REVIEW NOTES IN CLINICAL CHEMISTRY Flashcards
: the act of obtaining a blood sample from a vein using a needle attached to a syringe or a stoppered evacuated tube; it is the most common way to collect blood specimens
VENIPUNCTURE
THE MAJOR VEINS FOR VENIPUNCTURE are in the antecubital fossa, the area of the arm in front of the elbow. The H pattern is displayed by approximately 70% of the population and includes the following veins:
Median
Cephalic
Basilic
Located near the center of the antecubital fossa
Median cubital vein
• Preferred vein because it is typically large, closer to the surface and the most stationary
Median cubital vein
Easiest and least painful to puncture
Median cubital vein
• Least likely to bruise
Median cubital vein
Second-choice vein
Cephalic vein
• Often harder to palpate than medial cubital vein
Cephalic vein
• Fairly well-anchored
Cephalic vein
• Often the only vein felt in obese patients
Cephalic vein
Last choice
Basilic vein
Not well anchored and rolls easily
Basilic vein
• Increased risk of puncturing a median cutaneous nerve branch or the brachial artery
Basilic vein
• Not recommended unless no other vein in either arm is more prominent
Basilic vein
OTHER VEINS
Although antecubital veins are used most frequently, veins on the [?] may also be used for venipuncture. Veins on the [?], however, should never be used for venipuncture. [?] are sometimes used but not without permission of the patient’s physician, due to a potential for significant medical complications.
back of the hand and wrist
underside of the wrist
Leg, ankle, and foot veins
• normally a clear, pale yellow fluid
Serum
• non-fasting serum can be cloudy due to lipids
Serum
• separated from clotted blood by centrifugation (approx. 10 minutes at an RCF of 1,000 to 2,000g)
Serum
• many chemistry tests are performed on serum
Serum
• normally a clear to slightly hazy, pale yellow fluid
Plasma
• separates from the cells when blood in an anticoagulant tube is centrifuged
Plasma
• contains fibrinogen (serum does not because it was used in clot formation)
Plasma
• Stat and other tests requiring a fast turnaround time (TAT) are often collected in tubes containing heparin anticoagulant because they can be centrifuged immediately to obtain plasma
Plasma
• contains both cells and plasma
Whole blood
• must be collected in an anticoagulant tube to keep it from clotting
Whole blood
• used for most hematology tests and many point-of-care tests (POCTs), especially in acute care and stat situations.
Whole blood
preferred method because blood is collected directly from the vein into a tube, minimizing the risk of specimen contamination and exposure to the blood.
Evacuated tube system (ETS)
• discouraged by CLSI due to safety and specimen quality issues
Needle and syringe
• sometimes used on small, fragile, or damaged veins
Needle and syringe
• can be used with the ETS or a syringe
Butterfly set
• often used to draw blood from:
Butterfly set
infants and children; hand veins; in other difficult-draw situations
Butterfly set
Fibrin degradation products
Light blue
Inhibits thrombin formation
Light green/black
Green
Tan (glass)
Royal blue (heparin)
Inhibits glycolysis
Gray
WBC preservative
Yellow
Clot activator
Yellow/gray and orange
Silica clot
Red/gray and gold
CAPILLARY PUNCTURE Length of lancet should be
less than 2.0 mm to avoid penetrating bone
CAPILLARY PUNCTURE Sites:
Palmar surface of 3rd and 4th fingers Lateral plantar heel surface (newborns)
Earlobes
Syringes are used instead of evacuated tubes because of the pressure in an arterial blood vessel.
ARTERIAL PUNCTURE
ARTERIAL PUNCTURE Preferred anticoagulant:
Heparin
Collect without a tourniquet
ARTERIAL PUNCTURE
ARTERIAL PUNCTURE Primary arterial sites (in order of preference):
radial, brachial and femoral arteries
ARTERIAL PUNCTURE Major complications of arterial puncture:
thrombosis, hemorrhage, and possible infection
done before the collecting arterial blood from radial artery
Modified Allen Test
to determine whether the ulnar artery can provide collateral circulation to the hand after the radial
arter puncture
Modified Allen Test
Albumin, ALP (↑older), phosphorus, cholesterol
Age
(↑older)
ALP
: Albumin, ALP, creatine, calcium, uric acid, CK, AST, PO4, BUN, magnesium, bilirubin, cholesterol
↑ males
: Fe, cholesterol, gamma-globulins, a-lipoproteins
↑ females
Peaks 4-6 AM; lowest 8 PM-12 AM; 50% lower at 8 PM than at 8 AM
Cortisol
Lower at night
ACTH, Plasma renin activity, Aldosterone, Insulin
Lower at night; higher standing than supine
Plasma renin activity
Higher in afternoon and evening
Acid phosphatase, Growth hormone
Higher levels at 4 and 8 AM and at 8 and 10 PM
Prolactin
Peaks early to late morning; decreases up to 30% during the day
Iron
≥20% for ALT, bilirubin, Fe, TSH, triglycerides
Day-to-day variation
↑ Glucose, insulin, triglycerides, gastrin, ionized calcium
Recent Food Ingestion
↓ chloride, phosphorus, potassium, amylase, ALP
Recent Food Ingestion
: albumin, cholesterol, aldosterone, calcium
↑ when standing
: CK
↑ in ambulatory patients
: lactic acid, creatine, protein, CK, AST, LD, thyroxine
↑ with exercise
: cholesterol and triglycerides
↓ with exercise
↑ACTH, cortisol, catecholamines, prolactin
Stress
: ↑TP, ↓ albumin
Black
: ↑CK/LD
Black males
: IgG ↑40% and IgA ↑20%
Black male vs white male
: ↑cholesterol and triglycerides
White & >40 years old
FBS, GTT, Triglycerides, lipid panel, gastrin, insulin, aldosterone/ renin
Require fasting
Lactic acid, ammonia, blood gas (if not cooled = ↓ pH and pO2)
Require Ice (Immediate Cooling)
↑ potassium, PO4, Fe, magnesium, ALT, AST, LD, ALP, catecholamines, CK
Hemolysis
CK
(marked hemolysis)
: method of determining the concentration of substance in solution by measuring the amount of light absorbed by that solution after appropriate treatment.
SPECTROPHOTOMETRY
- photons travelling in waves
Electromagnetic radiation
- distance between two peaks
Wavelength
- distance between peak and trough
Amplitude
Visible light:
400-700nm
states that the concentration of a substance is directly proportional to the amount of light absorbed or
inversely proportional to the logarithm of the transmitted light
BEER-LAMBERT LAW (BEER’S LAW)
Light Source
= most common source of light for work in the visible and near-infrared regions
a. Incandescent tungsten or tungsten-iodide lamp
= most commonly used for ultraviolet (UV) work
Deuterium - discharge lamp & Mercury - arc lamp
= most commonly used Monochromator
Diffraction gratings
= advantage over round cuvets in that there is less error from the lens effect, orientation in the spectrophotometer, and refraction.
Square
= used for applications in the visible range
Glass
= for applications requiring UV radiation.
Quartz
= least expensive
Barrier - layer cell or photo cell
= used in instruments designed to be extremely sensitive to very low light levels and light flashes of very short duration
Photomultiplier (PM) tube
PRINCIPLE: measures the quantity of light reflected by a liquid sample that has been dispensed onto a grainy or fibrous solid support
REFLECTOMETRY
COMPONENTS are very similar to those of a photometer
REFLECTOMETRY
APPLICATION:
urine dipstick analysis
dry slide chemical analysis
REFLECTOMETRY
PRINCIPLE: measurement of concentration is done by detecting the absorption of electromagnetic by atoms rather than molecules. When a ground-state atom absorbs light energy, an excited atom is produced. The excited atom ther returns to the ground state, emitting light of the same energy as it absorbed.
ATOMIC ABSORPTION SPECTROPHOTOMETRY
COMPONENTS
Hollow-cathode lamp
Flame
Monochromator
ATOMIC ABSORPTION SPECTROPHOTOMETRY
- usual light source
• Hollow-cathode lamp
- breaks chemical bonds and form free, unexcited atoms; serves as sample cells (instead of a cuvet)
Flame
- used to isolate the desired wavelength; also protects photodetector from excessive light emanating from flame emissions.
Monochromator
APPLICATION: measurement of unexcited trace metals e.g. calcium and magnesium
ATOMIC ABSORPTION SPECTROPHOTOMETRY
APPLICATION: measurement of unexcited trace metals e.g. calcium and magnesium
ATOMIC ABSORPTION SPECTROPHOTOMETRY
PRINCIPLE: measurement of light emitted by excited atoms
FLAME PHOTOMETRY
APPLICATION: Widely used before to determine the concentration of Na*, K+ or Lit
FLAME PHOTOMETRY
PRINCIPLE: measurement of the concentration of solutions that contain fluorescing molecules
FLUOROMETRY
COMPONENTS
Xenon lamp - most common light source
FLUOROMETRY
APPLICATION: is used to measure small particles, such as drugs.
FLUOROMETRY
PRINCIPLE: Chemical energy generated in a chemiluminiscent reaction produces excited intermediates that decay a ground state with the emission of photons; no excitation is required unlike in fluorometry
CHEMILUMINESCENCE
PRINCIPLE: measurements are made with a spectrophotometer to determine concentration of particulate matter in sample. The amount of light blocked by a suspension of particles depends not only on concentration but also on size.
TURBIDIMETRY
APPLICATIONS
1. Detection of bacterial growth and bacterial culture
2. Antibiotic sensitivity
3. Coagulation studies
4. Protein concentration in CSF and urine
TURBIDIMETRY
PRINCIPLE: light scattered by small particles is measured at an angle to the beam incident to the cuvet
NEPHELOMETRY
measure particles which are too large for spectrophotometry, such as antibody-antigen complexes formed in enzyme immunoassays.
NEPHELOMETRY
PRINCIPLE: involves measurement of the current or voltage generated by the activity of specific ions. techniques include potentiometry, coulometry, voltammetry, and amperometry.
ELECTROCHEMISTRY
Measurement of potential (voltage) between two electrodes in a solution to measure analyte concentration
Potentiometry
pH, pCO2, Nat, Ca?, K, NH4*
Potentiometry
measurement of the current flow produced by an oxidation-reduction reaction
Amperometry
pOz (Clark electrode), glucose, peroxidase
Amperometry
Electrochemical titration in which the titrant is electrochemically generated
Coulometry
Cl
Coulometry
Potential is applied to an electrochemical cell and the resulting current is measured
Voltammetry
Anodic stripping voltametry (for lead and iron)
Voltammetry
PRINCIPLE: separation of charged compounds based on their electrical charge
ELECTROPHORESIS
COMPONENTS
1. A driving force (electrical power)
2. Support more (electrical power)
a. Filter paper
b. Agarose
c. Cellulose acetate
d. Polyacrylamide
3. Buffer
4. Sample
5. Detecting system
ELECTROPHORESIS
: movement of buffer ions and solvent relative to the fixed support
ELECTROENDOSMOSIS
- most common and reliable way for quantitation of separated protein fractions
DENSITOMETRY
PRINCIPLE: separation of complex mixtures on the basis of different physical attractions between the individual compounds and the stationary phase of the system
CHROMATOGRAPHY
COMPONENTS
1. Mobile phase (gas or liquid)
2. Stationary phase (solid or liquid)
3. Column
4. Eluate
CHROMATOGRAPHY
: carries the complex mixture
- Mobile phase (gas or liquid)
: substance through which the mobile phase flows
- Stationary phase (solid or liquid)
: holds the stationary phase
- Column
: separated components
- Eluate
CHROMATOGRAPHIC PROCEDURES
- Thin-Layer Chromatography
- High-Performance Liquid Chromatography (HPLC)
- Gas Chromatography (GC)
- uses pressure for faster separations
- High-Performance Liquid Chromatography (HPLC)
- separate mixtures of compounds that are volatile or can be made volatile
- Gas Chromatography (GC)
PRINCIPLE: Sample in a MS is first volatilized and then ionized to form charged molecular ions and fragments that are separated according to their mass-to-charge (m/Z) ratio
MASS SPECTROMETRY
Allows definitive identification when used on samples eluting from GC or HPLC
MASS SPECTROMETRY
Gold standard for drug testing when coupled with GC
MASS SPECTROMETRY
PRINCIPLE
Two-step procedure: (1) MALDI, then (2) Time-of-Flight (TOF) Mass
MALDI-TOF MS (Matrix-Assisted Laser Desorption lonization Time-of-Flight) analysis
• A laser pulse irradiates the sample, causing desorption and ionization of both the matrix and the sample.
MALDI-TOF MS (Matrix-Assisted Laser Desorption lonization Time-of-Flight) analysis
• lons from the sample are focused into the mass spectrometer.
MALDI-TOF MS (Matrix-Assisted Laser Desorption lonization Time-of-Flight) analysis
The molecular weight of the proteins acquired by mass spectrometry is used to determine the identity of the sample and is helpful in determining posttranslational modifications that may have occurred.
MALDI-TOF MS (Matrix-Assisted Laser Desorption lonization Time-of-Flight) analysis
APPLICATION: used for the analysis of biomolecules, such as peptides and proteins
MALDI-TOF MS (Matrix-Assisted Laser Desorption lonization Time-of-Flight) analysis
- process by which lab ensures quality results by closely monitoring preanalytical, analytical, & postanalytical stages of testing.
QUALITY ASSURANCE
- everything that precedes test performance,
• Preanalytical
e.g., test ordering, patient preparation, patient ID, specimen collection, specimen transport, specimen processing.
• Preanalytical
- everything related to assay,
• Analytical
e.g., test analysis, quality control (QC), reagents, calibration,
preventive maintenance.
• Analytical
- everything that comes after test analysis, e.g., verification of calculations & reference ranges, review of results, notification of critical values, result reporting, test interpretation by physician, follow-up patient care.
• Postanalytical
- part of analytical phase of quality assurance
QUALITY CONTROL (QC)
process of monitoring results from control samples to verify accuracy of patient results.
QUALITY CONTROL (QC)
- most frequently used measure of variation
b. Standard deviation (SD)
- an index of precision used to compare the dispersion of two or more groups of data with different units / concentrations
c. Coefficient of variation (CV)
- used to determine if there is a significant difference between the MEANS of two groups of data
T-test
- used to determine if there is a significant difference between the SD of two groups of data
F-test
describes many continuous laboratory variables and
deviation shares several unique characteristics
The Gaussian Distribution (Normal Distribution)
the mean, median, and mode are [?];
the distribution is [?] — meaning half the values fall to the left of the mean, and the other half fall to the right (the symmetrical shape is often referred to as a “bell curve.”)
The total area under the gaussian curve is [?].
identical
symmetric
1.0, or 100%
summarizes the above relationships between the area under a Gaussian distribution and the SD.
“68-95-99 Rule”
“68-95-99 Rule”
In other words, given any Gaussian distributed data
of the data fall between ‡1 SD from the mean
of the data fall between +2 SDs from the mean
fall between ‡3 SDs from the mean
68%
295%
99%
- nearness or closeness of assayed values to the true value
- Accuracy
- nearness or closeness of assayed values to each other
- Precision (Reproducibility)
- ability of an analytical method to maintain accuracy and precision over an extended period of time
- Reliability
- degree by which a method can easily be repeated
- Practicability
- ability to measure the smallest concentration of the analyte of interest
- Analytical sensitivity
- ability to measure only the analyte of interest
- Analytical specificity
- also known as linearity; range of values over which lab can verify accuracy of test system
- Reportable range
Formerly called normal value.
- Reference interval
Can vary for different patient populations (age, gender, race).
- Reference interval
Established by testing minimum of 120 healthy subjects & determining range in which 95% fall.
- Reference interval
Verifying a reference interval (transference) can required as few as 20 study individuals
- Reference interval
Reporting a positive result in a patient who has the disease
True positive (TP)
Reporting a positive result in a patient who doesn’t have the disease
False positive (FP)
Reporting a negative result in a patient who doesn’t have the disease
True negative (TN)
Reporting a negative result in a patient who has the disease
False negative (FN)
% of population with the disease that test positive
Diagnostic sensitivity
ability of the analytical method to detect the proportion of individuals with the disease
Diagnostic sensitivity
% of population without the disease that test negative
Diagnostic specificity
ability of the analytical method to detect the proportion of individuals without the disease
Diagnostic specificity
Number individuals without the disease with a negative test × 100%
Diagnostic Specificity (%)
% of time that a positive result is correct
Positive predictive value (PPV)
% of time that a negative result is correct
Negative predictive value (NPV)
• Assayed on a regular schedule to verify that a laboratory procedure is performing correctly
QC SAMPLES
• Generally, two different concentrations are necessary for adequate statistical QC
QC SAMPLES
• Chemically & physically similar to unknown specimen & is tested in exactly the same manner
QC SAMPLES
• New instrument or new lot of reagents: analyze QC materials for 20 days
QC SAMPLES
CHARACTERISTICS OF IDEAL QC MATERIALS
- Must resemble human samples
- Inexpensive and stable for long periods
- No communicable disease
- No known matrix effects
- With known analyte concentrations (for assayed controls)
- Convenient packaging for easy dispensing and storage
Also called a Shewart plot
LEVEY-JENNINGS CONTROL CHART
• Most common presentation for evaluating QC results
LEVEY-JENNINGS CONTROL CHART
shows each QC result sequentially over time
LEVEY-JENNINGS CONTROL CHART
- control values increasing or decreasing for six consecutive runs
Trend
Trend Main cause:
DETERIORATION OF REAGENTS
- six consecutive control values on the same side of the mean
Shift
Shift Main cause:
IMPROPER CALIBRATION OF INSTRUMENT
- highly deviating values
Outliers
control result outside established limits
Outliers
1 control >‡ 2s from mean.
1(2S)
1 control >‡ 2s from mean.
1(2S)
Warning flag of possible change in accuracy or precision.
1(2S)
Initiates testing of other rules (warning rule). If no violation of other rules, run is considered in control.
1(2S)
1 control >‡ 3s from mean
1(3S)
2 consecutive controls >2s from mean on same side
2(2S)
Random; Rejection rule
Systematic; Rejection rule
2 consecutive controls differ by >4s
R(4S)
4 consecutive controls > 1s from mean on same side
4(1S)
10 consecutive controls on same side of mean
10x
Present in all measurements; due to chance; no means of predicting it
- Random error
Error that doesn’t recur in regular pattern
- Random error
Associated with violations of the 12s, 13s and R4s Westgard rules
- Random error
• Error that influences ALL observations consistently in one direction
- Systematic error
Recurring error inherent in test procedure
- Systematic error
• Associated with violations of the 22s and 41s Westgard rules
- Systematic error
• Also known as external quality assessment
PROFICIENCY TESTING
• consists of evaluation of method performance by comparison of results versus those of other
PROFICIENCY TESTING
• laboratories for the same set of samples
PROFICIENCY TESTING
PROFICIENCY TESTING • Basic procedure:
PT providers circulate a set of samples among a group of
laboratories.
Each laboratory includes the PT samples along with patient samples in the usual assay process.
Results for the PT samples are reported to the PT provider for evaluation.
Error due to dirty glassware
RANDOM ERRORS
Dirty photometer
SYSTEMATIC ERRORS
Use of wrong pipet
RANDOM ERRORS
Faulty ISE
SYSTEMATIC ERRORS
Voltage fluctuation
RANDOM ERRORS
Evaporation or contamination of standards or reagents
SYSTEMATIC ERRORS
Sampling error
RANDOM ERRORS
Anticoagulant or drug interference
RANDOM ERRORS
• Comparison of patient data with previous results.
Delta checks
• Detects specimen mix-up & other errors.
Delta checks
• When limit is exceeded, must determine if due to medical change in patient or lab error.
Delta checks
Test results that indicate a potentially life-threatening situation.
Critical values
List typically includes glucose, Na+, K+, total CO2, Ca2+, Mg2+, phosphorus, total billrubin (neonates), blood gases
Critical values
• Patient care personnel must be notified immediately.
Critical values
Critical values• Example:
Serum glucose
<40 mg/dL
>500 mg/dL
Schedule of maintenance to keep equipment in peak operating condition; must be documented & must follow manufacturer’ s specifications & frequencies.
Preventive maintenance
CARBOHYDRATES
1. Contain C, H and 0; Empiric formula: (CH20)n
3. Can be reducing or non-reducing sugars ; Can be classified according to the number of sugar units
CARBOHYDRATES
CARBOHYDRATES Functions:
Major energy source (?)
Storage form of energy e.g. [?]
Components of cell membranes e.g. [?]
Structural component in plants, bacteria, insects (e.g. ?)
glucose
glycogen
glycoproteins
chitin, cellulose
- one sugar unit e.g. glucose, fructose, galactose
- 2 sugar units linked together by a glycosidic bond e.g. sucrose, lactose, maltose
- 3 to 10 sugar units
- more than 10 sugar units (e.g. starch, glycogen, cellulose)
a. Monosaccharides
b. Disaccharides
Oligosaccharides
d. Polysaccharides
Glucose + fructose
Sucrose-Sucrase
Glucose + galactose
Lactose-Lactase
Glucose + glucose
Maltose-Maltase
Metabolism of glucose molecule to pyruvate or lactate for production of energy
Glycolysis
Formation of glucose-6-phosphate from non-carbohydrate sources
Gluconeogenesis
Breakdown of glycogen to glucose for use as energy
Glycogenolysis
Conversion of glucose to glycogen for storage
Glycogenesis
Conversion of carbohydrates to fatty acids
Lipogenesis
Decomposition of fat
Lipolysis
Carbohydrates in the diet constitute about 50% of the calories in the average diet:
- 60%
- 30% C.
- 5%
- 5%
(part of dietary fiber)
a. Starch and dextrins
b. Sucrose
C. Lactose
d. Other sugars
e. Cellulose
breakdown polymers to dextrins and disaccharides.
Salivary amylase (ptyalin) and pancreatic amylase (amylopsin)
are further hydrolyzed into monosaccharides by specific enzymes (disaccharidases)
Disaccharides
are absorbed by the gut via active transport (glucose and galactose) or facilitated diffusion (fructose).
Monosaccharides
They are then transported into the liver through the portal circulation.
Monosaccharides
is the only carbohydrate to be used directly for energy.
Glucose
After glucose enters the cell, it undergoes phosphorylation into glucose-6-phosphate through the action of
hexokinase or glucokinase.
Glucose-6-phosphate is then shunted into the following metabolic pathways:
a. Glycolysis (Embden-Meyerhof pathway)
b. Glycogenesis
c. Hexose-Monophosphate shunt
• Produced by the beta cells of the islets of Langerhans (pancreas)
Insulin
→ insulin
• Preproinsulin → proinsulin
• Target: most cells of the body
Insulin
Increases utilization of glucose by the cells by increasing cellular uptake and hepatic glycolysis
Insulin
Increases glycogenesis and inhibits glycogenolysis; Inhibits gluconeogenesis
Insulin
Stimulates lipogenesis while inhibiting lipolysis
Insulin
Stimulates protein synthesis and stimulates uptake of amino acids into muscles
Insulin
• Produced by the alpha cells of the islets of Langerhans
GLUCAGON
• Target: liver
GLUCAGON
• Target: liver
GLUCAGON
Promotes liver glycogenolysis
GLUCAGON
Increases gluconeogenesis
GLUCAGON
Inhibits glycolysis
GLUCAGON
Increases gluconeogenesis
Cortisol (Glucocorticoids)
Decreases glucose uptake and utilization by extrahepatic tissues
Cortisol (Glucocorticoids)
Stimulates glycogenolysis
Catecholamines
Increases glucose absorption in the small intestines
Thyroid hormone
Inhibit glucagon and insulin secretion
Somatostatin
Increases liver gluconeogenesis
Growth hormone
Inhibits glucose transport
Growth hormone
- heterogeneous group of multifactorial, polygenic syndromes characterized by an elevated fasting blood glucose caused by a relative or absolute deficiency in insulin
DIABETES MELLITUS
- characterized by an absolute deficiency of insulin caused by an autoimmune attack on the beta cells of the pancreas
- Type 1 DM
- characterized by a combination of insulin resistance and dysfunctional beta cells
- Type 2 DM
Juvenile Onset DM; Insulin Dependent DM; Most common in children and young adults
TYPE 1 DIABETES
Adult Onset DM; Non-insulin Dependent DM; Most common with advancing age
TYPE 2 DIABETES
5-10%
TYPE 1 DIABETES
90-95%
TYPE 2 DIABETES
Genetic, autoimmune, environmental (e.g. viral infection)
TYPE 1 DIABETES
HLA DR3/4 ; Autoantibodies
-Anti-islet cell cytoplasmic antibody
-Insulin autoantibodies
-Anti-GAD (glutamic acid decarboxylase)
TYPE 1 DIABETES
Genetic, obesity, sedentary lifestyle, race/ethnicity
TYPE 2 DIABETES
Destruction of pancreatic beta cells, usually autoimmune
TYPE 1 DIABETES
No autoimmunity; Insulin resistance and progressive insulin deficiency
TYPE 2 DIABETES
Very low or undetectable c-peptide
TYPE 1 DIABETES
Detectable c-peptide
TYPE 2 DIABETES
Low to absent plasma insulin
TYPE 1 DIABETES
High in early disease; low to absent in disease of long duration plasma insulin
TYPE 2 DIABETES
Prone to ketoacidosis and diabetic complications
TYPE 1 DIABETES
Not prone to ketoacidosis
TYPE 2 DIABETES
Insulin absolutely necessary; muliple
TYPE 1 DIABETES
Oral agents (insulin sometimes daily injections or insulin pump indicated)
TYPE 2 DIABETES
None known therapy
TYPE 1 DIABETES
Lifestyle, oral medicines
TYPE 2 DIABETES
- associated with secondary conditions
E.g. genetic defects of beta cell function; pancreatic disease; endocrine disease; drug or chemical induced; insulin receptor abnormalities; other genetic syndromes
Other specific types of DM
Glucose intolerance with onset or first recognition during pregnancy
Gestational Diabetes Mellitus (GDM)
• Due to metabolic and hormonal changes
Gestational Diabetes Mellitus (GDM)
• Large % of patients develop DM Within 5 to 10 years
Gestational Diabetes Mellitus (GDM)
• Infants born to mothers with diabetes are at increased risk for RDS, hypocalcemia, hyperbilirubinemia and other complications
Gestational Diabetes Mellitus (GDM)
• Screening: 2-hour OGTT using a 75 g glucose load
Gestational Diabetes Mellitus (GDM)
Random plasma glucose
≥200 mg/dL (211.1 mmol/L), +symptoms of DM
Fasting plasma glucose
≥126 mg/dL (27.0 mmol/L)
Two-h plasma glucose
≥200 mg/dL (≥11.1 mmol/L)
N.B. In absence of unequivocal hyperglycemia, these criteria should be confirmed by repeat testing on a different day. The third measure (OGTT) is not recommended for routine clinical use.
Normal Fasting plasma glucose
<100 mg/dL
<5.6 mmol/L
Normal 2-hour plasma glucose level (after 75 g load)
<140 mg/dL
<7.8 mmol/L
Pre-diabetes HbA1c
5.7-6.4 %
Impaired fasting glucose Fasting plasma glucose
100-125 mg/dL
5.6-6.9 mmol/L
Impaired glucose tolerance 2-hour plasma glucose level (after 75 g load)
140-199 mg/dL
7.8-11.0 mmol/L
Diabetes mellitus Fasting plasma glucose
≥126 mg/dL
≥7.0 mmol/L
Diabetes mellitus 2-hour plasma glucose level (after 75 g load)
≥200 mg/dL
≥11.1 mmol/L
Diabetes mellitus HbA1c
≥6.5%
Use: detection of GDM
ORAL GLUCOSE TOLERANCE TEST (OGTT)
Before an OGTT is performed, individuals should ingest [?]
preceding the test.
At least 150g/day of carbohydrates for the 3 days (no restriction of diet)
No limitation in physical activity
ORAL GLUCOSE TOLERANCE TEST (OGTT)
Test should be performed after an overnight 8- to 14-hour fast.
ORAL GLUCOSE TOLERANCE TEST (OGTT)
The individual should not eat food, drink tea, coffee, or alcohol, or smoke cigarettes during the test, and should be seated.
ORAL GLUCOSE TOLERANCE TEST (OGTT)
Venous glucose samples are preferably collected in gray-top tubes containing fluoride and an anticoagulant (Henry’s, 23rd ed)
ORAL GLUCOSE TOLERANCE TEST (OGTT)
FBG is measured right before the administration of the glucose load. A FBG of greater than 140 mg/dL necessitates that the test be stopped immediately. Proceed with the glucose load if FBG is less than 140 mg/dL.
ORAL GLUCOSE TOLERANCE TEST (OGTT)
ORAL GLUCOSE TOLERANCE TEST (OGTT)
• Glucose load for adults
• Glucose load for children
• Pregnant women
75 g
1.75g/kg bw (max: 75 g)
75 g or 100 g
Patient should finish glucose load within 5 - 15 minutes.
ORAL GLUCOSE TOLERANCE TEST (OGTT)
ORAL GLUCOSE TOLERANCE TEST (OGTT)
Patient should NOT vomit. If patient vomits, [?]
Discontinue the test
DIAGNOSIS OF GESTATIONAL DIABETES MELLITUS
Gestational diabetes mellitus is diagnosed if [?] plasma glucose levels are exceeded (American Diabetes Association, 2004)
≥2
• Rate of formation of Hbac is proportional to the average blood glucose concentration over the previous 3 months
GLYCOSYLATED HEMOGLOBIN (HbA1c)
• For every 1% increase in Hba1c, there is a corresponding 35mg/dL change in plasma glucose
GLYCOSYLATED HEMOGLOBIN (HbA1c)
• The ADA also recommends that it be tested at least twice a year to monitor long-term glycemic control.
GLYCOSYLATED HEMOGLOBIN (HbA1c)
• Spn: EDTA-WB → hemolysate
GLYCOSYLATED HEMOGLOBIN (HbA1c)
• False decrease: decreased RBC lifespan
GLYCOSYLATED HEMOGLOBIN (HbA1c)
• Monitoring glucose control over past 2-3 weeks
FRUCTOSAMINE (Glycated Albumin)
• Albumin has a life span of 20 days in circulation
FRUCTOSAMINE (Glycated Albumin)
• Affected by albumin levels; false decrease in patients with hypoalbuminemia
FRUCTOSAMINE (Glycated Albumin)
100 g OGTT plasma glucose/75 g OGTT plasma glucose
Fasting
≥95 mg/dL
≥5.3 mmol/L
100 g OGTT plasma glucose/75 g OGTT plasma glucose
1 hour
≥180 mg/dL
≥10.0 mmol/L
100 g OGTT plasma glucose/75 g OGTT plasma glucose
2 hour
≥ 155 mg/dL
≥8.6 mmol/L
100 g OGTT plasma glucose
3 hour
≥140 mg/dL
≥7.8 mmol/L
HYPOGLYCEMIA
The plasma glucose concentration at which glucagon and other glycemic factors are released is between [?]. At about [?], observable symptoms of hypoglycemia appear. Warning signs and symptoms are all related to the central nervous system
65 and 70 mg/dL
50 to 55 mg/dL
HYPOGLYCEMIA
Most causes are secondary to other illnesses and resolve themselves when the primary disorder is treated.
Examples:
Insulinoma
Various liver disorders
Gastrointestinal disorders and surgery
HYPOGLYCEMIA
Possible specimens =
WB, serum, plasma, urine, CSF, serous fluid, synovial fluid
HYPOGLYCEMIA Standard clinical specimen
Fasting venous plasma
HYPOGLYCEMIA Fasting blood sugar should be obtained after
8 - 10 hours of fasting
HYPOGLYCEMIA Whole blood glucose levels
10-15% lower vs plasma levels
HYPOGLYCEMIA Glucose is metabolized at room temperature at a rate of
7 mg/dl/hour
HYPOGLYCEMIA At 4°C, glucose decreases by approximately
2 mg/dl/hour
HYPOGLYCEMIA Evacuated tube
Gray top (NaF)
HYPOGLYCEMIA CSF glucose levels
60-70% of plasma levels (decreased in bacterial meningitis)
HYPOGLYCEMIA As little as 10% contamination with 5% dextrose (D5W) will elevate glucose in a sample by
500 mg/dL or more
Principle: Glucose and other carbohydrates are capable of converting cupric ions in alkaline solution to cuprous ions.
Chemical Methods
GLUCOSE: Chemical Methods
a. Oxidation-reduction method
i. Alkaline Copper Reduction Method
Folin-Wu
Nelson-Somogyi
Neocuproine method
Benedict’s method
ii. Alkaline Ferric Reduction Method (Hagedorn-Jensen)
a. Condensation method (Dubowski)
GLUCOSE: Enzymatic Methods
a. Glucose oxidase method
i. Colorimetric method
ii. Polarographic method
b. Hexokinase method
is the most specific enzyme reacting with only B-D-glucose
Glucose oxidase
- uses a side reaction that consumes H202
i. Colorimetric method
- measure the rate of disappearance of oxygen using an oxygen electrode
ii. Polarographic method
• More accurate than glucose oxidase methods because the coupling reaction using G6PDH is highly specific; therefore it has less interference than the coupled glucose oxidase procedure
b. Hexokinase method
• NADPH has a strong absorbance at 340 nm
b. Hexokinase method
Generally accepted as the reference method
b. Hexokinase method
• Not affected by ascorbic acid or uric acid
b. Hexokinase method
Most common cause of lactose intolerance
Lactase deficiency
Lactose is not digested at a normal rate and accumulates in the gut, where it is metabolized by bacteria. Bloating, abdominal cramps, and watery diarrhea result
Lactase deficiency
result of the deficiency of a specific enzyme that causes an alternation of glycogen metabolism
Glycogen storage diseases
Glycogen storage diseases Most common congenital form:
Von Gierke Disease
i. Autosomal recessive disease
Von Gierke Disease
ii. Characterized by hepatomegaly, severe hypoglycemia, metabolic acidosis, ketonemia, and elevated lactate and alanine
Von Gierke Disease
• Building blocks of lipids
FATTY ACIDS
• Hydrocarbon chains with a terminal COO- group
FATTY ACIDS
• 3 fatty acid molecules attached to one molecule of glycerol by ester bonds
TRIGLYCERIDES
• Serves as main storage form of energy, insulator, shock absorber and integral part of cell membrane
TRIGLYCERIDES
• Similar to triglycerides except that the third position on the glycerol backbone contains a phospholipid head group
PHOSPHOLIPIDS
• Contains polar and non-polar end
PHOSPHOLIPIDS
• Constituent of cell membranes
PHOSPHOLIPIDS
• Serves as part of cell membranes and as parent chain for cholesterol-based hormones, e.g. aldosterone, cortisol and the sex hormones
CHOLESTEROL
CHOLESTEROL• Exists in two forms
- approximately 70% of total cholesterol
- approximately 30% of total cholesterol
• Cholesterol esters
• Free cholesterol
Typically spherical in shape with sizes ranging from 10 to 1200 nm
LIPOPROTEIN
Composed of lipids and proteins, called apolipoproteins
LIPOPROTEIN
Size particle correlates with its lipid content
LIPOPROTEIN
originally separated through ultracentrifugation
LIPOPROTEIN
• located on the surface of lipoprotein particles maintain structural integrity of lipoproteins
Apolipoproteins
• serve as ligands for cell receptors
Apolipoproteins
Apolipoproteins• Important types:
- largest
-least dense - highest TG content
- postprandial turbidity
- fxn: transports exogenous / dietary triglycerides
CHYLOMICRONS
- 2nd largest
-2nd least dense - 2nd highest TG content
- fasting hyperlipidemic turbidity
- fxn: transports endogenous/hepatic triglycerides
VERY LOW DENSITY LIPOPROTEIN (VLDL)
- small –> can cross BV walls
–> deposition of lipid - highest cholesterol content
- fxn: transports cholesterol to peripheral tissues
→inc LDL –> in atherosclerosis - target for cholesterol lowering therapy
LOW DENSITY LIPOPROTEIN (LDL)
- smallest but densest
- highest protein content
- fxns: reverse transport cholesterol (peripheral tissues –> liver)
- inc HDL –> dec atherosclerosis
HIGH DENSITY LIPOPROTEIN (HDL)
• Chylomicrons accumulate as a floating “cream” layer and can be detected visually. The presence of chylomicrons in fasting plasma is considered to be abnormal.
Standing Plasma Test
• A plasma sample that remains turbid after standing overnight contains excessive amounts of VLDL; if a floating
“cream” layer also forms, chylomicrons are present as well. (Henry, 23rd ed)
Standing Plasma Test
Floating Beta-lipoprotein
- Beta-VLDL
Increased in familial dysbetalipoproteinema
- Beta-VLDL
• Sinking pre-beta lipoprotein
- Lp(a)
• LDL - like particle
- Lp(a)
Increased risk of premature coronary heart disease and stroke
- Lp(a)
Seen in patient with biliary cirrhosis or cholestasis and in patients with mutations in the enzyme lecithin: cholesterol acyltransferase (LCAT)
- Lpx
happens in the intestines
Absorption Pathway
CM transports exogenous TG
Exogenous Pathway
VLDL and hDL transports endogenous TG
Endogenous Pathway
LDL transports Cholesterol
Reverse Cholesterol Transport Pathway
was used to characterize lipid disorders; used electrophoresis and a standing plasma test for CM to correlate clinical disease syndromes with laboratory phenotypes. Note that each phenotype is not a specific disease but rather a variety of disorders that affect the same lipoproteins and therefore express the same lipid pattern.
FREDRICKSON CLASSIFICATION OF LIPID DISORDERS
-Hyperchylomicronemia
-Familial LPL deficiency
Type 1
-Familial Hypercholesterolemia
Type 2a
-Familial Combined Hyperlipidemia
Type 2b
Familial Dysbetalipoproteinemia
Type 3
Familial Hypertriglyceridemia
Type 4
Low cardiac risk; eruptive xanthoma; recurrent pancreatitis
Type 1
High cardiac risk; xanthelasma; tendon xanthoma; corneal arcus; hypothyroidism and nephrotic syndrome
Type 2a
High cardiac risk
Type 2b
Eruptive and palmar xanthomas
Type 3
Low cardiac risk
Type 4
Low cardiac risk; eruptive xanthoma, may be associated
with pancreatitis
Type 5
Fasting = 12 hours before venipuncture
LIPIDS AND LIPOPROTEINS
• Can be measured non-fasting = TC and HDL-C
LIPIDS AND LIPOPROTEINS
• Prolonged tourniquet application = causes hemoconcentration
LIPIDS AND LIPOPROTEINS
Reclined patients = decreased values
LIPIDS AND LIPOPROTEINS
Preferred sample = Serum or plasma but plasma preferred in electrophoresis and ultracentrifugation
LIPIDS AND LIPOPROTEINS
Capillary blood samples = generally lower values
LIPIDS AND LIPOPROTEINS
Lipemic samples = seen when triglyceride levels exceed 4.6 mmol/L (400 mg/dL).
LIPIDS AND LIPOPROTEINS
Initial extraction with zeolite to remove sterols
- Abell-Kendall method
Redissolving of cholesterol
- Abell-Kendall method
Hydrolysis of cholesterol esters to cholesteriol
- Abell-Kendall method
Liebermann-Burchard reagent
glacial acetic acid
sulfuric acid
acetic anhydride
(+) formation of product which strongly absorbs at 410 nm
- Abell-Kendall method
Recently, the reference method has changed to a [?] that now specifically measures cholesterol and does not detect related sterols. (Bishop 7h ed.)
GC-MS method
- definitive method
- Isotope Dilution Mass Spectrometry (IDMS)
Hydrolysis of glycerol is accomplished using alcoholic KOH
- Chemical methods (triglyceride)
Oxidation of glycerol by periodic acid, forming formaldehyde and formic acid
- Chemical methods (triglyceride)
Formaldehyde combines with a variety of reagents:
- Chemical methods (triglyceride)
Reagent: Chromotropic acid → blue colored compound
a. Van Handel & Zilversmit (Colorimetric method)
Reagent: acetylacetone (aka diacetyl acetone; reactant of choice)
Hantzsch (Fluorometric method)
Product has a strong absorption maximum at 412 nm and also has good fluorescence
Hantzsch (Fluorometric method)
- new reference method for triglyceride measurements; involve the hydrolysis of fatty acids on triglycerides and the measurement of glycerol.
GC-MS method
: range in density observed among lipoprotein classes is a function of lipid and protein content and enables fractionation by density using ultracentrifugation
- Ultracentrifugation
: takes advantage of differences in size and charge
- Electrophoresis
: depends on particle size, charge and differences in the
apolipoprotein content; primarily used in research labs only
- Chemical precipitation
Uses polyanions (heparin sulfate, dextran sulfate and phosphotungstate) and divalent cations such as magnesium, calcium andmanganese e.g. HDL - dextran sulfate + magnesium
- Chemical precipitation
: uses antibodies specific to apolipoproteins to bind and separate lipoprotein classes
- Immunoassays
: takes advantage of size differences in molecular sieving methods or composition in affinity methods e.g. gel chromatography or affinity chromatography
- Chromatographic methods
• The term [Plasma TG]/5 is used when concentrations are expressed in mg/dL.
K. FRIEDEWALD CALCULATION
It has been reported that the factor [Plasma TG]/2.825 gives a more accurate estimate of VLDL-C (DeLong, 1986). This is equivalent to Plasma TG/6.5, when concentrations are expressed in mg/dL.
K. FRIEDEWALD CALCULATION
a. Involves large- and medium-sized arteries (e.g. abdominal aorta, coronary artery, popliteal artery, internal carotid artery)
- Atherosclerosis
b. LDL - increased risk ; HDL - decreased risk
- Atherosclerosis
c. Complications
Narrowing of blood vessels result in impaired blood flow and ischemia leading to:
(i) Peripheral vascular disease
(ii) Angina
(iii) Ischemic bowel disease
- Atherosclerosis
Plaque rupture → thrombosis → myocardial infarction and stroke
Plaque rupture → embolization atherosclerotic embolism
Weakening of blood vessel wall results in aneurysm
- Atherosclerosis
An extreme form of hypoalphalipoproteinemia (isolated decrease in circulating HDL)
- Tangier Disease
Associated with HDL cholesterol concentrations as low as 1-2 mg/dL (0.03-0.05 mmol/L) in homozygotes, accompanied by total cholesterol concentrations of 50 to 80 mg/dL (1.3-2.1 mmol/L).
- Tangier Disease
Associated with increased risk of premature coronary heart disease (CHD).
- Tangier Disease
Linear polymers of amino acids; Perform diverse functions
PROTEIN
Regulate metabolism
PROTEIN
• Facilitate contraction in the muscle
PROTEIN
Provide structural framework
PROTEIN
Shuttle molecules in the bloodstream
PROTEIN
