LEMAR Review Hub 1

CLINICAL CHEMISTRY
Christine Joy M. De Leon, RMT, MLS (ASCPi) CM

Outline: Checklist:
I. Quality Control
II. Analytical methods/ Instrumentation  LEMAR Notes
III. Specimen collection and Handling  Board of Certification Exam Questions
IV. Carbohydrates  Henry’s, Bishop Clinical Chemistry Book
V. Lipids  Polansky Review Cards
VI. Proteins
 Medical Laboratory Science Review by
VII. Kidney Function
Robert Harr
VIII. Liver Function
IX. Enzymes  Clinical Laboratory Science Review by
X. Electrolytes Theriot-Jarreau
XI. Blood gas measurement
XII. Endocrine hormones

I. QUALITY CONTROL
Objective of Quality Control
1. To check the stability of the machine
2. To check the quality of reagents
3. To check technical (operator) errors

a. Parameters of Quality Control

Analytical sensitivity Ability of a method to detect small quantities of an analyte

Analytical specificity Ability of a method to detect only the analyte it is designed to determine
Ability of a method to measure only the analyte of interest
Reliability Ability of the method to maintain accuracy and precision over an extended
period of time

Precision: measure of the closeness of the results

obtained when analysis is repeated
Accuracy: how close the answer is to the “true”
value

Diagnostic sensitivity • Ability of a test to detect proportion of individual with the disease
• SCREENING TEST require high sensitivity so that no case is missed
Diagnostic specificity • Ability of a test to correctly identify the proportion of individuals without
the disease
• CONFIRMATORY TEST requires high specificity to be certain of the
diagnosis
100% sensitivity and specificity indicate that the test or method detects every patient with the
disease and that the test is negative for every patient without the disease.

b. Kinds of Quality Control

Intralab/ Internal QC Interlab/ External QC
• QC within the laboratory • Proficiency testing programs to
• Analysis of control samples together participating laboratories (Gold standard:
with patient specimens College of American Pathologist
• Essential for daily monitoring of Proficiency Testing)
accuracy and precision • Significant in maintaining long term
• Detects random and systematic accuracy of the analytical method
error • Difference >2 in the result means that the
laboratory is not in agreement with other
laboratories included in the program.

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College of American Pathologist (CAP) Proficiency Program: GOLD STANDARD for clinical laboratory
EXTERNAL QC testing

c. Variations
Errors encountered in the collection, preparation and measurement of samples, including transcription
and releasing of laboratory results

Random Error
(Imprecision, indeterminate, unpredictable)

CAUSES OF RANDOM ERROR PARAMETERS

• Mislabelling of sample Standard Deviation
• Pipetting error Coefficient of Variation
• Voltage/temperature fluctuations
• Improper mixing of sample and reagents
• Dirty optics

Systematic Error
(Inaccuracy, predictable, determinate)

CAUSES OF SYSTEMATIC ERROR PARAMETERS

✓ Improper calibration: MOST COMMON cause of SHIFT Mean
✓ Deterioration of reagent: MOST COMMON cause of TREND
✓ Contaminated solution
✓ Sample instability/ unstable reagent blank
✓ Diminishing lamp power
✓ Leaky ISE
✓ Failing instrumentation
✓ Incorrect sample and reagent volume
✓ Poorly written SOP

a. Constant error
o It refers to a difference between the target value and assayed value
o Independent of sample concentration
o It exists when there is a continual difference between the comparative method and the test method
regardless of concentration
b. Proportional/Slope/Percent Error
o It results in greater deviation from the target value due to higher sample concentration
o It exists when the difference between the test method and the comparative method values is
proportional to the analyte concentration

Proportional Type of systemic error where the magnitude changes as a percent of the Slope
systematic analyte present; error dependent on analyte concentration.
error
Constant Type of systemic error in the sample direction and magnitude; the magnitude y-
systematic of change is constant and not dependent on the amount of analyte. intercept
error

You validate a new assay using linear regression to compare assay calibrator results with the distributor’s
published calibrator results. The slope is 0.99 and the y-intercept is +10%. What type of error is present?
A. No error
B. Constant Systematic error
C. Proportional systematic error
D. Random error

Answer: ______________

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Clerical Error

Is the highest frequency of clerical errors occurs with the use of handwritten labels and request forms.

PRE-ANALYTICAL ERRORS ANALYTICAL ERRORS POST-ANALYTICAL ERRORS

1. Selection and implementation 1. Laboratory staff competence 1. Accuracy in transcription and
of assay relative to the patient 2. Assay and instrument selection filing of results
need 3. Assay validation (including 2. Content and format of laboratory
2. Patient identification and linearity, accuracy, precision, and narrative report
preparation analytical limits, specificity) 3. Reference interval and
3. Specimen collection technique 4. Internal quality control therapeutic range
and apparatus 5. External quality assessment 4. Timeliness in communicating
4. Specimen transport, critical values
preparation, and storage 5. Patient and physician satisfaction
5. Monitoring of specimen 6. Turnaround time
condition 7. Cost-analysis
6. Blood culture contamination
7. Adequacy of specimen
information

d. Statistics
Science of gathering, analyzing, interpreting and presenting data

Measure of Dispersion/ Spread Calculate the coefficient of variation for a set

of data where mean (x) = 89 mg/dL and 2
Standard Deviation Dispersion of values from the mean standard deviation = 14 mg/dL.
Coefficient of Index of precision a. 7.8
Variation b. 7.9
Range Simplest expression of spread c. 12.7
d. 15.7

Answer: ________
Measure of Central Tendency

Mean Most commonly used measure of center

Median Middle point of data and is often used with skewed data
Mode Most frequently occurring value in dataset

e. Quality Control Charts

Used to observe values of control materials over time to determine reliability of the analytical method
o Shewhart Levey-Jennings Chart: Most widely used chart that allows laboratorians to apply multi-rules
o Cumulative Sum Graph: Earliest indication of systematic errors (trend); Disadvantage: needs to be
computerized
o Youden Twin Plot: Used to compare results obtained from different labs, used in EQAs
o Gaussian Curve: Bell-shaped curve, Normal Distribution Curve

Which combination best describes a ± 1 SD ± 2 SD ± 3SD

Gaussian (normal) distribution? 68.3% 95.4% 99.7%

a. Median > mode TAM t test Accuracy, mean

b. Mean = median = mode SPF f test Precision,
c. Median > mean Standard deviation
d. Mode > mean (SD)

Answer: ________

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SHIFT
• Abrupt, sudden and sustained change in one
direction in control sample values
• 6 or more consecutive daily values that
distribute themselves on one side of the
mean value line, but maintain a constant
level (e.g. an increase shift)
• May be observed with the sudden
malfunction of an instrument

TREND (DRIFT)
• Gradual change in the control sample result
• Values for the control that continue to either
increase or decrease over a period of 6
consecutive days (Turgeon, at least 3 days)
• Progressive problem with the testing system
or control sample, such as deterioration of
reagents or control specimen

WESTGARD MULTIRULES

1:2s One control observation exceeding the mean ± 2s Warning rule that initiates testing of
control data by other rules.
1:3s One control observation exceeding the mean ± 3s Random error
R:4s One control exceeding the +2s and another exceeding the −2s Random error
2:2s Two control observations consecutively exceeding the same +2s or −2s Systematic error
4:1s Four consecutive control observations exceeding +1s or −1s Systematic error
10:x Ten consecutive control observations falling on one side or the other of Systematic error
the mean
 LEMAR Review Hub 5

II. ANALYTICAL METHODS/ INSTRRUMENTATION

BEER’S LAW
Concentration of unknown is directly proportional to the absorbance/optical density and inversely
proportional to the transmitted light.

Absorbance= abc = 2-log%T

Where: a=molar absorptivity
b= length of light
c=concentration of soln.

Spectrophotometry • Chemical reaction produces colored substance that absorbs light of a specific wavelength.
• Amount of light absorbed is directly proportional to concentration of analyte.
• Measurement of light transmitted by a solution to determine the concentration of light
absorbing substance

REAGENT BLANK:
Corrects absorbance caused by color of reagent
SAMPLE BLANK:
Measure absorbance of sample and reagent in the absence of the product

Single beam: simplest

Double beam in space: uses two photodetectors
Double beam in time
PARTS OF Remember: UV <400 nm | Visible 400-700 nm | Infrared >700nm
SPECTROPHOTO-
METER A. LIGHT SOURCE
1. Tungsten Iodide lamp-most common; for Visible and infrared
2. Deuterium discharge lamp- UV only
3. Xenon Discharge lamp- UV and Visible
4. Mercury Arc lamp- UV and Visible
5. Hydrogen lamp- UV only
B. ENTRANCE SLIT: minimizes stray light
C.MONOCHROMATOR: isolates specific wavelength
1. Diffraction gratings- most common; used in spectrophotometer
2. Glass and interference filters; used in photometers
D. EXIT SLIT: controls the bandpass
E. CUVE: analytical/sample cell
1. Quartz: for UV
2.Aluminum silica glass: most commonly used
F. PHOTODETECTOR
1. Photomultiplier tube: most common; excellent sensitivity and rapid response
G. READ-OUT DEVICE

Flame Emission • Excitation of electrons from lower to Measures:

Photometry higher energy state K- violet flame
• Light source: flame Na- yellow flame
• Internal std: Lithium/Cesium (corrects Magnesium- blue flame
variation in flame and atomizer Lithium, Rubidium- red
characteristics)
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Atomic Absorption • Measures light absorbed by atoms Measures:

Spectrophotometry dissociated by heat. Calcium
• Light source: Hollow Cathode Lamp Magnesium
• No internal standard needed
• Atomizer (nebulizer/granite furnace): Lanthanum and strontium chloride-
convert ions to atoms reduce interference by phosphate
• Chopper: modulate the light source

Turbidimetry • Determines the amount of light blocked by Measuring abundant large particles and
a particulate matter in a turbid solution bacterial suspensions
Depends on specimen concentration and
particle size
Nephelometry • Determines amount of scattered light by a Measuring antigen-antibody complexes
particulate matter suspended in a turbid
solution Depends on wavelength and size
Capillary • Molecules separated by electro-osmotic
Electrophoresis flow.
Gas Chromatography • Separation of compounds that are Barbiturates, steroids, blood, alcohol and
naturally volatile or can be easily lipids
converted into volatile form.
Tandem Mass • Can detect 20 inborn errors of metabolism IEM
Spectroscopy in a single blood spot
GC-MS GOLD STANDARD FOR DRUG TESTING
High Performance Liquid • Uses pressure for fast separations, Fractionation of drugs, hormones, lipids,
Chromatography controlled temperature, in-line detectors carbohydrates, proteins; separation and
and gradient elution techniques. quantitation of various hemoglobin assoc.
with diseases; rapid glycosylated
hemoglobin test
Fluorometry/ Molecular • Determines amount of light emitted by a Measures:
Luminescence molecule after excitation by Porphyrins
electromagnetic radiation Magnesium
• Light source: Mercury arc or Xenon lamp Calcium
• Uses 2 monochromators (Primary filter: Catecholamine
selects best absorbed wavelength,
Secondary filter: prevents incident light
from striking the photodetector)

• Atoms absorb light of specific wavelength and emit light of longer wavelength (lower energy)
• 1000 times more sensitive than spectrophotometry
• Subject to QUENCHING (decreased fluorescence)
Chemiluminescence • Chemical reaction yields and electronically Used in immunoassays
excited compound that emits light as it
returns to its ground state
• More sensitive than fluorescence
• No light source and monochromator are
required
Potentiometry • Measurement of electrical potential due to pH and pCO2
the activity of free ions- change in voltage
indicates activity of each analyte.
pCO2 electrode
• PCO2 electrode for measuring the partial pressure of carbon dioxide (pCO2) is actually a pH electrode immersed
in a bicarbonate solution
• A modified pH electrode with CO2 permeable membrane covering the glass membrane surface
• The bicarbonate solution is separated from the sample by a membrane that is permeable to gaseous CO2 but
not to ionized substances such as H+ ions. When CO2 from the sample diffuse across the membrane, it dissolves,
forming carbonic acid and thus lowering the pH
• Change in pH is proportional to the concentration of dissolved CO2 in the blood

Ion Selective Electrode • Ionic selectivity depends of membrane or Glass Aluminum Silicate: Na
barrier used Valinomycin gel: K
Monactin/Nonactin: Ammonium
Organic liquid membrane ion exchangers:
Ca and Li

Coulometry • Measurement of amount of electricity (in Chloride test (CSF, serum, sweat)
coulombs) at a fixed potential

Amperometry • Measurement of the current flow produced pO2, glucose, chloride and peroxidase
Polarography by an oxidation-reaction determination
Voltammetry • Measurement of current after which a Anodic stripping voltammetry- for Lead and
potential is applied to an electrochemical Iron testing
cell

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III. SPECIMEN COLLECTION

a. Arterial puncture
o Use: blood gas analysis and pH measurement
o Sites: radial, brachial, femoral, scalp, umbilical
artery
o Blood sample collection without tourniquet

Before blood is collected from the radial artery, Modified

Allen Test should be done to determine whether the ulnar
artery can provide collateral circulation to the hand after
the radial artery puncture.

b. Venipuncture

o Median cubital vein is the best site for venipuncture 1st choice: Median Cubital vein
2nd choice: Cephalic vein
because it is the largest and the best anchored vein. 3rd choice: Basilic vein
o Basilic vein- close to brachial artery Other sites:
o Venous blood not specimen of choice for blood gas Veins at the wrist
Veins at the back of the hand
measurement, because it usually reflects the acid-base Veins at the foot
status of an extremity and not the body as a whole.

Phlebotomy Complications STOPPER COLOR ANTICOAGULANT

1. Vascular (most common) 1. Yellow SPS (Blood Culture)
2. Infection 2. Light Blue Trisodium Citrate
3. Red With or without clot activator or gel separator
3. Cardiovascular 4. Green Heparin (Lithium, Sodium, Ammonium)
4. Anemia 5. Lavender/Purple EDTA
5. Neurological 6. Gray NaF and K oxalate/iodoacetate and heparin
6. Dermatological

Important!
✓ TDM samples not collected in tubes with gel-separator or serum separator tubes because some gels
absorb certain drugs (phenytoin, phenobarbital, lidocaine, quinidine, and carbamazepine causing
falsely low result.
✓ A gray top tube containing fluoride oxalate should be used for lactate sample collection, as it blocks
further glycolysis.
✓ Sodium fluoride tubes are used to collect ethanol specimen to prevent either a decrease in alcohol
concentration due to glycolysis or an increase due to fermentation by bacteria.

c. Skin puncture

o Fingerstick to obtain blood for routine laboratory analysis is usually preferred for children older than
one year old.
o Length of lancet: 1.75mm (avoid penetrate bone)
o Depth of incision: <2.0 mm for infants and children, <2.5 mm for adults
Preferred sites:
Lateral plantar heel surface (newborns)
Palmar surface of fingers (3rd or 4th)
Plantar surface of big toe
Earlobes – least site

IV. CARBOHYDRATES
Hormonal regulation of glucose

INSULIN GLUCAGON
✓ Primary hormone responsible for entry of ✓ Primary hormone responsible for increasing
glucose into the cell glucose- hyperglycemic agent
✓ Normally released when glucose levels are high ✓ Released during stress and fasting state
✓ Serum insulin measurements may be falsely low ✓ Fasting plasma glucagon: 25 to 50 pg/mL.
in the presence of hemolysis.

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Other hormones that tend to increase glucose concentration

✓ Cortisol and corticosteroids ✓ Thyroid hormones
✓ Catecholamines ✓ Adrenocorticotrophic hormone
✓ Growth hormone ✓ Somatostatin

Clinical conditions of Carbohydrate Metabolism

Hyperglycemia Hypoglycemia
o Increase in blood glucose concentration o Imbalance between glucose utilization and
o It is toxic to beta cell function and impairs insulin production
secretion. o Warning signs and symptoms are related to
o Causes: stress, severe infection, dehydration or central nervous system
pregnancy, pancreatectomy, hemochromatosis,
insulin deficiency or abnormal insulin receptor
Laboratory findings in hyperglycemia Diagnosis of hypoglycemia should not be made
✓ Increased glucose in plasma and urine unless a patient meets criteria of Whipple’s triad:
✓ Increased urine specific gravity ✓ Low blood glucose concentration
✓ Ketones in serum and urine ✓ Typical symptoms
✓ Decreased blood and urine pH (acidosis) ✓ Symptoms alleviated by glucose
✓ Electrolyte imbalance administration
o Decrease sodium
o Increase potassium
o Decrease bicarbonate

Diabetes Mellitus
• Group of metabolic disorders characterized by hyperglycemia resulting from defects in insulin
secretion, insulin receptors or both.
• Fasting plasma glucose of >/= to 126 md/dl on more than one testing are diagnostic of DM.
TYPE I DM TYPE II DM
Insulin Dependent DM/ Juvenile Onset DM/ Brittle Non-insulin Dependent DM/ Maturity Onset DM/
Diabetes/ Ketosis Prone DM Stable Diabetes/ Ketosis Resistant DM/ Receptor-
Deficient DM
B-cell destruction Insulin resistance
C peptide: decreased or undetectable C peptide: Detectable
Auto-antibodies present Auto-antibodies not present
Anti-GAD65: common in adults
Insulin autoantibodies: common in children
Ketosis: common Ketosis: Rare
Treated by parenteral insulin Treated by oral agents/metformin

• C-peptide levels are measured in hypoglycemic states to help identify the cause of the hypoglycemia.
o insulinoma patients: high insulin and C-peptide levels
o hypoglycemia from injected or exogenous: high insulin levels and low C-peptide levels.

Gestational Diabetes Mellitus

• Hyperglycemia that occurs during pregnancy and disappears after delivery. Hormones secreted by the
placenta block the action of insulin, resulting in insulin resistance and hyperglycemia
• Leads to TYPE II DM in 5- 10 years.

Glucose Methodologies
A. Copper Reduction Methods
Nelson- Glucose method reduces copper in hot alkaline solution to cuprous ion Greenish-blue
Somogyi which in turn reduces arsenomolybdic acid in a greenish blue complex complex
Folin-Wu Phosphomolybdic acid Blue complex
Neocuproine Neocuproine (2, 9 – dimethyl-1, 10 – phenanthroline hydrochloride) Yellow color
method
B. Condensation method

ORTHO-TOLUIDINE is the ONLY CHEMICAL METHOD still used widely and is based on the condensation of
aldosaccharides such as glucose, with an aromatic amine and glacial acetic acid.

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C. Enzymatic methods

1. Glucose oxidase converts beta-D-glucose to gluconic acid.

Mutarotase may be added to the reaction to facilitate the conversion of alpha-D-glucose to beta-D-glucose.
2. Hexokinase method is the most specific glucose method; reference method.

TAKE NOTE!
10% contamination with 5% dextrose will increase glucose level by 500 mg/dL.
Whole blood fasting blood glucose level is 10-15% lower than plasma.
Glucose measurement by reduction methods is 5 to 15 mg/dL HIGHER than enzymatic methods.
Venous blood glucose is 7 mg/dL lower than capillary and arterial blood

Glucose metabolized at RT 7 mg/dL per hour

Glucose metabolized at Ref Temp 2 mg dL per hour
Glucose level and hyponatremia For every 100 mg/dL increase in glucose, Sodium is lowered by
1.6 mmoL/L

Glucose can be tested if serum is separated from cells within ___ minutes. 30 minutes
SAMPLES FOR GLUCOSE MEASUREMENT
1. Random Blood Sugar For emergency cases
Requested during insulin shock
For patients with diabetic coma
2. Fasting Blood Sugar Measure of overall glucose homeostasis
Achieved after at least 8 hours of fasting
3. 2-hour Post Prandial Blood Sugar 2 hours after eating
Measures how well the body metabolizes glucose
4. Glucose Tolerance Test Multiple blood sugar test
Performed to diagnose gestation DM (100g)
5. Glycosylated hemoglobin Reflects the average blood glucose level over the previous 2-4 months
For every 1% change in HBA1C, there is 35 mg/dL (2mmol/L) change in
plasma glucose
6. Fuctosamine/ Glycated albumin For monitoring DM patients with Chronic Hemolytic Anemia, Hgb S or C,
Shortened RBC lifespan.
1. FBS- screening
ROUTINE TESTS FOR DM 2. HBA1C – monitoring
3. Microalbuminuria – early indicator of glomerular dysfunction

To convert mg/dL to mmol/L: Normal Sugar Level Impaired Glucose Tolerance Diagnosis for Diabetes
multiply by 0.0555
FBS 70- <100 mg/dL 100-125 mg/dL ≥126 md/dL
OGTT <140 mg/dL 140-199 mg/dL ≥ 200 mg/dL
2-hour Post Prandial
HBA1C <5.7% 5.7-6.4 % ≥6.5 %

V. LIPIDS
Major lipids:
• Phospholipids
• Cholesterol
• Triglyceride
• Fatty Acid
• Fat soluble vitamins (ADEK)
a. Cholesterol
✓ It evaluates the risk for atherosclerosis, Diagnostic significance:
myocardial and coronary arterial occlusions Increased Cholesterol:
✓ Direct relationship between elevated serum 1. Hyperlipoproteinemia types II,III,V
cholesterol and myocardial infarction 2. Biliary cirrhosis
✓ It is essential in the diagnosis and management 3. Nephrotic syndrome
of lipoprotein disorders 4. Poorly controlled DM
✓ Used to monitor effectiveness of lifestyle 5. Alcoholism
changes and stress management 6. Primary hypothyroidism
Decreased Cholesterol:
1. Severe hepatocellular disease
2. Malnutrition
3. Severe burns
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4. Hyperthyroidism
5. Malabsorption syndrome

Lecithin-cholesterol acyl transferase (LCAT)

✓ Catalyzes the esterification of cholesterol (HDL) by promoting the transfer of fatty acids from lecithin
to cholesterol which results in the formation of lysolecithin and cholesterol ester.
✓ Apo A1 is the activator if LCAT
✓ It enables HDL to accumulate cholesterol as cholesterol ester

The simplest approach for quantification of LIEBERMANN Cholestadienyl GREEN END

cholesterol is the one-step direct method, known as –BURCHARDT Monosulfonic COLOR
the: REACTION Acid
A. Liebermann-Burchardt procedure SALKOWSKI Cholestadienyl RED END COLOR
B. Abell-Kendall method REACTION Disulfonic Acid
C. van Handel and Zilversmith method
D. Hantzch condensation
Answer: ________
One- Colorimetry Pearson, Stern and
step Mac Gavack
Three-step method for cholesterol determination: Two- C + Extraction Bloors
A. Pearson, Stern and Mac Gavack step
B. Bloors Three- C+E+ Abell-Kendall
C. Abell-Kendall step Saponification
D. Schoenheimer Sperry, Parekh and Jung
Four- C+E+S+ Schoenheimer Sperry,
Answer: ________
step Precipitation Parekh and Jung

RECOMMENDED CUT-OFF POINTS FOR SERUM CHOLESTEROL

RECOMMENDED CUT-OFF POINTS FOR SERUM CHOLESTEROL

AGE MODERATE RISK (mg/dL) HIGH RISK (mg/dL)
2-19 >170 >185
20-29 >200 >220
30-39 >220 >240
≥ 40 >240 >260

CDC reference method (Abell, Levy and Brodie Method)

It uses hexane extraction after hydrolysis with alcoholic KOH followed by reaction with Liebermann-
Burchardt color reagent.
b. Triglyceride/Triacylglycerol (Neutral fat)

Diagnostic significance: Reference value:

✓ It evaluates suspected atherosclerosis and <150 mg/dL – normal
measures the body’s ability to metabolize fat 150-199 mg/dL – borderline high
✓ Fasting TAG ≥ 200 mg/dL are at risk fir coronary 200-499 mg/dL – high TAG
artery disease because of atherogenic VLDL > 500 – very high TAG (acute and recurrent
remnants pancreatitis)
✓ TAG and cholesterol are the most important
lipids in the management of CAD.
✓ Fasting requirement: 12 to 14 hours

c. Lipoproteins

✓ Are large macromolecular complexes of lipids with specialized proteins known as apolipoproteins.
✓ Main purpose is to transport TAG and cholesterol to sites of energy storage and utilization
Apolipoproteins
• Helps to keep the lipids in solution during circulation through the blood stream.
Which apoprotein is inversely related to risk for
coronary heart disease? REMEMBER!
A. Apo-A1 Apo-A1 = HDL
B. Apo-B100 Apo-B100 = LDL, VLDL
C. Apo-B48 Apo-B48 = Chylomicron
D. Apo-C
Answer: ________
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MAJOR LIPOPROTEINS

1. Chylomicrons
o Largest and least dense
o Produced in the intestine from dietary fat, completely cleared within 6 to 9 hrs post prandial
o Transports exogenous/dietary TAG to liver, muscle, fat tissues.
2. VLDL/ Pre-beta Lipoprotein
o Transports endogenous TAG from the liver to muscle, fat depots and peripheral tissues
o Prolonged consumption of high fat diet leads to elevated TAG in VLDL particles
3. HDL/ Alpha lipoprotein
o Smallest but most dense
o Transports excess cholesterol from the tissues and return it to the liver (reverse cholesterol transport)
o HDL2 transports effectively the lipids to the liver and more cardioprotective
CDC reference method: Ultracentrifugation (precipitation with heparin-MnCl2 and Abell-Kendal assay)
4. LDL/ Beta Lipoprotein
o Major end product from catabolism of VLDL
o Transports cholesterol to the peripheral tissues
o Most cholesterol rich and most atherogenic
o Primary marker for CHD risk

What best differentiates Chylomicron and VLDL from LDL?

Protein and Triglyceride

Triglyceride Cholesterol Ester Phospholipid Protein
Chylomicron 80-95 % 2-4% 3-6% 1-2%
VLDL 45-65% 16-22% 15-20% 6-10%
LDL 4-8% 45-50% 18-24% 18-22%
HDL 2-7% 15-20% 26-32% 45-55%

FORMULA LDL-c Calculation

LDL-c = Total Cholesterol – HDL – VLDL Friedewald formula

Friedewald formula: (indirect, not valid for triglycerides over 400mg/dL)
VLDL: TAG/2.175= mmol/L
TAG/5.0 = mg/dL
De Long Formula:
VLDL: TAG/2.825 = mmol/L
TAG/6.5 = mg/dL

A patient is admitted to the hospital with intense chest pains. The patient’s primary care physician
requests the emergency department doctor to order several tests, including a lipid profile with cholesterol
fractionation. Given the patient’s results provided below, what would be the LDL-c for this patient?
Total cholesterol = 400 mg/dL
Triglycerides = 300 mg/dL
HDL-C = 100 mg/dL
Answer: ________
MINOR LIPOPROTEINS
Intermediate Density Lipoprotein (IDL) Lipoprotein a (Lpa)
✓ Product of VLDL catabolism (VLDL remnant) ✓ Known as sinking pre-B lipoprotein – due to
✓ Converted to LDL (subclass of LDL) electrophoretic mobility same as VLDL but
✓ Migrates either in pre B or B region in density like LDL
electrophoresis ✓ Independent risk factor for atherosclerosis

Serum Lipoprotein
Electrophoresis

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ABNORMAL LIPOPROTEINS

Lipoprotein X B-VLDL (floating B lipoprotein)

✓ Found in obstructive jaundice and LCAT ✓ “Abnormally migrating B-VLDL”- has the density
deficiency of VLDL by ultracentrifugation but migrates with
✓ Specific and sensitive indicator of cholestasis LDL in the B region during electrophoresis
✓ Found in type III hyperlipoproteinemia or
dysbetalipoproteinemia

Friedrickson Classification
TYPE LIPOPROTEIN PATTERN
I Familial LPL deficiency Extremely elevated TG due to the presence of chylomicrons
IIa Familial Hypercholesterolemia Elevated LDL
IIb Mixed Defect – Familial Combined Elevated LDL and VLDL
Hyperlipidemia
III Familial Dysbetalipoproteinemia Elevated cholesterol, TG; presence of -VLDL
IV Familial Hypertriglyceridemia Elevated VLDL
V Elevated VLDL and presence of chylomicrons

Which of the following liver conditions show an A type IIA hyperlipoproteinemia phenotype is
increase in conjugated bilirubin and ALP, manifests associated with an increased level of:
with anti-mitochondrial antibodies, and shows a Chylomicrons
characteristic lipoprotein X on electrophoresis? A. VLDL
A. Hemochromatosis B. IDL
B. Primary biliary cirrhosis C. LDL
C. Alcoholic fatty liver Answer: ________
D. Hepatic tumors
Answer: ________

VI. PROTEINS
Functions:
1. Repair body tissues
2. Important in blood coagulation and immunologic function
3. For transport of metabolic substances
4. Maintenance of osmotic pressure
5. Maintenance of blood pH (buffers)
6. Biocatalyst

PLASMA PROTEINS
1. Pre-albumin (transthyretin)
✓ Used to detect malnutrition and individual’s response to dietary supplementation
✓ Inc: alcoholism, chronic renal failure, steroid treatment
✓ Dec: poor nutrition
✓ Ref: 18-45 mg/dl
2. Albumin
✓ Highest concentration in plasma
✓ General transport protein
✓ Maintains osmotic pressure, indicator of nutritional status
✓ Negative acute phase reactant
✓ Sensitive and highly prognostic marker in cystic fibrosis
✓ Lowest level seen in nephrotic syndrome
✓ Ref: 3.5-5.0 g/dL
3. Globulin
✓ Measurement: Total protein – Albumin = Globulin
✓ Ref: 2.3 – 3.5 g/dL
Alpha 1 antitrypsin Major inhibitor of protease activity
Neutralizes trypsin like enzymes
Inc: inflammation, pregnancy, contraceptive use
Alpha 1- fetoprotein Maternal serum AFP used as a screening test for neural tube defects and
down syndrome
Specimen: maternal serum, amniotic fluid, serum (for cancer screening)
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Alpha 1- acid Negatively charged even in acid solution

glycoprotein Greatest affinity with progesterone, binds quinidine drugs
(orosomucoid) Inc: pregnancy, cancer, pneumonia, RA, and cell proliferation
Alpha 1- Inactivates PSA
antichymotrypsin Associated with pathogenesis of Alzheimer’s disease- it is a vital component
of amyloid deposits in brain
Hemopexin Binds heme released by degradation of hemoglobin- has the strongest
affinity for heme
Diagnosis of early hemolysis
Haptoglobin Prevents loss of hemoglobin and its constituent iron into the urine
Evaluates degree of intravascular hemolysis
Inc: stress, myoglobinuria
Dec: IV hemolysis, hemoglobinuria
Ceruloplasmin Copper binding
Marker for Wilson’s Disease (deposition of copper in skin, liver, brain and
cornea – Kayser Fleisher rings)
Alpha 2- Largest major non-immunoglobulin
macroglobulin Increases 10x in nephrosis- loss into urine is prevented by its large size
Beta 2- Light chain component of major human leukocyte antigen
microglobulin Has tendency to fold into B sheet configuration resulting to amyloid
formation- common cause of dialysis-associated amyloidosis.
Transferrin Used to determine the cause of anemia
Prevents loss of iron thru the kidneys
Inc: hemochromatosis, IDA
Dec: liver disease, malnutrition, nephrotic syndrome
Immunoglobulins IgG- most abundant
IgM- first antibody to appear
IgA- in mucous secretions
IgD- on surface of B cells
IgE – allergic and anaphylactic reaction
Lipoprotein Transports cholesterol, TAG and phospholipids
Fibrinogen Most abundant coagulation factor
Complement Natural defense mechanism, serves as link to inflammatory response
C reactive protein Undetectable in blood of healthy individuals
(CRP) Cardiac marker- early warning test to persons at risk of CHD
Rapid test for presumptive diagnosis of bacterial vs. viral infection

FRACTION SPECIFIC PROTEINS

Albumin Albumin
Alpha1 globulin Alpha1 antitrypsin, lipoproteins
Alpha2 globulin Ceruloplasmin, haptoglobin, alpha2 macroglobulin, lipoproteins
Beta globulin Transferrin, hemopexin, complement system, lipoproteins
Gamma globulin Immunoglobulins

Serum proteins have a net negative charge and migrate toward the anode, with ALBUMIN TRAVELING THE
FARTHEST, followed by alpha1 -globulins, alpha2-globulins, beta-globulins, and gamma-globulins

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Ceruloplasmin and haptoglobin migrate Which of the proteins listed migrates in the beta
electrophoretically as: region at pH 8.6?
A. Alpha1 globulins A. Orosomucoid
B. Alpha2 globulins B. Haptoglobin
C. Beta globulin C. Ceruloplasmin
D. Gamma globulins D. Transferrin
Answer: ________ Answer: ________

Clinical correlation
Liver cirrhosis Polyclonal ↑ (all fractions) in
gamma with beta-gamma
bridging

Nephrotic syndrome ↓ albumin, ↑ alpha-2

Alpha 1 antitrypsin ↓ alpha-1

deficiency

Acute inflammation ↑ alpha-1 and alpha-2

Chronic inflammation ↑ alpha-1, alpha-2, and gamma

Hypogammaglobulinemia ↓ gamma

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Monoclonal Sharp ↑ in 1 immunoglobulin

gammopathy (“M spike”). ↓ in other fractions

VII. KIDNEY FUNCTION TEST

Functions of the kidney:
1. Elimination of waste products
2. Maintenance of blood volume
3. Maintenance of electrolyte balance
4. Maintenance of acid-base balance
5. Endocrine function (erythropoietin secretion)
Three processes in urine formation:
1. Glomerular Filtration
2. Tubular Reabsorption
3. Tubular Secretion

A. Tests for Glomerular Filtration Rate

Glomerular filtration rate

o Measure of the clearance of normal molecules that are not bound to protein and are freely filtered by
the glomeruli
o Neither reabsorbed nor secreted by the tubules
o Best overall indicator of level of kidney function
1. Clearance
o Is the removal or substance from plasma into the urine over a fixed time
o Expressed in ml/minute, representing the volume of plasma that would be totally cleared of the solute
in one minute
o Plasma concentration and clearance is inversely proportional

Formula of clearance:
𝑚𝑙 𝑈 1.73
𝐶𝑙𝑒𝑎𝑟𝑎𝑛𝑐𝑒 ( )= x Volume (ml/min) x
min 𝑃 𝐴

Where:
U – concentration of analyte in the urine
P – concentration of analyte in plasma
Volume – volume of urine in milliliter in 24 hours
Minutes – time required to collect urine (1440 minutes)
1.73 – constant; average body surface area in adult
A – body surface of patient obtained from nomogram
Let’s try!
Using urine creatinine of 120 mg/dL (U), plasma creatinine of 1.0 mg/dL (P), and urine volume of 1440
mL obtained from a 24-hour specimen (V), calculate the GFR.
Computation:

Answer: ________

Clearance Tests
a. Inulin Clearance
✓ Reference method
✓ Not routinely done because need for continuous IV infusion
✓ extremely stable substance that is not reabsorbed or secreted by the tubules
b. Creatinine Clearance
✓ Excellent measure of renal function- creatinine is freely filtered by the glomeruli but not
reabsorbed
✓ Measures of the completeness of 24 hour urine collection
✓ Ref: Male (85-125 ml/min) Female ( 75-112 ml/min)
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INCREASED DECREASED
High cardiac output Impaired kidney function
Pregnancy Shock, dehydration
Burns Hemorrhage
Carbon monoxide poisoning Congestive heart failure
Clearance Tests

c. Urea Clearance
✓ Can demonstrate progression of renal disease or response to therapy
✓ Not reliable – freely filtered but variably reabsorbed by the tubules
✓ In advanced renal failure, urea clearance approaches unity with GFR and is a better predictor of
GFR than creatinine clearance.
As the renal function declines, the fraction of urea reabsorbed declined progressively, whereas tubular
secretion of creatinine increases progressively.

2. Cystatin C
o A low molecular weight protease inhibitor and produced at a constant rate by all nucleated cells.
o Completely reabsorbed and catabolized by the PCT, hence its presence in urine denotes damage of
that tubule – serum level is an indirect estimate of GFR.
o Specimen: serum or plasma
o Inc: acute and chronic renal failure, diabetic nephropathy
3. Beta trace protein
o Isolated primarily from the CSF- plasma BTP originates from the brain and is freely filtered at the
glomerulus, then is reabsorbed completely and catabolized by the proximal tubule.
o Increased: renal disease (because of reduced filtration in the presence of constant production)

B. Test for Renal Blood Flow

1. Blood Urea Nitrogen
o Major end product of protein and amino acid catabolism – 45% of NPN
o Synthesized in the liver from CO2 and ammonia
o First metabolite to elevate in kidney diseases
o Not easily removed by dialysis
o LOW BUN are not generally considered abnormal renal function
o Ref: 8-23 mg/dl
o BUN:Creatinine ratio
o 10:1 – 20:1
INCREASED DECREASED
1. Chronic renal disease 1. Poor nutrition
2. Stress 2. Hepatic disease
3. Burns 3. Impaired absorption (celiac disease)
4. High protein diet 4. Pregnancy
5. Dehydration

2. Creatinine

o End product of muscle catabolism

o Not affected by protein diet and not easily removed by dialysis
o Ref:
o Male = 0.1-1.3 mg/dl
o Female = 0.6-1.1 md/dl
o
Clinical Disorders of Impaired Kidney Function
Syndrome Significant Laboratory Findings
Nephrotic syndrome ✓ Striking proteinuria
✓ Decrease in serum albumin and total protein
✓ Relative increase in alpha2 and beta-globulin fractions
✓ Increases in serum creatinine, BUN and uric acid
✓ Lipids in urine
Glomerulonephritis ✓ Elevated urine protein
✓ Increases in serum creatinine and BUN
✓ Red cells and casts in urine
Renal failure ✓ Decrease in urine output
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✓ Increases in serum BUN and creatinine

✓ Electrolyte and acid/base alterations
Tubular defects ✓ Specific alterations in excretion of amino acids, electrolytes, or other specific
biochemicals

PRE-RENAL AZOTEMIA RENAL AZOTEMIA POST-RENAL AZOTEMIA

Result of poor perfusion of the Caused primarily by diminished Result of any type of obstruction in
kidneys and therefore diminished glomerular filtration as a which urea is reabsorbed into the
glomerular filtration consequence circulation
of acute or chronic renal
disease
Dehydration Renal failure Renal calculi
Shock Glomerular nephritis Tumors of the bladder, prostate
Diminished blood volume Tubular necrosis
Congestive heart failure Other intrinsic renal disease

Uremia vs. Azotemia

Uremia Abnormally high urea nitrogen in the blood
Azotemia Significant increase in the plasma concentrations of urea and creatinine, in kidney insufficiency

VIII. LIVER FUNCTION TEST

Functions:
o Synthetic function
o Conjugation function
o Detoxification and Drug metabolism
o Excretory and Secretory Function
o Storage function
FUNCTION EXAMPLES
Synthesis Proteins – albumin, cholinesterase, coagulation proteins, cholesterol, bile salts and glycogen
Metabolism Glucose to acetyl-CoA, gluconeogenesis, amino acid conversions, fatty acids
Detoxification Bilirubin, drugs, ammonia
Excretion Bile acids

Albumin/Globulin Ratio
o Determined to validate if globulin is higher than albumin
o INVERTED A/G RATIO (High globulin, Low albumin)
o Cirrhosis
o Multiple myeloma
o Waldenstrom’s macroglobulinemia
o Reference values: 1:3 – 3:1

Bilirubin
Indirect bilirubin (B1) Direct bilirubin (B2)
Unconjugated bilirubin Conjugated bilirubin
Water insoluble Water soluble
Non-polar bilirubin Polar bilirubin
Indirect reacting Direct reacting
Hemobilirubin Cholebilirubin
Slow reacting One-minute/Prompt bilirubin
Prehepatic bilirubin Post-hepatic bilirubin/Hepatic
bilirubin/Obstructive and regurgitive bilirubin

Elevation in Bilirubin fractions

Unconjugated only (B1) Hemolytic anemias, newborns, hereditary alteration of rate of conversion,
medications
Conjugated only (B2) Bile duct obstruction, some cases of hepatitis, medications
Both fractions Hepatitis
(B1 and B2)

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JAUNDICE
 LEMAR Review Hub 18

PRE HEPATIC JAUNDICE ✓ This condition produces increased serum unconjugated bilirubin.
HEPATIC JAUNDICE ✓ Exhibits increases in both unconjugated and conjugated bilirubin levels.
POST HEPATIC ✓ Causes increased conjugated bilirubin levels in serum and urine, but
JAUNDICE low urobilinogen levels in urine and colorless stool.

BILIRUBIN ASSAY
Evelyn-Malloy Methanol Acid pH 560 nm Red, reddish-purple
Jendrassik-Grof Caffeine, sodium benzoate Alkaline pH 600 nm Blue

Other liver function tests

Serum bilirubin level Overall capacity to transport bile
Ratio of direct and total bilirubin Patency of biliary ducts; hepatocellular metabolism of bilirubin
Serum bile acids (salts) Overall patency of biliary ducts
Fecal color and fact content Patency of biliary ducts
Fecal urobilinogen Patency of biliary ducts; quantity of bilirubin processed
Urine urobilinogen Patency of biliary ducts; quantity of bilirubin processed;
hepatocellular excretory capacity
Serum ALP and other Abnormality of bile duct epithelium
“obstructive” enzymes
Excretion of BSP (rarely used Hepatocellular function and patency of the bile ducts
Urine bilirubin Patency of biliary ducts, hepatocellular bilirubin metabolism
The danger of kernicterus is a certainty at bilirubin levels exceeding ___ mg/dL. Henry
A. > 5 mg/dL
B. > 10 mg/dL
C. > 15 mg/dL
D. 20 mg/dL
Answer: _________

IX. ENZYMES
Factors Affecting Enzymatic Reactions
1. Enzyme concentration
2. Substrate concentration
3. Cofactors
4. Inhibitors
5. Temperature
o Temperature coefficient (Q10) means for every 10 deg increase in temperature, there will be
two-fold increase in enzyme activity.
6. pH
7. Hemolysis- mostly increase enzyme concentration
8. Lactescence or milky specimen- decreases enzyme concentration

Enzyme categories
CATEGORY EXAMPLES
Oxidoreductase Lactate dehydrogenase
Glucose-6-phosphate dehydrogenase
Glutamate dehydrogenase
Transferase Aspartate aminotransferase
Alanine aminotransferase
Creatine kinase
Gamma glutamyltransferase
Glutathione-S-transferase
Glycogen phosphorylase
Pyruvate kinase
Hydrolase Alkaline phosphatase
Acid phosphatase
Amylase
Triacylglycerol lipase
Cholinesterase
Chymotrypsin
Elastase-1
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5-nucleotidase
Trypsin
Lyases Aldolase
Isomerases Triosephosphate isomerase
Ligases Glutathione synthetase

Which condition produces the highest Serum LD levels are highest in PA, reaching 10–50 times
elevation of serum lactate the upper reference limit (URL) as a result of intramedullary
dehydrogenase? hemolysis.
A. Pernicious anemia Moderate elevations (5–10 × URL) usually are seen in acute
B. Myocardial infarction MI, necrotic liver disease, and muscular dystrophy.
C. Acute hepatitis Slight increases (2–3 × URL) are sometimes seen in
D. Muscular dystrophy obstructive liver disease
Answer: _________

The isoenzymes LD-4 and LD-5 are LD ISOENZME TISSUE SOURCE

elevated in: LD 1 and LD 2 Heart and RBC
A. Acute pancreatitis LD 3 Lung, Lymphocytes, Spleen and Pancreas
B. Hemolyzed specimen
LD 4 and LD 5 Liver and skeletal muscle
C. Hepatic injury or inflammation
D. Myocardial infarct LD 6 Alcohol dehydrogenase (arteriosclerotic
Answer:_________ cardiovascular failure)

What particular fraction of LD Loss of activity occurs more quickly at 4°C than at 25°C.
isoenzymes is destroyed by freezing?
A. LD 1 Serum samples for LD isoenzyme analysis should be stored
B. LD 2 at 25°C and analyzed within 24 hours of collection.
C. LD 4
D. LD 5
Answer:_________
Tissue Specificity of Enzymes
Which of the following enzymes has high specificity for High ACP Erythrocytes,
the pancreas? specificity prostate
A. Amylase ALT Liver
B. Lipase Amylase Pancreas, salivary
C. ALT gland
D. AST Lipase Pancreas
Answer:_________ Moderate AST Liver, heart, skeletal
specificity muscles
Which of the following enzymes has high specificity for CK Heart, skeletal
the pancreas and salivary gland? muscles, brain
A. Amylase Low ALP Lover, bone, kidney
B. Lipase specificity LD All tissues
C. ALT
D. AST The presence of this ALP isoenzyme in
Answer:_________ serum depends on the blood group and
secretor status of the individual – B or O
Which of the following enzymes has moderate blood group, isoenzyme increases after
specificity for the liver, heart and skeletal muscles? consumption of a fatty meal.
A. ALT A. Liver ALP
B. AST B. Bone ALP
C. CK C. Placental ALP
D. LD D. Intestinal ALP
Answer:_________ Answer:_________

ALP TECHNIQUES
Heat Temperature control difficult
inactivation
Inhibitors Isoenzyme inhibitions overlap, somewhat useful when employed with electrophoresis
Electrophoresis Can distinguish major fractions, not quantitative
Immunological Data not reproducible
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Which isoenzyme of ALP are inhibited by L-

phenylalanine? Source of ALP INHIBITION BY ORDER
A. Intestinal and placental L- Heat or ANODAL
B. Bone and intestinal phenylalanine Urea (%) MIGRATION
C. Liver and placental (%)
D. Renal and liver Liver 10 60 1
Answer:_________ Bone 10 90 2
Intestine 75 60 4
Which isoenzyme of ALP is most heat Placenta 80 0 3
stable? Regan 80 0 3
A. Bone (carcinoma)
B. Liver
C. Intestinal
D. Placental
Answer: _________ The highest elevations of ALP activity occur in: Bishop
A. Rickets
Which of the following conditions can B. Paget’s disease (osteitis deformans)
“physiologically” elevate serum alkaline C. Osteomalacia
phosphatase? D. Hyperparathyroidism
A. Hyperparathyroidism Answer:_________
B. Diabetes
C. Third-trimester pregnancy
D. Nephrotic syndrome Increased ACP Decreased ACP
Answer:_________ ✓ Failure to use ✓ Prolonged storage,
What is the effect of hemolysis in ACP value: anticoagulant, release of changes in pH
A. Falsely increased enzyme from platelets ✓ Prolonged storage,
B. Falsely decreased ✓ Fluoride, oxalate and changes in pH
C. Variable heparin
D. No effect
Answer:_________ Amyloclastic Measures the disappearance of
starch substrate
For the determination of amylase, which assay Saccharogenic Measures the appearance of the
method is based on the measurement of the product
quantity of reducing substances formed? Chromogenic Measures the increasing color from
A. Amyloclastic production of product coupled with a
B. Saccharogenic chromogenic dye
C. Amylometric Continuous- Coupling of several enzyme systems
D. Chronometric monitoring to monitor amylase activity
Answer:_________
ENZYME CLINICAL UTILITY IN LIVER
The appearance of the LD1/LD2 flipped pattern and
DISORDERS
the presence of CK-MB isoenzyme on
Alkaline Elevated primarily in
electrophoresis highly suggestive of:
phosphatase obstructive processes
A. Acute pancreatitis
Aminotransferase Elevated in variety of liver
B. Cirrhosis
diseases; ALT more sensitive
C. Muscular dystrophy
indicator
D. Myocardial infarction
Gamma- Some increase in liver diseases;
Answer:_________
glutamyltransferase sensitive indicator of ethanol
Hepatocellular damage may be best assessed by intake
which of the following parameters? Cholinesterase Normally quite high; values
A. Serum ALT and AST levels decrease in liver disorders
B. GGT and ALP Lactate Elevated in a wide variety of
C. Bilirubin, GGT and ALP dehydrogenase situations; clinical utility low in
D. Ammonia and urea absence of isoenzyme studies
Answer:_________
Hepatocellular ALT and AST
Which of the following enzymes is useful in damage
diagnosing hepatobiliary obstructive disorders? Hepatobiliary damage ALP and GGT
A. ALP
B. ALT
C. GGT
D. LD
Answer:_________
 LEMAR Review Hub 21

In pesticide poisoning, cholinesterase activity is: Select the most sensitive marker for alcoholic liver
A. Increased disease:
B. Decreased A. ALP
C. Variable B. ALT
D. No change C. LD
Answer:_________ D. GGT
Answer:_________

Which of the following enzymes exhibit a IMPORTANT:

decreased serum level in liver disease? PSEUDOCHOLINESTERASE DEFICIENCY
A. Alkaline phosphatase ✓ Pseudocholinesterase deficiency causes an
B. Alanine aminotransferase increased sensitivity to certain muscle relaxants,
C. Lactate dehydrogenase called choline esters.
D. Cholinesterase ✓ Affected individuals can suffer from prolonged
Answer:_________ apnea and muscle paralysis after the
administration of these drugs.

X. ELECTROLYTES
Functions of Electrolytes
1. For volume and osmotic regulation – Na, K, Cl
2. For myocardial rhythm and contractility – K, Mg, Ca
3. Important cofactors in enzyme activation – Mg, Zn, Cl, K (2018)
4. For the regulation of ATPase ion pumps – Mg
5. For neuromuscular excitability – K, Mg, Ca
6. For the production and use of ATP from glucose - Mg, PO4
7. Maintenance of acid-base balance – HCO3, K, Cl, PO4
8. Replication of DNA and the translation of mRNA- Mg
1. Sodium
✓ Major extracellular cation
✓ Major contributor to osmolality
✓ Principal osmotic particle outside the cell
✓ All confirmed serum sodium abnormalities must be followed up with urinalysis (including urine sodium
and urine osmolality) on patient, who should be fluid restricted
✓ Reference: 135-145 mmol/L
✓ Threshold critical value:
o 160 mmol/L (hypernatremia) o 120 mmol/L (hyponatremia)
HYPONATREMIA HYPERNATREMIA
Increased Sodium Loss Excess Water Loss
a. Diuretic use a. Diabetes insipidus
b. Saline infusion b. Renal tubular disorder
Increased Water retention c. Prolonged diarrhea
a. Renal Failure d. Profuse sweating
b. Nephrotic syndrome e. severe burns
c. Aldosterone deficiency f. Vomiting
d. Cancer g. Fever
e. SIADH h. Hyperventilation
f. Hepatic cirrhosis Decreased water intake
g. Primary polydipsia Increased intake or retention
h. CNS abnormalities- meningitis, encephalitis, a. Hyperaldosteronisms (Conn’s)
multiple sclerosis b. Sodium bicarbonate infusion
i. myxedema c. Increased oral or IV intake of NaCl
d. Ingestion of sea water
✓ Pseudohyponatremia can occur when Na+ is measured using indirect ISE in a patient who is
hyperproteinemic or hyperlipidemic.
✓ An indirect ISE dilutes the sample prior to analysis and as a result of plasma/serum water
displacement; the ion levels are falsely decreased
HORMONAL CAUSES OF FLUID AND ELECTROLYTE IMBALANCE
INCREASED ADH Fluid retention, low serum sodium
DECREASED ADH Fluid loss, high serum sodium (in D.I)
INCREASED ALDOSTERONE Hypertension, low serum potassium
DECREASED ALDOSTERONE Low serum sodium, high serum potassium
INCREASED RENIN Hypertension
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2. Potassium
✓ Major intracellular cation
✓ Concentration in RBC is 105 mmol/L (23x its concentration in plasma)
✓ Single most important analyte in terms of an abnormality being immediately life threatening.
✓ Function: heart contraction, neuromuscular excitability, ICF volume regulation, hydrogen ion
concentration
✓ Reference value: 3.5 – 5.3 mmol/L
o 6.5 mmol/L (Hyperkalemia)
o 2.5 mmol/L (hypokalemia)
HYPOKALEMIA HYPERKALEMIA
Gastrointestinal loss Decreased Renal Excretion
a. Gastric suction and laxative abuse a. Acute or Chronic Renal Failure
b. Intestinal tumor and malabsorption b. Severe dehydration
c. Cancer and radiotherapy c. Addison’s disease
d. Vomiting and diarrhea Extracellular shift
Renal Loss a. Acidosis
a. Diuretics use (thiazides) b. Muscular/cellular injury
b. Hyperaldosteronisms c. Chemotherapy
c. Cushing syndrome d. Vigorous exercise
d. Leukemia e. Digitalis intoxication
e. Bartter’s syndrome Increased intake- oral or IV
f. Gitelman’s syndrome Use of immunosuppressive drugs (tacrolimus and
g. Liddle’s syndrome cyclosporine)
h. Malignant hypertension
Intracellular shift- alkalosis and insulin overdose
✓ Potassium is not a threshold substance.
o Unlike sodium, potassium exhibits no renal threshold, being excreted into the urine even in
K+-depleted states. (Ciulla)
3. Chloride

✓ Major extracellular anion- chief counterion of sodium in ECF

✓ Promotes maintenance of water valance and osmotic pressure in conjunction with sodium
✓ Only anion to serve as an enzyme activator
✓ Specimen consideration:
✓ Marked hemolysis- decreased level due to dilutional effect
✓ Slightly lower in post-prandial specimen
✓ Low serum values observed in conditions with high HCO3
✓ Ref: 98-107 mmol/L
4. Calcium
✓ Present almost exclusively in plasma BLOOD URINE
✓ Involved in coagulation, enzyme activity, excitability of 50% Ionized 85% Ionized
skeletal muscle and cardiac muscle and maintenance 40%Protein-bound 15% Complexed
of blood pressure 10% Complexed
Neuromuscular irritability, which may become clinically apparent as irregular muscle spasms, called
TETANY is associated with:
A. Hyponatremia
B. Hypernatremia
C. Hypocalcemia
D. Hypercalcemia
Answer:_________

ANION GAP

✓ Difference between the unmeasured cations (Sodium and Potassium) and unmeasured anions
(Chloride and Bicarbonate)
AG = Na – (Cl + HCO3-) Reference range:
7 to 16 mmol/L
AG = (Na + K) – (Cl + HCO3) Reference range:
10 to 20 mmol/L

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Determine the anion gap given the serum electrolyte data: Sodium 132 mmol/L, Chloride 90 mmol/L ,
Bicarbonate 22 mmol/

Answer:_________

XI. BLOOD GAS ANALYSIS

Select the anticoagulant of choice for blood gas Ninety percent of the carbon dioxide present in the
studies: blood is in the form of:
A. Sodium citrate, 3.2% A. Bicarbonate ions
B. Lithium heparin B. Carbonate
C. Sodium citrate, 3.8% C. Dissolved CO2
D. Ammonium oxalate D. Carbonic acid
Answer:_________ Answer:_________
What is the normal ratio of bicarbonate to Which of the following is directly measured by
dissolved carbon dioxide in arterial blood? blood gas analyzers?
A. 1:10 A. pH, pCO2, pO2
B. 10:1 B. pH, PO2 and oxygen saturation
C. 20:1 C. pH, HCO3-
D. 1:20 D. pH and pCO2 only
Answer:_________ Answer: ________

✓ Most blood gas analyzers measure pO2, pCO2, and pH by ion-specific electrodes and calculate
bicarbonate concentration by the Henderson-Hasselbalch equation.

pH: Sanz electrode

pO2: Clarke electrode
pCO2: Severinghaus electrode

IMPORTANT!

Parameters Measurement of Reference range (arterial blood)

pH [H+] 7.35-7.45
pCO2 Partial pressure of CO2 35 – 45 mmHg
pO2 Partial pressure of O2 80 – 100 mmHg
HCO3- Bicarbonate 22 – 26 mmol/L
Total CO2 Bicarbonate + Carbonic acid 23 – 27 mmol/L

CONDITION pH pCO2 HCO3- Compensation to Re-establish 20:1 ratio

Respiratory acidosis Low High Normal Kidneys retain HCO3-, excrete H+
Metabolic acidosis Low Normal Low Hyperventilation (blow off CO2)
Respiratory alkalosis High Low Normal Kidney excrete HCO3-, retain H+
Metabolic alkalosis High Normal High Hypoventilation (retain CO2)

Determine the acid-base balance derangement

Patient XYZ had the following blood gas analysis results:
pH= 7.20
pCO2= 47 mmHg
HCO3= 28 mmoL/L
Is it fully, partially or uncompensated?

COMPENSATION PH PC02 HCO3

Fully compensated Normal Acidic (↑) Basic (↑)
Normal Basic () Acidic ()
Partially compensated Close to Normal Acidic (↑) Basic (↑)
Close to Normal Basic () Acidic ()
Uncompensated Abnormal Normal Basic/Acidic

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Which of the following changes will occur with Open tube Entry of Increased pO2 and pH;
a blood gas sample exposed to room air? oxygen decreased pCO2
A. pH increased Closed Oxygen Decreased pO2 and
B. pCO2 increased tube utilized by pH; increased pCO2
C. pO2 decreased cells
D. Ionized calcium increased
Answer: ___________

The blood pH should be maintained within the The kidney exercises its metabolic control over blood
range of: pH by altering the retention or excretion of:
A. 7.00 to 7.50 A. Bicarbonate
B. 7.25 to 7.35 B. Carbonic acid
C. 7.35 to 7.45 C. Carbon dioxide
D. 7.35 to 7.60 D. Sodium chloride
Answer: ___________ Answer: ___________

An excessive loss of carbon dioxide through TYPE CAUSE COMPENSATION

hyperventilation is the primary cause of: Respiratory Excess CO2 Kidneys retain
A. Respiratory acidosis acidosis accumulation HCO3−, excrete H+
B. Respiratory alkalosis (hypoventilation)
C. Metabolic acidosis Respiratory Excess CO2 loss Kidneys excrete
D. Metabolic alkalosis alkalosis (hyperventilation) HCO3−, retain H+
Answer: ___________ Metabolic Excess H+ Hyperventilation
acidosis production (blow off CO2)
Metabolic Excess H+ loss or Hypoventilation
alkalosis excess alkali intake (retain CO2)

A patient with emphysema who has fluid Respiratory Respiratory diseases: pneumonia,
accumulation in the alveolar sacs (causing acidosis emphysema, chronic obstructive lung
decreased ventilation) is likely to be in which disease), myasthenia gravis
of the following acid-base clinical states? Respiratory Anxiety, some CNS disorders
A. Respiratory acidosis alkalosis
B. Respiratory alkalosis Metabolic Ketoacidosis (produced by starvation or by
C. Metabolic acidosis acidosis DM), lactic acidosis, severe diarrhea (loss of
D. Metabolic alkalosis bicarbonate)
a.
Answer: ___________ Metabolic Treatment for peptic ulcer, vomiting
alkalosis
Which of the following is a cause of metabolic
In salicylate poisoning, what is the first acid-base
alkalosis?
disturbance present?
A. Late stage of salicylate poisoning
A. Metabolic acidosis
B. Uncontrolled diabetes mellitus
B. Metabolic alkalosis
C. Renal failure
C. Respiratory acidosis
D. Excessive vomiting
D. Respiratory alkalosis
Answer: ___________
Answer: ___________
✓ If the salicylate anion is ingested, RESPIRATORY ALKALOSIS OCCURS FIRST, followed by MILD
METABOLIC ACIDOSIS.

XII. ENDOCRINE HORMONES

a. Thyroid hormones
Functions:
✓ For tissue growth
✓ For mental development
✓ For CNS development
✓ Elevate heat production
✓ Control oxygen consumption
✓ Influence carbohydrate and protein metabolism
✓ Energy conservation

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CONDITION TSH FT3 FT4

Primary hyperthyroidism Decreased Increased Increased
Primary hypothyroidism Increased Decreased Decreased
Subclinical hyperthyroidism Decreased Normal Normal
Subclinical hypothyroidism Increased Normal Normal
Plummer’s Disease (T3 thyrotoxicosis) Decreased Increased Normal

In a patient with suspected primary hyperthyroidism associated with Graves’ disease, one would expect the
following laboratory serum results: T4 by radioimmunoassay __________, T3 resin uptake ____________
and thyroid stimulating hormone __________.
A. Increased, decreased, decreased
B. Decreased, increased, normal
C. Increased, decreased, increased
D. Increased, increased, decreased
Answer: ___________

In patients with primary hypothyroidism, TSH levels In patients with developing subclinical
will likely be _____________, and T4 will be likely hypothyroidism, TSH levels will likely be ___, and
________. fT4 will likely be ____.
A. Decreased, increased A. Decreased, increased
B. Increased, decreased B. Increased, decreased
C. Decreased, normal C. Decreased, normal
D. Increased, normal D. Increased, normal
Answer: ___________ Answer: ___________
b. Antidiuretic hormone (ADH)

✓ Major function: maintain osmotic homeostasis by regulating water balance

✓ Decreases production of urine by promoting reabsorption of water by distal convoluted and collecting
tubules of the kidneys
✓ Potent pressor agent and affects blood clotting by promoting factor VII release from hepatocytes and
von Willebrand factor release from endothelium

Antidiuretic hormone (ADH) or vasopressin acts on Effect(s) of an increase in antidiuretic hormone:

the: A. Fluid loss
A. Collecting duct B. Fluid retention
B. Henle’s loop C. Low serum sodium
C. Proximal convoluted tubule D. Both B and C
D. Bowman’s capsule Answer: ___________
Answer: ___________

HORMONAL CAUSES OF FLUID AND ELECTROLYTE IMBALANCE

INCREASED ADH Fluid retention, low serum sodium
DECREASED ADH Fluid loss, high serum sodium (in D.I)
INCREASED ALDOSTERONE Hypertension, low serum potassium
DECREASED ALDOSTERONE Low serum sodium, high serum potassium
INCREASED RENIN Hypertension

c. Cortisol

✓ Principal glucocorticoid
✓ Stimulates gluconeogenesis in the liver resulting in hyperglycemia (anti-insulin effect)
✓ Has anti-inflammatory and immunosuppressive actions
✓ Secretion is diurnal and associated with person’s sleep wake cycle
o Highest secretion in the morning (8-10AM)
o Lowest at night (10-12MN)
Condition ACTH Cortisol
Adrenal Cushing’s Syndrome Low High
Pituitary Cushing’s Syndrome (Cushing’s Disease) High High
Congenital Adrenal Hyperplasia High Low

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The secretion of cortisol is such that blood Peak for cortisol is in the Trough values at 10 to
specimens should be drawn: morning 8 to 9 AM 11 PM
A. In early morning Laboratory requests for cortisol assays should
B. In late evening include evaluation of both morning and late
C. Any time of the day or night evening samples to document the presence or
D. Both A and B absence of the cyclic production of this steroid.
Answer: ___________ Patients undergoing significant stress often lose the
rhythmic production and exhibit elevated cortisol
levels with no discernible cycle of synthesis.
d. Human Chorionic Gonadotropin (HCG)
✓ Produced by the trophoblast cells of the placenta during pregnancy
✓ Has an alpha subunit and a unique beta subunit
✓ It serves to maintain progesterone production by the corpus luteum during pregnancy
✓ Indicative of pregnancy/ trophoblastic disease

Quantification of human chorionic gonadotropin (HCG) is All of the following are associated with
useful in which of the following clinical findings? false positive urine HCG test, except:
1. Threatened abortions A. Increased protein
2. Hydatidiform moles B. Penicillin
3. Chroiocarcinoma C. Barbiturate
4. Testicular tumors D. Dilute urine
A. 1 and 3 Answer: ___________
B. 2 and 4
C. 1, 2 and 3
D. 1, 2, 3 and 4
Answer: ___________

False negative urine HCG assay Dilute urine

False positive urine HCG assay Increased amount of protein in urine
Phenothiazines, barbiturate, chlorpromazine, methadone, penicillin
False positive blood HCG assay Chorioepithelioma, hydatidiform mole, or excessive ingestion of
aspirin (Turgeon)

e. Prolactin
✓ Pituitary lactogenic hormone; stress hormone
✓ Functions in the initiation and maintenance of lactation
✓ Acts in conjunction with estrogen and progesterone to promote breast tissue development
✓ Inhibited by dopamine
✓ Excess prolactin causes hypogonadism
Select the hormone that is associated (a) Hypersecretion causes galactorrhea or lactation
with galactorrhea, pituitary adenoma and and is associated with infertility and amenorrhea in
amenorrhea. women and impotence in men. It usually is induced by
A. Estradiol pituitary adenoma.
B. Progesterone (b) Hyposecretion leads to the lack of lactation in
C. Follicle-stimulating hormone postpartum women.
D. Prolactin
Answer: ___________

f. Estrogen

The most potent estrogen and considered Estrone (E1) Most- abundant in post-
to be the true ovarian hormone is: menopausal women
A. Estrone Estradiol (E2) Most abundant in pre-
B. Estradiol menopausal women
C. Estriol Most potent, major estrogen
D. Epiestriol Estriol (E3) Major estrogen during pregnancy
Answer: _________

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g. Testosterone

The biologically most active, naturally Testosterone (the most potent of the androgens)
occurring androgen is:
✓ induces growth of the male reproductive
A. DHEA
B. Androstenedione system, prostate gland, and development of
C. Epiandrosterone male sex characteristics, including hypertrophy
D. Testosterone of the larynx and initiation of spermatogenesis.
Answer: _________

References:

Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2013). Clinical chemistry: principles,
techniques, and correlations. 7th ed. Philadelphia: Wolters Kluwer Health/Lippincott
Williams & Wilkins.

Henry, J. B., McPherson, R. A., & Pincus, M. R. (2011). Henry's clinical diagnosis and
management by laboratory methods. 22nd ed. Philadelphia, PA: Elsevier/Saunders.

Clinical Chemistry Handbook: For Medical Technology Review Students by Dean Maria
Teresa T. Rodriguez

No part of this material may be reproduced, distributed, or transmitted in any form or by any means, including
photocopying, or other electronic or mechanical methods, without the prior written permission of the owner.
Unauthorized distribution or reproduction is subject to law.