CC Flashcards
UNITS OF MEASURE
Système International (SI) and Selected Non-SI Units
QUANTITY NAME SYMBOL
Mass ______________ ______
Amount of substance ____________ _____
Length ____________ _____
Time ____________ _____
Luminous intensity
Electric current
Thermodynamic temperature
SELECTED NON-SI AND DERIVED UNITS
Volume
Frequency
Force
Pressure
Electrical potential
Energy
Catalytic activity
Temperature
Temperature Conversions: formula?
kilogram (kg)
mole (mol)
meter (m)
second (s)
Candela (cd)
Ampere (A)
Kelvin (K)
Cubic meter m3/Liter L
Hertz Hz
Newton N
Pascal Pa
Volt V
Joule J
Katal Kat
Degree Celsius °C
°C = (°F - 32) / 1.8
°F = 1.8°C + 32
– number of moles of solute per liter of solution
– number of moles of solute per kilogram solvent
– number of equivalent weights of solute per liter of solution
– amount of solute per 100 total units of solution; expressed as % w/v, w/w, or v/v
– expression of relative concentration
*__ – volume of sample or stock divided by total volume of solution
*__ – done when a weaker reagent concentration is needed than the stock substance available
*__ – done when the analyte concentration is too high and exceeds the detection limit of the method
– amount of something relative to another; expressed as part per part or part per whole
Molarity
Molality
Normality
Percent solution
Dilution
Simple dilution
Dilution of stock solution
Dilution of sample
Ratio
UNITS AND CONVERSION FACTORS FOR SELECTED CHEMISTRY ANALYTES:
Glucose
Urea
BUN
Uric acid
Creatinine
Bilirubin
T4
T3
Vitamin B12
Glucose - 0.0555
Urea - 0.167
BUN - 0.357
Uric acid - 0.0545
Creatinine - 88.4
Bilirubin - 17.1
T4 - 12.9
T3 - 0.0154
Vitamin B12 - 0.738
B. LABORATORY STATISTICS
1. MEASURES OF CENTER
a. ___ – average or arithmetic mean
b. ___ – midpoint of a data set after the values have been rank-ordered
c. ___ – most frequently occurring value in a data set
Mean (x̅)
Median
Mode
- MEASURES OF SPREAD
a. ___ – distribution of data points around the mean [variance (s2)]
b. Coefficient of Variation – best indicator of ________
c. ___ – difference between the highest and lowest values - ___
▪ Data points are distributed symmetrically around the mean (bell-shaped curve) with most values close to the center
▪ Mean, median, and mode are identical
▪ Follows the empirical rule: __________
Standard Deviation (s)
precision
Range
GAUSSIAN/NORMAL DISTRIBURION
68-95-99.7% rule
- REFERENCE INTERVAL STUDIES
a. ___ – pair of medical decision points that span the limits of results expected for a
defined healthy population
b. ___ - done to confirm the validity of an existing or published RI for an analyte; requires at least ____ study individuals; RI is adopted if _____ of the subjects fall outside the range
c. ___ - done when there is no existing RI for an analyte or when transference studies fail; requires at least _______ study individuals; RI is set based on the _______
Reference Interval
Verifying a reference interval; 20; ≤10%
Establishing a reference interval; 120;95% CJ = x̅ ± 25
- METHOD EVALUATION:
a. ___ – first step in method evaluation; usually done by running two control materials twice a day over a 10-day period
b. ___ – involves spiking a sample with a known amount of an analyte and determining how much of it can be detected by the method in the presence of other compounds in the matrix
c. Inferential statistics (MNEMONIC)
1) t test – compares the __ of two groups of data or the __ of two methods
2) F test – compares the __ of two groups of data or the __ of two procedures
3) ___ – used to compare two methods using the best fit line through the data points (x = ______; y = _________)
Precision study
Recovery study
mean values; accuracy
SD; precision
Linear regression
SPF MAT
(SD, precision F test); (mean, accuracy, T test)
independent variable/reference method; dependent variable/new method
d. ___ – ability of a method to detect the smallest concentration of an analyte
e. ___ – ability of a method to detect only the analyte of interest
f. Diagnostic Efficiency:
1) ___ – ability of a test to detect a given disease or condition; proportion of individuals with the disease who have a positive test result
2) ___ – ability of a test to detect the absence of a given disease or condition; proportion of individuals with no disease who have a negative test result
3) ___ – probability that a positive test result indicates disease; proportion of
individuals with a positive result who truly have the disease
4) ___ – probability that a negative test result indicates absence of disease; proportion of individuals with a negative result who truly do not have the disease
FORMULAS!!
Analytical sensitivity
Analytical specificity
Diagnostic sensitivity
Diagnostic specificity
Positive predictive value
Negative predictive value
%se = TP/(TP+FN)x100 [w dse]
%sp = TN/(TN+FP)x100 [w.o dse]
%PPV = TP/(TP+FP)x100 [neg row]
%FPV = TN/(TN+FN)x100 [pos row]
C. QUALITY MANAGEMENT SYSTEM
1. APPROACHES TO QUALITY MANAGEMENT
a. __ – most widely used approach to quality improvement in the healthcare
PDCA cycle (plan-do-check-act)
C. QUALITY MANAGEMENT SYSTEM
1. APPROACHES TO QUALITY MANAGEMENT:
c. __ – based on statistics and quantitative measurements; applied in the clinical laboratory to reduce the frequency of test errors or the number of DPMOs.
▪ Indicators: Improved performance, improved quality, improved bottom line, improved customer, and employee satisfaction
▪ 5 steps: DMAIC ____________________
d. Lean – designed to reduce waste, increase efficiency, and improve customer satisfaction; often used in clinical laboratories to improve ____.
Categories of Wastes: (mnemonic)
e. Lean-Six Sigma Colored Belt Designations: (mnemonic)
▪ ______ – understands the basics of Lean-Six Sigma
▪ ______ – team members who contribute approximately 20% of their time to QI projects while delivering their normal job functions
▪ ______ – team leaders who dedicate as much as 100% of their time to QI projects
▪ ______ – black belt with at least two years of experience; Lean-Six Sigma advisers/coaches
Six sigma
Define, Measure, Analyze, Improve, Control
TAT
(DOWN TIME); defects, overproduction, waiting, non-utilized talent, transportm inventory, motion. excess processing
(you go boy!)
yellow
green
black
master
- QUALITY CONTROL:
a. __ – ability to maintain accuracy and precision over an extended period of time during which
equipment, reagents, and personnel may change
b. __ – closeness of the result to the true or actual value
c. __ – ability to produce a series of results that agree closely with each other; commonly expressed in terms of coefficient of variation; also called __
d. Internal Quality Control:
▪ Involves the analysis of at least ____ levels of control every ______
▪ Initial control limits are established by analyzing controls for at least ____ consecutive days or runs
▪ Important for the daily monitoring of accuracy and precision of analytical methods
e. ___: Involves testing blind samples (unknown concentration of analytes) sent periodically by regulatory agencies to participating laboratories
▪ Important for maintaining the long-term accuracy of analytical methods
▪ SDI = (Lab Result − Group Mean)/Group SD
Reliability
Accuracy
Precision; repeatability (w/i run) or reproducibility (interlab)
2; 24 hrs
20
External Quality Assessment/Proficiency Testing
- ANALYTICAL VARIATION:
a. Types of Error in Laboratory Testing
____:
Due to chance or an unpredictable cause; does not recur in regular pattern; Usually a one-time error; Affects ___; indicated by violation of the ___________ Westgard rules; Causes: instrument instability e.g. temperature and voltage fluctuations, dirty glassware, sampling error, improper mixing of sample and reagent, operator variability e.g. pipetting error, anticoagulant or drug interference
____:
Recurring error inherent in test procedure; influences observations in one direction; Affects all results; May be constant or proportional
Affects ___; indicated by shift, trend, or violation of the ___________ Westgard rules; Causes: deterioration of reagents, change in reagent lot, unstable reagent blanks, calibration error, changes in standard concentration, contaminated control solutions, diminishing lamp
power, dirty photometer, faulty ISE, and pipettor maintenance error
Random Error; precision
12s, 13s, R4s!!!
Systematic Error; accuracy
22s, 41s, 81s, 10x̄
b. Patterns in QC charts that indicate errors:
1) ___ – abrupt change in the distribution of control values such that they accumulate on one side of the mean for 6 consecutive days; major cause ______
2) ___ – gradual change in the distribution of control values such that they continue to increase or decrease over a period of __ consecutive days passing through the mean; major cause ______
3) Outliers – highly deviating control values caused by random or systematic errors
Shift; calibration error, error in std prep
Trend; 6; deterioration of reagent
c. Interpretation of Quality Control Results:
1) ___– prepared by plotting the – points or the algebraic sum of the difference between each QC result and the mean on the y axis and the run number the x axis.
2) ___ – used for interlaboratory comparison of monthly means by plotting the mean value for one sample on the y axis and the mean value for another on the x axis; effective method for comparing both within-laboratory and between-laboratory variability
3) ___ – most commonly used QC chart
CUSUM chart
Tonks-Youden plot
Shewhart Levey-Jennings chart
- BIOHAZARDS
a. Hand washing – most important means of preventing the spread of infection
▪ CDC Guidelines (Strasinger, 7th): During handwashing, thoroughly clean between your fingers and under your fingernails for at least __ and rinse your hands in a ___ position to prevent recontamination
b. Personal Protective Equipment – gloves, gown, face shield or goggles, and mask or respirator
▪ Sequence of donning: ______
▪ Sequence of doffing: _______
20 sec; downward
gown/ mask/ goggle/ gloves
glove/goggles/gown/mask
Sterilizes only the air to be exhausted. Does not protect the
work surface.
Class I
Most commonly used in microbiology; also called vertical laminar flow BSC Sterilize air that flows over the infectious material as well as air to be exhausted.
A1 = __% recirculated, __% exhausted
A2 = same as A1 but with higher
_______________________
B1 = ___% recirculated, ___% exhausted
B2 = ___% exhausted
Class II
70%; 30%
intake air velocity (100 lfm)
30%; 70%
100%
Completely enclosed with glove ports. Provides the highest level of personnel protection. For extremely hazardous organisms.
Class III
d. Decontamination of Body Fluid Spills
▪ Decontaminating solution: ___
▪ Protocol for managing spills in a clinical laboratory:
1) Wear gloves and a laboratory coat.
2) Absorb the blood with disposable towels. Remove as much liquid blood or serum as possible before decontamination.
3) Using a diluted bleach solution, clean the spill site of all visible blood.
4) Wipe down the spill site with paper towels soaked with diluted bleach.
5) Place all disposable materials used for decontamination into a biohazard container. Decontaminate non-disposable equipment by soaking overnight in a diluted bleach solution and rinsing with methyl alcohol and water before reuse. Disposable glassware or supplies that havecome in contact with blood should be autoclaved or incinerated.
5.25% NaOCl or 10% chlorine bleach (1:10 dilution)
- RADIATION HAZARD
___ - <400 nm Germicidal lamps used in biologic safety cabinets
___ - 400–700 nm General illumination
___ - >700 nm Heat lamps, lasers, vein selectors
___ - 3 μm–3 mm Microwave energy beam used to accelerate tissue staining
___- >1 cm Radiofrequency coil in ICP–MS - CHEMICAL HAZARD
a. ___ – used for materials that give off harmful vapors; provides personnel protection only
b. ___ – formerly MSDS, a major source of information about chemicals written by manufacturers; revised format consists of __ as follows: - FIRE HAZARD
▪ Actions to take in the event of fire: __
▪ Steps in operating a fire extinguisher: __
Ultraviolet
Visible spectrum
Infrared
Microwaves
Low frequency
Fume hood
SDS; 16 sections
Rescue, Alarm Contain Extinguish/Evacuate
Pull pin, Aim nozzle, Squeeze trigger, Sweep nozzle
E. BLOOD COLLECTION AND SPECIMEN CONSIDERATIONS
1. TYPES OF BLOOD SPECIMENS USED IN CLINICAL CHEMISTRY
____: Liquid portion of clotted or anticoagulated blood prepared by
centrifugation at _____ for ______. Preferred for most clinical chemistry tests
Serum value > plasma value: __________________
Plasma value > serum value: __________________
____: Anticoagulated venous, capillary, or arterial blood
WB value < plasma value: ______________
CB value > VB value: _____________________
VB value > CB value: _____________________
*Separate serum or plasma from cells within ___ of collection (except centrifuged gel tubes).
*Samples should be analyzed within ___; otherwise, refrigerate for 48 hours or freeze at –20°C for longer storage.
Serum & Plasma
1000-2000 for 10 mins
AST, CK, K, PO4, HCO3
glucose and albumin
Whole blood
glucose (10-15% lower)
glucose (post-prandial), K
TP, Ca
2 hours
4 hours
- VENIPUNCTURE:
a. General precautions and techniques
▪ Sites of puncture: Veins in the antecubital fossa ___________; Veins in the dorsal part of the hand; wrist, ankle, and foot veins
▪ Tourniquet application: _____ above the puncture site; should not be left applied longer than ____; If blood pressure cuff is used, inflate to ______
▪ Antiseptics:
Routine: ________
Culture: _________________
Ethanol assay: ____________
▪ Angle between needle and vein: ______________
median>cephalic>basilic
3-4 inches (7.62-10.16cm)
1 minute
40-60mmHg
70% isopropyl alc
70% isopropyl alc->iodophor, povidone iodine, chlorhexidine
bantzalkonium chloride (zephiran)
15!!-30°
b. Venipuncture methods
1) ___ – not recommended by CLSI due to safety and specimen quality issues
2) ____ – for infants and children, hand veins, and difficult-draw situations
3) ___ – consists of double-headed needle, adapter, color-coded evacuated tube
Needle and Syringe
Butterfly/Winged Infusion Set
Evacuated Tube System
c. Venipuncture needles (colors?)
20: For large-volume tubes or large-volume syringes used on patients with normal-size veins.
21: Standard needle gauge for routine venipuncture on patients with normal veins
22: For older children and adult patients with small veins or for syringe draws on difficult veins.
23: Used on infants and children, difficult veins, or hand veins of adults.
25: Used to collect blood from scalp or tiny veins of premature infants; prone to hemolysis
*Needle length: hypodermic/ETS___; butterfly__
yellow
green
black
blue
orange
1-1.5 inches; 0.5-1 inch
d. Categories of additives:
Anticoagulants: color?
EDTA; WB: HbA1c, TDM
Plasma; Lipids and LPs
Citrate; Not routinely used in CC
Oxalate; Not routinely used in CC
Heparin; Arterial blood: ABG, NH3; Plasma: Most CC tests, special CC tests
Clot activators: ___ for STAT serum tests
Thixotropic gel separator: __
Antiglycolytic agents: __ for glucose, lactate ethanol
lavender, pink
light blue, black
gray
green
Silica (red plastic) & Thrombin (orange)
Gel + silica (gold, red/black) & Gel + heparin (light green, green/gray)
Sodium fluoride/iodoacetate (gray)
f. Factors that Affect Evacuated Tube Quality
1) Ambient Temperature: high temperature =____ draw volume; low temperature = ____ draw volume
2) Altitude: high altitude (>5000 ft) = __________ draw volume
3) Humidity: high humidity = migration of water vapor inside a tube; low humidity = escape of water vapor from a tube containing a wet additive
4) Light: affects CTAD tube (citric acid, theophylline, adenosine, and dipyridamole), a photosensitive additive for coagulation testing which minimizes platelet activation after blood collection
dec; inc
dec
- CAPILLARY PUNCTURE:
a. Indications: Newborn Screening (heelstick), POCTs, DM monitoring
b. General Precautions and Techniques
▪ Sites of puncture: heel, 3rd or 4th finger, big toe, ear lobe
▪ Recommended length of the lancet blade for heel sticks: ______ mm
▪ Consequences of squeezing the site: ____________
c. Capillary Order of Draw?
1.75mm
dilution with tissue juice
Blood gas specimen>Slides/smears>EDTA>Other additive tubes>Serum tubes
- ___
a. Indications: arterial blood gas analysis, absence of palpable veins
b. General Precautions and Techniques
▪ Sites of puncture: ________________
▪ ___ should be done before using the radial artery
▪ Anticoagulant ___________
ARTERIAL PUNCTURE
radial>brachial>femoral artery
Modified Allen test
lyophilized/liquid heparin (0.05 heparin per mL of blood)
- SPECIAL SITUATIONS IN PHLEBOTOMY
a. __ – Use opposite arm or perform fingerstick, if possible; otherwise, have nurse turn off IV for 2 min
b. CVA – ______________________
c. Cannula/fistula, mastectomy – _______________
d. Burns, edema, scars, sclerosed veins, tattoos – _____________
e. Hematoma – _________
IV
indwelling catheter - discacrd first 3-5 mL, blood culture, additive tubes, clot tube
use opposite arm away from problem sites
draw below the hematoma
- PHLEBOTOMY COMPLICATIONS:
a. __ – bleeding from the venipuncture site and hematoma formation
b. __ – second most common complication
c. __ – results from blood loss for testing; particular problem with pediatric patients
d. __ – post-phlebotomy seizure or pain
e. __ – vasovagal syncope, orthostatic hypotension, and cardiac arrest. *Vasovagal syncope can be prevented by ensuring that the patient is lying down with feet elevated before
beginning the blood draw.
f. __ – allergic reaction to iodine, adhesives, or latex
Vascular
Infections
Anemia (iatrogenic)
Neurologic
Cardiovascular
Dermatologic
- PREANALYTICAL VARIABLES
a. Uncontrollable Factors: age, gender, race, underlying conditions
b. Controllable Factors: biological/physical factors, technical factors
___:
increases in AM: cortisol, ACTH, Aldosterone, Fe
PM: GH, PTH, TSH
Posture:
Standing: ↑ albumin, cholesterol, Ca2+
Supine to upright: ↑ __________________________________
Stress: ↑ __________
Incorrect tube additive or incorrect order of draw:
___: false inc - Na, K; false dec - Ca, Mg, Fe, CK, LD, ALP, AMS
___: F↑ Na, Li, NH3
___: F↓ trace metals, drugs
Diurnal variation
albumin, enzymes, protein bound substances
catecholamines, cortisol, ACTH, prolactin
EDTA, Citrate, or Oxalate
Na fluoride
SST and PST
F. LABORATORY EQUIPMENT AND SUPPLIES
1. TYPES OF GLASS MATERIAL
a. ___– tolerates heating and sterilization for lengthy periods; subject to scratching and may cloud with strong alkali
b. ___ – 6x stronger than borosilicate; better able to resist scratching and alkali attack
c. ___ – heat, chemical, and electrical tolerance and excellent optical properties; used for high thermal, drastic heat shock, and extreme chemical treatment with acids and alkali
d. ___ – low thermal resistance; can be used with strong acids and alkali
e. Flint glass – soda lime glass; most inexpensive; releases alkali causing errors in certain determinations
f. ___ – amber-colored glass; reduces light transmission; used to contain photosensitive substances
Borosilicate (Pyrex, Kimax)
Aluminosilicate (Corex)
High silica (Vycor)
Soft glass
Low actinic glass
- TYPES OF PLASTIC MATERIALS
a. __ – excellent temperature tolerance and chemical resistance
b. __ – resistant to most chemicals; withstands high temperatures, becomes discolored by solvents; used for pipet tips, test tubes
c. __– stronger than polypropylene (resistant to shattering) and better temperature tolerance, but chemical resistance not as good; used for centrifuge tubes, graduated cylinders
d. __ –resistant to most chemicals (except concentrated acids); for storage of alkaline solutions; used for disposable transfer pipets, test tubes, bottles
e. __ – rigid, clear; used for test tubes, graduated tubes
f. __ – soft and flexible but porous; frequently used as tubing
Teflon
Polypropylene
Polycarbonate
Polyethylene
Polystyrene
Polyvinyl chloride
- GLASS PIPETTES
a. Design!!
▪ __ – holds but does not deliver the exact volume
▪ __ – delivers the exact volume it holds
b. Drainage characteristics!!
▪ __– characterized by etched ring/band near the mouth
▪ __ – allowed to drain by gravity
a. __: single capacity mark; Nonviscous samples; standards
b. __: single capacity mark; Viscous fluids
c. __: with graduation down to tip!! Serial dilution; measuring reagents
d. __: graduated bet 2 marks; Serial dilution; measuring reagents
To contain (TC)
To deliver (TD)
Blow-out
Self-draining
Ostwald-Folin (TP - transfer pipet)
Volumetric (TP)
Serologic (maesuring)
Mohr (measuring)
- SEMI-AUTOMATIC MICROPIPETTORS
a. Types
1) ___ – uses suction to draw sample into a disposable polypropylene tip; piston does not come in contact with the liquid
2) ___ – operates like a hypodermic syringe; tips must be rinsed out; No air cushion; constant aspiration force; for high density or very viscous samples; Used for sampling in most discrete automated systems
b. Calibration
1) Frequency: every ___
2) Gravimetric method – weight of _____ delivered; most accurate!!
3) Spectrophotometric method – absorbance of colored solution -= K dichromate/p-nitrophenol delivered
Air displacement
Positive displacement
6 months/biannually
distilled water
- CENTRIFUGES
a. Types:
1) ___ – tubes attain a horizontal position during spinning and a vertical position when at rest
2) ___ – tubes are at fixed angle when rotating; capable of higher speeds with much less heat build-up
3) ___ – used to separate layers of different specific gravities; usually
refrigerated to counter the heat produced due to friction
4) ___ – for body fluid cell counts
b. Units used: rpm, RCF (g), Svedberg (S)
Formula for Converting rpm to RCF:
c. QC Parameters: _____________________
Frequency: every ___
Horizontal or swinging bucket
Fixed-angle or angle-head
Ultracentrifuge
Cytocentrifuge
RCF (g) = 1.118 x 10^-5 x r (in cm) x rpm^2
stopwatch (timer), speed (tachometer)
3 months (quarterly)
G. REAGENTS
1. CHEMICALS:
a. __ – high degree of purity suitable for use in most analytical procedures
b. __ – for specific procedures such as chromatography, AAS, immunoassays, molecular diagnostics, and standardization techniques
c. __ – impurity limitations not stated; not acceptable for research and laboratory techniques unless further purification is carried out or a reagent blank is included during analysis
d. __ – primarily used to manufacture drugs and
purity standards may not meet assay requirements.
e. __ – should not be used in the clinical laboratory
Analytic Reagent Grade (ACS)
Ultrapure Grades
Chemically Pure Grade
United States Pharmacopeia/National Formulary Grade (NFG?)
Technical or Commercial Grade
- REFERENCE MATERIALS:
a. __ – highly purified chemical that can be measured directly to produce a substance of exact known concentration and purity
▪ ACS Primary Standard – purity tolerance value of _____________
▪ ___ – certified for use in clinical chemistry laboratories
b. ___ – substance of lower purity with its concentration determined by comparison with a primary standard or SRM - REAGENT WATER
a. __ – purest type; recommended for __________; CC: <10
b. __ – acceptable for most laboratory procedures including reagent preparation; CC: <10^3
c. __ – can be used for some qualitative tests but not for routine analyses and reagent preparation; water source for the preparation of Type I or II water and for washing glassware; CC not specified
Primary standard
100±0.02%
Standard Reference Materials
Secondary standard
Type I, standard prep
Type II
Type III
H. ANALYTICAL TECHNIQUES AND INSTRUMENTATION
1. SPECTROPHOTOMETRY
a. Regions in Electromagnetic Spectrum
▪ Ultraviolet light ____; Visible light _____; Infrared _____
▪ Planck’s formula: E = hν
▪ Relationship between wavelength (λ) and energy (E) _______
b. ___: The concentration of a substance is directly proportional to the amount of light absorbed and is inversely proportional to the logarithm of transmitted light
A = abc (a or ɛ =molar absorptivity; b =light path; c =concentration)
To compute the absorbance value given the % transmittance: _______
To determine the concentration of an unknown analyte: _______
<400nm; 400-700nm; >700nm
inversely proportional
Beer-Lambert’s Law
A = 2-log%T
Cu = (AuCs)/As
Csample = (A sample/Astd)xCstd
c. Solutions used in Spectrophotometry:
1) __ – distilled water, reagent, or sample used to subtract absorbances not due to the analyte of interest; sets the spectrophotometer to 0 absorbance
▪ __ – corrects for absorbance caused by the color of reagents; used to zero theinstrument before measuring test samples and other blanks
▪ __ – used to subtract the intrinsic absorbance caused by hemolysis, icterus, turbidity, or drug interference during sample analysis
2) __ – substance of known purity and concentration used to determine the concentration of the unknown analyte
3) __ – solution containing various analytes with known target values; analyzed with patient samples to monitor analytical performance
▪ Characteristics: Commutable, stable, no matrix effects, with known analyte concentrations spanning clinically important range at appropriate decision levels
▪ Assayed – values provided by the manufacturer
▪ Unassayed – values determined by the laboratory
Blank
Reagent blank
Sample blank
Standard
Control
d. Spectrophotometer Quality Assurance
1) __ – the wavelength set is the actual one selected by the monochromator; checked using didymium glass or holmium oxide
2) __ – done using glass filters and solutions that have known absorbance values
3) __ – a change in concentration results in a straight-line calibration curve (Beer’s Law); determined using neutral density filters and dichromate solution
4) __ – any wavelength outside the band of interest; detected using sharp cut off filter!!
e. Components of a Single-beam Spectrophotometer:?
Wavelength accuracy
Absorbance check
Linearity
Stray light
light source, entrance slit, monochromator, exit slit, sample cuvette, PM tube, A/D, display
___: Provides polychromatic light which the sample will modify or attenuate by absorption;
Deuterium/Hydrogen - __
Xenon/Mercury - __
Tungsten - __
LASER - __
__: Prevents stray light from entering the monochromator system
Effects of stray light: absorbance error and loss of linearity
__: Wavelength selector; isolates a portion of the spectrum emitted by the source and focuses it on the sample
__: – continuous, non-linear spectrum better separation of high-frequency light
__: – continuous, linear; uniform separation of wavelengths
__: Controls the bandpass; allows only a narrow fraction of the spectrum to reach the cuvette
__: – range of wavelengths transmitted; calculated as width at more than half the maximum transmittance
Light source
UV
UV to visible
visible to near IR
visible to near IR
Entrance slit
Monochromator
Prism
Diffraction gratings
Exit slit
Bandpass
- DOUBLE-BEAM SPECTROPHOTOMETRY
▪ designed to compensate for variations in intensity of the light source by splitting the light beam from the lamp and directing one portion to a reference cuvet and the other to the sample cuvet
▪ 2 types:
Double-beam-in-space – ______________________
Double-beam-in-time – ___________________
beam splitter (+2 photodetectors)
chopper; 1 photodetector
- ___
▪ Principle: Measurement of the amount of light emitted by excited molecules
▪ Important components: gas discharge lamps; 2 monochromators or filters; PM tube
▪ Advantages: high specificity and sensitivity
▪ Causes of quenching or decreased fluorescence:inc temp, prolonged exposure to UV, highly concentrated samples/undiluted samples, pH changes and presence of contaminating chemicals
FLUOROMETRY
- ____
▪ Principle: Measurement of light emission caused by a chemical, biochemical, or electrochemical reaction, and not by photo illumination
▪ ___: – emission of light caused by oxidation of organic compounds catalyzed by an enzyme, a metal, or hemin
▪ ___: – a special form of chemiluminescence where an enzyme-catalyzed chemical reaction produces light emission; involves the use of natural substrates
▪ ___: – emission of light caused by a reaction generated electrochemically on the surface of an electrode
LUMINOMETRY
Chemiluminescence
Bioluminescence
Electrochemiluminescence
- AUTOMATION
a. Basic Approaches
1) __ – pumps each specimen in a batch through a system of continuous tubing at the same rate and is subjected to the same analytical reactions; significant carry-over problems and wasteful use of continuously flowing reagents e.g. SMA, Technicon
▪ Air bubbles ___________
▪ Coiled tubing/glass coils _____________________
2) __ – uses centrifugal force to transfer liquids in separate cuvets for measurement; capable of batch analysis e.g. Cobas-Bio (Roche)
3) __ – places each sample and accompanying reagents in separate containers; batch analysis, random access or stat capabilities e.g. OCD Vitros 350, Beckman Unicel DXC, Dupont ACA, Abbott Architect, Cobas 6000, Siemens Vista
Continuous flow
separating and cleaning media
mixing sample with reagent
Centrifugal analysis
Discrete analysis
- AUTOMATION
b. Analyzer Configurations
▪ ___ – each specimen in a batch enters the analytical process one after another, and each result or set of results emerges in the same order as the specimens are entered.
▪ ___ – all specimens are subjected to a series of analytical processes at the same time and in a parallel fashion.
▪ ___ – many specimens are grouped in the same analytical session; large number of specimens in one run
▪ ___ – each specimen is analyzed for a different selection of tests; capable of analyzing stat specimens out of sequence on an as-needed basis
▪ ___ – requires the reagent to be in a unique container or format provided by the manufacturer.
▪ ___ – the operator is able to change the parameters related to an analysis and prepare in-house reagents or use reagents from a variety of suppliers. - __:
▪ Advantages: reduced turnaround time and connectivity (electronic documentation of testing)
▪ Blood glucose monitors (glucometers) – most commonly used POCT; use enzymatic methods coupled with photometric or electrochemical detection; should not be used to diagnose diabetes mellitus
Sequential analysis
Parallel analysis
Batch analysis
Random-access analysis
Closed-system analyzer
Open-system analyzer
POINT OF CARE TESTING (POCT) DEVICES
- ___:
▪ detection of scintillations (flashes of light) using a PM tube and counting of the electrical impulses
▪ Types:
Crystal scintillation I-135; I-131;
Liquid scintillation H-3, C-14 - ___:
▪ measurement of the amount of light blocked (__) or of the amount of light scattered (__) by a suspension of particles - ___:
▪ involves fragmentation and ionization of molecules using a suitable source of energy followed by separation of the ions by mass-to-charge ratio and counting the number of ions of each type
▪ Applications: MALDI TOF MS, GCMS or HPLC-MS, Tandem MS (MS/MS), IDMS - ___:
▪ non-destructive method for determining the structure of organic compounds; used in lipoprotein particle measurements
SCINTILLATION COUNTING
TURBIDIMETRY AND NEPHELOMETRY.
MASS SPECTROMETRY
NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
CARBOHYDRATES
A. CLASSIFICATION OF CARBOHYDRATES
1. ___ – polysaccharides, disaccharides, monosaccharides
2. ___ – trioses, tetroses, pentoses, hexoses
3. ___ – aldoses and ketoses
4. ___ – D and L enantiomers
B. GENERAL PROPERTIES
* Form glycosidic bonds with other carbohydrates and with noncarbohydrates
* Modify proteins and their function by glycosylation
* Some are reducing substances (sugars with hemiacetal group)
Number of sugar units
Number of carbons
Location of the CO functional group
Stereochemistry of the compound
CARBOHYDRATES:
C. HORMONAL REGULATION OF GLUCOSE METABOLISM
what are the sources and effect on PG (plasma glucose)
Insulin
Glucagon
Somatostatin
Cortisol
Epinephrine
ACTH
GH
Thyroxine
hPL
beta cells; dec
alpha cells
delta cells
adrenal cortex
adrenal medulla
anterior pituitary
anterior pituitary
thyroid
placenta
ALL ELSE INC PG
D. DISORDERS OF GLUCOSE METABOLISM
1. HYPOGLYCEMIC DISORDERS
a. Types
▪ ___ – occurs after 10 hours without food; secondary to hyperinsulinism (insulinoma), hormonal deficiencies, genetic disorders, autoimmunity or drug-induced
▪ ___ – occurs usually within 4 hours after eating a meal
b. Symptoms
▪ Neurogenic – tremulousness, palpitations, anxiety, diaphoresis, hunger, and paresthesias
▪ Neuroglycopenic – dizziness, tingling, blurred vision, behavioral changes, seizure, and coma
c. Diagnosis
▪ Whipple’s triad of hypoglycemia: ___
▪ Diagnostic Criteria for Insulinoma:
Change in glucose level ___ (under controlled fasting condition) coincident with:
___ insulin (≥41.7 pmol/L)
___ proinsulin (≥5 pmol/L)
___ C-peptide (≥0.2 nmol/L)
___ β-hydroxybutyrate (≤2.7 mmol/L)
Post-absorptive/Fasting
Post-prandial/Alimentary/Reactive
~
symptoms of hypoglycemia, low plasma glucose level, and relief of symptoms with correction of hypoglycemia
≥25 mg/dL
↑
↑
↑
↓
- DIABETES MELLITUS
a. ADA Classification
I. ___ – β cell destruction leading to absolute insulin deficiency
a) immune-mediated - 99%
b) idiopathic
II. ___ – insulin resistance with progressive insulin deficiency
III. Other types
a) genetic defects of β cell function
b) genetic defects in insulin action
c) diseases of the exocrine pancreas (pancreatitis, pancreatectomy, pancreatic cancer/neoplasia,
cystic fibrosis, hemochromatosis, fibrocalculous pancreatopathy
d) endocrinopathies
e) drug- or chemical-induced
f) infections
g) uncommon forms of immune-mediated diabetes
h) other genetic syndromes
IV. Gestational – glucose intolerance during pregnancy that disappears post-partum but may convert to type 2 DM in 30-40% of cases within 10 years; due to metabolic and hormonal changes
Type 1
Type 2
b. Characteristics of Type 1 and Type 2 Diabetes Mellitus
10% have this; childhood/juvenile onset
Autoimmunity - autoantibodies +
Genetic predisposition - HLA: DR3/DR4
Therapy: Insulin injection
Acute complications: Diabetic ketoacidosis
Type 1
b. Characteristics of Type 1 and Type 2 Diabetes Mellitus
>90% have this; adulthood onset
* Family history of DM, high-risk minority population (e.g., African American, Latino, Native American, Asian American, and Pacific Islander)
* PCOS, GDM or delivering a baby weighing >9 lb, pre-diabetes (IFG, IGT, HbA1C 5.7-6.4%)
* Obesity or overweight tendencies (BMI _______), habitually physically inactive, hypertension (BP ___), dyslipidemia (HDL __;TG __), history of CVD
Therapy: Lifestyle changes, oral hypoglycemic agents; may require insulin
Acute complications: Hyperglycemic hyperosmolar non-ketotic coma
Type 2
≥25kg/m2
140/90;
≤35mg/dL; ≥250mg/dL
DM MELLITUS:
Lab Findings ↑ ______________________ ↓ ___________________________
Symptoms: (3Ps)___; pruritus, poor wound healing
Long-term Complications:
Microvascular complications: nephropathy, retinopathy, neuropathy
Macrovascular complications: CAD, CVA
↑ PG. plasma osm., urine gluc, urine SG↓
↓ pH, Na
Polyuria, polydipsia, polyphagia
E. TESTS AND DIAGNOSTIC CRITERIA FOR DM
Screening: All adults ≥45 years old ______; individuals with ≥1 risk factor ______
every 3 years; every year
E. TESTS AND DIAGNOSTIC CRITERIA FOR DM
Important considerations:
In conjunction with symptoms of DM
Normal: <200 mg/dL
Pre-diabetes: –
DM: ≥200 mg/dL
RPG
E. TESTS AND DIAGNOSTIC CRITERIA FOR DM
Fasting for at least ________ (no food or drink except water)
Normal: <100 mg/dL
Pre-diabetes: ___
DM: ___
FPG; 8 hours
100-125mg/dL
≥126 mg/dL
E. TESTS AND DIAGNOSTIC CRITERIA FOR DM
CHO intake ≥150g/day for 3 days prior
Fasting ___
Standard load; 75g/1.75g/kg
Normal: <140 mg/dL
Pre-diabetes: ___
DM: ___
2-hr PG (OGTT)
8-14 hrs
140-199mg/dL
≥200 mg/dL
E. TESTS AND DIAGNOSTIC CRITERIA FOR DM
Specimen: EDTA whole blood (not affected by fasting)
Normal: <5.7%
Pre-diabetes: 5.7%-6.4%
DM: >6.5%
HbA1c/Glycated hemoglobin
F. TESTS AND DIAGNOSTIC CRITERIA FOR GDM
All nondiabetic pregnant women should be screened for GDM at ___ of gestation.
24-28 weeks
G. TESTS FOR MONITORING GLYCEMIC CONTROL
1. ____
▪ most commonly analyzed using POCT devices at home or at the patient’s bedside; 3-4 times daily for type 1 DM patients according to ADA guidelines; about ___% lower than plasma glucose; ADA glycemic goals 70-130mg/dL; <180 mg/dL
2. ____
▪ Hb A with glucose irreversibly attached to one or both N-terminal valines of the β-chains
▪ provides an index of average blood glucose levels over the past ____ months
▪ unreliable in patients with hemolytic disorders
▪ ADA glycemic goal: ___ Poor control: ____ eAG = (28.7*A1c) – 46.7
3. ____
▪ used to assess glycemic control over the past ______ weeks in cases when HbA1c is unreliable
▪ has the advantage of using serum samples but is unreliable when serum albumin level is _______
▪ Reference range: 205-285umol/L
4. ____
▪ early indicator of diabetic nephropathy
▪ persistent albuminuria in two out of three urine collections of _____ μg/min, 30-300 mg/24 h, or a albumin–creatinine ratio of 30-300 μg/mg or 3.5–30 mg/mmol within a 3- to 6-month period
WHOLE BLOOD GLUCOSE
10-15%
GLYCATED HEMOGLOBIN/ HbA1C
2-3
<7%; >12%
FRUCTOSAMINE
2-3 weeks
≤ 3g/dL
MICROALBUMINURIA
20-200
H. METHODS OF GLUCOSE MEASUREMENT
1. SPECIMEN CONSIDERATIONS
a. Use of NaF or SST to prevent glycolysis. Otherwise, glycolysis will take place at a rate of 7mg/dL/h at RT or 2mg/dL/h at 4°C
b. Effect of dextrose contamination ____% contamination will inc PG by ___
2. CHEMICAL METHODS
a. Copper Reduction:
1) Folin-Wu - blue
2) Nelson-Somogyi - blue
3) Neocuproine - yellow-orange
b. Ferric Reduction
c. Condensation
10% = 500 mg/dL
- ENZYMATIC METHODS
a. Glucose oxidase
Detection Methods:
▪ ___ – subject to many interferences
▪ ___ – measurement of the degree of O2 consumption using a pO2 (Clark) electrode; requires addition of molybdate and iodide or catalase and ethanol to prevent reformation of O2
b. ___
▪ reference method; coupling reaction is highly specific
▪ effect of hemolyzed/icteric sample _______
c. ___
▪ Tetrazolium dye may be used to enable colorimetric-spectrophotometric measurement:
▪ highly specific for glucose, not subject to interference from substances normally found in serum
▪ not commonly used except in glucose POCT (glucometer)
Peroxidase-coupled/Trinder reaction
Polarographic
Hexokinase; false decreased
Glucose Dehydrogenase
I. INBORN ERRORS OF CARBOHYDRATE METABOLISM
1. ____: due to a deficiency of one of three enzymes involved in galactose metabolism: galactose-1-phosphate uridyl transferase (GALT), galactokinase (GALK), or uridine diphosphate galactose-4-epimerase (GALE); affected children present with mental retardation, failure to thrive, and galactosuria
2. ____:
a. Hepatic glycogenoses
▪ usually manifest with hepatomegaly, hypoglycemia, and growth retardation
▪ ___– most common; presents with hypoglycemia, lactic acidosis, and hyperuricemia
b. ___ – manifest with exercise intolerance, muscle cramps, fatigue, and weakness
Galactosemia
Glycogen Storage Diseases/Glycogenoses
von Gierke disease
Muscle glycogenoses
A. COMPONENTS OF LIPOPROTEINS:
1. __
* Apolipoproteins
* Phospholipids
* Non-esterified cholesterol
2. Core: (3)
Surface
- Triglycerides
- Cholesterol esters
- Free fatty acids
A-I and A-II
which has LCAT activator?
A-IV
B-100 *LDL-R ligand
B-48
C-I, C-II, C-III
*which LPL cofactor and LPL inhibitor
E - Recognition factor that targets chylomicron
and VLDL remnants to hepatic receptor
HDL
A-I
CM, VLDL, HDL
VLDL, IDL, LDL
CM; remnant receptor ligand
CM, VLDL, HDL
C-II - cofactor; C-III inhibitor
CM, VLDL, IDL, remnants and HDL
C. MAJOR CLASSES OF LIPOPROTEINS
Density: <0.93
Electrophoretic mobility: ___
Major component: TG (exogenous)
Apolipoproteins: __
Other comments: transports exogenous lipids
Chylomicrons
origin
B48, C, E
C. MAJOR CLASSES OF LIPOPROTEINS
Density: 0.43-1.006
Electrophoretic mobility: ___
Major component: TG (endogenous)
Apolipoproteins: ___
Other comments: transports endogenous lipids
VLDL
pre-B
B-100, C, E
C. MAJOR CLASSES OF LIPOPROTEINS
Density: 1.019-1.063
Electrophoretic mobility: __
Major component: Cholesterol (>50%)
Apolipoproteins: ___
Other comments: transports cholesterol to peripheral tissues
LDL
B
B100
C. MAJOR CLASSES OF LIPOPROTEINS
Density: 1.063-1.21
Electrophoretic mobility: __
Major component: Protein (~50%; phospholipid)
Apolipoproteins: __
Other comments: reverse cholesterol transport
order of LPPs from lightest to dense in:
Ultracentrifugation: ___
Electrophoresis: ___
HDL
α
A-I, A-III, C, E
chylo, (b-vldl), VLDL, (idl), LDL, HDL
chylo, LDL, VLDL, HDL
D. MINOR AND ABNORMAL LIPOPROTEINS:
1. ___ – lipolytic product of VLDL catabolism taken up by the liver or converted to LDL; density, electrophoretic mobility, protein and lipid contents are intermediate between those of VLDL and LDL
2. ___ – floating beta lipoprotein; high cholesterol content; seen in type 3 hyperlipoproteinemia or dysbetalipoproteinemia
3. ___ – sinking pre-beta lipoprotein; contains the prothrombotic Apo(a) which is homologous with plasminogen; associated with stroke, MI, CHD
4. ___ – abnormal lipoprotein associated with obstructive biliary disease and LCAT deficiency!;
consists mostly of phospholipids and non-esterified cholesterol
IDL
β-VLDL
Lp(a)
LpX
E. FREDRICKSON CLASSIFICATION OF HYPERLIPOPROTEINEMIAS:
CM
Familial chylomicronemia
Eruptive xanthoma; acute pancreatitis
Type 1
E. FREDRICKSON CLASSIFICATION OF HYPERLIPOPROTEINEMIAS:
LDL
Familial hypercholesterolemia
Xanthelasma; premature CHD
LDL, VLDL
Familial combined hyperlipoproteinemia
Isolated xanthelasma; premature CHD
Type 2a
Type 2b
E. FREDRICKSON CLASSIFICATION OF HYPERLIPOPROTEINEMIAS:
IDL, β-VLDL
Familial dysbetalipoproteinemia Eruptive xanthoma; premature CHD
VLDL
Primary hypertriglyceridemia
Eruptive xanthoma; may be associated with premature CHD
CM, VLDL
Mixed hyperlipidemia
Eruptive xanthoma; may be associated with pancreatitis and premature CHD
Type 3
Type 4
Type 5
F. HYPOLIPOPROTEINEMIAS
1. Abetalipoproteinemia – also known as _______; autosomal recessive disorder involving mutations in the MTTP gene with absolute nonexistent levels of apoB48 and apoB100, absence of ApoB-containing lipoproteins; total cholesterol very low, triglyceride level nearly undetectable
2. ___ – autosomal dominant disorder caused by nonsense or missense mutations in the apoB gene; low total cholesterol level and normal to low triglyceride level
3. ___ – rare autosomal recessive disorder characterized by low to undetectable HDL due to a mutation in the ABCA1 gene
4. ___ – common autosomal dominant disorder characterized by HDL-C levels <30 mg/dL (men) or <40 mg/dL (women)
Bassen-Kornzweig Syndrome
Hypobetalipoproteinemia
Tangier disease
Hypoalphalipoproteinemia
G. PLASMA LIPID ANALYSIS
1. Analytical Methods
a. Cholesterol - what is the reagent?
Saponification: Hydrolysis of cholesterol esters
Extraction: Removal of protein interference
Purification: Precipitation of free cholesterol
Colorimetry: Formation of colored compound
color of endpoint:
Liebermann-Burchardt: cholestadienyl monosulfonic acid
Salkowski: cholestadienyl disulfonic acid
*Modified Abell-Kendall – CDC reference method which involves saponification with alc. KOH,
extraction with n-Hexane & petroleum ether, and colorimetry using the ______
2) Enzymatic method – peroxidase-catalyzed dye oxidation
alcoholic KOH
Bloor’s reagent
digitonin
H2SO4, acetic anhydride (L-B) or Fe3+
L: green
S: red
L-B reaction
b. Triglyceride (N:<150mg/dL)
1) Chemical methods
Colorimetric: ___ (blue or pink)
Fluorometric: ____ (yellow)
2) Enzymatic methods
* Peroxidase-catalyzed dye oxidation: quinonemine (red/red-purple)
* Measurement of ↑ absorbance at 340 nm
van Handel-Zilversmit
Hantzsch
c. Standing plasma test
▪ ___ plasma in a 10×75 mm test tube is allowed to stand at 4°C undisturbed __.
▪ __ accumulates as a floating cream layer. ___ makes the sample remain turbid after standing.
2 mL; overnight
CM
VLDL
d. Lipoprotein Cholesterol
1) HDL-C
* ___ – involves adjusting the sample to a density of 1.063 (potassium bromide) followed by centrifugation at high speed for 24 h
* ___ (direct assay) – immunologic assay involving blockage of non-HDL lipoproteins using an antibody to apo B-100
* ___ of apoB-containing lipoproteins by polyanion-divalent cations (e.g. heparin sulfate- Mn2+, dextran sulfate-Mg2+, sodium phosphotungstate-Mg2+, heparin-Ca2+) followed by cholesterol determination on the supernatant using the POD-coupled method
* CDC reference method – ___, ___, and ___
- Reference values: <40 mg/dL high risk for CHD > 60 mg/dL desirable
Ultracentrifugation
Homogeneous
Precipitation
ultracentrifugation; heparin-Mn2+ precipitation
Abell-Kendall assay
2) LDL-C
* ___ – involves ultracentrifugation (to separate VLDL and chylomicrons) and precipitation (to remove HDL)
* ___ (direct assay) – uses detergents or other chemicals to block or solubilize non-LDL
lipoprotein classes to allow for quantitation of LDL
* Calculation: LDL = TC – (HDL + VLDL); VLDL is estimated from TG using the following equations: Friedewald; De Long; Martin-Hopkins
ANALYTE; CV (mnemonic)
Cholesterol:
Triglyceride:
LDL-Cholesterol:
HDL-Cholesterol:
β Quantification
Homogeneous
choles-TREE-ol; triglyce-FIVE
≤3%
≤5%
≤4%
≤4%
NONPROTEIN NITROGEN COMPOUNDS:
A. ___
1. PHYSIOLOGY
▪ NPN present in highest concentration in the blood; formed in the liver from CO2 and ammonia
▪ major excretory product of the metabolism of proteins and other nitrogen-containing chemicals
▪ concentration is expressed in terms of nitrogen content (BUN): _________
UREA
urea/2.14
- CLINICAL APPLICATION: UREA (45-50% NPNs)
a. Analytical Methods
1) Reference method _____
2) Chemical (Fearon Method)/DAM/Direct (yellow)
3) Enzymatic
a) Colorimetric - color endpoint?
- Nesslerization
- Berthelot
b) GLDH-Coupled Enzymatic: dec A @340nm
c) Conductimetric – measurement of the increase in conductivity due to NH4+ and CO3
2-ions
IDMS
yellow-orange
blue
b. Reference values: BUN 6–20 mg/dL BUN/Creatinine ratio _____
c. Clinical significance of abnormal values
1) ↑ BUN
▪ Prerenal Azotemia: dehydration, ↓ blood volume, high-protein diet, ↑ protein catabolism
▪ Renal Azotemia: ↓ excretion (glomerulonephritis, renal failure)
▪ Postrenal Azotemia: urinary tract obstruction (nephrolithiasis, tumors, severe infection)
*___ – very high plasma urea concentration accompanied by renal failure
2) ↓ BUN – repeated dialysis, liver disease, low protein diet
10-20:1
Uremia/Uremic syndrome
B. ___ (5% of total NPNs)
1. PHYSIOLOGY
▪ formed from creatine, a substance synthesized from ____________
▪ excreted into plasma at a constant rate proportional to an individual’s muscle mass
▪ removed from the circulation by glomerular filtration; concentration is inversely related to GFR
- CLINICAL APPLICATION
a. Analytical Methods
1) ___ – colorimetric, endpoint; non-specific; subject to positive bias due to ascorbic acid, glucose, glutathione, ketoacids, uric acid, cephalosporins
▪ Techniques to improve the specificity of the Jaffe reaction:
a) ___ – timed rate reaction
b) Use of adsorbents: ____________________
2) Enzymatic:
Creatininase–POD coupled
Creatininase–CK coupled
CREATININE
methionine, arginine, glycine
Chemical (Jaffe)
Kinetic
Lloyd’s reagent (NaAl silicate) or Fuller’s Earth reagent (MgAl silicate)
B. CREATININE: CLINICAL APPLICATION
b. Clearance Tests and GFR Estimates
▪ Creatinine clearance: __
▪ Cockgroft-Gault formula: __
▪ MDRD formula: __
(diff of new one from old is nawala yung BUN sa new)
C=(UV/P) x (1.73/A)
C=(140-age)(LBW)/(72 x ser. crea in mg/dL)
*LBW - lean body weight
eGFR (mL/min/1.73 m^2) = 186 (or 175 if IDMS-traceable) × Cr-1.154 ×Age-0.203 × 1.212 (if Black) × 0.742 (if female)
Stage; Description: what is the GFR?
1 Kidney damage with normal or ↑ GFR
2 Kidney damage with normal or ↓ GFR
3 Moderate ↓ GFR
4 Severe ↓ GFR
5 Kidney failure
> 90
60-89
30-59
15-29
<15
C. ___
1. PHYSIOLOGY
▪ product of the catabolism of purines in nucleic acids or nucleoproteins
▪ relatively insoluble in plasma; can be deposited in the joints and tissues at concentrations >6.8 mg/dL
2. CLINICAL APPLICATION
a. Analytical Methods: all have allantoin in product
1) Chemical (Phosphotungstic Acid Method)
2) Enzymatic (__)
Detection methods: Measurement of the ↓A293nm
Peroxidase-coupled colorimetric reaction
URIC ACID
Uricase Method
D. ___
1. PHYSIOLOGY
▪ produced from the catabolism of amino acids
▪ toxic compound metabolized exclusively in the liver (via the Krebs-Henseleit or urea cycle)
2. CLINICAL APPLICATION
a. Analytical Methods
1) Chemical
a) __ – measurement of pH change as NH3 diffuses through a selective membrane
b) Colorimetric: (bromphenol blue) -> blue dye
2) Enzymatic (___): prod - glutamate + NAD
b. Reference range: 19–60 μg/dL
c. Clinical significance of hyperammonemia: hepatic failure, cirrhosis, ____ syndrome, urea cycle enzyme def, hepatic coma, hepatoencephalopathy
AMMONIA
Potentiometric
GLDH Method
Reye syndrome
PROTEINS
A. GENERAL PROPERTIES
▪ Building blocks: ___
▪ Net charge and electrophoretic mobility – determined by the acidic and basic amino acid monomers
▪ ____ – the pH at which a particular protein has net charge equal to zero
▪ ____: lowest at pI; hydrophilic and more soluble when charged
▪ Nitrogen content of plasma proteins: approx __
▪ Levels of Structure:
__ – number and types of amino acids in a specific amino acid sequence
__ – regularly repeating structures stabilized by hydrogen bonds between the amino acids
__ – overall shape or conformation of the protein molecule
__ – results from the interaction of more than one protein molecule or subunit
Amino acids
Isoelectric point (pI)
Solubility
16%
Primary
Secondary
Tertiary
Quaternary
B. ___
➢Serum samples are applied close to the cathode end of a support medium that is saturated with an alkaline buffer (pH _____) at which pH proteins carry a _________ charge and migrate toward the _________
➢Support media: agarose, cellulose acetate, polyacrylamide gel
➢ ___ – uses a higher voltage coupled with a cooling system and a more concentrated buffer; separates proteins into 12 or more bands; detects unusual monoclonal bands
➢Stains used: Coomassie brilliant blue, Ponceau S, Amido black, Lissamine green
➢___ – specialized colorimeter designed to scan and quantitate electrophoretic pattern
PROTEIN ELECTROPHORESIS
8.6
negative
anode
High-resolution protein electrophoresis
Densitometer
C. SERUM PROTEIN FRACTIONS
Indicator of malnutrition; Binds/transports T4 as TBPA/transthyretin
Forms a complex with retinol (Vit A) binding protein; Forms a distinct band in CSF electrophoresis
Major contributor to plasma/osmotic pressure; General transport protein; Negative APR, decreased in inflammation
↓ level in nephrotic syndrome, PLE, liver disease, malnutrition
___ - absence of albumin
___ - two albumin bands
prealbumin
Albumin
Analbuminemia
Bisalbuminemia
C. SERUM PROTEIN FRACTIONS: a-1 proteins!!
↑ level in amniotic fluid leads to neural tube defects;
↑ level in adult serum leads to hepatocellular carcinoma
90% of a1 band; positive APR; Protease inhibitor; Major component of the α1 region
Deficiency is associated with SERPINA1 gene mutation and causes juvenile hepatic cirrhosis & pulmonary emphysema (flat a1 peak/curve)
positive APR; Binds progesterone and some drugs
Negatively charged even in acid pH; has a very high carbohydrate content
Protease inhibitor; positive APR;
Binds prostate-specific antigen
Transports vitamin D
↓ level may lead to abnormal calcium levels
α-Fetoprotein
α1-Antitrypsin
α1-Acid glycoprotein/orosomucoid
α1-Antichymotrypsin
Gc-globulin
C. SERUM PROTEIN FRACTIONS: a-2 proteins!!
Protease inhibitor; Largest non-Ig protein
↑ ten-fold in nephrotic syndrome & protein losing enteropathy (loss of proteins in GI tract)
positive APR; Binds hemoglobin to preserve iron
↓ level seen in hemoytic disorders (IVH)
positive APR; Copper-binding protein; Has oxidase activity
↓ serum level in Wilson’s disease (excess Cu deposition)
α2-Macroglobulin
Haptoglobin
Ceruloplasmin
C. SERUM PROTEIN FRACTIONS: B globulins!!!
Transports iron
↑ level seen in IDA (pseudoparaproteinemia); may be estimated using TIBC
Immune response
Most abundant complement component
Binds heme
↓ level in intravascular hemolysis
Component of MHC or HLA molecules
Used to measure GFR
~ other bands of protein:
positive APR; Enhances phagocytosis in inflammatory disease
↑ up to 1000 times in inflammatory states; hsCRP used as marker for cardiovascular risk
Antibodies produced by plasma cells
Include IgG, IgM, IgA, IgE, and IgD
~ ➢ Extra bands will be seen if the specimen used is: plasma __
hemolyzed serum ____
Transferrin
C3 complement
Hemopexin
β2-Microglobulin
~
C-reactive protein (Y migrating protein)
Immunoglobulins
fibrinogen (bet B and Y)
hemoglobin (bet a2 & B; ↑B)
E. CLINICAL SIGNIFICANCE OF ABNORMAL PROTEIN LEVELS
1. Hypoproteinemia
a. Increased protein loss – nephrotic syndrome, protein-losing enteropathy, blood loss, extensive burns
b. Malnutrition/malabsorption
Types of malnutrition: undernutrition (wasting, stunting, underweight), inadequate vitamins or minerals,
overweight, obesity, and resulting diet-related noncommunicable diseases.
c. Decreased synthesis – liver disease e.g. hepatic cirrhosis
d. Increased catabolism – infection, inflammation, burns, trauma
Positive APRs: a, AT, a1, AG, Hap, Cer, Fib, CRP
Negative APRs: _____________________
e. Immunodeficiency – hypogammaglobulinemia, agammaglobulinemia
pre-alb, alb, RBP (retinol binding protein), transferrin, antithrombin
E. CLINICAL SIGNIFICANCE OF ABNORMAL PROTEIN LEVELS
2. Hyperproteinemia; a/↓Y - agama/hypogammaglobulin - ___
a. ___ – multiple myeloma, Waldenstrom’s macroglobulinemia (absolute increase)
b. Dehydration (relative increase)
Bruton’s agammaglobulinemia
Monoclonal gammopathy
F. MISCELLANEOUS PROTEINS OF MEDICAL IMPORTANCE
1. ___: Negative predictor of AMI; O2-carrying protein in skeletal and cardiac muscles; detected sooner than troponin but not cardiac specific
2. ___: Gold standard for ACS Cardiac-specific and show sustained elevation; TnI with greater cardiac specificity than TnT
- ___: BNP and NT-proBNP; used as markers for CHF; Structurally related neurohormones that affect body fluid homeostasis and blood pressure
- ___: Endogenous marker of GFR; Useful in cases where creatinine measurement is not appropriate
- ___: Endogenous marker of GFR; Correlates with serum cystatin C and urine microproteins; not influenced by glucocorticoid therapy
Myoglobin
Cardiac Troponins
Natriuretic Peptides
Cystatin C
β Trace Protein
F. MISCELLANEOUS PROTEINS OF MEDICAL IMPORTANCE
6. ___: Biomarker of bacteremia and sepsis; Increases early in infection (3-6 hours) and has a greater specificity for infection than CRP.
- ___: Early predictor of acute kidney injury (AKI); 25-kDa protein expressed by neutrophils and epithelial cells including those of the proximal tubule
- ___: Plasma FN – marker for nutritional status; Demonstrates a wide variety of cellular interactions, e.g. cell adhesion, differentiation, growth, and wound healing
- ___: ↓ value = ↑ risk of metabolic syndrome; Inversely correlated with BMI
- ___: Biochemical marker of bone resorption (osteoporosis); Proteolytic fragments of collagen I
- ___: Biomarkers of Alzheimer’s disease; ↑ NTP, ↓ Aβ42, ↑ Tau
Procalcitonin
Neutrophil gelatinase-associated lipocalin
Fibronectin
Adiponectin
Cross-linked C Telopeptides
Neuronal thread protein, Amyloid β42, Tau protein
G. LABORATORY DETERMINATION OF PROTEINS
1. ___ – ultimate reference method based on the measurement of N content (16%)
2. ___ – measurement of refractive index due to proteins in solution
3. ___ – measurement of the change in optical density following precipitation with TCA, SSA, benzalkonium chloride
4. ___ – measurement of absorbance at 280nm owing mostly to Trp, Tyr, and Phe
5. ___ – detection of peptide bonds!! forming a violet!! color (540 nm); Reagent composition: CuSO4, Rochelle salt (Na, K, Tartrate), NaOH, KI (potassium iodide)
Kjeldahl method
Refractometry
Turbidimetry
UV absorption
Biuret method
G. LABORATORY DETERMINATION OF PROTEINS
6. ___ – oxidation of phenolic groups producing a blue color
7. ___ – formation of violet color in the presence of amino groups
8. ___ – measurement of the shift in absorbance when proteins bind to a dye
a. Dye used for total protein _______
b. Dyes used for albumin:
1) ___ – nonspecific (globulins may also bind)
2) ___ – less sensitive; subject to interferences
3) ___ – most commonly used dye
4) ___ – specific, sensitive, precise
~Normal Albumin: 3.5-5.5 g/dL
A/G ratio = ? (↑ is normal, ↓ is inverted (monoclonal gammopathy)
Folin-Ciocalteau (Lowry) method
Ninhydrin method
Dye-binding technique; coomassie brilliant blue
Methyl orange
2,4-HABA
BCG
BCP
TP = A + G
G = TP - A
ASSESSMENT OF LIVER FUNCTION
A. FUNCTIONS OF THE LIVER
1. Storage – glycogen, fat-soluble vitamins _____________
2. ___ – lipids, carbohydrates, proteins (bile, C, L, P, TP, Alb, glob)
3. ___ – drugs, poisons, and metabolic products (drugs, poisons, NH3)
4. Secretory and excretory – conjugated bilirubin
gly, vitamin A, D, E, K
Synthetic
Detoxification
B. FORMS OF BILIRUBIN IN PLASMA: of B1 & B2?
Origin:
Structure:
Solubility:
Polarity:
Reaction:
Excreted in urine:
Affinity for CNS:
Origin
Structure
Solubility
Polarity
Reaction
Excreted in urine
Affinity for CNS
___:
Conjugated bilirubin covalently; bound to ______; Long half-life ___;
Seen in cases of bile duct destruction!!!; Reaction with diazo reagent _______; Separated from other fractions byHPLC
B1/uncon: pre-hepatic/hemolytic; unconjugated, insoluble; non-polar; indirect; no; yes, high
B2/conju: post-hepatic/obstructive; conjugated, soluble; polar; direct; yes; no, low
Delta bilirubin
albumin
17 days
direct
C. DERANGEMENTS OF BILIRUBIN METABOLISM
1. Physiological Classification of Jaundice
a. ___ – ↑ B1 due to ↑ production, ↓ delivery and uptake, or ↓ conjugation
b. ___ – ↑ B2 due to intra- or extrahepatic cholestasis
c. ___ - ↑ B1 and B2 due to a combination of defects
Unconjugated hyperbilirubinemia
Conjugated hyperbilirubinemia
Mixed hyperbilirubinemia
D. BILIRUBIN DETERMINATION
1. Specimen Considerations: _____
2. Methods
a. Colorimetric-Spectrophotometric
▪ based on the _____ reaction (diazotization with incorporated accelerator/solubilizer)
b. Direct spectrophotometry (Bilirubinometry)
▪ based on the absorption of light by bilirubin at _______ nm; correction for oxyhemoglobin is achieved by measuring absorbance at two wavelengths
▪ appropriate only for neonatal serum; not done on adult serum due to the presence of ___
c. Enzymatic
▪ involves oxidation of bilirubin to biliverdin by __ at different pH levels followed by further oxidation to a colorless product and measurement of the decrease in absorbance
avoid exposure to light and avoid hemolysis
Van den Bergh
450nm
carotenoid pigments
bilirubin oxidase
___:
Diazo reagents
▪ Diazo A = 0.1% sulfanilic acid in HCl
▪ Diazo B = 0.5% sodium nitrite
▪ Diazo blank = 1.5% HCl
___:
Diazo reagents
Sodium acetate
Ascorbic acid
Alkaline tartrate (Fehling’s reagent)
Accelerator: __
pH of the reaction: __
Color reaction: __
Absorbance maximum: __
Evelyn-Malloy
- methanol, acid, red-purple, 560nm
Jendrassik-Grof
- caffeine, alkaline, blue, 600nm
INTRODUCTION TO ENZYMOLOGY
A. GENERAL PROPERTIES
* ___: Biological protein catalysts that accelerate biochemical reactions without being consumed or changed in composition
* Act by lowering the activation energy level that the reactants must reach for the reaction to occur
* Found in all body tissues and frequently appear in the serum following cellular injury
* Exhibit certain degrees of specificity:
___ – combines with only one substrate and catalyzes only the corresponding reaction
___ – combines with all substrates containing a particular chemical group
___ – breaks or forms a solitary type of covalent bond
___ – combines with only one optical or geometric isomer of a compound
enzymes
Absolute specificity
Group specificity
Bond or reaction specificity
Stereoisomeric/geometric specificity
B. CLASSES OF ENZYMES
1 __ Oxidation–reduction; __
2 __ Transfer of functional groups; __
3 __ Hydrolysis; __
4 __ Removal of groups to form double bonds; __
5 __ Interconversion of isomers; Triose phosphate isomerase
6 __ Bond formation coupled with ATP hydrolysis; Glutathione synthetase
Oxidoreductases; LDH, GLDH, G-6-PD
Transferases; kinases, CK, PK, GGT, ALT, AST
Hydrolases; AMS, LPS, trypsin, ALP, ACP, 5’NT, ACE
Lyases; aldolase
Isomerases
Ligases
C. ENZYME KINETICS
1. Enzymatic Reaction/Catalytic Mechanism: E+S↔ES↔P+E
2. Effect of Substrate and Enzyme Concentration
▪ First-order kinetics: _________
▪ Zero-order kinetics: _________
Michaelis-Menten equation: V = (Vmax x [S])/(Km + [S])
Lineweaver-Burke plot – double reciprocal plot of the Michaelis-Menten equation
1/V = (Km/Vmax) x (1/[S]) + (1/Vmax)
enzyme > substrate (substrate dep)
substrate > enzyme (enzyme dep)
D. MEASUREMENT OF ENZYME ACTIVITY
1. Activity Units
a. __ – amount of enzyme that catalyzes the reaction of 1umol of substrate per minute
b. __ – amount of enzyme that catalyzes the reaction of 1mol of substrate per second
c. Conversions: 1 IU = _____ μkat = _____ nkat 1 kat = _____ IU
2. Methods
a. Fixed-time – ________; the reactants are combined, the reaction proceeds for a designated time, the reaction is stopped followed by measurement of the amount of reaction that has occurred
b. Continuous-monitoring – __________; multiple measurements of absorbance change as a function of time, either at specific time intervals or continuously by a continuous-recording spectrophotometer
IU
Katal
0.0167; 16.7; 6x 10^7
endpoint
kinetic
E. OTHER FACTORS THAT INFLUENCE ENZYMATIC REACTIONS
1. pH. Most reactions occur in the pH range of ____ except __________
2. Temperature
* For each 10 °C increase in temperature, the rate of the reaction doubles.
* Denaturation is usually significant at ___________
* Incubation temperatures in enzyme assays should not deviate by ±0.1°C
3. Cofactors: Nonprotein entities that must bind to particular enzymes before a reaction occurs.
a. Activators –
▪ may be essential for the reaction or may only enhance the reaction rate
▪ include metallic and nonmetallic ions e.g. Mg, Ca, Zn, Cl
b. Coenzymes – ___
▪ serve as second substrates for enzymatic reactions
▪ called prosthetic groups when bound tightly to the enzyme
▪ Examples: OR/DH (oxidation rxn, dehydrogenase)2 (NAD/NADH, NADP/NADPH)
7.0-8.0; ACP (pH 4-5) and ALP (pH 9-10)
40-50°C
inorganic cofactors;
organic:
E. OTHER FACTORS THAT INFLUENCE ENZYMATIC REACTIONS
4. Inhibitors
__ - binding is on the active site and effect of adding [S] reverses the inhibition
__ - binding is on the allosteric sites and adding [S] has no effect
__ - binding is in the ES complex and adding [S] increases the inhibition
F. __
▪ High-molecular-mass forms of the serum enzymes that can be bound to either an immunoglobulin (macroenzyme type 1) or a nonimmunoglobulin substance (macroenzyme type 2).
▪ Enzymes with macroforms: CK, AMS, LD, AST, ALT, ALP, ACP, LPS, GGT
Competitive
Non-competitive
Uncompetitive
~
MACROENZYMES
CLINICAL ENZYMOLOGY
A. ___
1. Tissue sources: _______________
2. Diagnostic significance
* Pronounced elevation: Duchenne’s muscular dystrophy, polymyositis, dermatomyositis, myocardial infarction
* Mild to moderate elevation: acute agitated psychosis, alcoholic myopathy, severe exercise, delirium tremens, severe ischemic injury, pulmonary infarction, intramuscular injections, hypothyroidism, muscular trauma
3. Isoenzymes
a. __ – elevated in CNS damage, tumors, presence of macro-CK
b. __ – present in significant amounts in the cardiac tissue; <6% of total CK in serum
c. __ – major isoenzyme found in striated muscle and normal serum (94-100%)
CREATINE KINASE
brain, myocardium, skeletal muscle
CK-BB (CK1) - most labile
CK-MB (CK2)
CK-MM (CK3)
CLINICAL ENZYMOLOGY
A. CREATINE KINASE
4. CK Determination
a. Specimen considerations: avoid prolonged storage, avoid chelators, avoid gross hemolysis
b. Methods
___: uses LDH to make Lactate + NAD⁺ anaerobically
___: uses G-6-PD to make 6PG + NADPH aerobically
Tanzer-Gilvarg (forward reaction)
Oliver-Rosalki (reverse reaction)
B. LACTATE DEHYDROGENASE
1. Tissue sources and isoenzymes
___: Heart, RBCs, renal cortex
___: Lungs, lymphocytes, spleen, pancreas LD3
___: Liver, skeletal muscles
~fastest? slowest?
▪ Normal serum ratio: ___
▪ Flipped ratio: ___
▪ __ – associated with arteriosclerotic cardiovascular failure signifying grave prognosis and impending death
LD1 (HHHH) & LD2 (HHHM)
LD3 (HHMM)
LD4 (HMMM) & LD5 (MMMM)
fast LD1; slow LD5
2,1,3,4,5
1,2,3,4,5
LD-6
- Diagnostic significance
* Pronounced elevation: ___, renal infarction, systemic shock and hypoxia, hepatic metastases, hepatitis
* Moderate elevation: ___, hemolytic conditions, pulmonary infarction, muscular dystrophy, delirium tremens, leukemias, IM
* Slight elevation: Most liver diseases, nephrotic syndrome, hypothyroidism, cholangitis - LD Determination
a. Specimen considerations; avoid cold storage, avoid hemolysis and avoid use of chelators
b. Methods both uses LDH
1) ____; product: pyruvate + NADH + H+
2) ____; product: lactate + NAD+
Megaloblastic/pernicious anemia
Myocardial infarction
Wacker (forward reaction)
Wroblewski-Ladue (reverse reaction)
Summary of MI Markers: (mnemonic fast to slow)
starts: peaks normalizes
1-3 hrs; 5-12 hrs; 18-30 hrs
3-4 hrs; 14-20 hrs; 5-10days
4-6 hrs; 12-24 hrs; 2-3 days
6-8 hrs; 18-24 hrs; 4-5 days
12-24 hrs; 38-72 hrs; 10 days
MICAL:
Myoglobin
Troponin I
CK-MB
AST
LD
C. AMINOTRANSFERASES/TRANSAMINASES
___:
1. Tissue sources _______
2. Diagnostic significance:
* Pronounced ↑: acute hepatocellular disorders, circulatory collapse, myocardial infarction
* Moderate ↑: muscular dystrophy, hepatic tumor, biliary obstruction, CHF, cardiac arrhythmia
* Slight ↑: cirrhosis, pericarditis, pulmonary infarction, cerebrovascular accident
___:
1. Tissue sources _______
2. Diagnostic significance:
* acute hepatocellular disorders (more specific, higher and more sustained elevation)
- Isoenzymes Cytoplasmic AST - AHCDs
Mitochondrial AST - ↑ in alcoholic liver disease & fulminant hepatic failure
____: AST:ALT ratio
AST/SGOT; liver, heart, skeletal muscles
ALT/SGPT; liver
De Ritis Ratio
C. AMINOTRANSFERASES/TRANSAMINASES
4. AST/ALT Determination
a. Specimen considerations ______________________________________________________________
b. Methods
1) ___- uses 2,4-dinitrophenylhydrazine and 0.4 N NaOH; product: 2,4− dinitrophenylhydrazone
2) ___ – based on the Karmen method; product: malate (lactate)+ NAD+
avoid hemolysis and prolonged storage
Reitman-Frankel
Coupled Enzymatic Reaction
D. ALKALINE PHOSPHATASE
1. Tissue sources: __________________________
2. Diagnostic significance
* Pronounced elevation: Bile duct obstruction, biliary cirrhosis, Paget’s disease, osteogenic sarcoma, hyperparathyroidism
* Moderate elevation: Granulomatous or infiltrative diseases of the liver, IM, metastatic bone tumors, rickets, osteomalacia
* Slight elevation: Viral hepatitis, cirrhosis, healing bone fractures, growing children, pregnancy
liver, bone, placenta, intestine
D. ALKALINE PHOSPHATASE
3. Isoenzyme analysis
* Electrophoresis ______; Heat stability: ______
(Placental ALP resists heating at ______; Bone ALP, <20% activity at _____)
* Chemical inhibition:
3M Urea __________
Phenylalanine ________
Levamisole ___________
liver, bone, placenta, intestine (lover boy, putang ina)
P>I>L>B (pota i love boners)
65°C for 30 mins
56°C for 10 mins
bone
P & I (putang ina)
L & B (lover boy)
D. ALKALINE PHOSPHATASE
*___________________ – isoenzyme that is more likely to be found in B or O secretor individuals; bound by group A erythrocytes; level increases after consumption of a fatty meal
* Carcinoplacental Isoenzymes:
Regan – detected in lung, breast, ovarian, colon, ovarian, and gynecologic cancers; inhibited by ________
Nagao – detected in carcinoma of pleural surfaces, pancreas, and bile duct; inhibited by _____
Kasahara – associated with _______
- ALP Determination
a. Specimen considerations: avoid chelators, hemolysis and long storage
b. Reference method: __________
c. Other methods: Bessy, Lowry, Brock (PNPP), King-Armstrong (phenylphosphate), Moss (α-naphthylphosphate), Bodansky, Sinowara, Jones, Reinhart (β-glycerophosphate), Huggins-Talalay
intestinal ALP
phenylalanine
leucine
GI and hepatic tumors
Bower’s-McComb (C/S)
E. PANCREATIC ENZYMES
___:
1. Tissue sources _________
2. Diagnostic significance: Acute pancreatitis (↑ 5-8 h, peaks at 24 h, normalizes in 3-5 days); Mumps, parotitis, macroamylasemia
___:
1. Tissue sources _________
2. Diagnostic significance: Acute pancreatitis (↑ 4-8 h, peaks at 24 h, normalizes in 7-14 days)
- Isoenzymes: pancreatic amylase, salivary amylase or __ (inhibited by __)
- Amylase Determination
a. Specimen considerations: avoid painrelievers. and lipemic serum
b. Methods
1) ___ – decrease in color of starch-iodine complex
2) ___ – ↑ reducing sugars as starch is hydrolyzed by AMS
3) ___ – ↑color intensity of soluble dye-substrate solution
4) ___ – ↑ Abs of NADH at 340 nm
Amylase; pancreas, salivary glands
Lipase; pancreas
sAMS; wheat germ lectin
Amyloclastic method
Saccharogenic method
Chromogenic method
Coupled-enzymatic/Continuous-monitoring method
E. PANCREATIC ENZYMES
5. Lipase Determination
a. Specimen considerations ______________
b. Methods
1) ___ – uses olive oil as substrate: TG + H2O LPS ↔ glycerol + fatty acids
2) ___ – measures the rate of clearing as fats are hydrolyzed by LPS
3) ___ – based on coupled reactions with glycerol kinase and peroxidase
avoid hemolysis
Cherry-Crandall method
Turbidimetric method
Colorimetric methods
F. MISCELLANEOUS ENZYMES
1. ___ - Hepatobiliary disorders, alcoholic liver disease, microsomal induction by drugs and alcohol; used to identify the source of ↑ ALP
○ ___: γ-Glutamyl-p-nitroanilide + Glycylglycine → γ-Glutamyl-glycylglycine + p-nitroaniline
2. ___ - Intrahepatic cholestasis; used to identify the source of ↑ ALP
3. ___ - Major sources: prostate, RBCs
○ Significance: prostatic carcinoma, hyperplasia, rape
○ Roy method: substrates: thymolphthalein monophosphate
○ Chemical inhibition: ___
Prostatic ACP = Total ACP – ACP after tartrate inhibition
4. ___ - skeletal/muscle disorders
Gamma glutamyl transferase (GGT); Szasz method
5’Nucleotidase
Acid Phosphatase;
○ tartrate (-) prostatic ACP; Cu & formaldehyde (-) RBC, ACP
Aldolase
F. MISCELLANEOUS ENZYMES
5. Glutamate dehydrogenase & Glutathione-S-transferase - MDH disorders
7. ACE - BP regulation
8. Glycogen phosphorylase - AMI
9. ___ - pancreatic insufficiency
11. ___ - G-6-PD deficiency → drug-induced hemolytic anemia
12. ___ - hemolytic anemia secondary to emmbrane abnormality (echinocytosis)
13. ___ - pesticide poisoning, genetic variants (abnormal muscle relaxants), liver disease
Trypsin & Chymotrypsin/Elastase 1
G-6-PD
Pyruvate kinase
Pseudocholinesterase
ELECTROLYTES AND BLOOD GASES
A. REGULATORY FACTORS
1. ADH/AVP – stimulates ________
2. ANP – released from the myocardial atria in response to volume expansion, promotes _____
3. RAAS: __ (promotes vasodilation of afferent, vasoconstriction of efferent; Na reabsorption in DCT; (+) aldosterone secretion;
H2O reabsorption
Na excretion
angiotensinogen→angiotensin I (by renin)→angiotensin II (by ACE)
B. FUNCTIONS OF ELECTROLYTES
1. Volume and osmotic regulation _____
2. Myocardial rhythm and contractility and neuromuscular excitability ___
3. Cofactors in enzyme activation __
4. Regulation of ATPase-ion pumps __
5. Blood coagulation ___
6. Production and use of ATP from glucose __
7. Maintenance of acid-base balance ____
- Na, Cl, K
- K, Mg, Ca
- Mg, Ca, Zn, Cl
- Mg
- Ca, Mg
- Mg, PO4
- HCO3-, Cl, K
C. INSTRUMENTATION
1. Photometric Techniques
a. ___ – measurement of light emitted by atoms following excitation by heat energy; traditionally used to determine the concentration of Na, K, and Li; employs an internal standard
b. ___ – measurement of the amount of light absorbed by ground state atoms; light source hollow cathode lamp
Flame photometry
Atomic absorption spectrophotometry
C. INSTRUMENTATION
2. Electrochemical Techniques
a. ___ – measurement of electrical potential (voltage) due to the activity of free ions
○ ___ – H+ sensitive glass electrode containing Ag/AgCl wire in electrolyte of known pH and reference electrode (Hg/Hg2Cl2)
○ ___ – pH electrode covered with membrane permeable to CO2, with bicarbonate buffer between membrane and electrode
○ ___ – used to measure Na, K, Cl, ionized Ca
b. ___ – measurement of the amount of electricity in coulombs at a fixed potential
▪ ___ – used in serum and sweat chloride analysis; endpoint is increase in conductivity measured by amperometry
c. ___ – measurement of the current flow produced by an oxidation-reduction reaction at a fixed applied potential to the electrodes
▪ ___ – uses a platinum cathode and Ag/AgCl anode covered with gas-permeable membrane
which allows dissolved O2 to pass through
d. ___ – measurement of current produced at gradually increasing voltage applied between two electrodes in contact with a solution containing the analyte
e. ___ – measurement of current flow as a metal is stripped off the anode of a polarographic cell; used for Pb and Fe testing
Potentiometry
○ pH electrode
○ pCO2 electrode
○ Ion-selective electrodes
Coulometry
○ Cotlove chloridometer
Amperometry
pO2 electrode
Polarography
Anodic stripping voltammetry
C. INSTRUMENTATION
3. ___ – measurement of osmolality indirectly by measuring one of the colligative properties of solution which change proportionally with the number of dissolved particles present.
Colligative property; Effect of 1 mole of solute
Boiling point ___ 0.52 °C
Osmotic pressure ___ 1.7 × 109 mm Hg
Freezing point ___ 1.86 °C
Vapor pressure ___ 0.3 mm Hg at 25°C
Osmometry
↑
↑
↓ (most common)
↓
D. IMPORTANT ELECTROLYTES
▪ Major extracellular cation
▪ Major contributor to ____
▪ Renal threshold:_________
▪ Reference range: 136–145 mmol/L
▪ Panic values: ≤ 120; ≥ 160 mmol/L
Methods
a. ISE - most common glass electrode
▪ Direct - no sample dilution
▪ Indirect - with sample dilution
b. Flame photometry: ___ Na
c. Albanese-Lein
Clinical Significance
a. Hyponatremia
▪ ↑ Na+ loss: Addison’s disease, salt-losing nephropathy, ketonuria (DKA), prolonged vomiting or diarrhea, severe burns, diuretics
▪ ↑ water retention: renal failure, CHF, nephrotic syndrome, hepatic cirrhosis
▪ Water imbalance: increased water intake, SIADH, pseudohyponatremia
b. Hypernatremia
▪ ↑ Na+ intake or retention: NaHCO3 excess, hyperaldosteronism
▪ ↑ water loss: dehydration, profuse sweating, DI, renal tubular disorder
- SODIUM
plasma osmolality
110-130mmol/L
yellow
D. IMPORTANT ELECTROLYTES
▪ Major intracellular cation
▪ __ higher inside the cells
▪ Reference values: 3.5-5.1 mmol/L (serum); 3.5-4.5 mmol/L (plasma)
▪ Panic values: ≤2.8; ≥6.8mmol/L
Methods
a. ISE - valinomycin
b. Flame photometry - ___
c. Lockhead-Purcell
Clinical Significance
a. Hypokalemia
▪ Cellular shift: alkalosis, insulin overdose
▪ GI loss: vomiting, diarrhea, gastric suction, laxatives, malabsorption
▪ Renal loss: renal tubular acidosis, hyperaldosteronism, thiazide diuretics
b. Hyperkalemia
▪ Cellular shift: acidosis, chemotherapy, muscle injury, leukemia, hemolysis
▪ ↑ intake: oral or IV potassium replacement
▪ ↓ renal excretion: renal failure, hypoaldosteronism, K+-sparing diuretics
- POTASSIUM
20x
violet light
D. IMPORTANT ELECTROLYTES
▪ Major extracellular anion
▪ Passively follows Na+
▪ Inverse relationship with HCO3-, chloride shift
▪ ___ – uses pilocarpine iontophoresis to stimulate sweat production, with subsequent collection of sweat for analysis
▪ Reference values:
Serum, 98–107 mmol/L; Sweat, <40 mmol/L
▪ Panic values: Serum, ≤80; ≥120mmol/L; Sweat, >60mmol/L
Methods
a. __ – ion exchange membrane (tri-n-octylpropylammonium chloride decanol)
b. ___ - coulometric-amperometric titration
c. ___ – mercurimetric titration using mercuric nitrate and diphenylcarbazone indicator (blue-violet color)
d. ___ – spectrophotometric assay using mercuric thiocyanate (red complex)
Clinical Significance
a. Hypochloremia
▪ aldosterone deficiency, salt-losing nephropathy, DKA, prolonged vomiting or diarrhea, severe burns, diuretics
▪ metabolic alkalosis, compensated respiratory acidosis
b. Hyperchloremia
▪ renal tubular acidosis, GI loss of HCO3-
▪ metabolic acidosis, compensated respiratory alkalosis
- CHLORIDE
Gibson and Cooke’s method
ISE
Cotlove chloridometry
Schales-Schales
Whitehorn titration
D. IMPORTANT ELECTROLYTES
▪ Second major extracellular anion
▪ Comprises >90% of the total CO2 at physiologic pH
▪ Reference values: TCO2, 23–27 mmol/L; HCO3, 22–26 mmol/L
▪ Panic values: ≤10, ≥40mmol/L
Methods (Total CO2)
a. ___ – involves acidification of the sample ff by electrode-based detection of released CO2
b. ___ – involves alkalinization to convert all CO2 and H2CO3 to HCO3- followed by coupled enzymatic reaction: (uses malate dehydrogenase)
Clinical Significance
a. ↓ HCO3: metabolic acidosis, compensated respiratory alkalosis, RTA, GI loss of HCO3
b. ↑ HCO3: metabolic alkalosis, compensated respiratory acidosis
- BICARBONATE
ISE
Enzymatic
D. IMPORTANT ELECTROLYTES
▪ Cofactor of >300 enzymes
▪ 10× higher in RBCs
▪ Distribution:
_____ ionized
_____ bound to proteins
_____ bound to ions
▪ Reference values: 1.26-2.1 mEq/L; (1.51-2.52 mg/dL)
▪ Panic values: ≤1; ≥4.7mg/dL
Methods
a. AAS - ref mtd
b. Dye-binding
▪ ___ – most commonly used; Other dyes used: formazan, methylthymol blue, xylidyl blue
c. ___ – Titan yellow, Clayton yellow, or thiazole yellow dye
Clinical Significance
a. Hypomagnesemia
▪ ↓ intake: poor diet, starvation, prolonged Mg-deficient IV therapy, chronic alcoholism
▪ ↓ absorption: prolonged vomiting, diarrhea, nasogastric suction, laxatives, malabsorption syndrome, intestinal resection
▪ ↑ excretion: renal, endocrine, drug-induced
b. Hypermagnesemia
▪ ↑ intake: antacids, enemas, cathartics
▪ ↓ excretion: renal failure,hypoaldosteronism
- MAGNESIUM
55%
30%
15%
Calmagite
Dye-lake method
D. IMPORTANT ELECTROLYTES
▪ Plasma level regulated by ___: stimulates bone resorption, Vit D activation, CA2+ reabs in kidney
○ Active Vit D (1,25-[OH]2-D3 or ___): intestinal absorption of Ca; Ca2+ reabsorption
○ ___: inhibits PTH actions = ↓ Pl. Ca
▪ Distribution:
_____ ionized
_____ bound to proteins
_____ bound to ions
▪ Reference values (total): 4.3-5 mEq/L (8.6-10 mg/dL)
▪ Panic values: ≤6; ≥13mg/dL
Methods
a. AAS
b. ISE – consists of PVC membrane impregnated with a calcium ion exchanger; used to measure ionized calcium
c. Dye-binding - O-cresolphthalein complexone (CPC) method – requires addition of _______ to prevent Mg interference; Arsenazo III – high affinity for Ca2+ and inhibits Mg2+
d. Clark-Collip oxalic acid precipitation - purple
e. Ferro-Ham chloranilic acid precipitation - purple
Clinical Significance
a. Hypocalcemia-
▪ primary hypoparathyroidism
▪ secondary hyperparathyroidism, chronic renal failure, hypomagnesemia, ↓ albumin, pseudohypoparathyroidism, vitamin D deficiency, pancreatitis
b. Hypercalcemia
▪ primary hyperparathyroidism
▪ hypercalcemia of malignancy, multiple myeloma, vitamin D excess, prolonged immobilization
- CALCIUM
PTH
calcitriol
Calcitonin
50%
40%
10%
8-hydroxyquinoline
D. IMPORTANT ELECTROLYTES
▪ Major intracellular anion
▪ Component of several essential biomolecules
▪ Usually measured as inorganic phosphate
▪ Reference range: 2.4–4.4 mg/dL
▪ Panic values: ≤ 1; ≥8.9mg/dL
Method
___: PO4 + ammonium molybdate → Ammonium phosphomolybdate
Clinical Significance
a. Hypophosphatemia: diabetic ketoacidosis, long-term TPN, IBD, anorexia nervosa, alcoholism, hyperparathyroidism, vitamin D deficiency
b. Hyperphosphatemia: renal failure, milk or laxatives, neoplastic disorders, intravascular hemolysis, lymphoblastic leukemia
- PHOSPHATE
Fiske-Subbarow
D. IMPORTANT ELECTROLYTES
▪ By-product of anaerobic glycolysis
▪ Accumulation in the blood indicates ___
▪ Converted back to glucose by the ____
▪ Reference values (venous blood):
Colorimetric, 8.1-15.3 mg/dL; UV, 4.5-19.8 mg/dL
Methods
a. POD-coupled reaction
b. Enzymatic-UV
Lactate + NAD →(LD)→ pyruvate + NADH
Clinical Significance
a. Type A lactic acidosis: Shock, MI, severe CHF, pulmonary edema, or severe blood loss
b. Type B lactic acidosis: Diabetes mellitus, severe infection, leukemia, liver or renal disease, and toxins (ethanol, methanol, or salicylate poisoning)
- LACTATE
hypoxia
liver (gluconeogenesis)
E. OSMOLALITY AND OSMOLAL GAP
1. __ – physical property of a solution that is based on the concentration of solutes (expressed as milliosmoles) per kilogram of solvent; affected only by the number of dissolved particles present
2. Specimen: __________
3. Methods
a. ___ (measured osmolality) – based on the measurement of a decrease in freezing point (or vapor pressure)
b. Calculated Osmolality = 2Na (mmol/L)+(gluc mg/dL/20)+(BUN/3)
c. Osmolal gap = ___ (formula); effective screening method in
detecting the presence of toxic compounds
4. Reference values: Serum osmolality: 275–295 mOsm/kg Osmolal gap: 5–10 mOsm/kg
5. Clinical significance (OG>12 mOsm/kg): uremia, diabetic ketoacidosis, lactic acidosis, intoxication with alcohols, ethylene glycol, salicylate
Osmolality
serum (plasma causes F↑)
Osmometry
Osmolal gap = Osmm-Osmc
▪ Used as a form of quality control for _____
▪ May be used to distinguish between normochloremic and hyperchloremic metabolic acidosis
1. Formulas: AG = (Na+ + K+) – (Cl- + HCO3) or AG = Na+ – (Cl- + HCO3-)
2. Reference values: 10-20 mmol/L or 7-16 mmol/L
↑ Anion Gap:
↑ Unmeasured Anions - mnemonic
↓ Unmeasured Cation - hypocalcemia
Laboratory Error - F↑ Na; F↓ Cl or HCO3
↓ Anion Gap
↓ Unmeasured Anions _____
↑ Unmeasured Cations _____
Laboratory Error: F↓ Na, F↑ Cl or HCO3
ANION GAP
electrolyte analyzers
MUDPILES:
Methanol, Uremia, Diabetic ketoacidosis, Propylene glycol, Isoniazid, Iron, Lactic acidosis, Ethylene glycol, Salicylates
hypoalbuminuria
hypercalcemia
G. ACID-BASE HOMEOSTASIS
1. Basic concepts
▪ __ – substance that can yield hydrogen ions (H+) when dissolved in water (Arrhenius); proton donor (Bronsted and Lowry)
▪ __ – substance that can yield hydroxyl ions (OH−); proton acceptor
▪ __ – combination of a weak acid and its salt or conjugate base that resists changes in pH
- Buffer systems
▪ __ – second most important blood buffer; contains 38 histidine residues that bind H+
▪ __ – have free carboxyl and amino groups that are able to bind H+
▪ __ – uses HPO42– and H2PO4− to minimize pH changes in plasma and erythrocytes
▪ __ – most important buffer system in the blood - Henderson-Hasselbalch equation: __
▪ The numerator (HCO3–) denotes function of the _____ and is ____ to pH.
▪ The denominator (pCO2, which represents H2CO3) denotes function of the ___ and is ___ to pH.
▪ When the ratio between HCO3– and H2CO3 is 20:1, the pH is ____.
Acid
Base
Buffer
Hemoglobin
Plasma proteins
Phosphate buffer system
Bicarbonate/carbonic acid buffer system
pH = pKa + log (HCO3/H2CO3)
H2CO3 = pCO2 x 0.03
kidneys; proportional
lungs; inversely proportional
7.4
H. ARTERIAL BLOOD GAS ANALYSIS
1. Specimen considerations
▪ Specimen: Arterial blood (required if pO2 will be measured) collected __ in 1- to 3-mL sterile glass syringes. Venous and capillary samples may be acceptable.
▪ Anticoagulant: ___ (preferred) or 0.05 mL liquid heparin (1000 IU/mL) per mL of blood
▪ Handling: Anaerobic collection, kept on __ to retard WBC metabolism, and assayed within 15 minutes
without tourniquet
Lyophilized heparin
ice
Reference ranges:
pH
pCO2
pO2
HCO3–
Total CO2 (HCO3−+ H2CO3 + CO2)
Base excess
O2 saturation
7.35-7.45
35-45 mmHg
80-110 mmHg
22-26 mmol/L
23-27 mmol/L
-2 to +2
>95%
- Factors that affect ABG values
a. Oxyhemoglobin Dissociation Curve
b. Temperature:
▪ Electrode chambers of blood gas analyzers are
thermostatically controlled at __________.
▪ For every 1°C increase in body temperature,
pCO2 increases by _____; pO2 decreases by _____; and
pH decreases by _________ unit.
c. Specimen exposure to air: bubbles, ↓pCO2, ↑pO2 and pH
d. Prolonged storage of specimen: ↑pCO2, ↓pO2 and pH
e. Excess anticoagulant:
▪ ↑ dry heparin – ___________
▪ ↑ liquid heparin – __________ (dilutional effect)
37±0.1°C
3%; 7%
0.015
↓pH
↓pCO2
I. MICRONUTRIENTS
1. ___: component of heme-containing substances
* transport protein____; storage forms ___
a. Clinical significance
1) Iron deficiency: Anemia due to blood loss, decreased intake, decreased release from ferritin
2) ___: Hemochromatosis (primary/genetic or acquired)
transferrin; ferritin, hemosiderin
Iron overload
I. MICRONUTRIENTS
b. Iron Determination
1) ___
Specimen considerations: early morning collection, void hemolysis and chelators
Procedure: Acidification with HCl to deconjugate Fe3+ from transferrin followed by reduction to Fe2+which then reacts with a chromogen (ferrozine, bathophenanthroline, or tripyridyltriazine) producing a blue complex.
Reference range: ________
2) ___
▪ Methods involve addition of sufficient Fe3+ to saturate transferrin followed by removal of excess iron and measurement of bound iron by the same procedure used for serum iron.
▪ Formula for estimating TIBC: ___
▪ Reference range: 250-350 μg/dL
Serum Iron
50-160mg/dL
Total Iron-Binding Capacity
TIBC (mg/dL) = Tf (mg/dL) x 1.43
TIBC (mg/dL) = UIBC + serum Fe
I. MICRONUTRIENTS: IRON DET
3) % Saturation or Transferrin saturation
▪ Formula ___
▪ Reference range: _______________________
4) ___
▪ Best test for iron deficiency; determined using immunoassay
▪ Reference values: 12-125 μg/L (women) 30-250 μg/L (men)
% sat = (Serum Fe/TIBC) x 100
20-50%
Ferritin
- Other Essential Trace Metals
○ Cofactor for∼300 enzymes
○ Acrodermatitis enteropathica, Chronic liver and kidney disease, Alcoholism, diets low in zinc
♣ Administration of high doses; Exposure to ZnO fumes
○ Component of several metalloenzymes
○ Chronic copper loss; Aceruloplasminemia
○ ________ – failure of Cu absorption
♣ ________ – failure to excrete Cu in bile; excess Cu in liver, brain, eyes (cornea)
ZINC
COPPER
Menke’s syndrome
Wilson’s disease
- Fat-soluble vitamins:
___: Retinol; Component of retinal rod; night blindness
___: Cholecalciferol; Absorption of Ca and P; Rickets (C), osteomalacia (A)
___: Tocopherol; Antioxidant, RBC integrity ;Mild hemolytic anemia, red cell fragility, ataxia
___: Phylloquinone/menaquinone; Cofactor of procoagulants hemorrhage or bruising post-
traumatic bleeding
Vit A
Vit D
Vit E
Vit K
- Water-soluble vitamins
Vit C; ___; Hydroxylation of collagen; ___
Vit B1; ___; Coenzyme in decarboxylation reactions; _________, Wernicke-Korsakoff syndrome
Vit B2; ___; Converted to flavin coenzyme; _______, dermatitis, photophobia, neurologic changes
VIt B3; ___; Component of NAD and
NADP, redox reactions; ______
Vit B5; __; Incorporated in coenzyme A; Paresthesia, depressed immune system, muscle weakness
Ascorbic acid; Scurvy
Thiamine; Beri-beri
Riboflavin; Angular stomatitis
Niacin/ Nicotinic acid; Pellagra
Pantothenic acid
- Water-soluble vitamins
Vit B6; __; Coenzyme in many intermediary reactions; _________________, cheilosis, glossitis,
dermatitis, peripheral neuropathy
Vit B7; __; Cofactor in carboxylation reactions; Dermatitis, glossitis, hair loss, depression
Vit B9; __; DNA and amino acid synthesis; ___________
Vit B12; __; Folate metabolism and DNA synthesis; ___
Pyridoxine/pyridoxal phosphate; Facial seborrhea
Biotin
Folic acid/Pteroylglutamic acid; Megaloblastic anemia; Neural tube defects
Cyanocobalamin; Megaloblastic anemia
CLINICAL ENDOCRINOLOGY
A. ___
* chemical messengers synthesized and secreted by endocrine glands, organs, or isolated cells that have regulatory effects on target cells; generally produced at one site and exert their action(s) at distant sites
HORMONES
B. STRUCTURAL CLASSES OF HORMONES
___:
- Water-soluble; no carrier protein; short half-life
- Glycoproteins: FSH, LH, TSH, hCG
- Polypeptides: ACTH, GH, PRL, ADH, oxytocin, PTH, calcitonin, insulin, glucagon, gastrin
___:
- Synthesized from cholesterol; Lipid-soluble; require a carrier protein
- Adrenocortical hormones: cortisol, aldosterone, DHEA
- Gonadal hormones: estrogens, progesterone, testosterone
___:
- Synthesized from tyrosine
- Thyroid hormones behave like steroids
- Catecholamines behave like protein hormones
- Thyroxine (T4), T3, epinephrine, norepinephrine
PROTEINS/PEPTIDES
STEROIDS
AMINES
C. THE HYPOTHALAMIC-PITUITARY-TARGET ORGAN (HPT) AXIS
1. Feedback Loop
a. ___ – a releasing factor acts on the pituitary gland which in turn releases tropic hormones that act on a specific target gland
b. ___ – elevated levels of a target cell hormone turn off the secretion of a stimulating hormone from the pituitary gland and a releasing hormone from the hypothalamus
- Disorders of Hormone Secretion
a. Tertiary disorders refer to a defect in the ______________________
b. Secondary disorders refer to a defect in the _______________________________
c. Primary disorders refer to a defect in the _______________________________
Positive feedback
Negative feedback
hypothalamus
anterior pituitary
target organ
D. HYPOTHALAMUS
HORMONE; ACTION
Gonadotropin-releasing hormone Stimulates release of __________
Corticotropin-releasing hormone Stimulates release of __________
Thyrotropin-releasing hormone Stimulates release of __________
GH-releasing hormone Stimulates release of ___________
Somatostatin Inhibits release of _____________
Dopamine (PIF) Inhibits release of ______________
FSH, LH
ACTH
TSH
GH
GH, TSH
PRL-prolactin
E. PITUITARY GLAND
* Previously referred to as the master gland; connected to the hypothalamus by the __
* Endocrine systems that are independent of the pituitary gland: ________________
- ANTERIOR PITUITARY GLAND
* 5 distinct populations of glandular cells: somatotrophs (secrete GH), lactotrophs (secrete PRL), thyrotrophs (secrete TSH), gonadotrophs (secrete FSH and LH), and corticotrophs (secrete ACTH)
pituitary stalk/infundibulum
RAAS, glucose-insulin axis, calcitonin-PTH axis
- ANTERIOR PITUITARY GLAND; target tissue?
FSH: __ Follicle development; secretion of
estrogen; sperm production
LH: __ Stimulates ovulation and secretion of
progesterone/ testosterone
ACTH: __ Release of cortisol
TSH: __ Release of thyroid hormones
PRL: __ Production of milk
GH: __ Growth of muscles and long bones;
stimulates release of _________________
~ * ___ – synthesized in the liver in response to stimulation by GH; mediates the metabolic actions of GH; negatively feeds back on the pituitary gland to inhibit GH secretion
gonads
gonads
adrenal cortex
thyroid
breasts
whole body
IGF-1/Somatomedin C
- ___
* Serves as repository of hormones produced by the supraoptic and paraventricular nuclei of the
hypothalamus and releases them on demand
2 hormones and its target tissues
__: Increases water reabsorption and BP; ↑ in SIADH, ↓/x in DI
__: Uterine contraction, ejection of milk
POSTERIOR PITUITARY GLAND
ADH/AVP; renal tubules, arterioles
Oxytocin; uterus & breasts
F. THYROID GLAND
The thyroid gland is a small __ organ made up of two lobes that rest on each side of the trachea bridged by a band of tissue called ________
Follicles are spheres of thyroid cells surrounding a core of a viscous substance termed ______ which mainly consists of _____, a glycoprotein manufactured exclusively by thyroid follicular cells.
Main cell types:
* __: produce T4 and T3 which are stored in the colloid of the follicles
* __: produce calcitonin which is important in calcium homeostasis
Thyroid hormone fractions:
* __ – 100% of thyroidal origin
* __ – 20% of thyroidal origin; 80% from
monodeiodination of T4 in nonthyroidal tissues.
* Bound forms: 70% of T4 is bound to ____; 20%
bound to TBPA/transthyretin; 10% bound to __
* Free forms: 0.04% of T4 and 0.4% of T3
butterfly-shaped
isthmus
colloid
thyroglobulin
Follicular cells
Parafollicular (C) cells
T4
T3
TBG
albumin
- Thyroid Hormones
___: Regulation of oxygen consumption, heat production, growth, sexual maturity, metabolism
○ Primary hyperthyroidism: ________
○ Primary hypothyroidism: ________
Reverse T3 (3,3’,5’-Triiodothyronine); ↑ non-thyroidal illness/Euthyroid sick syndrome
__: Regulation of calcium level; Medullary Thyroid Carcinoma
- Laboratory Evaluation
FTI formula: FTI = TT4 x (Px T3U/Mean x N x T3U)
__ - biologically active form of T4.
__ - Most important test of thyroid function. 1st test for screening. If normal, no further testing.
__ - Reflects the quantity of TBG present and the quantity of hormone attached to it
__ - biologically active form of T3. Usually only tested when TSH is ____ and FT4 is _________.
__ - Measures thyroidal uptake of iodine after an oral dose of __________; useful in the evaluation and treatment of thyroid cancer.
__ - Detected as thyroid microsomal antibodies using agglutination or immunofluorescence; very sensitive marker for _______
__ - Thyroid-stimulating antibodies that mimic the action of TSH; Positive in 85% of patients with ________.
Thyroxine (T4) & 3,5,3’–Triiodo-thyronine (T3)
Grave’s disease, T3 thyrotoxicosis
Hashimoto’s thyroiditis, congenital hypothyroidism (cretinism)
Calcitonin
FT4
TSH
T3 Uptake (T3U)
FT3; ↓; N
RAIU; I-131
Anti-TPO; Hashimoto’s Thyroiditis
TRAb/TSHRAb; Grave’s disease
H. ADRENAL HORMONES: source and target sites?
Aldosterone (mineralocorticoid); Blood pressure and electrolyte regulation (under the control of RAAS)
Cortisol (glucocorticoid); Carbohydrate, protein, and lipid metabolism
Sex steroids; Precursor to active androgens; growth and maturation
Epinephrine; Stimulates sympathetic nerves;
promotes glycogenolysis and lipolysis
Norepinephrine; Stimulates sympathetic nerves
Zona glomerulosa; kidneys
Zona fasciculata; general
Zona reticularis; gonads
Medulla; SNS; liver, SM, adipose
Medulla; SNS
Clinical significance of adrenal hypersecretion and hyposecretion:
Urine free cortisol collected over 24 hours
Midnight salivary cortisol
Dexamethasone suppression test (LD = 1 mg overnight or 2 mg over 48 hours; HD = 8 mg)
Plasma ACTH – requires use of __
Cushing’s syndrome/Cushing’s disease
pre-chilled tube
Clinical significance of adrenal hypersecretion and hyposecretion:
___: 21-Hydroxylase, plasma 17hydroxyprogesterone and pregnanetriol, urinary pregnanetriol and 17-ketosteroids
*___ – development of overt male secondary sexual features
*___ – non-typically appearing female external genitalia
*___ – excessive hair growth due to ↑ ovarian androgens in women; Scale used for grading hirsutism: _________
Congenital adrenal hyperplasia
○ Virilization
○ Genital ambiguity
○ Hirsutism; Ferriman-Gallwey Scale (mabalbon na babae sa gateway)
I. MALE GONADAL/REPRODUCTIVE HORMONES
___:
* Seminiferous tubules – contain germ cells and ____
* Interstitium – contains ____ (synthesize testosterone)
Hormones:
* Principal androgen synthesized in the testes (95%);<5% derived from adrenal precursors
* 50% bound to albumin, 45% is bound to SHBG, 2-3% free; Metabolites: ___________
Testes
sertoli cells
Leydig cells
Testosterone
estradiol, DHT (dihydrotestosterone)
Clinical significance of abnormal secretion
Primary hypergonadism – _______
Primary hypogonadism – ________
Secondary hypergonadism – _____
Secondary hypogonadism – ____
FSH, LH; testosterone
testicular tumor; ↓; ↑
Klinefelter syndrome; ↑; ↓
Precocious Puberty; ↑; ↑
Panhypopituitarism; ↓; ↓
J. FEMALE GONADAL/REPRODUCTIVE HORMONES
Ovaries
* ___– approx. 400,000 in neonates, each containing an immature ovum
* ___ – selected from a cohort of follicles; releases ovum during the luteal phase giving rise to corpus luteum
Placenta
* ___ – vascular projections of fetal tissue surrounded by chorion!!
* ___ – consists of the outer syncytiotrophoblast!!, which is in direct contact with maternal blood, and the inner cytotrophoblast
Primordial follicles
Dominant Graafian follicle
Chorionic villi
Chorion
HORMONES:
Estrogens
* ____ – principal and most potent estrogen in premenopausal women;
* ____ – metabolite of estradiol or produced from androstenedione
* ____ – produced by the ovaries and the placenta
__ - androstenedione, dehydroandrostenedione, testosterone, and DHT
__ – produced by the corpus luteum and the placenta; prepare the endometrium for implantation and maintains pregnancy
__ – structurally similar to PRL & GH; stimulates development of mammary gland; increases maternal plasma glucose levels
___:
* produced by the syncytiotrophoblast cells of the placenta; stimulates the ovary to produce progesterone, which in turn, prevents menstruation
* dimeric hormone: α subunit - same as ______, β subunit - unique
* increases from less than 5 mIU/mL to >100,000 mIU/mL during the first trimester of pregnancy, reaching a peak at about 16 weeks’ gestation
Estradiol (E2)
Estrone (E1)
Estriol (E3)
Androgens
Progesterone
hPL
hCG
FSH, LH & TSH
Disorders of abnormal secretion of female hormones:
Primary hypergonadism – ______
Primary hypogonadism – _____
Secondary hypergonadism – _____
Secondary hypogonadism – ___
FSH, LH; Estradiol:
ovarian tumor; ↓; ↑
menopause/Turner Syndrome; ↑; ↓
Gonadotropin secreting tumors; ↑; ↑
Sheehan’s Syndrome; ↓; ↓
K. MISCELLANEOUS GLANDS AND HORMONES
target: Hypothalamus; action: Induction of sleep
target: Cardiovascular, respiratory, and gastrointestinal systems; brain; action: Neurotransmitter; stimulation or inhibition of various smooth muscles and nerves
target: Stomach; action: Secretion of gastric acid,
gastric mucosal growth
target: Muscle, liver; action: Increases fatty acid oxidation; suppresses glucose formation
target: Hypothalamus; action: Inhibition of appetite, stimulation of metabolism
source and clin sig
Melatonin; pineal gland
Serotonin; pineal gland, GIT; inc in Agentaffinoma/Carcinoid syndrome
Gastrin; GIT; inc in Zollinger-Ellison Syndrome
Adiponectin; adipose
Leptin; adipose
___
A. CLINICAL UTILITIES
* Cancer screening or diagnosis, tumor staging, monitoring therapy, predicting patient outcomes, detecting cancer recurrence
B. CHARACTERISTICS OF AN IDEAL TUMOR MARKER
* Cancer-sensitive, cancer-specific, and tissue-specific
* Readily detectable in the blood
* Shows elevation at the early stage of cancer
* Serum levels correlate with tumor burden
TUMOR MARKERS
TUMOR MARKERS; what is their type?
AFP
CEA
CA 125
CA 72-4
CA 19-9
CA 15-3, 27.29
ER, PR, HER-2/neu
Prostate-specific antigen
Neuron-specific enolase
CYFRA 21-1
Nuclear matrix protein
Thyroglobulin S
Calcitonin
B-hCG
HVA, VMA
MN, nMN
liver
colorectal
ovarian
gastric
pancreatic
breast
breast
prostatic
neuroendocrine
lung
urinary bladder
thyroid
medullary thyroid
ovarian, testicular
neuroblastoma
phaeochromocytoma
TDM AND TOXICOLOGY
A. THERAPEUTIC DRUG MONITORING
1. BASIC PHARMACOLOGIC CONCEPTS
a. __ – highest point; amount of drug absorbed and distributed is greater than the amount metabolized and excreted
b. __ – lowest concentration achieved just before the next dose
c. __ – time required for the concentration of the drug to decrease by half
d. __ – amount of drug absorbed and distributed equals amount metabolized and excreted; usually reached after 5-7 half-lives
e. __ – lowest concentration of drug in the blood that will produce adverse response
f. __ – lowest concentration of drug in the blood that will produce desired effect
g. __ – range of values between the MTC and MEC that produce therapeutic effect
h. __ – drug dose that produces beneficial effect in 50% of the population
i. __ – drug dose that produces adverse effect in 50% of the population
j. __ – drug dose that produces death in 50% of individuals.
k. Therapeutic index – _________
Peak
Trough
Half-life
Steady state
MTC
MEC
Therapeutic range
ED50
TD50
LD50
TD50/ED50
- ANALYTICAL TECHNIQUES
a. __ – used as a semi-quantitative screening test; interpretation of results utilizes Rf values of solutes in comparison to aqueous standards
▪ __ – distance migrated by a sample component / distance migrated by the solvent
b. __ – for insufficiently volatile and thermolabile compounds
▪ __ – type of liquid chromatography where the mobile phase is a liquid that is passed over the stationary phase of the column
▪ __ – polar stationary phase and nonpolar mobile phase
▪ __ – nonpolar stationary phase and polar mobile phase
c. __ – useful for compounds that are naturally volatile or can easily be converted into a volatile form (various organic molecules including many drugs)
▪ Tr retention time – time it takes for a compound to elute; used as basis for identification of a solute
▪ Peak area/height – proportional to the amount of solute present; used for quanti
d. ___ – common detector system in GC or HPLC; produces a signal for identification and quantification of the solutes
e. ___
▪ MEIA – uses microparticles as solid phase, an enzyme label, and a fluorogenic substrate
▪ EMIT – a homogenous assay in which the activity of an enzyme as a label is inhibited by the binding of antibody to antigen conjugated with enzyme
▪ FPIA – uses polarized light to excite a fluorescent label; rate of rotation is inversely proportional to the degree of polarization and analyte conc
Thin-layer Chromatography
○ Rf
Liquid Chromatography
○ HPLC
○ Normal-phase LC
○ Reverse-phase LC
Gas Chromatography
Mass spectrometry
Immunoassays
- __ – drug activity or fate of drugs in the body as influenced by:
a. __ – through the GI tract for orally administered drugs; dependent on many factors and is related to the drug’s bioavailability
▪ __ – amount of drug absorbed relative to the quantity given; affected by first-pass metabolism, which reflects the activity of metabolic enzymes in the intestine and liver.
b. __ – diffusion out of the vasculature into interstitial and intracellular spaces; dependent on the lipid solubility of the drug
c. __ – hepatic uptake and enzymatic biotransformation in the hepatic microsomes via the cytochrome P450 enzyme system (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4)
d. __ – elimination through hepatic or renal clearance or a combination of the two - ___ – biochemical and physiological effects of drugs and the mechanisms of their actions; involves receptor binding, post-receptor effects, and chemical interactions
PHARMACOKINETICS
Absorption
Bioavailability
Distribution
Metabolism
Excretion
PHARMACODYNAMICS
- SPECIMEN CONSIDERATIONS
a. Steady state must be established before sampling.
b. STAT sampling when toxicity is suspected.
c. Trough specimen ___________
d. Peak specimen – __ after oral administration; __ after completion of IV administration
e. Appropriate specimens/Collection tubes
▪ ________ – preferred for most drug assays
▪ Heparinized plasma – suitable for most drugs except _______
▪ SST and PST – may falsely ↓ tricyclic antidepressants, anti-arrythmics due to drug absorption by the gel
▪ EDTA whole blood – appropriate for immunosuppressants
▪ ______ – recommended for the impedance method of aspirin determination
before the next dose is administered
1 hour; 0.5 hour
serum
Li and free drug assays
Hirudin
- DRUGS THAT REQUIRE TDM
Digoxin – a cardiac glycoside used to treat _______; inhibits Na+, -K+, -ATPase, causing decrease in intracellular K+ and increase in intracellular Ca2+ resulting in improved contraction of cardiac muscle
___ – used to treat tachyarrythmias
CHF (congenital heart failure)
Antiarrythmics
Antiarrhythmics: (MNEMONIC)
Class I; Lidocaine, Quinidine, Disopyramide, Procainamide
Class II; Propranolol
Class III; Amiodarone
Class IV; Verapamil
So, Be it, Pota, Ca!
sodium channel blockers
beta blokers
potassium channel blockers
calcium channel blockers
First-generation (classical) anticonvulsants
__ – slow-acting barbiturate with a long half-life;
inactive form _______
○ Benzodiazepines
○ Phenytoin (Dilantin)
○ Carbamazepine (Tegretol)
○ Ethosuximide
○ Valproic acid (Depakote)
New generation anticonvulsants – Tiagabine, Vigbatrin, Gabapentin, Topiramate, Felbamate
Antiepileptics/anticonvulsants
Phenobarbital
primidone
β-adrenergic agonists
Theophylline – used to treat asthma and COPDs
Caffeine – minor metabolite of theophylline; treatment of neonatal apnea
Bronchodilators
___ – major tranquilizers used to treat Schizophrenia
○ Phenothiazines – chlorpromazine
○ Butyrophenones – haloperidol (Haldol)
○ Newer drugs – risperidone (Risperdal), olanzapine (Zyprexa), quetiapine (Seroquel), and aripiprazole (Abilify)
Antimanic agents
○ __ – used to treat bipolar/manic depressive disorder
Antidepressants – used to treat clinical depression
○ Tricyclic antidepressants – amitriptyline, imipramine, doxepin
○ Selective serotonin reuptake inhibitors – fluoxetine (Prozac)
○ Monoamine oxidase inhibitors – clorgyline, selegiline
Psychoactive drugs/Antidepressants
Neuroleptics
Lithium
___ (tobramycin, amikacin, gentamicin, kanamycin) – used to treat gram (-) infections by inhibiting protein synthesis; toxic effects include nephrotoxicity & autotoxicity
Vancomycin – used to treat ____________; may cause erythemic flushing of extremities called ______
Antibiotics
Aminoglycosides
gram (+) infections; red man syndrome
Immunosuppressants – used to prevent _____
___ – block T lymphocyte function
○ Cyclosporine – fat-soluble cyclical peptide isolated from Trichoderma
○ Tacrolimus – macrolide lactone isolated from Streptomyces; 100x more potent than cyclosporine (administered in much lower doses); associated with thrombus formation in patients with toxic levels
Proliferation signal inhibitors
○ Sirolimus (Rapamycin), Everolimus, Mycophenolate, Leflunomide
graft rejection and GVHD
Calcineurin inhibitors
Methotrexate – inhibits DNA synthesis; requires administration of leucovorin to rescue host cells from methotrexate inhibition
Busulfan – alkylating agent used to treat leukemias and lymphomas before bone marrow transplantation
Antineoplastics
B. TOXICOLOGY
___– associated with a single, short-term exposure to a substance, the dose of which is sufficient to cause immediate toxic effects
___ – results from repeated frequent exposure for extended periods at doses insufficient to cause an immediate response; may affect different systems
Acute toxicity
Chronic toxicity
- THERAPEUTIC DRUGS COMMONLY ABUSED
Acetylsalicylate (__) – used as an analgesic, antipyretic, and anti-inflammatory; exhibits antiplatelet activity through inhibition of ________; toxic effects include mixed acid-base disorder & Reye’s syndrome
Acetaminophen (__) – preferred over aspirin in patients with a bleeding disorder; toxic effect hepatotoxicity
Analgesics
Aspirin; cyclooxygenase
Tylenol
- ILLICIT DRUGS
__ – local anesthetic; primary metabolite; short half-life of 1-2hrs
___ – used to treat narcolepsy “SHABU”
___ – euphoria-producing synthetic phenylethylamines
MDMA ______
Stimulants *Dopaminergic pathway stimulants
Cocaine
Amphetamines (Methamphetamine)
Designer drugs
ecstasy
Sedative/ hypnotics
○ Barbiturates – Secobarbital, Pentobarbital, Amobarbital, Phenobarbital
○ Benzodiazepines – Diazepam (Valium), Chlordiazepoxide, Lorazepam
○ Methaqualone (Quaalude) – not structurally similar to but has many of the properties of barbiturates
___ – used for sedation, analgesia, anesthesia
○ Naturally occurring – ___ (3)
○ Chemically modified – __, hydromorphone (Dilaudid), and oxycodone (Percodan)
○ __ – meperidine (Demerol), methadone (Dolophine), propoxyphene (Darvon), pentazocine (Talwin), and fentanyl (Sublimaze)
Depressants
Opiates/Narcotics
opium, morphine, and codeine
heroin
Fully synthetic
___ – primary cannabinoid component THC;
major urinary metabolite __________________
___ – structurally similar to serotonin; acts as serotonin agonist; produces panic reactions and undulating vision
Dissociative anesthetics
○ __ (angel dust or angel hair) – a tricyclic compound with varied actions and paradoxical symptoms
○ __ – commonly used as an anesthetic for short surgical procedures in pediatric medicine.
Hallucinogens/Psychedelics
Marijuana (Cannabis sativa)
THC-COOH
Lysergic acid diethylamide (LSD)
Phencyclidine
Ketamine
___ – derivatives of serotonin with hallucinogenic properties e.g. dimethyltryptamine and psilocin/psilocybin
Piperazines – produce effects similar to those of the amphetamines
___ – psychoactive designer drugs that
produce amphetamine-or cocaine-like effects
Sleep aids – non-benzodiazepine agents e.g. Zolpidem and Zopiclone
___ – CNS depressants used in the pain management of musculoskeletal conditions e.g. Carisoprodol and meprobamate
Miscellaneous classes
Tryptamines
Synthetic cathinones (Bath salts)
Muscle relaxants
- OTHER TOXIC SUBSTANCES
a. __ – most commonly abused substance in the world; chronic exposure is associated with toxic hepatitis and cirrhosis
▪ Methods:
1) Enzymatic: Ethanol + NAD (ADH) → acetaldehyde + NADH
2) Gas-liquid chromatography (GLC) - ref mtd
3) Osmometry - screening test (osmolal gap)
Ethanol
▪ Stages of impairment:
LEVEL OF IMPAIRMENT
___: No obvious impairment
___: Mild euphoria; some impairment of motor skills; >0.1 %w/v - cut off for legal intoxication (USA)
___: Loss of critical judgment; memory impairment
___: Mental confusion; strongly impaired motor skills
___: Impaired consciousness
___: Complete unconsciousness; deep, possibly fatal, coma
___: Respiratory arrest
BAC (% w/v); INFLUENCE
0.01-0.05; subclinical
0.03-0.12; euphoria
0.09-0.25; excitement
0.18-0.30; confusion
0.27-0.40; stupor
0.35-0.50; coma
>0.45; death
- OTHER TOXIC SUBSTANCES
b. ___ – causes acidosis, blindness, and death due to formation of _______
c. ___ – produces severe, acute ethanol-like symptoms that persist for a long period of time
d. ___ – ingestion produces severe metabolic acidosis and renal tubular damage
e. ___ – 245x greater affinity for Hb compared to oxygen; shifts the oxyhemoglobin dissociation curve to the _____
Methanol; formaldehyde & formic acid
Isopropanol
Ethylene glycol
Carbon monoxide; left (hypoxia)
- OTHER TOXIC SUBSTANCES
f. ___ – commonly used as rodenticide/ insecticide; characteristic odor of _________; binds to heme iron and mitochondrial cytochrome oxidase inhibiting oxidative phosphorylation and causing cellular hypoxia
g. Caustic Agents:
* ___ is associated with pulmonary edema and shock, which can rapidly progress to death.
* ___ causes metabolic acidosis or alkalosis and may produce lesions or perforations in the esophagus and GI tract resulting in hematemesis, abdominal pain, and possibly shock.
Cyanide; bitter almonds
○ Aspiration
○ Ingestion
h. Heavy metals
1) ___ – component of insecticides, pesticides, and herbicides; high affinity for _______; binds to sulfhydryl groups of proteins; characterized by _____ odor and metallic taste; associated with transverse white striations in the nail beds called Mees lines
2) ___ – metal food containers or industrial exposure; may cause renal tubular function (ATN) __
3) ___ – inhibits many enzymes and affects vitamin D metabolism and heme synthesis pathway
▪ Enzymes inhibited in the heme synthesis pathway ALA dehyrdratase/PBG synthase ↑delta-ALA in urine
▪ Routes of exposure – ___________
4) ___ – acquired through inhalation and ingestion; may also cause renal tubular damage
Forms of mercury compounds:
▪ ___ – metallic form (Hgo); liquid at room temperature (non-toxic)
▪ ___ – mercurous (Hg+) and mercuric (Hg2+) forms (moderately toxic)
▪ ___ – alkyl, aryl, and alkoxyalkyl forms (environmental pollutants; extremely toxic)
Arsenic; keratin; garlic breath
Cadmium; acute tubular necrosis
Lead; ferrochelatase/heme synthase
Mercury
Elemental
Cationic/inorganic
Organic
b. __ - provides both a management philosophy or organizational development and a management process for improvement of quality in all aspects of work.
__ is concerned with broader measures of laboratory performance including TAT, patient identification, specimen collection, and test utility.
TQM
QA
__ is necessary to standardize the remedy, establish measures for performance monitoring, and ensure that the performance achieved satisfies quality requirements.
__ involves statistical control procedures as well as nonstatistical checks, e.g. linearity checks, temperature monitors, reagent and standard checks
QP
QC
__ include processes, policies, practices, and procedures that define how all aspects of work are done.
__ provides a structured problem-solving process to help identify the root cause of a problem and a remedy for that problem.
QLP
QI
__: may be round or square
Quartz/Fused silica - __
Plastic - __
Glass - __
__: Converts the transmitted light energy into an equivalent amount of electrical energy
__: – simplest type
__:– requires external voltage source
__: – excellent linearity
__: - most sensitive, commonly used
__: Processes the electrical signal, performs mathematical perations, and displays the output. Digital meters, d’Arsonval meters, recorders, LEDs, CRTs, LCDs
Cuvette
UV to IR
UV to visible
visible only
Photodetector
Barrier layer cell
Phototube
Photodiode
Photomultiplier tube
Read-out device
