1

Interpretation of Laboratory Tests:

A Case-Oriented Review of Clinical
Laboratory Diagnosis
Roger L. Bertholf, Ph.D.
Associate Professor of Pathology
University of Florida Health Science Center/Jacksonville
2
Case 1: Oliguria and hematuria
R. Bertholf American Society of Clinical Pathologists 3
Case 1: Oliguria and hematuria
A 7-year-old boy was brought to the pediatrician because of vomiting
and malaise. On physical examination he was slightly flushed, and had
some noticeable swelling of his hands and feet. The patient was
uncomfortable, and complained of pain in his tummy. He had a slight
fever. Heart was normal and lungs were clear. His past medical history
did not include any chronic diseases. The mother noted that he had a
severe sore throat about two weeks ago, but that it had cleared up on
its own. The child was not taking any medications. There were no
masses in the abdomen, and lymphadenopathy was not present. The
child had some difficulty producing a urine specimen, but finally was
able to produce a small amount of urine, which was dipstick-positive
for blood and protein.

R. Bertholf American Society of Clinical Pathologists 4
Questions. . .
What is the differential diagnosis in this case?

What laboratory tests might be helpful in
establishing the diagnosis?
R. Bertholf American Society of Clinical Pathologists 5
What do the kidneys do?
Regulate body fluid osmolality and volume
Regulate electrolyte balance
Regulate acid-base balance
Excrete metabolic products and foreign substances
Produce and excrete hormones
R. Bertholf American Society of Clinical Pathologists 6
The kidneys as regulatory organs
The kidney presents in the highest degree the phenomenon
of sensibility, the power of reacting to various stimuli in a
direction which is appropriate for the survival of the organism;
a power of adaptation which almost gives one the idea that its
component parts must be endowed with intelligence.

E. Starling (1909)
R. Bertholf American Society of Clinical Pathologists 7
Review of Renal Anatomy and Physiology
The kidneys are a pair of fist-sized organs that are
located on either side of the spinal column just
behind the lower abdomen (L1-3).

A kidney consists of an outer layer (renal cortex)
and an inner region (renal medulla).

The functional unit of the kidney is the nephron;
each kidney has approximately 10
6
nephrons.
R. Bertholf American Society of Clinical Pathologists 8
Renal anatomy
Cortex
Medulla
Pelvis
To the bladder
Capsule
R. Bertholf American Society of Clinical Pathologists 9
The Nephron
Renal artery
Glomerulus
Bowmans capsule
Proximal tubule
Distal tubule
Collecting duct
Henles Loop
Afferent arteriole
R. Bertholf American Society of Clinical Pathologists 10
Glomerular filtration
Glomerlular
capillary
membrane
Vascular space Bowmans space
Mean capillary blood
pressure = 50 mm Hg
BC pressure = 10 mm Hg
Onc. pressure = 30 mm Hg
Net hydrostatic = 10 mm Hg
~ 2,000 Liters
per day
(25% of cardiac output)
~ 200 Liters
per day
GFR ~ 130 mL/min
R. Bertholf American Society of Clinical Pathologists 11
What gets filtered in the glomerulus?
Freely filtered
H
2
O
Na
+
, K
+
, Cl
-
,
HCO
3
-
, Ca
++
,
Mg
+
, PO
4
, etc.
Glucose
Urea
Creatinine
Insulin
Some filtered
|
2
-microglobulin
RBP
o
1
-microglobulin
Albumin
None filtered
Immunoglobulins
Ferritin
Cells


R. Bertholf American Society of Clinical Pathologists 12
Then what happens?
If 200 liters of filtrate enter the nephrons each day,
but only 1-2 liters of urine result, then obviously
most of the filtrate (99+ %) is reabsorbed.

Reabsorption can be active or passive, and occurs
in virtually all segments of the nephron.
R. Bertholf American Society of Clinical Pathologists 13
Reabsorption from glomerular filtrate
% Reabsorbed
Water 99.2
Sodium 99.6
Potassium 92.9
Chloride 99.5
Bicarbonate 99.9
Glucose 100
Albumin 95-99
Urea 50-60
Creatinine 0 (or negative)
R. Bertholf American Society of Clinical Pathologists 14
How does water get reabsorbed?
Reabsorption of water is passive, in response to
osmotic gradients and renal tubular permeability.
The osmotic gradient is generated primarily by
active sodium transport
The permeability of renal tubules is under the
control of the renin-angiotensin-aldosterone
system.
The driving force for water reabsorption, the
osmotic gradient, is generated by the Loop of
Henle.
R. Bertholf American Society of Clinical Pathologists 15
The Loop of Henle
Proximal tubule Distal tubule
D
e
s
c
e
n
d
i
n
g

l
o
o
p

A
s
c
e
n
d
i
n
g

l
o
o
p

I
n
c
r
e
a
s
i
n
g

o
s
m
o
l
a
l
i
t
y

Renal Cortex
Renal Medulla
Na
+

Na
+

Na
+

H
2
O
Na
+

1200 mOsm/Kg
300 mOsm/Kg
R. Bertholf American Society of Clinical Pathologists 16
Regulation of distal tubule Na
+

permeability
JGA
Renin
+ Na
+

+ BP
Angiotensinogen
Angiotensin I
Angiotensin II
Angiotensin III
vasoconstriction
Aldosterone
Adrenal cortex
Na
+
Na
+
R. Bertholf American Society of Clinical Pathologists 17
Regulation of H
2
O reabsorption
Pituitary
ADH (vasopressin)
Plasma
hyperosmolality
H
2
O H
2
O
Renal Medulla (osmolality ~1200 mOsm/Kg)
R. Bertholf American Society of Clinical Pathologists 18
Summary of renal physiology
Filtration - Reabsorption + Secretion = Elimination
GFR (Filtered but not reabsorbed or secreted)
TRPF (Filtered and secreted)
R. Bertholf American Society of Clinical Pathologists 19
Measurement of GFR
694 . 0
) 24 (

(
(

=
p
h u u
C
V C
Clearance
C
u
= Concentration in urine
V
u(24h)
= 24-hour urine volume
C
p
= Concentration in plasma
0.694 = 1000 mL/1440 min
R. Bertholf American Society of Clinical Pathologists 20
Compounds used to measure GFR
Should not be metabolized, or alter GFR
Should be freely filtered in the glomeruli, but neither
reabsorbed nor secreted
Inulin (a polysaccharide) is ideal
Creatinine is most popular
There is some exchange of creatinine in the tubules
As a result, creatinine clearance overestimates GFR by
about 10% (But. . .)
Urea can be used, but about 40% is (passively)
reabsorbed
R. Bertholf American Society of Clinical Pathologists 21
Relationship between creatinine and GFR
P
l
a
s
m
a

c
r
e
a
t
i
n
i
n
e

GFR (mL/min)
1
2
3
4
5
6
0
0 20 40 60 80 100 120 140
R. Bertholf American Society of Clinical Pathologists 22
Measurement of TRPF
Para-aminohippurate (PAH) is freely filtered in the
glomeruli and actively secreted in the tubules.

PAH clearance gives an estimate of the total
amount of plasma from which a constituent can be
removed.
R. Bertholf American Society of Clinical Pathologists 23
Creatinine
O
N
CH
3
HN
N
H
HO
O CH
3
NH
NH
2
- H
2
O
N
Creatine Creatinine
1-2% of creatine is hydrolyzed to creatinine each day
R. Bertholf American Society of Clinical Pathologists 24
Jaffe method for creatinine
O
N
CH HN
N
H
O N NO
NO
OH
OH
+
Janovsky Complex

max
= 490-500 nm
Max Eduard Jaffe (1841-1911), German physiologic chemist
R. Bertholf American Society of Clinical Pathologists 25
Modifications of the Jaffe method
Fullers Earth (aluminum silicate, Lloyds reagent)
adsorbs creatinine to eliminate protein interference
Acid blanking
after color development; dissociates Janovsky
complex
Pre-oxidation
addition of ferricyanide oxidizes bilirubin
Kinetic methods
R. Bertholf American Society of Clinical Pathologists 26
Kinetic Jaffe method
A
b
s
o
r
b
a
n
c
e

(

=

5
2
0

n
m
)

Time (sec)
0 80 20
F
a
s
t
-
r
e
a
c
t
i
n
g

(
p
y
r
u
v
a
t
e
,

g
l
u
c
o
s
e
,

a
s
c
o
r
b
a
t
e
)

S
l
o
w
-
r
e
a
c
t
i
n
g

(
p
r
o
t
e
i
n
)

At
AA
rate
t
A
=
A
A
creatinine (and o-keto acids)
R. Bertholf American Society of Clinical Pathologists 27
Enzymatic creatinine methods
Creatininase
creatininecreatineCKADPPKLD
Creatinase
creatininecreatinesarcosinesarcosine
oxidaseperoxideperoxidase reaction
Creatinine deaminase (iminohydrolase)
most common
R. Bertholf American Society of Clinical Pathologists 28
Creatinine deaminase method
Creatinine
Creatinine
iminohydrolase
+ H
2
O
N-Methylhydantoin
ATP
ADP
NMH amidohydrolase
N-Carbamoylsarcosine
H
2
O
Peroxidase
Oxygen receptor Colored product
Sarcosine
NCS
amidohydrolase
- NH
3
, CO
2

+ O
2

Sarcosine oxidase
H
2
O H
2
O
2

Formaldehyde + glycine
R. Bertholf American Society of Clinical Pathologists 29
Measurement of urine protein
Specimen
Timed 24-h is best
Urine protein/creatinine ratio can be used with
random specimen
Normal protein excretion is <150 mg/24h
50-60% albumin
Smaller proteins (o
1
-, |
2
-microglobulins)
Tamm-Horsfall (uromucoid, secreted by tubules)
IgA, tubular epithelial enzymes, and other non-
filtered components
R. Bertholf American Society of Clinical Pathologists 30
Dipstick method for urine protein
Method is based on protein association with pH
indicator
Test pad contains dye tetrabromphenol blue at
pH=3
If protein binds to the pH indicator, H
+
is displaced
and the color changes from yellow to green (or
blue)
Most sensitive to albumin (poor method for
detecting tubular proteinuria)

R. Bertholf American Society of Clinical Pathologists 31
What causes excess urinary protein?
Overload proteinuria
Bence-Jones (multiple myeloma)
Myoglobin (crush injury, rhabdomyolysis)
Hemoglobin
Tubular proteinuria
Mostly low MW proteins (not albumin)
Fanconis, Wilsons, pyelonephritis, cystinosis
Glomerular proteinuria
Mostly albumin at first, but larger proteins appear
as glomerular membrane selectivity is lost.

R. Bertholf American Society of Clinical Pathologists 32
Classification of proteinuria: Minimal
<1 gram of protein per day
Chronic pyelonephritis
Mild glomerular disease
Nephrosclerosis (usually due to hypertension)
Chronic interstitial nephritis (usually analgesic-
related)
Renal tubular disease
R. Bertholf American Society of Clinical Pathologists 33
Classification of proteinuria: Moderate
1.0 - 4.0 grams of protein per day
Usually associated with glomerular disease
Overflow proteinuria from multiple myeloma
Toxic nephropathies
R. Bertholf American Society of Clinical Pathologists 34
Classification of proteinuria: Severe
>4 grams of protein per day
Nephrotic syndrome (|GBM permeability)
Sx: edema, proteinuria, hypoalbuminemia,
hyperlipidemia
In adults, usually 2 to systemic disease (SLE,
diabetes)
In children, cause is usually primary renal disease
Minimal Change Disease (Lipoid Nephrosis)
Most common cause of NS in children
Relatively benign (cause unknown, not autoimmune)
R. Bertholf American Society of Clinical Pathologists 35
Proteinuria due to glomerulonephritis
Acute, rapidly progressive, or chronic GN can
result in severe proteinuria
Often the result of immune reaction (Circulating
Immune-Complex Nephritis)
Antigen can be endogenous (SLE) or exogeneous
Glomerular damage is mostly complement-
mediated
If antigen is continuously presented, GN can
become chronic
R. Bertholf American Society of Clinical Pathologists 36
How do red blood cells get in urine?
Hematuria can result from bleeding anywhere in
the kidneys or urinary tract
Disease, trauma, toxicity
Hemoglobinuria can result from intravascular
hemolysis
Disease, trauma, toxicity
R. Bertholf American Society of Clinical Pathologists 37
Dipstick method for hemoglobin
Ascorbic acid inhibits the reaction, causing a false
negative test
Depends on RBC lysis (may not occur in urine
with high specific gravity)
Detection limit approximately 10 RBC/L
H
2
O
2
+ chromogen* Oxidized chromogen + H
2
O
Heme
Peroxidase
*tetramethylbenzidine; oxidized form is green
R. Bertholf American Society of Clinical Pathologists 38
Microscopic examination of urine sediment
R. Bertholf American Society of Clinical Pathologists 39
Significance of RBC casts in urine
Indicative of blood crossing the GBM
Casts form in the distal tubules
Stasis produces brown, granular casts
RBC casts almost always reflect glomerular disease

R. Bertholf American Society of Clinical Pathologists 40
Brights Disease (acute glomerulonephritis)
Characterized by oliguria, proteinuria, and
hematuria
Most common cause is immune-related
Richard Bright (1789-1858)
R. Bertholf American Society of Clinical Pathologists 41
Primary Glomerulonephritis
Proliferative GN
Acute Post-infectious GN
Idiopathic or Crescentic GN
o-GBM disease
Membranoproliferative GN
Focal GN
IgA nephropathy
R. Bertholf American Society of Clinical Pathologists 42
Primary Glomerulonephritis, cont.
Idiopathic membranous GN
Histological diagnosis, probably immune complex
Chronic GN
Clinical Dx; many potential causes
Lipoid Nephrosis
Histological findings normal; Nephrosis
Focal Glomerular Sclerosis
Probably immune (IgM) related
R. Bertholf American Society of Clinical Pathologists 43
Secondary Glomerulonephritis
Systemic Lupus Erythematosus
Renal failure accounts for 50% of SLE deaths
Polyarteritis (inflammatory vasculitis)
Wegeners Granulomatosis (lung and URT)
Henoch-Schnlein Syndrome
Lacks edema assoc. with post-streptococcal GN
Goodpastures Syndrome (pulmonary hemorrhage)
Hemolytic-Uremic Syndrome
Progressive Systemic Sclerosis (blood vessels)

44
Case 3: Chest Pain
R. Bertholf American Society of Clinical Pathologists 45
Case 3: Chest Pain
A 63 year old male was brought to the emergency department
after complaining of severe chest pain that had lasted for two
hours. He had been mowing his lawn when the pain developed,
and he became concerned when the pain did not subside after
he stopped the activity. He had no previous history of heart
disease. On presentation he was moderately overweight, dia-
phoretic, and in obvious discomfort. He described his chest
pain as beginning in the center of my chest, then my arms,
neck, and jaw began to ache too.

Diagnostic procedures were performed.
R. Bertholf American Society of Clinical Pathologists 46
Questions
What is the most important consideration in the
triage of this patient?

What tests should be ordered?
R. Bertholf American Society of Clinical Pathologists 47
Chest pain
One of the most common reasons for seeking
medical attention
Characteristics of cardiogenic chest pain (angina)
induced by exercise
described as pressure
radiates to extremities
MI not relieved by rest or vasodilatory drugs (NG)
Only 25% of patients presenting with chest pain as
the primary complaint will ultimately be diagnosed
as MI (specificity=25%; sensitivity=80%)
R. Bertholf American Society of Clinical Pathologists 48
The Heart
Aorta
Superior vena cava
RA
LA
RV
LV
Pulmonary arteries
R. Bertholf American Society of Clinical Pathologists 49
The Heart (posterior view)
Aorta
Superior vena cava
Inferior vena cava
Pulmonary veins
Pulmonary arteries
R. Bertholf American Society of Clinical Pathologists 50
Cardiac physiology
R. Bertholf American Society of Clinical Pathologists 51
Cardiac conduction system
Sinoatrial (SA) node
Atrioventral (AV) node
His bundle
Right bundle branch
Left bundle branch
R. Bertholf American Society of Clinical Pathologists 52
Normal Electrocardiogram
P
Q
R
S
T
U
R. Bertholf American Society of Clinical Pathologists 53
Myocardial infarction
Right coronary artery
Left coronary artery
Anterior left ventricle
R. Bertholf American Society of Clinical Pathologists 54
ECG changes in myocardial infarction
S
P
R
T
Q+
S-T elevation
R. Bertholf American Society of Clinical Pathologists 55
Diagnostic value of ECG
ECG changes depend on the location and severity of
myocardial necrosis
Virtually 100% of patients with characteristic Q-
wave and S-T segment changes are diagnosed with
myocardial infarction (100% specificity)
However, as many as 50% of myocardial infarctions
do not produce characteristic ECG changes
(sensitivity ~ 50%)
ECG may be insensitive for detecting prognostically
significant ischemia
R. Bertholf American Society of Clinical Pathologists 56
History of cardiac markers
1975: Galen describes the use of CK, LD, and
isoenzymes in the diagnosis of myocardial infarction.
1980: Automated methods for CK-MB (activity) and
LD-1 become available.
1985: CK-MB isoforms are introduced.
1989: Heterogeneous immunoassays for CK-MB
(mass) become available.
1991: Troponin T immunoassay is introduced.
1992: Troponin I immunoassay is introduced.


R. Bertholf American Society of Clinical Pathologists 57
Enzyme markers
Aspartate transaminase (AST; SGOT)
2-Hydroxybutyrate dehydrogenase
Lactate dehydrogenase
Five isoenzymes, composed of combinations of H
(heart) and M (muscle) subunits
Creatine kinase
Three isoenzymes, composed of combinations of
M (muscle) and B (brain) subunits

R. Bertholf American Society of Clinical Pathologists 58
Lactate dehydrogenase (LD)
Pyruvate
LD activity is measured by monitoring absorbance
at = 340 nm (NADH)
Methods can be P L or L P
But. . .reference range is different
Total LD activity has poor specificity
Lactate
LD
NAD
+
NADH
R. Bertholf American Society of Clinical Pathologists 59
Tissue specificity of LD isoenzymes
LD isoenzyme Tissues
LD-1 Heart (60%), RBC, Kidney
LD-2 Heart (30%), RBC, Kidney
LD-3 Brain, Kidney
LD-4 Liver, Skeletal muscle, Brain, Kidney
LD-5 Liver, Skeletal muscle, Kidney
R. Bertholf American Society of Clinical Pathologists 60
LD isoenzyme electrophoresis (normal)
LD-1
LD-2
LD-3
LD-4
LD-5
LD-2 > LD-1 > LD-3 > LD-4 > LD-5
Cathode (-) Anode (+)
R. Bertholf American Society of Clinical Pathologists 61
LD isoenzyme electrophoresis (abnormal)
LD-1
LD-2
LD-3
LD-4
LD-5
LD-1 > LD-2
Cathode (-) Anode (+)
R. Bertholf American Society of Clinical Pathologists 62
Direct measurement of LD-1
Electrophoresis is time-consuming and only semi-
quantitative
Antibodies to the M subunit can be used to
precipitate LD-2, 3, 5, and 5, leaving only LD-1
Method can be automated
Normal LD-1/LD
total
ratio is less than 40%
R. Bertholf American Society of Clinical Pathologists 63
Sensitivity and specificity of LD-1
Sensitivity and specificity of the LD 1:2 flip, or
LD-1 > 40% of total, are 90+% within 24 hours of
MI, but. . .
May be normal for 12 or more hours after
symptoms appear (peak in 72-144 hours)
May not detect minor infarctions
Elevations persist for up to 10 days
Even slight hemolysis can cause non-diagnostic
elevations in LD-1

R. Bertholf American Society of Clinical Pathologists 64
Creatine Kinase (CK)
Phosphocreatine
ADP
HK
Glucose Glucose-6-phosphate
NADPH
=340 nm
NADP
+

GPD
6-Phosphogluconate
Oliver and Rosalki method (1967)
Creatine
CK
ADP ATP
Mg
++

R. Bertholf American Society of Clinical Pathologists 65
Tissue specificities of CK isoenzymes
Tissue
CK-1
(BB)
CK-2
(MB)
CK-3
(MM)
Skeletal muscle 0% 1% 99%
Cardiac muscle 1% 20% 79%
Brain 97% 3% 0%
R. Bertholf American Society of Clinical Pathologists 66
Measurement of CK isoenzymes
Electrophoresis (not used anymore)
Immunoinhibition/precipitation
Antibody to M subunit
Multiply results by 2
Interference from CK-1 (BB)
Most modern methods use two-site (sandwich)
heterogeneous immunoassay
Measures CK-MB mass, rather than activity
Gives rise to a pseudo-percentage, often called the
CK-MB index
R. Bertholf American Society of Clinical Pathologists 67
Sensitivity/specificity of CK-MB
Sensitivity and specificity of CK-MB for
myocardial infarction are >90% within 7-18 hours;
peak concentrations occur within 24 hours
CK is a relatively small enzyme (MW = 86K), so it
is filtered and cleared by the kidneys; levels return
to normal after 2-3 days
Sensitivity is poor when total CK is very high, and
specificity is poor when total CK is low
Presence of macro-CK results in false elevations
R. Bertholf American Society of Clinical Pathologists 68
CK isoforms
C-terminal lysine is removed from the M subunit--
therefore, there are three isoforms of CK-3 (MM)
t

: CK-MB
1
> CK-MB
2

Ratio of CK-MB
2
to CK-MB
1
exceeds 1.5 within six
hours of the onset of symptoms
Only method currently available is electrophoresis
CK-MB
2
(tissue) CK-MB
1
(circulating)
C-terminal lysine
Plasma carboxypeptidase
R. Bertholf American Society of Clinical Pathologists 69
Myoglobin
O
2
-binding cytosolic protein found in all muscle
tissue (functional and structural analog of
hemoglobin)
Low molecular weight (17,800 daltons)
Elevations detected within 1-4 hours after
symptoms; returns to normal after 12 hours
Nonspecific but sensitive marker--primarily used
for negative predictive value
Usually measured by sandwich, nephelometric,
turbidimetric, or fluorescence immunoassay
R. Bertholf American Society of Clinical Pathologists 70
Temporal changes in myoglobin and CK-MB
0
200
400
600
800
0 8 16 24 32 40 48
Time after symptoms
M
y
o
g
l
o
b
i
n

(
u
g
/
L
)
0
10
20
30
40
50
60
C
K
-
M
B

(
u
g
/
L
)
Myoglobin CK-MB
R. Bertholf American Society of Clinical Pathologists 71
Troponin
Thick Filament
Myosin
Tropomyosin Actin
TnC
TnT (42 Kd)
TnI (23 Kd)
R. Bertholf American Society of Clinical Pathologists 72
Tissue specificity of Troponin subunits
Troponin C is the same in all muscle tissue
Troponins I and T have cardiac-specific forms,
cTnI and cTnT
Circulating concentrations of cTnI and cTnT are
very low
cTnI and cTnT remain elevated for several days
Hence, Troponins would seem to have the
specificity of CK-MB (or better), and the long-term
sensitivity of LD-1
R. Bertholf American Society of Clinical Pathologists 73
Is cTnI more sensitive than CK/CK-MB?
-1
-0.5
0
0.5
1
1 8 40 66
Hours since presentation
l
o
g

X

n
o
r
m
a
l
CK
CK-MB
CK-MB Index
cTnI
79 y/o female with Hx of HTN, CHF, CRI, Type II diabetes
R. Bertholf American Society of Clinical Pathologists 74
Measurement of cTnI and cTnT
All methods are immunochemical (ELISA, MEIA,
CIA, ECIA)
Roche Diagnostics (formerly BMC) is the sole
manufacturer of cTnT assays
First generation assay may have had some cross-
reactivity with skeletal muscle TnT
Second generation assay is cTnT-specific
Also available in qualitative POC method
Many diagnostics companies have cTnI methods
R. Bertholf American Society of Clinical Pathologists 75
W.H.O. has a Myocardial Infarction?
A clinical history of ischemic-type chest discomfort
Changes on serially obtained ECG tracings
A rise and fall in serum cardiac markers
A patient presenting with any two of the following:
Source JACC 28;1996:1328-428
R. Bertholf American Society of Clinical Pathologists 76
Sensitivity/Specificity of WHO Criteria
0%
20%
40%
60%
80%
100%
Chest Pain ECG changes Serum
markers
Sensitivity
Specificity
R. Bertholf American Society of Clinical Pathologists 77
What Cardiac Markers do Labs Offer?
0
500
1000
1500
2000
2500
3000
3500
#

o
f

l
a
b
s

r
e
p
o
r
t
i
n
g
CK-MB
(ng/mL)
CK-MB
(IU/L)
cTnI cTnT
1997
1998