Scott Jenkinson, D.O. (class of 1983)
Associate Professor of Pathology
75 Hospital Dr., ste. 370
Athens, OH 45701
Pathology as a Medical Specialty
Pathology is the study of disease. A.T Still implied that finding disease was easy and
that looking beyond the conditions that hinder human achievement was the better goal
of a physician. At present, western medicine remains heavily disease based, but there
are recent encouraging signs that the maintenance of wellness may become just as
Pathologists are sometimes called the physician’s physician. They have medical
degrees and complete residencies lasting 4 years. The standard residency curriculum
includes 2 years of anatomic pathology and 2 years of laboratory medicine. These
subdivisions include the following disciplines:
Anatomic Pathology
Surgical pathology
Laboratory Medicine
Clinical chemistry
Transfusion medicine
An emerging discipline is molecular pathology that overlaps with both anatomic
pathology and lab medicine. It involves detecting genes and genetic alleles and gene
copy number using methods like fluorescent in situ hybridization, RNA microarray
hybridization and gene sequencing. Another discipline that doesn’t fit neatly in the
above dichotomy is forensic pathology, a field centered on autopsy pathology with the
intent of providing answers for the criminal justice and public health systems.
Fellowships of 1-3 years in all the pathologic disciplines and several other fields are
Types of Laboratory Tests
Qualitative. The result is positive or negative. An analyte is present in sufficient
concentration to be detected by the system.
Examples: pregnancy test, HIV antibody test.
Semiquantitiative. The result is expressed in arbitrary units for comparison, such as 0,
trace, 1+, 2+, 3+, 4+. This allows some degree of comparison when the analyte is
tested serially in a patient.
Examples: urinalysis dipstick, HER2 protein expression in tumor cell nuclei.
Quantitative. Reported as a numeric value, usually a concentration. These test results
lend themselves to statistical analysis.
Examples: plasma glucose concentration, blood hemoglogin concentration, CSF cell
Interpretive. The pathologist or clinical chemist collects and reviews data and
information and synthesizes an interpretation expressed in the lab report.
Examples: surgical pathology report of a breast biopsy, cause and manner of death
determination from an autopsy, pap smear cytologic diagnosis, serum protein
electrophoresis pattern interpretation.
Quantitative Test Reference Ranges
Sometimes called normal ranges, a test reference range expresses the most likely test
results in a group of people that are healthy and free of diseases that might influence
the test results. Reference ranges are created by doing a reference range study in
which a suitable number of subjects are tested and their test results analyzed
statistically. When test results from the study form a bell shaped curve, it is fairly
standard to define the reference range as the mean plus or minus two standard
deviations. Considering the area under the bell shaped curve, 95% of the test results in
the study group would be within the reference range. Laboratories are required to
include a reference range on test results and the laboratory must do a reference range
study with sufficient statistical power or verify a test kit manufacturer’s reference range
with an in-house study.
The statistical nature of reference ranges creates important caveats for physicians who
interpret test results in light of the patient’s history and physical exam.
2.5% of healthy, nondiseased people will have a test value below the reference
range and 2.5% will have a value above the reference range.
Test results much higher or lower than the reference range are more likely to be
true abnormal tests and thus indicate disease.
When analyzing an abnormal test result, always ask yourself whether the
reference range on the report matches the age, gender and ethnicity of your
patient. Some laboratories lack the data or the capability of reporting the correct
reference range.
Test results alone do not typically define disease. Tests have measurable
sensitivity and specificity for the detection of disease. It is possible for a patient
to have a disease and a false negative lab result and also possible for a healthy
person to have a false positive lab result.
Decision Levels and Critical Values
Some tests are reported with clinical decision levels rather than reference ranges.
Reference range reporting for some tests would provide misleading information for test
interpretation. Total cholesterol, for example, might have a reference range in a North
American population of adults of 150- 300 mg./dL. We know from data collected during
the Framingham Heart Studies decades ago that people with a total cholesterol above
240 mg./dL. are at high risk for a myocardial infarction, so a decision level of 240mg./dL.
is a more appropriate way of reporting.
Critical values are test values so high or low compared to reference ranges that the
patient’s life may be immediate danger. Laboratories have policies to notify health care
workers of these critical values as soon as they are discovered. This becomes very
challenging when the results are discovered after hours and on weekends and holidays.
Laboratory Testing
About half of all lab tests done on humans in the U.S. are performed in hospital,
independent and reference laboratories and the other half are done in physician office
labs. The Clinical Laboratory Improvement Amendments (CLIA) federal law enacted in
1988 regulates all human laboratory testing, regardless of location. If tests are very
simple to perform or results have minimal impact on patient care, then a CLIA certificate
of waiver applies. Waived tests can be performed by anyone as long as the test
manufacturers’ directions are followed. Moderate and high complexity testing is
generally performed by medical technologists in laboratories where detailed procedure
manuals are maintained, a quality management program includes regular quality control
testing and instrument calibration and preventive maintenance and lastly, participation
in a proficiency testing program.
Quality Control – a stabilized test material with known analyte concentrations is
tested along with a run of patient samples. If the control material results are
acceptable, then we have confidence that the assay system is functioning
properly and the patient test results are reliable. More than one QC material with
low and high analyte concentrations may be used with a patient test run to
ensure test system integrity over the linear range of the system.
Calibration – the test instrument is tuned to recognize samples of known
concentration (calibrators). If multiple calibrators are used for a particular test,
the instrument stores the calibration curve and uses it to determine patient test
results by interpolation or extrapolation. Calibration is done on a schedule
unique to the instrument and method; it is not usually done with each patient
sample run.
Proficiency Testing – the lab receives 2 to 5 test samples and performs the test
on each sample, then sends the results to the company providing the samples for
comparison to other participants or to an industry standard. A passing score for
proficiency testing is 80%. If a lab fails two consecutive proficiency test
challenges for a particular analyte or a category of tests, then they must
discontinue testing until they fix the assay and successfully complete repeat
proficiency testing.
Causes for Inaccurate Lab Test Results
Random lab error – examples: tester does not follow procedure and uses
variable quantities of sample or reagents, lab temperature varies day to day,
reagent not brought to room temperature before use.
Systematic lab error – examples: testers follow an incorrect procedure manual,
samples are collected in an incompatible tube, pathologist uses outdated criteria
for diagnosing biopsies.
Sink testing – the tester dishonestly makes up test results and throws samples
away in order to save time or sabotage medical care.
Interfering substances – the sample integrity is compromised by RBC hemolysis,
high bilirubin or paraproteins that interfere with spectrophotometric tests. Human
anti-mouse antibodies in the patient plasma can cause tests with mouse
antibody-containing reagents to have falsely elevated results. Patient’s may
intentionally add interfering substances to samples, such as urine drug screens.
Improper patient preparation or instruction – examples: patient not fasting when
it is required, patient fails to collect all urine in a 24 hr. period.
CMS (Centers for Medicare and Medicaid Services) Approved Test Panels
A few groups of lab tests are recognized by CMS as panels ( also called profiles) and
health care providers are reimbursed for the panel by a single payment. These panels
can be ordered only when each test in the panel is considered medically necessary for
diagnosis and treatment.
Electrolyte Panel
serum or plasma
Basic Metabolic Panel (BMP)
serum or plasma
Options – with or without total or ionized calcium
Comprehensive Metabolic Panel (CMP)
BMP plus
Total protein
Total bilirubin
Total calcium
Hepatic Function Panel
Alkaline phosphatase
Total bilirubin
Lipid Panel
serum or plasma
Alkaline phosphatase
serum or plasma
Conjugated bilirubin
serum or plasma
Total cholesterol
HDL cholesterol
Renal Function Panel
serum or plasma
Total calcium
Inorganic phosphate (HPO4)
Chemistry Tests
The major cation in all body fluids with predominant impact on serum osmolality. A
typical reference range is 135-150 meq/L. Hyponatremia can be caused by many
medical conditions with the commonalities of retention of water, salt wasting, and/or
excessive antidiuretic hormone release. It is often helpful to narrow the possibilities by
deciding,based on clinical exam and possibly other lab tests, whether the hyponatremia
is associated with increased body fluid volume, normal fluid volume or reduced body
fluid volume. Hypernatremia is often seen in conditions where fluid loss exceeds fluid
intake, such as dehydration from sweating, watery diarrhea, and diabetes insipidis.
Another less common cause is intake of excessive salt, such as drinking sea water or
ingesting lots of salty food.
This cation is much lower in concentration than sodium in the plasma; a typical
reference range is 3.5 – 5.0 meq/L. Body regulatory mechanisms keep serum K within
this narrow range as hypokalemia and hyperkalemia can produce dysfunction in nerve
conduction and muscle contraction. Hypokalemia can be caused by salt wasting
diuretics, metabolic alkalosis with renal potassium loss, diabetic ketoacidosis, adrenal
gland mineralocorticoid excess and ingestion of licorice.. Hyperkalemia can be caused
by impaired excretion in the urine as occurs in renal failure, mineralocorticoid deficiency
and the use the certain medications, and from excessive release from body tissues as
occurs in skeletal muscle necrosis and red blood cell hemolysis. If the hemolysis .
The major anion in body fluids. Hypochloremia seldom occurs as an isolated
electrolyte disturbance, but can be seen in severe vomiting, respiratory acidosis and
metabolic alkalosis.
Bicarbonate (Carbon Dioxide)
A component of the bicarbonate buffer system in plasma, bicarbonate ion is the
predominant form that CO2 is found in plasma. It is reduced when there is metabolic
acidosis or respiratory alkalosis an and it is increased when there is respiratory
acidosis, most often due to hypoventilation.
Anion Gap
The formula defining anion gap is serum sodium – (serum chloride + serum
bicarbonate). The upper limit of normal for anion gap is typically about 11 meq./L.
Conditions with excess unmeasured anions, such as lactic acidosis, diabetic
ketoacidosis, alcoholic ketoacidosis, ethylene glycol poisoning and others result in an
elevated anion gap. Keep in mind, however, that there is always electrical neutrality
maintained in the serum, so there really is no excess in cations, only a gap based on
limiting the calculation to the most abundant cation and the two most abundant anions
in serum.
This is the major carbohydrate available for glycolytic energy metabolism. Fasting
serum glucose is maintained at a steady state level by insulin secretion and to a lesser
extent by glucagon. Normal fasting serum glucose should be below 100 mg./dL.
Hypoglycemia is usually due to insulin excess, however transient it may be.
Hyperglycemia may be transient following food intake. Sustained, fasting
hyperglycemia is the hallmark of diabetes mellitus, which may be related to insufficient
insulin secretion or to insulin resistance at the tissue level. The lab test for glucose is
specific for glucose and does not identify other sugars that may be in excess
concentration in serum or urine in patients with metabolic defects in carbohydrate
Abbreviation for blood urea nitrogen. The nitrogenous waste compound measured in
this test is urea and only a carried over vestige of medical laboratory science explains
why we still call it BUN and not urea concentration. Since urea is excreted by the
kidney, renal disease or urinary obstruction can cause an elevation in BUN, also called
azotemia. Reduced blood flow to the kidneys, as in renal vascular disease or heart
failure, can also cause prerenal azotemia. Since urea represents the mammalian
excretion product for most organic nitrogens produced from amino acid catabolism,
BUN would be expected in transiently rise when a high protein meal or a gastrointestinal
hemorrhage occurs. A low serum BUN is seldom seen clinically but could occur in liver
failure since the enzymes for the urea cycle are primarily hepatic.
This nitrogenous compound is formed in skeletal muscle as a catabolic product of
creatine metabolism. The serum level is directly proportional to skeletal muscle mass,
so most labs report gender specific reference ranges. Creatinine is excreted in the
urine and at normal serum concentrations tends to be reabsorbed and secreted by renal
tubular epithelial cells to the same degree, making calculation of the creatinine
clearance a fairly good estimate of the glomerular filtration rate. (GFR). Calculation of a
classic creatinine clearance involves a 24 hr. urine collection, so spot urine creatinine
levels and estimation of the creatinine clearance using a formula using only the serum
creatinine can be reported.
The relatively steady formation of creatinine in muscle makes the urinary excretion rate
steady, so the variability seen in serum BUN due to protein load does not exist. Serum
creatinine will be increased in renal failure, but a noticeable increase requires loss of the
majority of functioning nephrons. It is, therefore, not a useful test for detecting early
renal disease.
AST and ALT (aspartate aminotransferase and alanine aminotransferase)
AST is found in many body tissues but is especially rich in the liver hepatocytes, heart
muscle and RBC. ALT is more hepatic specific. Both are commonly called
transaminases. Elevation in the transaminases is caused by hepatocyte necrosis with
release of cytoplasmic enzymes into the interstitial fluid which is then carried by
lymphatics to the venous system. In the case of AST, necrosis of cardiac muscle and
hemolysis of RBC could also cause an increase. A low serum AST/ALT would be seen
in patients with liver failure after the initial hypertransaminasemia had dissipated.
Bilirubin is the metabolic byproduct of heme catabolism. Heme is the oxygen carrying
component of hemoglobin. Senescent red blood cells are removed in the spleen and
bilirubin is created. Bilirubin is not water soluble, so it is bound to plasma proteins,
largely albumin, and it is transported to the liver. Bilirubin enters hepatocytes where it is
conjugated with glucuronic acid. The conjugated form of bilirubin is water soluble and it
is excreted in the bile. Once in the small and large intestine, some of the conjugated
bilirubin is reabsorbed and enters the bloodstream. Historic terms related to the
laboratory methods for measuring unconjugated and conjugated bilirubin are indirect
and direct bilirubin, respectively. The typical reference range for total serum bilirubin is
0.1 – 1.0 mg./dL. About 90% of the total serum bilirubin is unconjugated and 10% is
Hyperbilirubinemia of 2.5 or 3 mg./dL causes jaundice. Hyperbilirubinemia can be due
to several disparate diseases:
 RBC hemolytic diseases – increased bilirubin production.
 Bile duct obstruction – conjugated bilirubin builds up in liver and spills over into
lymphatics, which then goes to venous system.
 Hepatocyte conjugation ineffective – a common cause of neonatal jaundice.
Alkaline Phosphatase (ALP)
This enzyme is found primarily in the biliary duct lining cells of the liver and in bone
osteocytes. When bile ducts are under pressure as would occur when they are
obstructed, the bile duct lining cells secrete ALP which enters the venous system.
Although the source of an elevated alkaline phosphatase can be determined by ordering
ALP isoenzymes, it is usually fairly easy to distinguish bone from biliary diseases based
on clinical information. An elevated ALP has clinical significance whereas a low value
does not. Causes of elevation in alkaline phosphatase include:
Biliary obstruction, small or large ducts, such as from a gallstones, pancreatic or
duodenal tumors, intrahepatic masses, both primary and metastatic.
Biliary atresia.
Bone growth and proliferation, such as fracture healing, bone neoplasms.
Serum calcium concentration is tightly regulated by the secretion of parathyroid
hormone (PTH). If serum calcium becomes too low, the parathyroid glands secrete
PTH which stimulates osteoclasts in bone to resorb bone trabeculae and release
calcium into circulation. Low calcium can cause tetany, which is sustained muscle
contraction and hypercalcemia is associated with muscle weakness.
About 50% of total serum calcium is bound to serum proteins and 50% is free ionized
calcium, the form considered metabolically active. Lab tests for total and ionized
calcium are available.
Hypocalcemia can be due to:
Hyperphosphatemia, as occurs in renal failure
Congenital or surgical absence of the parathyroid glands.
Vitamin D deficiency.
Hypoalbuminemia (ionized form may remain normal, but total calcium is low)
Hypercalcemia can be due to:
 Parathyroid gland hyperfunction (adenoma, hyperplasia, carcinoma).
 PTH related protein release from malignant neoplasms.
 Hyperparathyroidism secondary to chronic renal failure.
Inorganic Phosphate
The anions PO4-3, HPO4-2 and H2PO4-2 exist in serum and form the phosphate buffer
system. The test for inorganic phosphate detects them all.
Causes of hyperphosphatemia include:
PTH deficiency;
Renal failure; since ingested phosphate cannot be excreted.
GI absorption of phosphate enemas.
Total Protein and Albumin
Hundreds of specific plasma proteins exist with functions varying from small molecule
transporters, clotting factors, hormones, fibrinolytic factors, inert proteins released by
somatic cells and proteins that maintain intravascular oncotic pressure. Many of them
are synthesized in the liver. They are generally classified as albumin and the globulins.
Albumin typically has a reference range of 3.5 – 5.0 g./dL. and as the plasma protein in
the highest concentration, it provides the majority of the intravascular oncotic pressure.
Globulins are further classified by what bands they migrate to in a serum protein
Alpha-1-globulins: alpha 1 antitrypsin is the major contributer to this band. It
functions as an antiprotease; protecting cells from errantly released inflammatory
cell proteases.
Alpha-2-globulins: two proteins make up most of this band; haptoglobin and
alpha 2 macroglobulin. Haptoglobin can bind tightly to hemoglobin that
abnormally could be free in the plasma to prevent it from damaging the kidneys.
Alpha-2-macroglobulin, as the name implies, is a large protein that can increase
in concentration to maintain oncotic pressure if serum albumin is chronically low,
as it often is in renal failure associated with nephrotic syndrome.
Beta globulins: three proteins make up this band: beta lipoprotein – an essential
component to lipid transport and metabolism; transferrin – a transport protein that
ferries iron ions absorbed in the gut to the bone marrow where they will be
incorporated into hemoglobin; complement proteins, especially C3.
Gammaglobulins: immunoglobulins of the IgG, IgM, IgA and IgE classes.
Because of varying antibody specificities related to unique amino acid sequences
in the Ig variable regions, these proteins migrate to slightly different locations on
the gel and produce a broad band.
Causes of hypoproteinemia:
Protein losses in the urine or GI tract.
Liver disease – inadequate protein synthesis
Causes of hyperproteinemia:
Monoclonal gammopathy – neoplastic formation of a monoclonal
A battery of chemical tests performed on a urine sample can provide a wealth of
information about the patient’s urinary tract, kidney and metabolic functions. Dipstick
chemical urinalysis is a waived CLIA test. A complete urinalysis includes a sediment
analysis, which involves centrifuging about 10 ml. of urine and examining the formed
elements in the urine (cells, crystals and casts) under a microscope. As a labor saving
practice, many labs will not perform a sediment analysis when the urine chemical test
results are normal, since it would be unusual to find significant pathology in the
sediment if the chemical tests are normal.
Urine dipsticks have variable numbers and types of test pads on them. The standard
test pads are described here and the limitations of the results are emphasized.
Specific gravity
The normal range is typically 1.001 to 1.030. The s.g. is increased in a concentrated
urine and in urines that have iodinated radiocontrast material in them from a recent
radiologic procedure. The kidney’s ability to form concentrated urine is an important
function that can be lost in some renal disorders. The dipstick s.g. can be falsely
elevated in highly alkaline urines.
Normal urine pH can vary from 4 – 9. Urine pH varies widely throughout the day
depending on the overall metabolic state of the patient, whether they have had a recent
meal and whether they have a urinary tract infection with an organism that can
metabolize urea and form ammonium compounds that can raise the urine pH. Patients
with a sustained metabolic or respiratory acidosis would be expected to have an acidic
urine. People with normal resting metabolic function and that are fasting should have a
slightly acidic urine with a pH of 6.
Blood is not normally present in urine, although it may present as a contaminant in
menstruating women. Blood can enter the urine at any level of the urinary tract, from
the renal glomerulus to the distal end of the urethra. The dipstick reagent that detects
blood relies on the peroxidase-like activity of hemoglobin. Red cells in the urine sample
are lysed on the reagent pad and the released hemoglobin produces a color change.
Another protein that has similar peroxidase activity is myoglobin. Myoglobin is found in
high concentration in skeletal muscle. Thus, patients suffering from necrosis of skeletal
muscle, also called rhabdomyolysis, may have myoglobin in their urine that will produce
a false positive blood result on urine dipstick.
Glucose will transiently enter the urine following a high carbohydrate meal until insulin
action reduces to resting levels. Glucose in the urine of a fasting person indicates
hyperglycemia. The test pad is specific for glucose; other reducing substances that
might be in the urine of patients with inborn errors of carbohydrate metabolism will not
be detected.
Ketones are produced metabolically glucose is in short supply, such as when glycogen
stores are depleted, during starvation and when insulin or insulin receptors are deficient.
They include betahydroxybutyrate, acetone and _______________.
Betahydroxybutyrate is the most plentiful ketone produced in diabetic ketoacidosis, but
the dipstick test pad is least sensitive to it, causing false negative results.
Bilirubin reaching the large intestine can be metabolized by gut bacteria to urobilinogen.
This compound is then absorbed in the gut and some is excreted in the urine, thus a
small amount of urobilinogen is normally present in urine. A complete obstruction of the
biliary tract would eventually lead to no urobilinogen in the urine. Elevated bilirubin
production, as in chronic hemolytic disorders, could increase urobilinogen production
and excretion in urine. Urobilinogen testing in urine is little used today now that serum
assays for conjugated and unconjugated bilirubin are readily available to sort out
jaundice is due to biliary disease or not.
Bilirubin is not normally present in the urine. When it is present, it is in the conjugated
form and may indicate hemolysis or biliary obstruction with associated jaundice and
Leucocyte esterase
This is a test for a neutrophil granule enzyme. A positive leucocyte esterase test in
urine indicates the presence of neutrophils in the urine. Neutrophils in urine could
indicate urinary tract infection, sexually transmitted diseases, kidney stones and
Nitrite is a surrogate marker for bacteria in the urine. Certain bacteria contain enzymes
that can change nitrate to nitrite. A urinary tract infection with one of these types of
organisms would produce a positive nitrite test. The test will be negative in cases of
non-nitrase producing bacteria and also when frequent urination precludes
accumulation of enough nitrite positive urine to be detected.
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