Lab Medicine Conference :
Amylase, Lipase, Pregnancy
Tests, Sedimentation Rate
Jim Holliman, M.D., F.A.C.E.P.
Professor of Emergency Medicine
Director, Center for International Emergency Medicine
M. S. Hershey Medical Center
Penn State University
Hershey, Pennsylvania, U.S.A.
Amylase Measurement
ƒ Named for Greek word for starch (amylone)
ƒ Amylase acts to split starches ; can cleave
amylopectin, amylose, glycogen, & their
hydrolyzed products
ƒ 2 types in humans :
–P type : in pancreas
–S type : mainly in salivary glands
ƒ also in lung, fallopian tubes, lactating
mammary glands, & sweat glands
ƒ Both have molecular weight 54,000
Netter’s diagram of
amylase
physiology
Activity of Amylase
ƒ Requires calcium for catalytic activity
ƒ Also nedds a halide (chloride, bromide, or
iodide) for activation
ƒ Maximum activity at temp. 50 OC & pH 6.9 to
7.0
ƒ Both P and S types have isoenzymes, but
differentiation not generally clinically useful
at present
Clearance of Amylase
ƒ P type cleared 80 % faster than S type
ƒ Serum half life of P type is 2 hours
ƒ Cleared by glomerular filtration without
tubular excretion or reabsorption
ƒ Some limited extrarenal clearance ( ? in
reticuloendothelial system)
Methods for Amylase Level
Quantification
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Saccharogenic : standard since 1938
Amyloclastic (Iodometric)
Chromolytic
Turbidimetric
Nephelometric
Saccharogenic Method for
Amylase Quantification
ƒ Measures hydrolyzed products of starch
by direct measurement of sugars created
by amylase activity
ƒ Measured in Somogyi units
–One unit is defined as amount of
enzyme in 100 ml of specimen that
liberates reducing substances
equivalent to 1 mg of glucose from
starch in 30 minutes at 40 OC
Amyloclastic Method for
Amylase Quantification
ƒ Measures color change of iodine reaction with
starch
–When iodine combines with polysaccharides
of more than 12 to 18 glucose residues, a
brown color appears
–No color change occurs when iodine is mixed
with polysaccharides of 12 glucose residues or
less
–Amount of amylase then correlated to amount
of color change by photometric analysis
Chromolytic Method for
Amylase Quantification
ƒ Starch is covalently bonded to a dye
molecule
ƒ Hydrolyzed products then measured
photometrically
ƒ Very sensitive
ƒ Easily performed in lab
Turbidimetric & Nephelometric
Methods for Amylase Quantification
ƒ Starch solutions are colloidal by nature
ƒ Amylase action decreases the colloidal
nature of a starch solution
ƒ Colloidal status can then be measured
photometrically
ƒ Both methods utilize measurement of
light reflection
–Nephelometric method more accurate
–Both methods can be automated
Reporting of Amylase
Measurements
ƒ Some current methods reported in
International Units (IU)
–One IU catalyzes transformation of one
micromole of substrate per minute
–Some methods using IU may not be as
accurate as those using Somogyi units
Macroamylase
ƒ Represents P or S type linked to an
immunoglobulin or complex
polysaccharides
ƒ Molecular weight 150,000 to 1 million
ƒ Not filtered at glomerulus ; stays in serum
ƒ Can occur with alcoholism, malabsorption, &
GI tract diseases
ƒ Serum amylase levels are 4 to 5 times
normal, but urine amylase is low or normal
Direct Pancreatic Causes of
Elevated Serum Amylase
ƒ Acute or chronic pancreatitis
ƒ Pancreatic pseudocysts
ƒ Pancreatic ascites
ƒ Mumps
ƒ Pancreatic or duodenal trauma
Salivary Causes of Elevated
Serum Amylase
ƒ Tumors
ƒ Salivary gland calculi
ƒ Sialadenitis
ƒ Head & neck surgery
ƒ Head & neck trauma
Miscellaneous Abdominal Causes of
Elevated Serum Amylase
ƒ Renal failure (up to 2X normal)
ƒ Perforated peptic ulcer
ƒ Bowel obstruction
ƒ Ruptured ectopic pregnancy
ƒ Mesenteric infarction
ƒ Afferent loop syndrome
ƒ Aortic aneurism / dissection
ƒ Cirrhosis
Other Miscellaneous Causes
of Elevated Serum Amylase
ƒ Macroamylasemia
ƒ Cerebral trauma
ƒ Burns
ƒ Generalized shock
ƒ DKA
ƒ Renal transplant
ƒ Pneumonia
ƒ Drugs & meds (on a later slide)
Amylase Levels in Acute
Pancreatitis
ƒ Elevation > 5X normal often indicates acute
pancreatitis
–sensitivity 70 to 98 %
–specificity 70 to 76 %
ƒ Peak levels reached in first 48 hours
ƒ Levels return to normal 5 to 7 days after
resolution of inflammation
ƒ Urine amylase levels peak later & can remain
elevated for days after symptoms are
resolved
Clinical Use of Amylase to
Creatinine Clearance Ratio
ƒ Used to help differentiate acute pancreatitis
from other causes of elevated amylase
ƒ (urine amylase X plasma creatinine) divided
by (urine creatinine X plasma amylase) X
100
ƒ Normal clearance ratio is < 5 %
ƒ Ratios > 10 % suggest acute pancreatitis
ƒ Ratio not affected by urine volume or rate
Limitations of Use of Amylase to
Creatinine Clearance Ratio
ƒ 30 % of patients with proven acute
pancreatitis may have normal ratio
ƒ Can be elevated in DKA, renal failure,
heart disease, peptic ulcer, or postop
ƒ Because of often sporadic secretion of
amylase, single measurement may be
unreliable
Mechanisms of Amylase Elevation
by Drugs and Medications
ƒ Induce spasm of Sphincter of Oddi
–e.g., narcotics
ƒ Direct inflammation of parotid gland
–e.g., phenylbutazone
ƒ Direct inflammation of pancreas
–e.g., alcohol, steroids, thiazides
List of Medications and Drugs
Causing Elevated Amylase Levels
ƒ Alcohols
ƒ Corticosteroids
ƒ Estrogens
ƒ Thiazide diuretics
ƒ Sulfonamides
ƒ Furosemide
ƒ Ethacrynic acid
ƒ Clofibrate
ƒ Indomethacin
ƒ Salicylates
ƒ Coumadin
ƒ Asparaginase
ƒ Amphetamines
ƒ Narcotics
ƒ Phenylbutazone
ƒ Rifampin
ƒ Tetracycline
ƒ Vitamin D
ƒ Carbon tetrachloride
ƒ Histamine
ƒ Phenformin
ƒ Acetominophen
ƒ Propoxyphene
ƒ Cimetidine
ƒ Valproic acid
ƒ Ciproheptadine
Interpretation of Elevated
Amylase Levels
ƒ Positive predictive value approaches 100 % at value of 1000
IU/L
ƒ At 300 IU/L, total amylase is 90 to 95 % sensitive, but only 71
to 80 % specific
ƒ Biliary pancreatitis causes markedly higher initial serum
amylase than for alcoholic or other causes
–with biliary : only 11 % of cases < 1000 IU/L
–with alcoholic : only 6 % of cases > 1000 IU/L
ƒ If hypertriglyceridemia present, can have pancreatitis with
normal amylase (mechanism is uncertain)
ƒ Magnitude of increase does NOT correlate with severity of
disease or prognosis
Lipase Physiology
ƒ Enzyme found mainly in pancreas
ƒ Molecular weight 48,000
ƒ Filtered at glomerulus but reabsorbed
ƒ Very little circulates in blood
ƒ Turbidimetric method of measurement :
–Colipase added
–this stabilizes the lipid - lipase - bile salt
complex from dissociation & permits reliable
assay
–liberated H2O2 oxidizes a leucodye that is read at 540 nm
Nonpancreatic Causes of Elevated
Serum Lipase (usually < 3X normal)
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Mumps
Types I & IV hyperlipoproteinemias
Peptic ulcer
Acute cholecystitis
Extrahepatic biliary obstruction
Mesenteric infarction
Bowel perforation
Acute renal failure
Bone fractures
Fat embolism syndrome
Crush injury
Post-cholecystectomy syndrome
Lipase Levels in Pancreatitis
ƒ Parallels amylase in onset & height of
elevation
ƒ Not elevated with macroamylasemia &
DKA
ƒ Levels > 3X normal have > 99 %
predictive value for acute pancreatitis
ƒ Generally : lipase assay is as reliable,
more specific, almost as sensitive, &
about same cost as amylase assay for
pancreatitis
Amylase and
lipase secretion in
acute pancreatitis
Measurement of Immunoreactive
Trypsinogen (IRT)
ƒ Radioimmunoassay can measure IRT ; is not affected by
serum proteases as are other methods
ƒ IRT is elevated in :
–Extrahepatic obstructive jaundice
–Renal failure
–Hypercalcemia
–Hypertriglyceridemia
–Liver cirrhosis
–Chronic pancreatitis
ƒ Can serve to confirm pancreatic origin of an elevated
amylase level, but offers no improvement in diagnostic
accuracy over amylase or lipase as a single test
Lab Charges at H.M.C. for
Pancreatic Lab Tests
Test
Routine
Stat
Amylase
$ 13
$ 39
Lipase
$ 22
$ 29
$ 13
$ 29
Triglycerides
Simplified Algorithm for Interpretation of
Pancreatic Enzyme Levels
ƒ If amylase > 1000, & renal function
normal, assume acute pancreatitis
ƒ If amylase < 1000 (even if normal) &
pancreatitis suspected, check lipase
–If lipase > 3X normal, assume acute
pancreatitis
–If lipase < 3X normal, consider diagnoses
on slide # 23
Categories of Pregnancy Tests
ƒ Bioassays (no longer used)
ƒ Immunoassays (variations of agglutination
tests)
–Radioimmunoassays (RIA)
–Enzyme immunoassays
–Radioreceptor assays (RRA)
–Each of these is based on detection of Human
Chorionic Gonadotropin (HCG) in urine or blood
–HCG is measured in International Units (IU)
Bioassay Pregnancy Tests
ƒ Developed in 1920's
ƒ Used toads, mice, frogs, or rabbits for
testing
ƒ Problems :
–High cost
–Need for frequent restandardization
–High false positive rate (from circulating
Leutinizing Hormone)
Characteristics of HCG
ƒ Is a glycoprotein produced by
trophoblastic tissues
ƒ Consists of 2 subunits : alpha & beta
ƒ Beta subunit is unique to HCG, but
similar to beta subunit of Leutinizing
Hormone (LH) so there can be some
cross-reactivity
ƒ HCG levels rise immediately after
implantation & are detectable in serum
within 24 hours of implantation
Patterns of HCG Levels
ƒ During first 6 weeks of normal pregnancy, HCG
levels should double every 48 hours
ƒ Mean serum HCG level at 6 weeks (from LMP) is
10,000 mIU/ml
ƒ Detection limit (& definition of nonpregnant
level) is 5 mIU/ml
ƒ HCG levels with ectopic pregnancy usually stay
< 6000 mIU/ml & do not show 2 day doubling
ƒ Postpartum levels fall to normal in 3 to 5 days
HCG Levels in Normal Pregnancy
Time Post-Conception
mIU / ml.
1 week
2 weeks
3 weeks
4 weeks
5 to 6 weeks
7 to 8 weeks
2 to 3 months
Second trimester
Third trimester
5 to 50
40 to 1000
100 to 5000
600 to 10,000
1500 to 100,000
16,000 to 200,000
12,000 to 300,000
24,000 to 55,000
6000 to 48,000
Days from onset of last menstrual
period
60 days
120 days
Before term
Comparison of Current HCG Tests
Lower Sensitivity
Limit (mIU / ml)
Approximate Days
Since Last Menses
Beta subunit RadioImmunoassay
<5
22
Serum Monoclonal
Antibody Test
< 10
22 to 24
Urine Monoclonal
Antibody Test
20
24 to 26
Home Monoclonal
Antibody Test
200
28
Use of Urine Monoclonal Antibody
Tests to Detect Ectopic Pregnancy
ƒ Serum & urine qualitative monoclonal antibody
pregnancy tests have almost equivalent ability to
detect ectopic pregnancy
ƒ < 3 % of ectopics have serum HCG < 40 mIU/ml
ƒ Ectopics with HCG < 40 mIU/ml are not at
imminent risk of rupture & may be undergoing
resorption ; repeat test at followup or quantitative
HCG (radioimmunoassay for beta HCG) will clarify
these cases
Causes of False Positive
Pregnancy Test Results
ƒ Increased LH
–Midcycle peak
–Menopause
ƒ Ectopic HCG production
–Trophoblastic tumors
(choriocarcinoma or
hydatidiform mole)
–Small cell lung cancer
–Ovarian cancer
ƒ Tubo-ovarian abscess
ƒ Test interface interference
–Proteinuria > 1 g / 24 hrs.
–Methadone
–Phenothiazine
–Promethazine
ƒ Missed abortion or
miscarriage
–Sometimes have levels <
200 IU/ml persistent for up
to 90 days
Relation of Expected HCG Levels with
Transvaginal Ultrasound Findings
Transvaginal Ultrasound Finding
Number of Days
Post-Ovulation
Beta-HCG Level
(mIU / ml)
Gestation Sac
First Seen
20
1500
Gestation Sac
Reliably Seen
21 to 24
3000
Fetal Pole Seen
25
5000
Fetal Heart Motion
Seen
30
15,000
Indications for Obtaining
Quantitative Serum HCG Level
ƒ Monitor trophoblastic disease
ƒ Positive monoclonal test but no fetal
sac or pole, & no mass in tube on
ultrasound
ƒ Uncertain missed abortion
ƒ Uncertain incomplete abortion
ƒ Negative monoclonal test but
suspected, and absolutely need to rule
out, very early pregnancy
Lab Charges at H.M.C. for
Pregnancy Testing
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Qualitative serum HCG : $ 28
Quantitative serum HCG : $ 68
Qualitative urine HCG : $ 24 (routine)
Qualitative urine HCG : $ 36 (stat)
Erythrocyte Sedimentation Rate (ESR)
General Principles
ƒ Diagnostically useful indicator of some
inflammatory conditions
ƒ Increased ESR due to any condition causing
red cell aggregation or rouleaux formation
ƒ Is an index of the suspension stability of
RBC's in citrated blood
ƒ Depends on the difference in specific gravity
between RBC's & plasma
ƒ A normal value does not exclude organic
disease
Factors Affecting the ESR Value
ƒ Depends mainly on concentration of
fibrinogen, but to a lesser degree on
alpha-2-globulin & gamma globulin
ƒ Is rapid (increased) in disorders where
fibrinogen or globulins are increased
Methods of Measurement of ESR
ƒ Wintrobe or Westergren
–Most common methods
–Require relatively larger sample
ƒ Microsedimentation
–Requires only a few drops of blood
–Linzenheimer / Landau / Adler modifications
ƒ Distance from top of tube to RBC column
meniscus at 1 hour (mm per hour) reported
Normal Westergren ESR Value
Ranges
ƒ Newborn : 0 to 2 mm/hr
ƒ Children : 1 to 10 mm/hr
ƒ Adults
–Males : 0 to 25 mm/hr
–Females : 0 to 30 mm/hr
ƒ Micromethods closely correlate,
especially at lower values
Zeta Sedimentation Ratio (ZSR)
ƒ Measures ability of RBC's to pack
under a standardized stress of
alternating dispersion & compaction
ƒ No real advantage over Wintrobe or
Westergren methods
ƒ For males, mean ZSR is 44 % & upper
limit of normal is 54 %
ƒ For females, mean ZSR is 45 % &
upper limit of normal is 56 %
Causes of High (> 100 mm/hr) ESR
ƒ Acute bacterial infections
–Meningitis
–Pneumonia
–Cholangitis
–Septic arthritis
–Osteomyelitis
–Pyelonephritis
–Abscesses
ƒ Polymyalgia rheumatica
ƒ Rheumatoid arthritis
ƒ S.L.E.
ƒ Viral encephalitis
ƒ Multiple myeloma
ƒ Leukemia
ƒ Lymphoma
ƒ Carcinomas
ƒ Drug hypersensitivity
reactions
ƒ Pulmonary infarction
ƒ Uremia
ƒ Open heart surgery
ƒ Cerebrovascular accident
ƒ Thrombophlebitis
ƒ Major orthopedic surgery
Causes of Moderately Elevated ESR
(50 to 100 mm/hr)
ƒ Tuberculosis
ƒ Viral hepatitis
ƒ Pelvic inflammatory disease
ƒ Infectious mononucleosis
ƒ Acute glmerulonephritis
ƒ Chronic infectious diseases
ƒ Rheumatic fever
ƒ Sarcoidosis
ƒ Rheumatic fever
ƒ Hypothyroidism
ƒ Drug fever
ƒ Liver metastases
ƒ Atrial myxoma
ƒ Macroglobulinemia
ƒ Regional enteritis
ƒ Myocardial infarction
ƒ Thyroiditis
ƒ Abdominal surgery
ƒ Menstrual period
ƒ Pregnancy after first
trimester
ƒ Ectopic pregnancy
Causes of Slightly Elevated ESR
(25 to 50 mm/hr)
ƒ Uncomplicated viral
diseases
ƒ Cholecystitis
ƒ Malaria
ƒ Typhoid fever
ƒ Pertussis
ƒ Cytomegalovirus
ƒ Toxoplasmosis
ƒ Rickettsial infections
ƒ Digital osteomyelitis
ƒ Localized infections
ƒ Osteoarthritis
ƒ Gout
ƒ Benign neoplasms
ƒ Cirrhosis
ƒ Peptic ulcer disease
ƒ Acute allergies
ƒ Ulcerative colitis
ƒ Pancreatitis
ƒ Drug fever
Causes of Very Low ESR (0 to 1
mm/hr)
ƒ High dose steroid or salicylate therapy
ƒ Severe anemia
ƒ Cachexia
ƒ Massive hepatic necrosis
ƒ D.I.C.
ƒ Polycythemia vera
ƒ Trichinosis
ƒ Chronic lymphocytic or myeloid leukemia
ƒ Hypofibrinogenemia
ƒ Macroglobulinemia (hyperviscosity syndrome)
ƒ "Chronic mononucleosis syndrome"
Situations Where ESR May Be
Diagnostically Helpful
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Cholangitis (> 50) vs. Cholecystitis (< 50)
P.I.D. (> 50) vs. Ovarian cyst (< 50)
Strep (< 50) vs. Infectious mono (> 50)
Trichinosis (< 50) vs. Polymyositis (> 50)
Rheumatoid (> 50) vs. Osteo (< 50) arthritis
Carcinoma (> 50) vs. Cachexia (< 50)
Temporal arteritis (> 50) vs. tension headache(< 50)
Synovitis (< 50) vs. Septic arthritis (> 50) of hip
Loose prosthesis (< 50) vs. Infected prosthesis (> 50)
Situations in Which ESR Is
NOT of Diagnostic Help
ƒ Rheumatoid arthritis vs. sarcoidosis
ƒ Tubercular arthritis vs. sarcoidosis
ƒ Rheumatoid arthritis vs. S.L.E.
ƒ Early prosthetic valve endocarditis vs. recent open heart
surgery
ƒ Inflammatory diskitis vs. septic diskitis
ƒ Osteogenic sarcoma vs. chronic osteomyelitis
ƒ Early orthopedic trauma vs. early infection
ƒ Drug fever vs. fever due to infection
ƒ Regional ileitis vs. ulcerative colitis
ƒ Appendicitis vs. pyelonephritis
ƒ Pulmonary infarction vs. pneumonia
Lab Charges at H.M.C. for ESR
ƒ Routine : $ 14
ƒ Stat : $ 37
ƒ Run off of a purple top tube