cell divisions and
even the remainder of the cell’s life, as well as for future generations of cells. However, because there is no change in the underlying DNA sequence of the organism, nongenetic factors cause
the organism’s genes to express themselves dierently.
44
Proteomics
e study of the full complement of proteins in a cell or tissue
is called proteomics and includes the comprehensive analysis
and characterization of all proteins, including their structure
and function that are encoded by the human genome. Proteinbased assays were among the rst assays to be approved by the
FDA, mostly using immunohistochemistry techniques. Most
important biological functions are controlled by signal transduction, which are processes governed by the enzyme activities
of proteins. Diseases such as cancer, while fundamentally the
result of genomic mutations, manifest as dysfunctional protein
signal transduction. Many pharmaceuticals are now being developed to aim at modulating the aberrant protein activity, not the
genetic defect.
Proteomics will eventually have a great impact in the practice
of medicine. Although the genome is the source of basic cellular
45-47
40 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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information, the functional aspects of the cell are controlled by
and through proteins, not genes. e main challenge to the study
of proteomics is due to the proteome’s complexity compared
with the genome. e human genome encodes approximately
23,000 genes, approximately 21,000 of which encode proteins.
However, the total number of proteins in human cells is estimated to be between 250,000 and 1 million. Furthermore, proteins are dynamic and constantly undergo changes, synthesis,
and breakdown. Currently, most of the FDA- approved targeted
therapeutics are directed at proteins and not genes.
Metabolomics
Similar to proteomics, metabolomics is a rapidly emerging eld
that combines multiple strategies and techniques to identify and
quantify metabolites. Metabolites are small molecule substrates
(ie, intermediates substances and the products of our metabolism). Like the Human Genome Project, which launched in
1990 and was completed in April 2003, the Human Metabo
lome Project, funded by Genome Canada, was launched in
2005 to describe and understand the complete collection of
small molecules in a sample, including endogenous and exogenous compounds. e project led to the development of the
Human Metabolome Database (https://hmdb.ca/) a freely avail-
able web- accessible database that contains detailed information
about small molecule metabolites found in the human body. To
date, the database contains more than 114,000 metabolite entries
with links to more than 5,700 protein sequences associated with
the metabolites. Metabolomics is useful for future identication
biomarkers that could be used as diagnostic or prognostic of any
number of diseases.
ARRAY-BASED COMPARATIVE
HYBRIDIZATION
Molecular proles of cells can now be determined using arraybased comparative hybridization.48 is technique is especially
useful in proling tumor cells. Until recently, changes occurring
in cancer cells were studied one at a time or in small groups
in small sets of tumors. New array comparative hybridization
or microarray technology (“gene chips”) has enabled investigators to simultaneously detect and quantify the expression
of large numbers of genes (potentially all genes) in dierent
tumors using mRNA levels. In this technique, samples are
obtained from tissues embedded in paran blocks, and serve
as the sources to prepare new blocks that may contain up to
thousands of tissue fragments. ese multiple samples are then
used to test the expression of potential tumor markers by mRNA
expression proling. e mRNA levels, however, do not always
correspond to changes in tumor cell proteins. e quantity of
protein within a cell depends not only on the amount and rate
of transcription and translation, but also on protein breakdown
and the rate of transport out of the cell. Although tissue used
for mRNA proling may include both tumor and stromal cells,
by adding immunohistochemistry methods, specic proteins in
tissue sections originating from both normal as well as tumor
cells can be identied.
As a specic example, several types of breast cancer cells,
which were previously identied only by morphology, are now
being studied by array- based comparative hybridization tech
niques. Combined with immunohistochemistry staining and
protein expression levels, new subtypes that were not previ
ously well dened have been identied (eg, the basal- like carcinomas).49 As a consequence, new treatment modalities have
been developed. Array- based comparative hybridization methods have also identied new subtypes of other tumors, such as
lymphomas and prostate cancer with potential for susceptibility
and prognosis.
50
NANOTECHNOLOGY
Nanotechnology refers to the emerging science that studies
interactions of cellular and molecular components at the most
elemental level of biology, typically clusters of atoms, molecules,
and molecular fragments. Nanoscale objects have dimensions
smaller than 100 nm. At this dimension, smaller than human
cells (which vary from 10,000 to 20,000 nm in diameter), small
clusters of molecules and their interactions can be detected.
Nanoscale devices smaller than 50 nm can easily enter most
cells, while those smaller than 20 nm can move out of blood
vessels, oering the possibility that these devices will be able to
enter biological chambers, such as the blood–brain barrier or the
gastrointestinal epithelium, and identify tumors, abnormalities,
and deciencies in enzymes and cellular receptor sites. Within
these biological chambers, they will be able to interact with an
individual cell in real time and in that cell’s native environment.
Despite their small size, nanoscale devices can also hold tens
of thousands of small molecules, such as a magnetic resonance
imaging contrast agent or a multicomponent diagnostic system
capable of assaying a cell’s metabolic state. A good example of
this approach will capitalize on existing “lab- on- a- chip” and
microarray technologies developed at the micron scale. Widely
used in biomedical research and to a lesser extent for clinical
diagnostic applications today, these technologies will nd new
uses when shrunk to nanoscale. (In some instances, nanotechnology has already taken advantage of previous clinically relevant technological developments on larger scales.)
Currently, innovative testing is available for many dierent
viruses, mutation analysis, and hematologic and solid tumors.
With continuing advances and developments in nanotechnology, it is impossible to speculate as to what this new area of testing holds for the future of the clinical laboratory.
SUMMARY
is chapter presents a brief overview of the more common
and some emerging laboratory methodologies, including their
potential advantages and pitfalls. Some historical methods
have been discussed to provide a basis and description of the
simple principles on which the more complex methods are
based. A summary of some of the most common assay methods
performed for routine laboratory tests is provided in Table2-2.
-
-
CHAPTER 2 • InTRoduCTIon To Common LAboRAToRy AssAys And TECHnoLogy 41
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Because of its simplicity and improved sensitivity, ISE has
replaced ame photometry as the principal method for measuring serum and urine electrolytes in clinical specimens. Some
methods, including turbidimetry, nephelometry, and spectro
photometry, are used in conjunction with other tests such as
immunoassays. With these methods, concentrations of sub
stances such as immune complexes are able to be determined.
Mass spectrometry is the gold standard for the identication of unknown substances, including drugs of abuse. Many
of the newest designer drugs and bath salts are only identiable based on this technique, as no other methodologies exist
to detect them in clinical specimens. e two principal forms
of chromatography are liquid and gas. Both types are similar
in that they depend on dierences in either solubilities or boiling points, respectively, to separate dierent analytes in a sample. Another group of important tests are the immunoassays:
EIA, EMIT, ELISA, and FPIA. ese methods depend on an
immunologically mediated reaction that increases sensitivity
and specicity over RIA. ese assays are commonly used to
determine routine clinical chemistries and drug concentrations.
PCR and other nucleic acid amplication techniques are used to
amplify specic DNA and RNA sequences, primarily in the areas
of microbiology and detection of genetic diseases. Finally, with
the potential advances envisioned in the area of nanotechnology,
the laboratory will be able to provide clinicians with information
and access to the patient’s cellular and molecular environments,
thus providing the ability to target therapies at the exact site of
the pathologic process.
e rapid technological advancement of laboratory instrumentation has led to the implementation of new and enhanced
clinical laboratory methodologies, including MS, cytometry,
laboratory automation, and POC testing. Although laboratory
medicine endeavors to keep pace with the burgeoning developments in biomedical sciences, especially with an increase in the
sophistication of the tests, it is essential that today’s clinicians
have a basic understanding of the more common and esoteric
tests to select the most appropriate one in each case. All of these
developments will translate directly into improved patient care.
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Immunochemistry. In: McPherson RA, Pinkus MR, eds. Henry’s Clinical
Diagnosis and Management by Laboratory Methods. 21st ed. Philadelphia,
PA: WB Saunders; 2001.
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29. Kitson FG, Larsen BS, McEwen CN. Gas Chromatography and Mass
Spectrometry: A Practical Guide. San Diego: Academic Press; 1996.
30. Siuzdak G. Mass Spectrometry for Biotechnology. San Diego: Academic
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31. Bowers LD, Ullman MD, Burtis CA. Chromatography. Burtis CA, and
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32. Van Bramer SE. An introduction to mass spectrometry (1997).
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316-321.PubMed
Primer on Drug Interferences
https://t.me/med1917
with Test Results
3
OBJECTIVES
After completing this chapter, the
reader should be able to
•
Distinguish between in vivo and
in vitro drug interferences with
laboratory tests
•
Identify suspected drug–laboratory
test interference in a logical,
systematic manner given a drug and
a laboratory test
•
Devise a stepwise process to conrm
that a drug is causing a clinically
signicant drug–laboratory test
interference
•
Distinguish among tertiary,
secondary, and primary literature
resources about drug–laboratory test
interferences
•
Apply a systematic process to search
and identify medical literature
relevant to a suspected drug–
laboratory test interference
Mary Lee
rough a variety of mechanisms, drugs can interfere with laboratory test results. If
the clinician who has ordered the laboratory test is not aware that the drug has altered
the results of the test, inappropriate management of the patient may follow, including unnecessary hospitalization, extra oce visits, or additional laboratory or clinical
testing— all of which may increase the cost of healthcare. is chapter addresses this
situation and provides resources that health professionals can use to better interpret
laboratory tests when a drug is suspected to interfere with test results.
IN VIVO AND IN VITRO DRUG INTERFERENCES
WITH LABORATORY TESTS
When a drug interferes with a laboratory test result, it alters the laboratory value.
Mechanisms for drug interference of clinical laboratory tests can be classied as either
in vivo or in vitro.1 In vivo drug interferences can also be called physiologic and can be
subclassied as pharmacological or toxicological. In vivo drug interferences account
for most eects of drugs on laboratory tests.2 In contrast, the term in vitro drug inter-
ferences is used synonymously with analytical or methodological interferences.
In Vivo Interference
An in vivo interference is an actual change in the analyte concentration or activity
prior to specimen collection and analysis. e assay measurement is actual and
accurate and reects a change in the measured substance that has occurred in the
patient. erefore, an in vivo interference will always change a laboratory test result,
independent of the assay methodology. A drug can produce an in vivo interference in
several ways. By a direct extension of its pharmacological eects, a drug can produce
changes in some laboratory test results. For example, thiazide and loop diuretics
will commonly cause increased renal elimination of potassium. erefore, decreased
serum potassium levels can occur in treated patients. In these patients, hypokalemia
is actual and accurate. Similarly, β adrenergic antagonists decrease renin and aldosterone secretion, which can lead to increased serum potassium levels.
Other drugs produce changes in laboratory test results by producing in vivo toxicological eects. As the drug damages a particular organ system, abnormal laboratory tests may be one of the rst signs of the problem. For example, as isoniazid and
rifampin produce hepatotoxicity, elevated hepatic transaminases will signal the onset
of liver inammation. Similarly, as a prolonged course of high- dose aminoglycoside antibiotic causes acute proximal tubular necrosis, serum creatinine and serum
trough aminoglycoside levels will increase steadily if the antibiotic is not stopped or
if the antibiotic dose is not reduced. In the face of cyclophosphamide- induced bone
marrow suppression, neutropenia will become evident 10 to 14 days aer a dose has
been administered.
2
DOI 10.37573/9781585286423.003
In Vitro Interference
Drugs in a patient’s body uid or tissue can directly interfere with a clinical laboratory
test during the in vitro analytical process. is type of drug–laboratory test interaction
is highly dependent on the laboratory test methodology, as the reaction may occur with
one specic assay method but not another. In vitro drug- laboratory test interactions are
43
44 BASIC SKILLS IN INTERPRETING LABORATORY DATA
https://t.me/med1917
common with radioimmunoassays for which cross reactions can
occur because of drug metabolites or drugs that are chemically
similar to the parent drug, or because of heterophilic antibodies that are similar to endogenous antibodies. False high or false
low laboratory test results occur.
2-5
For example, serum digoxin
levels are commonly determined using a radioimmunoassay, a
uorescent polarization immunoassay, or a TDx assay. However,
these assays are based on the three- dimensional structure of the
digoxin molecule, and many other drugs with a similar chemical
structure to digoxin (eg, spironolactone, estrogen replacement
products, cortisol, digoxin metabolites) can cross- react with the
ass ay.6 A falsely increased or decreased serum digoxin level can
result. To determine the true serum digoxin level in this situation, another assay technique (eg, high- pressure liquid chromatography [HPLC]) may be used. In addition, substances that are
prepackaged in or added to the in vitro system before or aer
sample collection can cause laboratory test interference in vitro.
As an example, test tubes sometimes contain lithium heparin
or sodium uoride. Heparin can interfere with aminoglycoside assays, and uoride can cause false increases in blood urea
nitrogen (BUN) when measured by the Ekatchem assay.
Alternatively, a drug may cause discoloration of the body
uid specimen, which may interfere with colorimetric, photometric, or uorometric laboratory- based assay methods. For
example, phenazopyridine causes an orange- red discoloration
of urine that may be mistaken for blood. Nitrofurantoin may
cause a brown discoloration of the urine that may alarm the
patient. ese types of drug interference with laboratory testing can be detected visually and appropriate attribution of the
abnormality should be made by knowledgeable clinicians and
clinical laboratory sta.
Other common mechanisms by which drugs cause in vitro
interferences with laboratory tests include the following:
•
A drug alters the specimen pH (usually urine) so that reagent
reactions are inhibited or enhanced. For example, acetazol-
amide produces an alkaline urinary pH that causes false-
positive proteinuria with reagent dip strips.
•
A drug chelates with an enzyme activator or reagent used
in the in vitro laboratory analysis. For example, large doses
of biotin, as are included in some over- the- counter nutri-
tional supplements, can compete for the biotin- streptavidin
complex which is a component of many radioimmunoas-
3,7-9
says.
Also, daptomycin interacts with rabbit or human
thromboplastin, which is associated with a dose- dependent
prolongation of prothrombin time and international normalized ratio.
•
A drug absorbs at the same wavelength as the analyte. For
10,11
example, methotrexate interferes with analytic methods using
high performance liquid chromatography and an absorbance
range of 340 to 410 nm.
•
A drug reacts with reagent to form a chromophore (eg, cefoxitin or cephalothin) with the Jae- based creatinine assay.
In addition to the parent drug, other drug- related compo-
nents may cause signicant interferences with laboratory tests.
Metabolites can cross- react with the parent drug in an assay,
such as in the case with cyclosporine. Its metabolites cross- react
with the parent drug in HPLC assays and can produce a falsely
high measurement of the concentration of cyclosporine.12 Contaminants in herbal products, which are subject to less regulation than medications in the United States, may interfere with
some laboratory tests.7 Inactive ingredients of some drug products, which includes excipients such as lactose or starch, preservatives, colorants, or avoring agents, may inuence assay
results. Although most manufacturers do report the inactive
ingredients in their products, little systematic research has been
performed to assess the impact of these substances on laboratory tests. Compounding these factors, many laboratory test
interferences are concentration related, and many drug metabolites and their usual plasma concentrations have yet to be identied. erefore, systematic study of all of these potential causes
of interactions is dicult to conduct and is not available in
many cases.
13
Simultaneous In Vivo and In Vitro Effects
Some drugs can aect an analyte both in vivo and in vitro. In
these situations, interpretation is extremely dicult because the
degree of impact in each setting cannot be determined easily.
For example, when a drug produces hemolysis in a patient with
glucose-6- phosphate dehydrogenase deciency who is exposed
inadvertently to ciprooxacin, hemolytic anemia may result.
Hemolyzed red blood cells produce a red discoloration of the
plasma or serum. e hemoglobin released from the damaged
red blood cells can interfere with analysis of alkaline phosphatase or γ- glutamyl transferase, both of which can be assayed
using a spectrophotometric analysis that depends on color
changes aer a chemical reaction.
13,14
Simultaneous in vivo and
in vitro drug interferences with laboratory tests can also occur
commonly when drugs increase bilirubin or when a drug causes
lipemia.
15
IDENTIFYING DRUG INTERFERENCES
Incidence of Drug Interferences
e true incidence of drug interferences with laboratory tests
is unknown. is is because many situations probably go undetected. However, as the number of laboratory tests and drugs
on the U.S. commercial market increase, it is likely that the
number of cases of in vivo interferences will also increase. As
a reection of this, consider the number of drug–laboratory
test interferences reported by D. S. Young, author of one of the
classic literature references on this topic. In the rst edition of
Eects of Drugs on Clinical Laboratory Tests, published in the
journal Clinical Chemistry in 1972, 9,000 such interactions were
included.16 In the second edition of the same publication, which
was published in 1975, 16,000 such interactions were reported.17
In 1997, this resource, which had been converted to an online
searchable database, included more than 135,000 interactions.18
In 2014, this resource included 171,000 interactions.
As for in vitro interferences, the number of drug– laboratory
test interferences may be moderated over time because of newer,
more specic laboratory test methodologies that minimize
19
CHAPTER 3 • PRimER on DRug inTERfEREnCEs wiTH TEsT REsulTs 45
https://t.me/med1917
cross- reactions with drug metabolites or drug eects on reagents
or laboratory reactions.
14,18
In addition, manufacturers of commonly used laboratory equipment systematically study the
eects of drugs on assay methods.17 erefore, this information
is oen available to clinicians who confront problematic laboratory test results in patients. is increased awareness reduces the
number of patients who are believed to have experienced newly
reported drug–laboratory test interferences.
Suspecting a Drug Interference
A clinician should suspect a drug–laboratory test interference
when an inconsistency appears among related test results or
between test results and the clinical picture. Specically, clinicians should become suspicious when the following occurs:
•
Test results do not correlate with the patient’s signs,
symptoms, or medical history.
•
Results of dierent tests— assessing the same organ anatomy
or organ function, or the drug’s pharmacologic eects—
conict with each other.
•
Results from a series of the same test vary greatly over a short
period of time and for no apparent reason.
• Serial test results are inconsistent.
No Correlation with Patient’s Signs, Symptoms,
or Medical History
As emphasized elsewhere in this book, when an isolated test
result does not correlate with signs, symptoms, or medical
history of the patient, the signs and symptoms should be considered more strongly than the test result. is rule is particularly
true when the test result is used to conrm suspicions raised by
the signs and symptoms in the rst place or when the test result
is used as a surrogate marker or indirect indicator of underly
ing pathology.
For example, serum creatinine is used in various formulae
to approximate the glomerular ltration rate, which is used to
assess the kidney’s ability to make urine. However, actual urine
output and measurement of urinary creatinine excretion is a
more accurate method of assessing overall renal function. If
a patient’s serum creatinine has increased from a baseline of
1 mg/dL to 5 mg/dL over a three- day period, but the patient
has had no change in urine output, urinary creatinine excretion, or serum electrolyte levels, then the serum creatinine level
may be elevated because of a drug interference with the laboratory test. Similarly, if a patient has a total serum bilirubin of
6 mg/dL, but the patient is not jaundiced or does not have scleral
icterus, then a drug interference with the laboratory test should
be considered.
Conflicting Test Results
Occasionally, pharmacological or toxicological eects of a drug
produce conicting results of two tests that assess the same
organ function. For example, a presurgical test screen shows a
serum creatinine of 4.2 mg/dL in an otherwise healthy 20- yearold patient with a BUN of 8 mg/dL. Usually, if a patient had true
renal impairment, BUN and serum creatinine would be elevated
in tandem. us, in this patient, a drug interference with the
laboratory test is suspected. Further investigation revealed that
the patient received cefoxitin shortly before blood was drawn for
the laboratory test. Cefoxitin can falsely elevate serum creatinine
concentrations. us, the elevated serum creatinine is likely due
to drug interference with the laboratory test and not to renal
failure. To conrm that this is the case, cefoxitin should be
discontinued and the serum creatinine repeated aer that. If it
is due to the drug, the elevated serum creatinine should return
to the normal range.
20
Varying Serial Test Results Over a Short Time Period
Typically, the results of a specic laboratory test should follow
a trend in a patient. However, in the absence of a new onset
of medical illness or worsening of existing disease, a sudden
change in the laboratory test result trend should cause examination of a possible drug interference with a laboratory test. For
example, prostate specic antigen (PSA) is a tumor marker for
prostate cancer. It is produced by glandular epithelial cells of
the prostate. e normal serum level is <4 ng/mL in a patient
without prostate cancer, and the level is typically elevated in
patients with prostate cancer. However, it is not specic for
prostate cancer. Elevated PSA serum levels are also observed in
patients with benign prostatic hyperplasia, prostatitis, or follow
ing instrumentation of the prostate. A 70- year- old male patient
with metastatic prostate cancer has a PSA of 30 ng/mL and has
decided not to undergo treatment. Four serial PSA tests over the
course of one year and done at three- month intervals show no
change. Despite the absence of any changes on pelvic computerized axial tomography, bone scan, or chest radiograph, his PSA
is 10 ng/mL at his most recent oce visit. Aer a careful interview of the patient, the urologist discovers that the patient has
been treated for androgenetic alopecia for the past six months
with nasteride. e patient received the prescription from
another physician for lower urinary tract voiding symptoms,
and nasteride lowered the PSA level.
21
Serial Test Results That Are Inconsistent with
ExpectedResults
Generally, repeated laboratory test results should show little
change over time assuming that the status of the medical condition or treatment for the medical condition in an individual
patient stays the same. However, when serial test results are
inconsistent with expected results, a drug–laboratory test interference should be suspected. For example, leuprolide, a luteinizing hormone- releasing hormone (LHRH) agonist, is useful in
the management of prostate cancer, which is an androgen dependent tumor. Persistent use of leuprolide causes down- regulation
of pituitary LHRH receptors, decreased secretion of luteinizing
hormone, and decreased production of testicular androgens.
Apatient with prostate cancer treated with leuprolide should
experience a sustained reduction in serum testosterone levels
from normal (280 to 1,100 ng/dL) to castration levels (<50 ng/dL)
aer 2 to 3 weeks. e serum testosterone level should remain
below 50 ng/dL as long as the patient continues treatment with
leuprolide, and as long as he makes returns to the clinic for
repeated doses on schedule. However, one of the adverse eects
of leuprolide is decreased libido and erectile dysfunction, which
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