Interpretation of Laboratory Tests: A Case-Oriented Review of Clinical Laboratory Diagnosis

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Interpretation of Laboratory TestsA
Case-Oriented Review of Clinical Laboratory
Diagnosis

• Roger L. Bertholf, Ph.D.
• Associate Professor of Pathology
• University of Florida Health Science
  Center/Jacksonville

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Case 1 Oliguria and hematuria
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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.
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Questions. . .

• What is the differential diagnosis in this case?
• What laboratory tests might be helpful in
  establishing the diagnosis?

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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

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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)
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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 106 nephrons.

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Renal anatomy
Cortex
Pelvis
Capsule
Medulla
To the bladder
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The Nephron
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Glomerular filtration
Glomerlular capillary membrane
Vascular space
Bowmans space
? 2,000 Liters per day (25 of cardiac output)
? 200 Liters per day
GFR ? 130 mL/min
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What gets filtered in the glomerulus?

• Freely filtered
• H2O
• Na, K, Cl-, HCO3-, Ca, Mg, PO4, etc.
• Glucose
• Urea
• Creatinine
• Insulin

• Some filtered
• ?2-microglobulin
• RBP
• ?1-microglobulin
• Albumin

• None filtered
• Immunoglobulins
• Ferritin
• Cells

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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.

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Reabsorption from glomerular filtrate
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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.

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The Loop of Henle
Proximal tubule
Distal tubule
Renal Cortex
Descending loop
Ascending loop
Renal Medulla
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Regulation of distal tubule Na permeability
JGA
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Regulation of H2O reabsorption
Pituitary
Renal Medulla (osmolality ?1200 mOsm/Kg)
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Summary of renal physiology
TRPF (Filtered and secreted)
Filtration - Reabsorption Secretion
Elimination
GFR (Filtered but not reabsorbed or secreted)
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Measurement of GFR
Cu Concentration in urine Vu(24h) 24-hour
urine volume Cp Concentration in plasma 0.694
1000 mL/1440 min
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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

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Relationship between creatinine and GFR
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5
4
3
Plasma creatinine
2
1
0
0
20
40
60
80
100
120
140
GFR (mL/min)
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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.

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Creatinine
Creatine
Creatinine
1-2 of creatine is hydrolyzed to creatinine each
day
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Jaffe method for creatinine
Janovsky Complex ?max 490-500 nm
Max Eduard Jaffe (1841-1911), German physiologic
chemist
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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

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Kinetic Jaffe method
Slow-reacting (protein)
Fast-reacting (pyruvate, glucose, ascorbate)
Absorbance (? 520 nm)
creatinine (and ?-keto acids)
0
Time (sec) ?
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Enzymatic creatinine methods

• Creatininase
• creatinine?creatine?CK?ADP?PK?LD
• Creatinase
• creatinine?creatine?sarcosine?sarcosine
  oxidase?peroxide?peroxidase reaction
• Creatinine deaminase (iminohydrolase)
• most common

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Creatinine deaminase method
Creatinine
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Measurement of urine protein

• Specimen
• Timed 24-h is best
• Urine protein/creatinine ratio can be used with
  random specimen
• Normal protein excretion is lt150 mg/24h
• 50-60 albumin
• Smaller proteins (?1-, ?2-microglobulins)
• Tamm-Horsfall (uromucoid, secreted by tubules)
• IgA, tubular epithelial enzymes, and other
  non-filtered components

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Dipstick method for urine protein

• Method is based on protein association with pH
  indicator
• Test pad contains dye tetrabromphenol blue at
  pH3
• 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)

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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.

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Classification of proteinuria Minimal

• lt1 gram of protein per day
• Chronic pyelonephritis
• Mild glomerular disease
• Nephrosclerosis (usually due to hypertension)
• Chronic interstitial nephritis (usually
  analgesic-related)
• Renal tubular disease

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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

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Classification of proteinuria Severe

• gt4 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)

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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

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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

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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

tetramethylbenzidine oxidized form is green
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Microscopic examination of urine sediment
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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

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Brights Disease (acute glomerulonephritis)

• Characterized by oliguria, proteinuria, and
  hematuria
• Most common cause is immune-related

Richard Bright (1789-1858)
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Primary Glomerulonephritis

• Proliferative GN
• Acute Post-infectious GN
• Idiopathic or Crescentic GN
• ?-GBM disease
• Membranoproliferative GN
• Focal GN
• IgA nephropathy

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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

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Secondary Glomerulonephritis

• Systemic Lupus Erythematosus
• Renal failure accounts for 50 of SLE deaths
• Polyarteritis (inflammatory vasculitis)
• Wegeners Granulomatosis (lung and URT)
• Henoch-Schönlein Syndrome
• Lacks edema assoc. with post-streptococcal GN
• Goodpastures Syndrome (pulmonary hemorrhage)
• Hemolytic-Uremic Syndrome
• Progressive Systemic Sclerosis (blood vessels)

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Case 3 Chest Pain
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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.
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Questions

• What is the most important consideration in the
  triage of this patient?
• What tests should be ordered?

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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 (specificity25 sensitivity80)

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The Heart
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The Heart (posterior view)
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Cardiac physiology
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Cardiac conduction system
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Normal Electrocardiogram
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Myocardial infarction
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ECG changes in myocardial infarction
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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

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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.

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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

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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

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Tissue specificity of LD isoenzymes
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LD isoenzyme electrophoresis (normal)
LD-2 gt LD-1 gt LD-3 gt LD-4 gt LD-5
Cathode (-)
Anode ()
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LD isoenzyme electrophoresis (abnormal)
LD-1 gt LD-2
Cathode (-)
Anode ()
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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/LDtotal ratio is less than 40

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Sensitivity and specificity of LD-1

• Sensitivity and specificity of the LD 12 flip,
  or LD-1 gt 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

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Creatine Kinase (CK)
Phosphocreatine
Oliver and Rosalki method (1967)
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Tissue specificities of CK isoenzymes
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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

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Sensitivity/specificity of CK-MB

• Sensitivity and specificity of CK-MB for
  myocardial infarction are gt90 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

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CK isoforms

• C-terminal lysine is removed from the M
  subunit--therefore, there are three isoforms of
  CK-3 (MM)
• t½ CK-MB1 gt CK-MB2
• Ratio of CK-MB2 to CK-MB1 exceeds 1.5 within six
  hours of the onset of symptoms
• Only method currently available is electrophoresis

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Myoglobin

• O2-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

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Temporal changes in myoglobin and CK-MB
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Troponin
TnT (42 Kd)
Tropomyosin
Actin
TnI (23 Kd)
TnC
Myosin
Thick Filament
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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

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Is cTnI more sensitive than CK/CK-MB?
79 y/o female with Hx of HTN, CHF, CRI, Type II
diabetes
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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

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W.H.O. has a Myocardial Infarction?
A patient presenting with any two of the
following

• A clinical history of ischemic-type chest
  discomfort
• Changes on serially obtained ECG tracings
• A rise and fall in serum cardiac markers

Source JACC 2819961328-428
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Sensitivity/Specificity of WHO Criteria
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What Cardiac Markers do Labs Offer?