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

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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

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Case 1: Oliguria and hematuria

R. Bertholf American Society of Clinical

Pathologists3

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.


Pathologists4

Questions. . .

• What is the differential diagnosis in this case?

• What laboratory tests might be helpful in establishing the diagnosis?


Pathologists5

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


Pathologists6

The kidneys as regulatory organs

“The kidney presents in the highest degree the phenomenonof 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)


Pathologists7

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.


Pathologists8

Renal anatomy

Cortex

Medulla

Pelvis

To the bladder

Capsule


Pathologists9

The Nephron

Renal artery

Glomerulus

Bowman’s capsule

Proximal tubule

Distal tubule

Collecting duct

Henle’s Loop

Afferent arteriole


Pathologists10

Glomerular filtration

Glomerlularcapillary

membraneVascular spaceVascular space Bowman’s spaceBowman’s space

Mean capillary bloodpressure = 50 mm Hg

BC pressure = 10 mm Hg

Onc. pressure = 30 mm Hg

Net hydrostatic = 10 mm Hg

2,000 Litersper day

(25% of cardiac output)

200 Litersper day

GFR 130 mL/min


Pathologists11

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


Pathologists12

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.


Pathologists13

Reabsorption from glomerular filtrate

% ReabsorbedWater 99.2

Sodium 99.6Potassium 92.9Chloride 99.5

Bicarbonate 99.9Glucose 100Albumin 95-99

Urea 50-60Creatinine 0 (or negative)


Pathologists14

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.


Pathologists15

The Loop of Henle

Proximal tubule Distal tubule

Descending loop A

scen

ding

loop

Increasing osmolality

Renal Cortex

Renal Medulla

Na+Na+

Na+Na+

Na+Na+

H2ONa+

1200 mOsm/Kg

300 mOsm/Kg


Pathologists16

Regulation of distal tubule Na+ permeability

JGA Renin Na+

BP

Angiotensinogen

Angiotensin I

Angiotensin II

Angiotensin III

vasoconstriction

AldosteroneAdrenal cortex

Na+ Na+


Pathologists17

Regulation of H2O reabsorption

Pituitary

ADH (vasopressin)

Plasmahyperosmolality

H2OH2O

Renal Medulla (osmolality 1200 mOsm/Kg)


Pathologists18

Summary of renal physiology

Filtration - Reabsorption + Secretion = Elimination

GFR (Filtered but not reabsorbed or secreted)

TRPF (Filtered and secreted)


Pathologists19

Measurement of GFR

694.0)24(

p

huu

C

VCClearance

Cu = Concentration in urineVu(24h) = 24-hour urine volumeCp = Concentration in plasma0.694 = 1000 mL/1440 min


Pathologists20

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


Pathologists21

Relationship between creatinine and GFRP

lasm

a cr

eati

nine

GFR (mL/min)

1

2

3

4

5

6

00 20 40 60 80 100 120 140


Pathologists22

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

O

N

CH3HN

NH

HO

O CH3

NH

NH2

- H2ON

Creatine Creatinine

1-2% of creatine is hydrolyzed to creatinine each day


Pathologists24

Jaffe method for creatinine

O

N

CH3HN

NH

O2N NO2

NO2

OH

OH -

+Janovsky Complexmax = 490-500 nm

Max Eduard Jaffe (1841-1911), German physiologic chemist


Pathologists25

Modifications of the Jaffe method

• Fuller’s Earth (aluminum silicate, Lloyd’s reagent)

– adsorbs creatinine to eliminate protein interference

• Acid blanking

– after color development; dissociates Janovsky complex

• Pre-oxidation

– addition of ferricyanide oxidizes bilirubin

• Kinetic methods


Pathologists26

Kinetic Jaffe methodA

bsor

banc

e (

= 5

20 n

m)

Time (sec) 0 8020

Fast

-rea

ctin

g(p

yruv

ate,

glu

cose

, asc

orba

te)

Slow

-rea

ctin

g(p

rote

in)

t

A

ratet

A

creatinine (and -keto acids)


Pathologists27

Enzymatic creatinine methods

• Creatininase

– creatininecreatineCKADPPKLD

• Creatinase

– creatininecreatinesarcosinesarcosine oxidaseperoxideperoxidase reaction

• Creatinine deaminase (iminohydrolase)

– most common


Pathologists28

Creatinine deaminase method

Creatinine

Creatinineiminohydrolase

+ H2O N-Methylhydantoin ATP

ADP

NMH amidohydrolase

N-Carbamoylsarcosine

H2O

PeroxidaseOxygen receptor Colored product

Sarcosine

NCSamidohydrolase

- NH3, CO2

+ O2

Sarcosine oxidase

H2O H2O2

Formaldehyde + glycine


Pathologists29

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 (1-, 2-microglobulins)

– Tamm-Horsfall (uromucoid, secreted by tubules)– IgA, tubular epithelial enzymes, and other non-

filtered components


Pathologists30

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)


Pathologists31

What causes excess urinary protein?

• Overload proteinuria– Bence-Jones (multiple myeloma)– Myoglobin (crush injury, rhabdomyolysis)– Hemoglobin

• Tubular proteinuria– Mostly low MW proteins (not albumin)– Fanconi’s, Wilson’s, pyelonephritis, cystinosis

• Glomerular proteinuria– Mostly albumin at first, but larger proteins appear as

glomerular membrane selectivity is lost.


Pathologists32

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


Pathologists33

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


Pathologists34

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)


Pathologists35

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


Pathologists37

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

H2O2 + chromogen* Oxidized chromogen + H2OHeme

Peroxidase

*tetramethylbenzidine; oxidized form is green


Pathologists38

Microscopic examination of urine sediment


Pathologists39

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


Pathologists40

Bright’s Disease (acute glomerulonephritis)

• Characterized by oliguria, proteinuria, and hematuria

• Most common cause is immune-related

Richard Bright (1789-1858)


Pathologists41

Primary Glomerulonephritis

• Proliferative GN

– Acute Post-infectious GN

– Idiopathic or Crescentic GN -GBM disease

– Membranoproliferative GN

• Focal GN

– IgA nephropathy


Pathologists42

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


Pathologists43

Secondary Glomerulonephritis

• Systemic Lupus Erythematosus– Renal failure accounts for 50% of SLE deaths

• Polyarteritis (inflammatory vasculitis)• Wegener’s Granulomatosis (lung and URT)• Henoch-Schönlein Syndrome

– Lacks edema assoc. with post-streptococcal GN• Goodpasture’s Syndrome (pulmonary hemorrhage)• Hemolytic-Uremic Syndrome• Progressive Systemic Sclerosis (blood vessels)

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Case 3: Chest Pain


Pathologists45

Case 3: Chest Pain

A 63 year old male was brought to the emergency departmentafter complaining of severe chest pain that had lasted for twohours. He had been mowing his lawn when the pain developed,and he became concerned when the pain did not subside afterhe stopped the activity. He had no previous history of heartdisease. On presentation he was moderately overweight, dia-phoretic, and in obvious discomfort. He described his chestpain as “beginning in the center of my chest, then my arms, neck, and jaw began to ache too.”

Diagnostic procedures were performed.


Pathologists46

Questions

• What is the most important consideration in the triage of this patient?

• What tests should be ordered?


Pathologists47

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%)


Pathologists48

The Heart

Aorta

Superior vena cava

RA LA

RV

LV

Pulmonary arteries


Pathologists49

The Heart (posterior view)

Aorta

Superior vena cava

Inferior vena cava

Pulmonary veins

Pulmonary arteries


Pathologists50

Cardiac physiology


Pathologists51

Cardiac conduction system

Sinoatrial (SA) node

Atrioventral (AV) nodeHis bundle

Right bundle branch

Left bundle branch


Pathologists52

Normal Electrocardiogram

PQ

R

S

T

U


Pathologists53

Myocardial infarction

Right coronary artery

Left coronary artery

Anterior left ventricle


Pathologists54

ECG changes in myocardial infarction

S

P

R

T

Q

S-T elevation


Pathologists55

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


Pathologists56

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.


Pathologists57

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


Pathologists58

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

LactateLD

NAD+NADH


Pathologists59

Tissue specificity of LD isoenzymes

LD isoenzyme TissuesLD-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


Pathologists60

LD isoenzyme electrophoresis (normal)

LD-1

LD-2

LD-3LD-4

LD-5

LD-2 > LD-1 > LD-3 > LD-4 > LD-5

Cathode (-) Anode (+)


Pathologists61

LD isoenzyme electrophoresis (abnormal)

LD-1

LD-2

LD-3LD-4

LD-5

LD-1 > LD-2

Cathode (-) Anode (+)


Pathologists62

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%


Pathologists63

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


Pathologists64

Creatine Kinase (CK)

Phosphocreatine

ADP

HKGlucose Glucose-6-phosphate

NADPH=340 nm

NADP+

GPD

6-Phosphogluconate

Oliver and Rosalki method (1967)

CreatineCK

ADP ATPMg++


Pathologists65

Tissue specificities of CK isoenzymes

TissueCK-1(BB)

CK-2(MB)

CK-3(MM)

Skeletal muscle 0% 1% 99%

Cardiac muscle 1% 20% 79%

Brain 97% 3% 0%


Pathologists66

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”


Pathologists67

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


Pathologists68

CK isoforms

• C-terminal lysine is removed from the M subunit--therefore, there are three isoforms of CK-3 (MM)

• t½: CK-MB1 > 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

CK-MB2 (tissue) CK-MB1 (circulating)

C-terminal lysine

Plasma carboxypeptidase


Pathologists69

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


Pathologists70

Temporal changes in myoglobin and CK-MB

0

200

400

600

800

0 8 16 24 32 40 48

Time after symptoms

Myo

glob

in (

ug/L

)

0102030405060

CK

-MB

(ug

/L)

Myoglobin CK-MB


Pathologists71

Troponin

Thick Filament

Myosin

Tropomyosin Actin

TnC

TnT (42 Kd)

TnI (23 Kd)


Pathologists72

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


Pathologists73

Is cTnI more sensitive than CK/CK-MB?

-1

-0.5

0

0.5

1

1 8 40 66

Hours since presentation

log

X n

orm

al CKCK-MBCK-MB IndexcTnI

79 y/o female with Hx of HTN, CHF, CRI, Type II diabetes


Pathologists74

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


Pathologists75

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


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Sensitivity/Specificity of WHO Criteria

0%

20%

40%

60%

80%

100%

Chest Pain ECG changes Serummarkers

SensitivitySpecificity


Pathologists77

What Cardiac Markers do Labs Offer?

0

500

1000

1500

2000

2500

3000

3500

# of

labs

rep

ortin

g

CK-MB(ng/mL)

CK-MB(IU/L)

cTnI cTnT

19971998

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