The Clinical Chemistry
Lab
Vicki S. Freeman, Ph.D.
Objectives
Upon completion of lecture, discussion, case studies and
laboratory, the student will be able to:
 Quality Control and CLIA Regulations
– Explain the importance of QC in the lab
– Define sensitivity, specificity, shift, trend, precision , accuracy
and reliability
– Describe the 6 aspects of quality control
– Classify procedures according to the CLIA regulations
– List the Quality Assurance requirements mandated in the CLIA
regulations

Carbohydrates
– Differentiate types of diabetes by clinical symptoms and
laboratory data
– Type 1
– Type 2
– Gestational diabetes
– Impaired glucose tolerance
Objectives (Con’t)
Upon completion of lecture, discussion, case studies
and laboratory, the student will be able to:
 Carbohydrates (Continued)
– Relate expected laboratory results and clinical symptoms
to the following metabolic complications of diabetes:
– Ketoacidosis
– Hyperosmolar coma
– Describe the used of hemoglobin A1C and microalbumin
in the long term monitoring of diabetes

Lipids and Cardiac Risk
– Discuss cholesterol, LDL cholesterol, HDL cholesterol,
lipoproteins and triglycerides as predictors of
cardiovascular risk
– Calculate a LDL cholesterol, given total cholesterol,
triglyceride and HDL results
Quality Control
 Purpose
of QC is to
– assure the reliability of patient data
obtained from a procedure
– monitor variables that can alter data
 Patient
data is unknown information
and “known” samples must be run
concurrently
Terms
 Precision
– the reproducibility of the result
 Accuracy
– the closeness of the measured result to
the true value
 Reliability
– the ability to maintain both precision
and accuracy
Quality Control

Precision

Accurate

Reliability
 Shift
– 6 or more consecutive values distributed
above or below the mean
 Trend
– A continual increase or decrease in 6 or
more consecutive values
Quality Control

Shift

Trend

Outliers
6 Aspects of Quality Control
 Sample
collection
 Method of analysis
 Proper control material
 QC monitoring
 Instrument maintenance
 Documentation
CLIA ‘88
 Waived
tests
 Moderate complexity tests
 High complexity tests
 Provider-performed microscopy
Waived Test Category
UA dipstick
 Spun hematocrits
 Hemoglobin
 Sedimentation rate
 Fecal occult blood
 UA pregnancy test

Ovulation tests
 Single analyte
instrument

– Hemacue
– Glucose
– Total cholesterol
Provider-performed
Microscopic

Wet mounts
– vaginal
– cervical
– skin
KOH preparations
 Pinworm exams
 Fern tests
 UA sediment exam


Postcoital direct
– vaginal and cervical
mucus
Nasal granulocytes
 Fecal leukocytes
 Qualitative semen
analysis

Common Tests in POLs
Hemoglobin
 Hematocrit
 Dipstick UA
 Occult Blood
 Strep A (ag)
 UA pregnancy

Glucose
 Cholesterol
 Triglycerides
 BUN
 Uric Acid
 Cholesterol
 HDL Cholesterol

Quality Assurance
Vicki S. Freeman, Ph.D.
Quality Assurance Program
 Written
laboratory procedure manual
 Specimen collection and
identification
 Methodologies
 Reference Ranges
 Quality control
 Preventive maintenance
 Record keeping
Profile Groups
Carbohydrates
 Lipids
 Enzymes
 Cardiac Function
 Liver Function
 Renal Function

Electrolytes
 Parathyroid
Function/ Calcium
Metabolism
 Acid/Base Balance
 Pancreatic Function
 Prostate

Carbohydrates
Hyper and Hypoglycemia
Classes of Hyperglycemia
 Diabetes
Mellitus
 Impaired Glucose Tolerance
 Gestational Diabetes
Diabetes
A
syndrome characterized by
inappropriate hyperglycemia with
chronic microvascular complications.
 Upper
limit of 100 mg/dl on the FPG
as the upper limit of normal blood
glucose
Diabetes Mellitus
 Types
– Type 1 (Type I) -Insulin Dependent
Diabetes Mellitus
 IDDM
– Type 2 (Type II) -Non Insulin
Dependent Diabetes Mellitus
 NIIDM
– Other Types - Secondary
TYPE 1
TYPE 2
Destruction
of the
B-cells
Resistance
to
Insulin
Absolute
Deficiency of
Insulin
Relative
Deficiency of
Insulin
Type 1 (Type I) - IDDM
 Characteristics
– Abrupt Onset
– Insulin Dependent
– Ketosis-prone
– Genetic related - HLA Dw4 related
– Islet Cell Antibodies
 10
- 20 % of all diabetes
Type 2 (Type II) - NIDDM
 Characteristics
– Little or no symptoms
– Does not exhibit the characteristics of IDDM
Have high basal insulin levels - develop insulin resistance
 Decreased # of insulin receptors in insulin-sensitive
tissues

– Subclasses
Non-obese
 Obese - 60 - 90 % of all diabetics
 Further divided by the type of treatment the patient
receives (Insulin, oral hypoglycemic, diet)

Other Types - Secondary
 Destroyed
pancreas
 Pituitary Hyperfunction
 Cushings Disease
Diagnosis of Diabetes
Mellitus

Classic symptoms & a casual plasma
glucose concentration >200 mg/dl
Fasting venous plasma glucose
concentrations >126 mg/dl
 2 hour post prandial >200 mg/dl

– Any of the 3 criteria must be
confirmed on a subsequent day by
any of the 3 methods.
Diagnosis of Diabetes Mellitus


Classic symptoms of diabetes and hyperglycemia
Laboratory Tests
– Preferred - Fasting venous plasma glucose
 > 126 mg/dl on more than one occasion
 Impaired fasting plasma glucose 100 mg/dl - 126
mg/dl
– Casual plasma glucose concentration >200 mg/dl
– Not recommended - Screening test - 2 hour post prandial
 OGTT value of >200 mg/dl in 2 hr sample
 Normal
<140 mg/dl
 Impaired GTT
140 - 199 mg/dl
 Abnormal
>200 mg/dl
– Any of the 3 criteria must be confirmed on
a subsequent day by any of the 3 methods
Glucose Curves
Gestational Diabetes
Diagnosed by any two of the following:
a fasting plasma glucose of more
than 105 mg/dl
a 1-hour glucose level of more than
190 mg/dl
a 2-hour glucose level of more than
165 mg/dl
or a 3-hour glucose level of more
than 145 mg/dl
Specimen

Overnight fast
– New evidence of diurnal variation with FPG higher in AM

Plasma
– NaFloride

if cannot be separated from cells within 60 minutes
– Anticoagulated (oxalate)



Whole blood values ~11% lower than plasma
Heparinized plasma values 5% lower than serum
Capillary vs venous blood
– Fasting state ~= (2 mg/dL difference)
– After a glucose load, capillary values ~20-25% higher
 Day - to - day control
Self
Monitoring Blood Glucose (SMBG)
 Long term
Hemoglobin
A 1C
Shows a direct relationship with the glucose level
over the preceding 2-3 months
Microalbumin
Monitors Monitors kidney function
 Urine glucose - obsolete
 Urine ketones - fat breakdown products
Glycosylated (glycated) Hemoglobin
(GHb or HgbA1c)

ADA Guidelines - Glycosylated hemoglobin
– Glucose attaches to tissues and proteins,
including hemoglobin
– Measures % of hgb that has been
modified by glucose
 Shows
a direct relationship with the glucose level over
the preceding 2-3 months
– A valuable tool for monitoring glycemia,
 Normal
levels range from 3%-6%
 Should be maintained at <7% (some sources say 6%)
 Re-evaluate treatment regimen if GHb >8%
 Should be measured at less than 2 x/year (if diabetic
is well controlled; otherwise, every 3 months)
Approximate correlation between A1C
level and mean plasma glucose levels
HbA1C
%
6
7
8
9
10
11
12
Mean plasma glucose
mg/dl
mmol/l
135
7.5
170
9.5
205
11.5
240
13.5
275
15.5
310
17.5
345
19.5
Microalbumin
– Diabetes is leading cause of end-stage real
disease
– Microalbumin- Monitors kidney function
 Also
a marker of increased risk of cardiovascular
morbidity and mortality
 Annual testing is recommended
– Microalbuminuria defined as excretion of
 30-300
mg of albumin/24hrs or
 20-200 g/min or 30-300 g/mg creatinine
 On 2 of 3 urine collections
Acute Complications

Ketoacidosis due to lack of insulin/stress,
can result in death (assoc. with Type I)
– B HCO3-, B pH, A glucose
Hypoglycemia - with too much insulin results in coma
 Hyperosmolar coma (assoc. with Type II)

– A blood osmolarity
– A glucose
Chronic Complications
Correct Management of diet and insulin,
 Avoid further complications of the disease

– Retinopathy - blindness 50% after 10 years
– Nephropathy - Renal disease

proteinuria, increased BUN and creatinine
– Neuropathy 
poor sensation, ulceration of skin, may lead to
amputation of limbs
– Accelerated macrovascular disease - CAD, CVA
Hypoglycemia

Decreased fasting glucose <50 mg/dl
–
Fasting
Caused by pituitary/adrenal insufficiency, pancreatic islet
cell hyperplasia, islet cell tumors
 Other causes may be from drugs, alcohol, insulin
 Triad of characteristics (Whipple's Triad)
– Hypoglycemic symptoms
– Simultaneous demonstration of decreased plasma
glucose
– Relief of symptoms with glucose administration

–
Postprandial (or reactive)
Seen after gastric surgery - food is absorbed too fast
 Idiopathic - following extreme stress (catecholamines)
 Spontaneous recovery

Diagnosis of Hypoglycemia
 Diagnosed
by looking for the cause
– Thrust of the clinical evaluation is to
rule out insulinomas.
– Hypoglycemia in insulinomas are related
to excessive and inappropriate
production of insulin - insulin levels are
important in making the diagnosis
Diabetes Case Study 1
A 40-year-old African American woman
(nonpregnant) has symptoms
suggestive of diabetes. On two
occasions, the fasting plasma
glucose (FPG) is 130 mg/dL and 135
mg/dL.
What is the next diagnostic or therapeutic
step?
Diabetes Case Study 2
A 35-year-old Caucasian female
(nonpregnant) has FPG
concentrations on two occasions of
120 mg/dL and 124 mg/dL without
symptoms suggestive of diabetes.
 How
would this patient would be
classified?
Diabetes Case Study 3

A 72 year old male presents with
numbness in the legs and frequent
urination. A 4 hour glucose tolerance is
ordered. The results are:
FPG
1/2 hr
1 hr

160 mg/dL 2 hr 220 mg/dL
205 mg/dL 3 hr 195 mg/dL
260 mg/dL 4 hr 165 mg/dL
A follow up Hgb A1c was ordered.
 Does
this gentleman have diabetes?
Lipids and Cardiac
Risk
Cholesterol Synthesis
Genomic Medicine: Cardiovascular Disease
New England Journal of Medicine Volume 349(1) 3 July 2003 pp 60-72
Lipid Profile and Cardiac Risk
 Cardiac
•
•
•
•
Risk Factors
Cholesterol, total
Triglycerides
HDL cholesterol
LDL cholesterol (calculated vs direct)
 NCEP
Guidelines ATPIII
– Fasting sample now required for:
 Total
cholesterol
 HDL-C
 LDL-C
 Triglycerides
Cholesterol
 Dependent
on many factors
– genetics, age, sex, diet, physical
activity, hormones
 American
Heart Association < 200
mg/dl
 Measurement
– Enzyme method most common
LDL-C

The villain - directly correlated with CHD
–
Carries cholesterol from its site of origin into
the blood vessels
Optimal <100 mg/dL
 Near or above optimal 100-129 mg/dL
 Borderline high 130-159 mg/dL
 High 160 – 189 mg/dL
 Very high > 190 mg/dL


Calculation
LDL = Total Cholesterol - (HDL + Triglycerides)
5
Triglycerides > 400 mg/dL causes problem in
calculation


Direct measurement (new)
HDL-Cholesterol

The hero - inversely correlated with CHD
– transfers cholesterol from cells back to the
liver
– New!! <45 male; <55 female
– Factors which increase HDL

estrogen (women), exercise, alcohol, blood pressure
medicine
– Factors which decrease HDL include:

progesterone, obesity, smoking, triglycerides and
diabetes
– Measurement

HDL Precipitation method
Non-HDL-C
 Other
lipid compounds including
– Lp(a), VLDL remnant are significant
in individuals with increased
triglycerides
 Non
HDL-C
 Triglycerides
<200 mg/dL and LDLC<100 mg/dL
– Should then look at non-HDL-C
 Total
cholesterol – HDL
Triglycerides
 Role
as a risk factor remains
unsettled
– Considered an independent risk factor
– Definite + association between triglycerides
and CHD. >150 mg/dL = risk
– While high levels may not cause
atherosclerosis, they may indirectly
accelerate atherogenesis by influencing the
concentration and composition of other
lipoproteins

Measurement - fasting > 12 hours required
Other lipid measurements

Lp(a)
– similar in structure to LDL
– a unique protein apo(a) linked to
apolipoprotein B-100 - has a structure similar
to plasminogen
– directly correlated with CHD - not affected by
lifestyle factors such as diet, exercise or
smoking
– levels >30 mg/dl

Apolipoprotein A
– Associated with HDL

Apolipoprotein B-100
– Associated with LDL
Major Risk Factors (Exclusive of LDL
Cholesterol) That Modify LDL Goals*





Cigarette smoking
Hypertension (blood pressure > 140/90 mm Hg
or on antihypertension medicine
Low HDL cholesterol (<40 mg/dL)
Family history of premature CHD
– males first degree relative <55 years
– Female first degree relative < 65 years

Age
– Men > 45 years
– Women > 55 years


Diabetes is regarded as a CHD risk equivalent
HDL cholesterol > 60 mg/dL counts as a “negative
risk factor
– Its presence removes 1 risk factor from the total count
High risk individuals
 Risk
for a diabetic is as high as
someone with existing heart disease
 Other individuals with >20% risk for
heart attack in 10 years
– Goal – reduce LDL-C to <100 mg/dL
Three Categories of Risk That
Modify LDL Cholesterol Goals
Risk Category
LDL Goal
(mg/dL)
CHD and CHD Risk <100
Equivalents*
Multiple (2+) risk
<130
factors
0-1 risk factor
<160
NON-HDL
Goal
(mg/dL)
<130
<160
<190
* Diabetes counts as a CHD risk equivalent because it confers a high
risk of new CHD within 10 years
Metabolic syndrome
 Previously
called Syndrome X
 A constellation of risk factors that
include:
– Abdominal obesity
– Atherogenic dyslipidemia (elevated
triglyceride concentration, small LDL
particles, low HDL-C, elevated blood
pressure, insulin resistance and
prothrombotic and proinflammatory
states)
Clinical ID of Metabolic Syndrome
Risk Factor
Defining Level
Waist Circumference
Men
Women
>40 in
>35 in
Triglycerides
HDL cholesterol
Men
Women
Blood Pressure
Fasting glucose
>150 mg/dL
<40 mg/dL
<50 mg/dL
> 130/> 85 mmHg
> 110 mg/dL
Clinical Guidelines
 With
new guidelines
– Perform a lipoprotein profile on every
adult at least every 5 years
– Annual profile on diabetics
– Estimate 1 in 5 individuals will be
treated with one of the statins (lipid
lowering drugs)
 More
frequent measurements for those on
therapy
Lipid Case Study
Below are the lab results of a 50 yr old male:
•
•
•
•


Glucose
Cholesterol
Triglycerides
HDL Chol
75 mg/dL
309 mg/dL
588 mg/dL
23 mg/dL
Calculate the LDL cholesterol value of this
patient.
The direct measurement of LDL Cholesterol is
240 mg/dL. Is there a discrepancy between the
measured and calculated LDL result? If so, why?
Clinical Chemistry
Part 2
Vicki S. Freeman, Ph.D.
Enzymes
Objectives

Discuss the use of enzymes as laboratory
aids in the following disorders:
– Myocardial infarction (LD, CK, AST, LD-1,
CKMB)
– Hepatocelluar disease (AST, ALT)
– Hepatobiliary disease ALP, GGT)
– Degenerative bone disease (ALP)
– Pancreatitis (amylase, lipase)
– Prostatic carcinoma (ACP, PSA)
– Dengerative muscle disease
Enzymes
 Diagnostic
Value
– Found in differing concentrations in
tissues
– Cellular damage and/or increased
intracellular synthesis results in
increased serum enzyme levels
– Isoenzyme forms of an enzyme may be
more specific to certain organ systems
Clinically Significant Enzymes
– Creatine kinase (CK)
– Lactate dehydrogenase (LD)
– Aspartate transaminase (AST)
– Alanine transaminase (ALT)
– Gamma glutamyltransferase (gGT)
– Alkaline phosphatase (ALP)
– Acid phosphatase (ACP)
– Amylase
– Lipase
Creatine Kinase (CK)
3
isoenyzmes (MM, MB, BB)
 Found in
– skeletal muscle (CK-MM)
– cardiac muscle (CK-MB)
– brain, nerve, intestine (CK-BB)
 Significance
– Skeletal muscle disease
– Cardiac disease
– Central nervous system
Lactate Dehydrogenase


5 isoenzymes (LD 1, 2, 3, 4, 5)
Found in
– skeletal muscle , erythrocytes, cardiac muscle, kidney,
lung, tumor cells, hepatocellular

Significance
– LDH-1 (heart) - myocardial infarction, RBC diseases,
kidney disease, and testicular tumors
– LDH-2 (RE system) - infections
– LDH-3 (lung) lung disease and certain tumors
– LDH-4 (kidney, placenta, and pancreas)- pancreatitis
– LDH-5 (liver and striated (skeletal) muscle) - liver
disease, intestinal problems, and skeletal muscle disease
and injury
– All LDH isoenzymes - Diffuse disease or injury (for
example, collagen disease, shock, low blood pressure) and
advanced solid-tumor cancers
Alanine Transaminase (ALT)
Aspartate Transminase (AST)
 Found
in
– skeletal muscle
– cardiac muscle
– hepatocellular tissue
– kidney, pancreas, erythrocytes
 Significance
– Liver disease
– Cardiac (AST only)
– Skeletal muscle (AST only)
Gamma glutamyltransferase
(gGT)
 Found
in
– kidney
– hepatobiliary
– tumors
 Significance
– Liver disease (particularly alcoholic
cirrhosis)
– Renal disease
– neoplasms or tumors
Alkaline Phosphatase
 Found
in
– hepatobiliary
– Bone (higher in children)
– placenta
– renal tubules, intestinal
 Significance
– Hepatobiliary disease (particularly
obstruction)
– Bone disease
Acid Phosphatase
 Found
in
– Prostate
– hepatobiliary
– breast tissue
– bone marrow, rbcs, plts, spleen
 Significance
– Prostatic cancer
– bone disease
– vaginal washings in rape investigations
Amylase and Lipase
 found
in
– Pancreas
– Salivary glands (amylase only)
 Significance
– Pancreatic Disease
– Mumps (amylase only)
Cardiac Function
Acute Coronary
Syndromes
Objectives
Discuss the changes in total serum CK,
LD, and AST after acute myocardial
infarction.
 Interpret cardiac markers in patients with
suspected acute myocardial infarction

– CK and CKMB
– LD and LD-1
– Troponin

Describe the clinical usefulness of
myoglobin, troponin and BNP versus CK
markers in assessing acute myocardial
injury.
Cardiac Markers

CK isoenzymes
–
–
CK-BB
CK-MB

–

CK-MB isoforms
– CKMB1
– CKMB2
CK-MM
Troponin
–
–
–
–
complex consists of
3 subunits:
troponin T (cTnT)
troponin I (cTnI)
troponin C




Myoglobin
B-type natriuretic
peptide (BNP)
or
N-terminal pro-BNP
(NT-proBNP)
hsC-Reactive Protein
(hsCRP)
Cardiac Injury Panel

Cardiac Injury
–
–
–
–
CK-MB
Troponin (T and I) (The preferred marker!!
Myoglobin
Others

old
– Total CK
– LD/LD1
– AST (SGOT)

New
– BNP
– hsCRP
Myocardial Infarction
Initial Evaluation
 Assess probability that
patient’s symptoms (i.e.
chest pain) are related to
acute coronary ischemia
 Assess the patient’s risk of
recurrent cardiac events
(including death and
recurrent ischemia)
 Cardiac biomarkers should
be used in conjunction with
clinical history, physical
exam, ECG interpretation
Troponin






Preferred marker for detection of cardiac injury and risk
stratification
Has isoforms that are unique to cardiac myocytes
Fewer false positive results (when concomitant with skeletal
injury)
Rises within 3-4 hours after onset
Remains elevated 10-14 days
Independent risk factor of death and ischemic events in
acute coronary syndrome
– 4 fold higher risk of death and recurrent MI in patients with an
elevated troponin (both T and I)
– Independent of other clinical indicators such as age, ST
deviation, and presence of heart failure
– Elevated troponin levels are associated with likelihood of poor
outcomes in angioplasty
CK-MB


CK-MB (by mass spectrometry) is an acceptable alternative
to troponin
Perform serial testing
– Upon presentation to hospital
– At at 6-8 hours
– Again at 12-24 hours




1-3% of CKMB comes from skeletal muscles
Begins to rise between 3-6 hours post MI
Falls to normal levels at 48-72 hours
Use of the serial measurements useful in the management
of the MI after diagnosis
– Release of CKMB from cardiac myocytes indicates myocardial
necrosis

Use for detection of recurring MIs
Myoglobin
An early marker of myocardial necrosis, if
performed during first 6 hours of onset of
symptoms
 High concentration also found in skeletal
muscle
 Because of small molecular size, is useful
for early detection
 Begins to rise 1 hour after onset of
myocyte damage
 Returns to normal within 12-24 hours

Other markers
 Total
CK, AST, b-hydroxybutric
dehydrogenase, or LD should not be
used as biomarkers for MI
– These are of low specificity
Serum Cardiac Markers





Cardiac troponin is the preferred marker for the diagnosis
of MI.
CK-MB subforms for diagnosis within 6 hrs of MI onset
cTnI and cTnT efficient for late diagnosis of MI
CK-MB subform plus cardiac-specific troponin best
combination
Myoglobin may be added
– as an early marker for MI
– for an early detecxtion of a reinfarction

CKMB – preferred marker for detection of re-infarction early
after MI
– Do not rely solely on troponins because they remain elevated
for 7-14 days and compromise ability to diagnose recurrent
infarction
Markers for Risk Stratification
–Troponin - the preferred marker
–hsC-Reactive Protein (CRP)
–B-type natriuretic peptide (BNP) or
N-terminal prohormone BNP (NTproBNP)
Markers of inflammation

hs-CRP
– Patients without biochemical evidence of
myocardial necrosis but who have an elevated
hsCRP level are at an increased risk of an
adverse outcome, especially those whose
hsCRP levels are markedly elevated

interleukin-6 & serum amyloid A - acute
phase reactant proteins
– Elevated levels have been shown to have a
similar predictive value of an adverse outcome
as CRP
BNP – (B-type natriuretic peptide)

Neurohormone synthesized predominantly
in ventricular myocardium
– Released from cardiac myocytes in response to
ventricular wall stress
– Strong relationship with mortality in patients
with unstable angina
– Rises after exercise in patients with coronary
disease
– Circulating levels of BNP correlate with
presence and severity of congestive heart
failure
Troponin and BNP
– a single measurement of B-type
natriuretic peptide, obtained in the first
few days after the onset of ischemic
symptoms, provides predictive
information for use in risk stratification
across the spectrum of acute coronary
syndromes (ACS)
– Low mortality rate found for patients
with negative troponin results and low
BNP levels
ACC Guidelines (ACC)

4 Categories
–
–
–
–

Noncardiac diagnosis
Chronic unstable angina
Possible ACS (acute coronary syndrome)
Definite ACS
Patient Management includes:
– Patient history
– 12 lead ECG
– Cardiac Markers - preferrably cardiac-specific
troponin
 ACC = American College of Cardiology
Rule of Thumb
Criteria for Diagnosis of MI





Serial increase, then decrease of plasma CK-MB,
with a change >25% between any two values
CKMB >10-13 U/L or >5% total CK activity
Increase in CKMB activity >50% between any two
samples, separated by at least 4 hrs
If only a single sample available, CK-MB elevation
>twofold
Beyond 72 hrs, an elevation of troponin T or I
Hepatic Function
Objectives
Identify laboratory tests commonly used
to diagnose liver disease
 Correlate expected results in pre-hepatic
(hemolytic jaundice), intrahepatic
(hepatitis and cirrhosis), and posthepatic
(obstructive jaundice) related disorders for
the following tests:
 Serum and urine bilirubin (total,
conjugated, unconjugated)
 Urine and stool urobilinogen
 Enzymes (AST, ALT, Alkaline Phosphatase,
GGT)

Hemoglobin Breakdown
The reticuloendothelial cells break down hemoglobin
into bilirubin:
Hemoglobin
B
Verdohemoglobin
B
Biliverdin + Fe + Globin
B
Bilirubin
Albumin
;B
Bilirubin - Albumin Complex
Bilirubin Conjugation
The bilirubin-albumin complex is transported
by the bloodstream to the liver where it is
conjugated:
Bilirubin-Albumin Complex
B to the liver
Bilirubin
B to parenchymal cells
Bilirubin + UDP-glucuronic acid
B
Bilirubin diglucuronide
Urobilinogen Formation

Bilirubin diglucuronide is excreted to the intestines
through the bile ducts where it is converted further
for excretion:
Bilirubin diglucuronide
B to intestine
Converted to urobilinogen by bacterial enzymes
B
B
B
50% reabsorbed
bloodstream
Rest converted to into
urobilin
Reabsorbed by liver
or excreted in urine
Excreted in feces
B
B
Hepatic Function
Prehepatic Jaundice
 Causes
– Hemolytic disease
– Neonatal physiologic
 Lab
Findings
– Bilirubin
 Serum:

A unconjugated Urine:
N - A conjugated
 Urobilinogen
 Stool:
A levels
Urine:
Negative
A levels
Hepatic
 Causes
– Conjugation failure due to enzyme deficiency
– Bilirubin transport failure
– Hepatic cell damage
 Lab
Findings
– Bilirubin
 Serum:

A unconjugated
A conjugated
Urine:
Positive
– Urobilinogen
 Stool:
Variable
Urine:
Variable
Posthepatic Jaundice
 Causes
– Obstruction of the common bile duct
 Lab
Findings
– Bilirubin
 Serum:

A unconjugated
A conjugated
Urine:
– Urobilinogen
 Stool:
B to negative
negative
Urine:
B to
Positive
Liver Function Profile
•
•
•
•

Bilirubin (total and direct)
AST
ALT
Alkaline phosphatase
gGT
Hepatocellular disease
 Damage
to the parenchymal cells of
the liver
 Laboratory Findings
– A serum bilirubin
– Marked
A AST and ALT
– A alkaline phosphatase
–
A gamma glutamyltransferase (gGT)
Cirrhosis
Cirrhosis is a condition in which the liver has
been progressively destroyed through a
disease process such as primary biliary
cirrhosis or alcoholism.
 Laboratory Findings
– A bilirubin

– A gamma glutamyltransferase (gGT)
– A alkaline phosphatase
– Mod A AST
– Normal to sl A ALT
Biliary Obstruction
A blockage of the biliary duct usually
caused by a gallstone or tumor.
 Laboratory Findings
– B to no urobilinogen

– A in conjugated bilirubin
– A markedly alkaline phosphatase
– Mild A in AST and ALT
– A gGT helps differentiate source of
ALP
Renal Function
Clinical Chemistry
Part 3
Vicki S. Freeman
Renal Function
Objectives
Renal Function
 Identify laboratory tests commonly used to diagnose renal
disease:



–
–
–
–
BUN (urea)
Creatinine
Creatinine Clearance
Ammonia
–
–
–
–
–
Glomerulonephritis
Nephrotic Syndrome
Renal tubular acidosis
Renal failure - acute and chronic
Renal transplants
Discuss the sensitivity and specificity of serum creatinine and BUN
as renal function tests.
Correlate kidney function tests with clinical findings in:
Correlate uric acid values with advanced chronic renal failure and
gout.
Renal Function Profile
 Electrolytes
 Anion
(NA, K, CL,HCO3)
gap
 BUN and Creatinine
 Creatinine clearance
 Glucose
 Ca, P, and Mg
 Protein and Albumin
 24 hr urine protein and creatinine
Renal Function
 Non-protein
– Urea (BUN)
– Creatinine
– Ammonia
– Uric Acid
Nitrogen Compounds
Azotemia
 Any
significant increase in NPN
compounds (usually BUN and
creatinine) in the blood
 Prerenal
 Renal
 Post renal
Blood Urea Nitrogen (BUN)
 Urea
H2N-CO-NH2
– end product of NH3 (protein and amino acid)
metabolism in liver
– 2 molecules of nitrogen per mole of urea
– secreted by the renal tubules at a rate that is
proportional to the glomerular filtration rate
(GFR)
– freely filtered by the glomeruli (90% is
excreted)
– BUN is an indirect measure of urea (convert to
urea by multiplying by 60/28 or 2.14)
BUN - Significance

Increased in
– Impaired kidney function
– Prerenal azotemia - any cause of reduced blood
flow
– Post azotemia - any obstruction of the urinary
tract

Decreased
– in low protein diet or increased utilization of
protein
– severe liver disease

Levels may vary with diet, sythesis in liver
and amount secreted by kidney
Creatinine
 Formed
by the muscle from creatine
 Amount proportional to muscle mass,
constant excretion rate
 Freely excreted by the kidney
glomerulus
 Better indicator of glomerular
function than BUN
– Less influenced by diet and prerenal and
post renal factors
Creatinine
 Increased
due to
– impaired renal function
 1/2-2/3
of function lost
– Prerenal azotemia
– postrenal azotemia
– Muscle disease
 Decreased
in pregnancy
– Serum creatinine levels are a direct
reflection of muscle mass and show little
response to diet
BUN/Creatinine Ratio
 Ratio
generally between 10:1 and
20:1
 Increased ratio indicates
– catabolic states of tissue breakdown
– compromised blood flow
 Decreased
ratio indicates
– acute tubular necrosis
– low-protein diet, starvation
– severe liver disease
Creatinine Clearance
Measure both blood and urine creatinine
 Timed collection

– usually 24 hours
– midperiod blood collection
the volume of plasma that contained
creatinine excreted into the urine per unit
volume (1 min) can be calculated
 Significance - indication of glomerular
filtration rate (GFR) as renal function fails,
creatinine clearance decreases

Pathological Conditions
BUN Creatinine Ratio


Prerenal
N
Caused by reduce blood flow or
cardiovascular failure


-N
Renal
Caused by diseases affecting the
glomerulus or tubule function


-N
Postrenal
Caused by obstruction of urine flow
Uric Acid
End product of purine (nucleic acid)
metabolism
 Serum uric acid depends on

–
–
–

Increased uric acid seen in
–
–
–

purine synthesis and metabolism
dietary intake and metabolism
renal function
gout
increased cell turnover
renal impairment
Uric acid is very insoluble and can form
kidney stones
Pathological Conditions
BUN Creatinine
Proteinuria
Acute
glomerulonephritis


+
Nephrotic syndrome
N
N
+++
Tubular disease
N
N
+
Acute Renal Failure


+
Renal Tubular Function Tests
Measures the concentrating and diluting
ability of the renal tubules
 Osmolality

– Measure of # of moles of particles/kg water
– Impairment of renal concentrating ability is
an early manifestation of chronic renal
disease

Specific gravity
– ratio of weight in grams/ml of body fluid
compared to water
Electrolytes
Objectives
Electrolytes and Acid-base Balance
 Identify the major electrolytes found in the body and the
relative distribution of each.
 Calculate an anion gap given a set of electrolyte values.
 Describe the use of a measured and calculated osmolality
result.
 Calculate an osmolality given a set of laboratory results.
 Identity the normal HCO3/H2CO3,
 Describe the laboratory parameters (pH, pCO2, and HCO3)
for the following acid/base disorders:
–
–
–
–
Respiratory acidosis
Respiratory alkalosis
Metabolic acidosis
Metabolic alkalosis
Electrolytes
 Cations
-
– positively charged ions
– includes major electrolytes
 Na+
 K+
Ca+
Mg+
– Trace elements
 Cu++
 Zn++
Fe++
Co++
Mn++
Br++
Li+
Electrolytes
 Anions
– Negatively charged ions
– Includes major electrolytes
 Cl-
 HCO2-
HPO4-SO4--
– Trace elements
 I Fl-
Electrolytes
 Extracellular
– Na - Major cation
– Cl - Major anion
 Intracellular
– K - major cation
– Others - Mg
Sodium: Major extracellular cation

Na+ levels are controlled by renal tubular
function and somewhat by aldosterone
(adrenocortical hormone from reninangiotensin system)
– Relates to plasma osmolality (2x Na+ ~
osmolality)
Hypernatremia: hypotonic dehydration,
renal failure, lack of ADH,
hyperaldosteronism, etc.
 Hyponatremia: over-hydration, renal
tubular dysfunction, hypoaldosteronism

Potassium: main intracellular cation

K+ levels controlled by renal tubular secretion/
reabsorption and affected by aldosterone
(inversely with Na), acid-base balance and
glucose transport under insulin influence.
– Plasma K+ falsely increased in hemolysis.
– K+ levels fall after insulin administered to control
hyperglycemia.
– K+ and H+ levels often correlate.


Hyperkalemia due to renal failure, ketoacidosis,
hypoaldosteronism.
Hypokalemia in renal tubular defects,
hyperaldosteronism, dietary deficiencies (esp.
when taking diuretics or laxatives), severe
vomiting
Chloride: main extracellular anion
Cl- is controlled by renal function with
aldosterone influence on tubular secretion
(Na+ and Cl- are reabsorbed as K+ and H+
secreted) in response to aldosterone.
 Hyperchloremia may be due to
dehydration, severe hyperaldosteronism,
renal failure, diabetes insipidus, etc.
Hypochloremia is found in overhydration,
severe vomiting, renal tubular
dysfunction, severe hypoaldosteronism

Total CO2 and Bicarbonate



CO2 (and HCO3-) is controlled by renal tubular
function based on plasma pH. It buffers H+
produced in metabolic functions or control acidbase disturbances.
Increased HCO3- is due to metabolic alkalosis
(vomiting, hypokalemia, overtreatment with
bicarbonate) or to compensate respiratory
acidosis (from hypercapnia and pulmonary
diseases).
Decreased HCO3- is due to metabolic acidosis
(from organic acid production, severe diarrhea,
renal tubular acidosis or renal failure) or to help
compensate for respiratory alkalosis in hypoventilation and hypocapnia
Anion gap



Anion gap = Na - (Cl + CO2)
Normal range 8 - 16
Gap due to excess unmeasured anions
– HPO4-- SO4-– organic and lactic acids


Increased anion gap usually due to decreased
anions, especially CO2 as in metabolic acidosis
from lactate, ketones, organic acid poisoning,
uremia.
Decreased anion gap is rarely due to pathologic
problem (such as increased proteins in
myeloma), almost always technical problem with
instrument.
Case of Electrolyte Imbalance






Case # 333333 15 year old nonresponsive diabetic female with
gastrointestinal virus over past few days
has the following results:
Na 144
K
4.5
Cl
98
CO2 15
Glucose
Lactate
Osmolality
Urea N
250 mg/dl
5 (0.5-2.2)
312 (275-295)
35 (6-20)
Controls were accepted for all analysis. CO2
results from earlier and subsequent patient
samples were relatively normal.
Osmolality
 Measure
of colligative properties
– properties that are directly affected by #
of solute particles per mass of solvent
 Major
contributions to plasma
osmolality are Na+, Glucose, BUN and
unmeasured organic substances such
as ethanol, methanol.
 Osmolality units are mOsmole/Kg
(plasma H2O).
Osmolality
 Can
be measured or calculated
 1.86
(Na) + Glucose
18
 Increased
–
in
Diabetes, renal disorders
 Decreased
–
+ BUN + 9
2.8
in
Lymphomas, shock, MI
 Osmolality
gap =
– measured osmolality - calculated osmolality
– normal < 10
Hyperosmolar Coma
 Hyperglycemic,
nonketonic
hyperosmolar,
– Due to a combination of severe
dehydration caused by inadequate fluid
intake and insulin deficiency
– Characterized by
 Blood
glucose above 600 mg/dl
 N - sl decreased pH
 Serum osmolality above 350 mOsm/kg
 Lethargy or coma
 BUN increased
Osmolality
– Measurement by
 Freezing
point depression
 Vapor pressure increase
 boiling point increase
 osmotic pressure increase
– Osmolal gap
 measured
osmo minus calculated osmo
– Gap increased in
 ketoacidosis
 lactic
acidosis
poisonings
renal tubular acidosis
methanol, etc.
Acid Base Balance
Acid/Base Balance
 pH
 pCO2
 HCO3
 pO2
– pH and HCO3- are directly related
– pH and pCO2 are inversely related
– Balance is maintained by ratio
Relationships

Normally measured
– Total CO2
– pCO2

Mathematical Conversions
– H2CO3 = pCO2 x 0.03
– Total CO2 - HCO3- + H2CO3

Henderson/Hasselbach Equation
pH = pKa + log [HCO3-]
[H2CO3]
Acid Base Components

HCO3– metabolic component
– Total CO2 (from electrolyte report in mMole/L)
relates closely to HCO3-

H2CO3
– respiratory component
– pCO2 is measured value relating to H2CO3 =
pCO2 x 0.031.

Neither HCO3- nor H2CO3 is directly
measured
Classifying Acid Base Balance
 Low
pH = acidosis High pH =
alkalosis
 Compare HCO3- to pH to determine
metabolic (should be directly related)
 Compare pCO2 to pH to determine
respiratory (should be inversely
related)
 Look for compensation.
Compensation
 Compensation
begins with the
unaffected component from the
HCO3-/ H2CO3 ratio.
 Compensation is evident when both
values in the ratio are increased or
decreased and pH is moving towards
normal
Acid-Base Disorders
pH pCO2 HCO3
Respiratory
Acidosis 

 -N
Alkalosis 

 -N
Acidosis 
 -N

Alkalosis 
 -N

Metabolic
Case Study
A patient has the following results
blood gas results:
Patient
Reference Range
pH 7.33
(7.35-7.45)
pCO2 65 mm Hg
(35-45)
tCO2 35 mM/L
(25-33)
What is the likely acid-base status?
Parathyroid Function
and Calcium Metabolism
Objectives
Parathyroid Function and Calcium Metabolism
 Using PTH and calcium assay results, differentiate between
– Hypoparathyroidism (primary vs secondary)
– Hyperparathyrodism (primary vs secondary)
– Vitamin D levels
Correlate serum alkaline phosphatase (ALP) with bone
disorders.
Thyroid Function
 Using T3, T4 and TSH levels, differentiate between:

– Hyperthyroidism (primary, secondary and tertiary)
– Hypothyroidism (primary, secondary and tertiary)

Describe the factors that affect thyroid binding globulin
levels.
Minor Electrolytes/Minerals
Physiologic Control of Calcium and
Phosphorus
 Parathyroid Hormone Secretion in
response to low plasma Ca++ by 4-6
glands in larynx region

– Maintains homeostasis but increased level will
increase serum calcium and urinary
phosphorus and decrease serum phosphorus.
Vitamin D increases calcium levels.
 Calcitonin: counters PTH effect on bone.

Physiologic Control of Minerals
PTH causes




bone resorption
(breakdown)
renal reabsorption
(into bloodstream)
intestinal absorption
of calcium
renal secretion of
phosphorus into urine.
Parathyroid Function
Ca PO4 PTH ALP
Hyperparathyroidism
Primary 
Secondary V





 or N




N
N



Hypoparathyroidism
Primary 
Secondary 
Vitamin D
Deficiency -N - N
Excess 
N
Thyroid Function
Thyroid Function

Primary

Secondary

Tertiary
Thyroid Function
T4 T3 FTI TSH TBG TRH
Hyperthyroidism
1o
2o








N
N


- 
N
 

-N

Hypothyroidism
1o

2o

Euthyroid
-N 
v

N

N
N
Thyroid Testing
Sensitive TSH (mU/L)
<0.3
0.3 - 5.0
FT4
Normal
No further test
T3 if FT4
normal
3rd generation
TSH if sTSH
<0.1 mU/L
>5.0
Microsomal
antibody
and FT4
Thyroid Function
 Thyroid-binding
globulin
– increased in
 estrogen
 pregnancy
 oral
contraceptives
– decreased in
 androgens
 malnutrition
 liver
disease
Tumor Markers
 Screen
in healthy or asymptomatic
population low false positive, low
false negative rate; specificity and
sensitivity issues:
– Example: colorectal cancer screen with
fecal occult blood
 Monitoring
in symptomatic patients
for diagnosis, follow-up to treatment;
most tumor markers fit in this
category
Diagnostic Relevance/Medical
Decision Levels for Clinical Sig.

Diagnostic Specificity: Absence of Tumors (disease)
– Negative Predictive Value
%
of patients with negative tumor marker (below
the cut-off point) who don’t have the tumor
 Specificity relates to % of true negatives

Diagnostic Sensitivity: Presence of Tumors
(disease)
– Positive Predictive Value
%
of patients with positive tumor marker (above the
cut-off point) who do have the tumor
 Sensitivity relates to % of true positives
Tumor Markers
 PSA
in conjunction with digital rectal
exam
 Fecal occult blood with colonoscopy
 Ca-15-3 with mammography