Clinical Chemistry 1 LAB_First Semester

Download as pdf or txt
Download as pdf or txt
You are on page 1of 46

CLINICAL CHEMISTRY LABORATORY

1st Semester | A.Y. 2024-2025


Lecturer: Ma’am Jane Ross Mendoza
By: Aimee G. Montes

Lesson 1: OVERVIEW infections occurring within their facilities


BIOSAFETY AND BIOSECURITY (e.g. Biosafety and Biosecurity officers)
Biosafety
 containment principles, technologies, and Mode of Transmission
practices implemented to prevent 1. Direct contact - the unprotected host touches
unintentional exposure to pathogens and the patient, specimen, or a contaminated object
toxins, or their unintentional release (reservoir)
 protecting the person o HIV – most common disease that can be
 Each institution MUST have at least 1 transmitted by direct contact
biosafety officer and ALL HEALTHCARE 2. Droplet - the host inhales material from the
WORKERS of an institution are REQUIRED reservoir (e.g.,aerosol droplets from a patient or
to attend biosafety training (w/ certificate & an uncapped centrifuge tube, or when
exam; paid or free training) specimens are aliquoted or spilled)
Biosecurity o common colds, coronavirus
 protection, control, and accountability for 3. Airborne - inhalation of dried aerosol particles
valuable biological materials within circulating on air currents or attached to dust
laboratories, in order to prevent their particles
unauthorized access, loss, theft, misuse, 4. Vector – from an animal or insect bite
diversion or intentional release. o dengue, malaria
5. Vehicle - ingestion of a contaminated
CHAIN OF INFECTION substance (e.g., food, water, specimen)
 requires a continuous link o amoebiasis
 breaking the link / chain of infection will
stop the harmful microorganisms from
 Once the chain of infection is complete, the
spreading
infected host then becomes another source able
 hand hygiene – most effective way/best
to transmit the microorganisms to others
practice to break the chain of infection
o hand washing MUST take 60 Universal Precautions (UP)
seconds (according to DOH)  instituted by CDC in year 19987
o use paper towel when turning off the  under UP, all patients are considered to
faucet after washing to avoid be possible carriers of blood-borne
recontamination pathogens
Infection Control
 all health-care facilities have developed Body Substance Isolation (BSI)
procedures to control and monitor  BSI guidelines are not limited to blood-
borne pathogens; they consider all body
fluids and moist body substances to be
potentially infectious.

In 1996, the CDC and the Healthcare Infection


Control Practices Advisory Committee (HICPAC)
combined the major features of UP and BSI
guidelines and called the new guidelines Standard
Precautions.

STANDARD PRECAUTIONS
 Hand Hygiene
 Gloves
 Mouth, nose and eye protection
 Gown
 Environmental Control
 Linen Management (focuses on theoretical
 Occupational Health and Blood-borne knowledge of biosafety)
pathogens  M2 – Technical training in Lab Biorisk
management (application of all things
PROPER DONNING AND DOFFING of PPE (DOH) learned in M1)
Link:  M3 – Essentials of BRAP; Importance
https://youtu.be/oO5Awp5LCNg?si=6gWZ0S6c_3rliecV of Biorisk

Lesson 2
RISK ASSESSMENT AND SPECIMEN
MANAGEMENT
Review
 Biosafety – protects people against bad bug 3. Specimen Receipt And Storage
 Biosecurity – protects bad bug against bad 4. Facility Design
people  one of the component in containment;
 Bioterrorism – using biological agent for  simply refers to the physical plant
intentional release to harm people  DOH inspects the physical plant or the
 Most important in containment – Good floor plan annually
Laboratory Practices or Good 5. Emergency/Incident Response Plan
Microbiological practices  the BRM Committee (Biorisk
management committee) - makes the
incidence response plan
6. Biosafety Cabinet And Laboratory
Equipment

LESSON 1
BIORISK ASSESSMENT  Biosafety cabinet – uses laminar air flow;
Biorisk Assessment – backbone of biosafety o laminar meant that air goes in one
Biosafety officer (BSO) direction only;
 called Biorisk management advisor o uses HEPA Filters (High efficiency
 conducts risk assessment and corrective particulate air) a filtering process of
measures upon receiving IR (incident biosafety cabinet as the BSC is used
report) when dealing with
biologically/potentially harmful
Core requirements for Biorisk Assessment substances/agents
1. Good Microbiological Practice and o Bacteriology section always have BSC,
Procedure and separate enclosed area/workplace;
 Two most important – Hand washing and All processes of samples in bacteriology
wearing PPE section are performed in BSC
2. Personal Competence And Training o working in BSC protects the user and
 laboratory workers must undergo training the environment
to keep up and adapt with the changes
in the guidelines and practices  Fume Hoods – do not use HEPA filter
 to be updated o basic compared to BSC
o with exhaust as ventilation systems
 BRAP – Biorisk Association of the
o commonly seen in clinical chemistry
Philippines
section
 provides Biosafety training: three-
module BRAP Credentialing and
Competency Program (BCCP) basic Fume Hoods BSC
biosafety course (on-site package Used for hazardous Used for solid materials
001); 1 month chemicals and volatile & infectious biological
 M1 – Biosafety, Biosecurity, and vapours. agents.
Orientation to Lab Biorisk Protects the user Protects the user,
environment & material.
No HEPA filter Must have HEPA filter Note:
Exhausts air outside the Does not exhaust o Sharps – thrown in puncture-resistant
building. outside but removes container
contaminants. o biohazard materials – thrown in trash
container with biohazard symbol
Biohazard symbol description:
o 3 overlapping circles with one
circle at the midldle
o yellow as background color

8. Occupational Health
 an area of work in public health to
promote and maintain highest degree of
physical, mental, and social well-being of
Note: Class III is used in facilities that workers in all occupations
processes extremely harmful samples; the  OSHA – Occupational Safety and
one that only uses two HEPA filters for added Health Administration
protection  OSHA Lab Standard – requires the
appointment of a chemical hygiene
7. Decontamination and Waste officer and the development of a
Management chemical hygiene plan to reduce or
 BMW – Biomedical Waste Management eliminate occupational exposure to
 4 Basic Waste-Disposal Techniques hazardous chemicals
 flushing down the drain to the sewer  a chemical hygiene officer with
system chemical hygiene plan
o lab have many sinks (lavatory)  STANDARD: “Treat every biologic
o DOH requires specific number of agent/bodily fluids should be treated
lavatory in the lab as potentially infectious.”
o urine – only specimen that can be  Two common pathogen in Lab
flushed down in the sink that will  Blood-borne pathogens – standard
not harm the environment precautions considers blood and other
 incineration body fluids from all patients as
o to burn potentially infective
o histopath specimen – waste  Air-borne pathogens – the purpose of
specimen that can be burned the guidelines is to encourage early
(e.g.amputated leg or diabetic foot) detection, isolation, and treatment
 landfill burial of active cases. e.g. TB (droplets of TB)
o general wastes are buried (papers,
plastics) 9. Personal Protective Equipment
 recycling
o not everything are disposed; (e.g. 10. Controlled or Limited Access to
in some hospitals urine container Laboratory
are washed to be reused)  not all parts of the lab is accessible for
everyone
 “No Entry” or “Authorized Person Only”
signage

11. Clean to Dirty Work Flow


 Separate place for donning and doffing
and correct sequence of execution to
AVOID RECONTAMINATION

RISK ASSESSMENT
 A systematic process of gathering
information and evaluating the likelihood
and consequences of exposure to or
release of workplace hazards and
determining the appropriate risk control
measures to reduce the risk to an Risk Group 3 pathogen usually causes
acceptable risk. High individual risk disease but it does not
and low community easily spread from one
Difference between risk and hazard risk; person to another (kaya
Risk low community risk)
 a combination of the likelihood of an Risk Group 4 pathogen that usually
incident and the severity of the harm causes disease and
(consequences) if that incident was to occur readily
 incident/accident and possible spread/transmitted
consequences from one individual to
Hazard another
 an object or situation that has the potential Note:
to cause adverse effects when an organism  Risk Group 1 to 3 has low community risk
or population is exposed to it  Risk Group 4 – high individual risk and high
 once exposed, there are potentially community risk
adverse effects
CHEMICAL SAFETY
MSDS Material Safety Data Sheet
RISK ASSESSMENT  a major source of safety information for
employees who may use hazardous materials in
1. Identification of hazardous characteristics their occupations
of the agent  employers are responsible for obtaining from
 Identify the agent, what is its the chemical manufacturer or developing an
characteristics (whether it is an acid, MSDS for each hazardous agent used in the
alkali metal, etc.) workplace
 must be easily seen and accessible in the
2. Identification of laboratory procedure laboratory
hazards  MSDS is created by the institution’s
 MSDS – Material Safety Data Sheet; a management and MUST BE accessed by their
compilation of all protocols in the lab personnel
(hazardous chemicals and agents)  Two types of Common Hazard in the
 consult with MSDS, gather info about Laboratory: Chemical and Fire Hazard
the hazardous agent, study about it.
Always read the MSDS.
CHEMICAL HAZARD
Types of Chemicals
3. Determine appropriate
1. Flammable/Combustible Chemicals
Biosafety Levels and
 Possible fire or explosion
select additional
 Classified according to flash point,
precautions
which is the temperature at which
sufficient vapour is given off to form an
4. Review RA with
ignitable mixture with air.
Biosafety Professionals
2. Corrosive Chemicals
and Subject Matter
 Injurious to the skin or eyes by direct
Experts
contact
 discuss and
 Example: acids; eye contact of acid
assess with
must be washed immediately and
colleagues; inform BSO through
continuous for 15 minutes
incident report (IR)
3. Reactive Chemicals
 under certain conditions, can
5. Evaluate staff proficiencies regarding safety.
spontaneously explode or ignite
6. Revisit RA regularly and verify risk
4. Carcinogenic Chemicals – cancer-causing ex:
management strategies.
histopath stains
Risk Group Classification
Risk Group 1 does not usually cause
No or low individual human disease
and community risk
Risk Group 2 Can cause disease but
Moderate individual unlikely to be serious
risk and low hazard to laboratory
community risk workers
FIRE HAZARD
Additional info:
Blue top tube is brought to hematology section as
it is for coagulation studies; and put on
centrifugation for 15 minutes.

Special Handling – for sensitive samples


 Analyte protection
 Commonly encountered tests in the
laboratory: potassium and bilirubin
 Bilirubin – photo/light sensitive;
o results are altered when exposed to
light;
o sample containers must be covered to
protect from exposure to light
 Potassium – very sensitive when sample
A A(o)rdinary combustibles – paper, clothes, is hemolyzed (serum is pinkish to reddish;
wood normal serum is yellowish);
B Basa - flammable liquids o when processed, will result to falsely
C Curyente - light electrical equipment elevated
D Matigas na D - combustible materials – metal o rationale:
– uses sand to extinguish the fire  PISO: Potassium In Sodium Out
K K(c)ommercial cooking equipment  In a hemolyzed sample, potassium
is inside the RBC, and when RBC
burst, lalabas si potassium

Specimen Suitability
All samples must be suitable or viable for running
Rejected samples:
 Hemolyzed sample – recollect for suitable
sample as it may alter the results
 Collection in the wrong tube – ex. purple top
for CBC; always check the request of patient to
prevent usage of wrong tube
 Failure to follow special timing or handling
requirements (FASTING)
 Quantity Not Sufficient (QNS)
 tubes has lines indicating the amount of
blood needed
 Clotting in whole blood or Plasma specimens

SPECIMEN HANDLING AND PROCESSING


Routine Handling
1. Inversion of Tubes
 must be done first to prevent clotted
sample
 e.g. blue top – 4 inversions (standard)
2. Proper Labelling
 name (last name, first name), age,
gender, birthday
3. Transport of Specimen
4. Separation/Centrifugation
5. Aliquot Preparation
 aliquot – small portion from the entire
sample
 Automated or manual processing
Lesson 3
BLOOD COLLECTION: VENIPUNCTURE
THE VASCULAR SYSTEM
1. Arteries
 have thick walls to withstand the
pressure of ventricular contraction,
which creates a PULSE that can be
felt, distinguishing them from veins.
 the pressure normallly causes blood
to pulse into the syringe under its
own power.
 BRIGHT RED in color
 arteries have thick walls compare to
veins because it pumps blood abd
gas greater pressure
2. Veins
 have thinner walls than the same-
size arteries because blood in them
is under less pressure
 they collapse more easily
 DARK RED in color WITH A
SLIGHT TINT OF BLUISH HUE
 veins tend to collapse because of Source and Composition of Blood Specimens
their thin walls Arterial Blood
3. Capillaries  difficult and potentially hazardous
 capillary bed in the skin can easily  primarily reserved for BLOOD GAS evaluation
be punctured with a lancet because  some institutions do not allow medtechs to
it is located subdermally extract arterial blood for ABG
 Arterial samples for ABG are processed and
VEIN PATTERNS released in pulmonary department
H Pattern Veins –
approximately Venous Blood
70% of the  Affected by metabolic activity of the tissue it
population drains
 Lower oxygen content
1. MEDIAN – near 
the center of the
antecubital fossa; Capillary Blood
preferred vein for  contains arterial and venous blood plus
venipuncture; TISSUE FLUID
most stationary and the least painful site of  first drop of capillary blood is wiped off to
extraction; most stable & least painful remove the tissue fluid and other contaminants
2. CEPHALIC – SECOND option of site; fairly well- that may alter the blood sample
anchored and often the only vein that can be felt in  warming the site increases it further
obese patients.
3. BASILIC – LAST choice of site; not well 3 Basic Methods to Collect Blood Sample
anchored and rolls easily 1. Evacuated Tube System (ETS)
 preferred method because blood is
collected directly from the vein into a
tube, minimizing the risk of specimen
contamination and exposure to the blood.
2. Needle and Syringe
 most commonly practiced
3. Winged Infusion Set (butterfly)
 often used to draw blood from infants and
children, from hand veins, and in other
difficult-draw situations
 purpose of the wings – to stabilize the
needle position
The order of draw is a special sequence of tube
Venipuncture Equipment collection that reduces the risk of specimen
contamination by:
 Microorganisms
 Additive carry-over
The most common tubes in the order of draw can
be remembered by recalling the phrase “stop,
light red, stay put, green light, go”

Stop Sterile Blood culture bottles


Note:
 When labelling a blood culture
bottle, include the site where the
sample is obtained (e.g. left
arm/right arm or right foot/left
foot)
 Physician ang magrerequest kung
ilan at saan ang site
1. Micropore
2. Evacuated tube  1 blood culture bottle per site
3. Multi-sample needle  usually two sites
4. Light Light Sodium citrate – coagulation
5. Winged infusion set Blue
6. Syringe Red Red Chemistry
7. Tourniquet Glass – plain red top tube
8. Alcohol swab Plastic – with clot activator
 How will you activate the clot?
9. Gauze pad
make a 1 complete inversion;
10. ETS Needle Adapter/Vacutainer tube holder
 Purpose: to hasten the clot
formation
 allows blood to clot (15 minutes)
before centrifugation
Stay SST  Serum separator tube
 Thixotropic gel – gel inside
the yellow top tube
 Serum is being
separated/obtained
 When allowed to clot, red cell
settles at the bottom; gel is
at the middle (less dense
compare to RBCs); and
serum at the top
Note:
 The liquid portion at the top is
serum if there is not
anticoagulant.
 Plasma if there’s anticoagulant
Put PST  Plasma separator tube

Green Green  Chemistry


Order of Draw and Additive Carry-over  POCT
 all results are STAT
Light Lavend  EDTA
er  Hematology
Go Gray  Sodium fluoride
 Have them sign a waiver or proof that they don’t
want to proceed with the procedure anymore.

Problem Sites
1. Burns, scars, and tattoos
 tattoos might contaminate the sample
2. Damaged veins (sclerosed or thrombosed)
 sclerosed vein – hard vein
 thrombosed vein – clotted vein
 ask the patient his/her medical history
 ask if may maintenance
3. Edema
 manas
 too much tissue fluid build-up
 troubleshoot – talian ng tourniquet tightly,
masssafe the site to move the fluid away from
Troubleshooting Failed Venipuncture the extraction site
Failure to draw blood can be caused by: 4. Hematoma
1. Bevel of the Needle is not Positioned  avoid; extract to the other side
Upwards 5. Mastectomy – lymph node removal
2. Loss of Tube Vaccum  surgical removal of the breast
 Check the expiration date as it causes loss  ask the clinical history
of vacuum  if both breast are removed, choose another site
 Discard the expired tube as expired tubes far from lymph nodes
can alter results.  if tinanggal ang breast, tanggal din ang lymph
 If not expired and no vacuum at all, open nodes kaya ang tendency, pag tinusok sa
mastectomy side, there’s a high chance of re-
the tube and transfer the sample
infection kasi immunocompromised na si
manually. patient due to the absence of her lymph nodes.
 Avoid the needle hub to touch the
side/wall of the tube when transferring Patient Conditions and Complications
sample.
1. Allergies to Supplies or Equipment
3. Improper position of the Needle in the Vein  patient may be allergy to latex gloves
4. Through-and-through 2. Excessive bleeding
 tumagos  to troubleshoot bleeder patients:
 troubleshoot action – slightly pull o remove the micropore and cotton, clean
backwards until flash of blood is seen the site
5. Needle not deep enough o put another cotton ball and instruct the
6. Tourniquet not tightly positioned patient to strictly put pressure for 10 to
7. Dehydrated patient 15 minutes
 have thinner veins 3. Fainting (syncope)
 discontinue the procedure
 (1) use winged infusion set
 allow and ensure proper ventilation for patient
 (2) use 1 cc syringe
4. Nausea or vomiting
 (3) capillary or skin puncture 5. Obese patients
8. Easy to collapse vein  position needle at high angle
 bulging vein indicates that the vein is 6. Pain
damaged or has collapsed  patient with very low pain tolerance
9. Patient is moving frequently during  assure the patient, do not sugarcoat na
extraction hindi masakit
 calm down the patient  tell them that there’ll be pain, but
 explain that upon movement, veins can be TOLERABLE
affected 7. Petechiae – harmless red spots
 tell and explain them the possible 8. Seizures/Convulsions
consequences: recollection another  discontinue the procedure
extraction to the other side/site  remove the tourniquet, followed by needle
10. Patient fainted or patient decided not to  alalayan ang ulo
proceed with the extraction
 Explain and persuade but DO NOT FORCE the Procedural Error Risks
patient. 1. Hematoma formation
 Persuasion tips: (1) hahanapin ng doctor,  causes: (1) upon puncture, na-damage na
kailangan ng doctor for accurate diagnosis; (2) ang vein pero hindi tinuloy; (2) too tight
SAYANG ANG BINAYAD tourniquet; (3) through-and-through; (4)
pressure applied after blood collection is not LESSON 4
enough
LABORATORY SUPPLIES & EQUIPMENT
2. Iatrogenic anemia
 infants are prone to IA because they are
smaller and thus has lesser liquid volume
 perform skin puncture
3. Arterial puncture
4. Infection of the site
 factor: Mastectomy
5. Nerve injury
6. Reflux
 bumalik ang dugo and you are using tube
with anticoagulant such as EDTA, sodium
citrate, and sodium fluoride additives
 Heparin additive is safe
7. Vein damage  Regardless of design, most laboratory supplies
must satisfy certain tolerances of accuracy.
 Vessels holding or transferring liquid are
Capillary Order of Draw
designed either to contain (TC) or to deliver
Specimens must be collected quickly to minimize (TD) a specified volume.
the effects of platelet clumping and microclot  Routinely used clinical chemistry glassware
formation and to ensure that an adequate amount should consist of high thermal borosilicate
of specimen is colcted before the site stops (Pyrex) or aluminosilicate (Corex) glass and
bleeding. meet the Class A tolerances recommended by
1. EDTA specimen the NIST (National Institute of Standards and
2. Other additive specimens Technology).
3. Serum specimen  PLASTICWARE is beginning to replace
glassware in the laboratory setting. The unique
Indication for Capillary Puncture high resistance to corrosion and breakage,
as well as varying flexibility, has made
 There are no accessible veins. plasticware most appealing. Relatively
 Available veins are fragile or must be saved for inexpensive, it allows most items to be
other procedures such as chemotherapy. completely disposable after each use.
 The patient has thrombotic or clot-forming
tendencies. LABORATORY VESSELS
 Blood is to be obtained for POCT procedures
such as glucose monitoring.
 Newborn screening tests.
 Infants:
 Due to small blood volume
 Difficult and can damage veins and
surrounding tissues
 Can be injured by the restraining
method.

 Class A volumetric flask is calibrated to hold


one exact volume of liquid (TC).
 When bringing the bottom of the meniscus to
the calibration mark, a pipet should be used
when adding the final drops of diluent to
ensure maximum control is maintained and the
calibration line is not missed.
 Erlenmeyer flasks and Griffin beakers are
designed to hold different volumes rather than
one exact amount and are often used in
reagent preparation.
 All laboratory utensils should be Class A
whenever possible to maximize accuracy and
precision and thus decrease calibration time.
BALANCE
 Analytic Balances are required for the
preparation of any primary standards.
 Many laboratories discontinued in-house
reagent preparation, balances may no longer be
as widely used.

DESICCATORS and DESICCANTS

 These materials make excellent drying


substances and are sometimes used as
desiccants (drying agents) to keep other
chemicals from becoming hydrated.
 If these compounds absorb enough water from
the atmosphere to cause dissolution, they are
called deliquescent substances.

CENTRIFUGE
 Centrifugation is a process in which
centrifugal force is used to separate solid
matter from a liquid suspension.
 The speed is expressed in revolutions per
minute (rpm), and the centrifugal force
generated is expressed in terms of relative
centrifugal force (RCF).
LESSON 5  To Deliver
PIPETTING  will dispense the volume indicated
 delivers
EXACT
amount
needed
Note:
o When
submerged in
beaker, NEVER
allow the pipet
to touch the
vessel wall.
o Pipet must
STAND
STRAIGHT,
VERTICALLY

Notes:
 In pipetting, it is important that volume is
accurate and precise.
 Blowout – using pipet and aspirator bulb BLOWOUT PIPETS
 Aspirator bulb – tool used to blow or suck
up liquid  Has a continuous
 In laboratory, most commonly used are the etched ring or two
Semi-automated or automated pipets small, close,
 Serologic (common/traditional) pipets are continuous rings
only used when semi-automated pipets has located near the top of
functional problems (e.g. not calibrated) the pipet.
PIPETS  This means that the
last drop of liquid
 Glass or plastic utensils used to transfer should be expelled into
liquids; the receiving vessel
 May be reusable or disposable.
Notes:
 Although pipets may transfer any volume, they
 A blowout pipet has 2 etched rings is located at the top
are usually used for volumes of 20 mL or less.  Aspirator bulb is pressed to expel all the liquid, even the
Note: last drop
 There are pipets that are disposable  Why all liquid must be expelled? To have an
 Micropipet volume: less than 1mL accurate/precise volume transferred in the vessel
 Aliquot – small part or a part from for an accurate results
an entire sample or a whole  Advantage: removes even the last drop of liquid
 To Contain and To Deliver
 The major difference is the amount SELF-DRAINING PIPETS
of liquid needed to wet the interior
surface of the ware and the amount  The user allows the contents of the pipet to
of any residual liquid left in the drain by gravity.
pipet tip.  The tip of the pipet should not be in contact
 To Contain with the accumulating fluid in the receiving
vessel during drainage.
 holds or contains a particular
volume but does not dispense the  The tip should remain in contact with the side
exact volume of the vessel for several seconds after the liquid
has drained
 does not completely dispense the
desired volume because there’s  Notes:
residual liquid left inside o With large diameter compare to blowout
pipet
o Dapat hindi nakadikit ang tip ng seropipet o Used with biologic fluids having a viscosity
sa fluid pero dapat nakadikit sa vessel wall greater than that of water.
para ma-drain completely by gravity (slightly o Blowout pipets, indicated by two etched
tilt the pipet) continuous rings at the top
Notes:
MEASURING OR GRADUATED PIPETS
 used for biologic fluids
 Capable of dispensing several different more viscous than water
volumes. Because the graduation lines located (malapot); ex: sputum
on the pipet may vary, they should be indicated  The only pipet with this
unique structure
on the top of each pipet. (bulging part)
 Measuring pipets are used to transfer reagents
and to make dilutions and can be used to
 Volumetric pipet
repeatedly to transfer a particular solution.
o It is designed to
 Mohr Pipet
dispense or transfer
o It does not have graduations to the tip.
aqueous solutions and
It is a self-draining pipet, but the tip
is always self-draining.
should not be allowed to touch the
o This type of pipet
vessel while the pipet is draining
usually has the
o Note: 2 things to remember when using
greatest degree of
self-draining: accuracy and precision
 Should not come in contact with and should be used
receiving fluid when diluting
 When draining, should be in standards, calibrators,
contact with the vessel wall or quality-control
material. They should
 Serologic Pipet
only be used once.
o Has graduation marks to the tip and is
Note:
generally a blowout pipet.
 has greatest accuracy and precision
 Best used in quantitative analysis, quality
 Micropipet control materials, calibrators, diluting standards
o A pipet with a total holding volume of (in form of powder)
less than 1mL  ang standards ay powder na nakalagay sa vial,
Notes: so need sya timplahin, dilute the powdered
 Sa first stop lang titigil kapag standard in distilled water
kukuha pa lang ng sample;  Has small diameter
kapag nakakuha na and to
be transferred to the  Pasteur Pipets
cartridge (similar o Do not have calibration marks and are used to
appearance sa preg test transfer solutions or biologic fluids without
kit), sa 2nd stop na titigil consideration of a specific volume. These pipets
(sagad na pag press) to should not be used in any quantitative analytic
deliver all the contents techniques
 Carefully press the stopper Note:
to prevent bubbles;
 we do not use this in
micropipet is prone for measuring accurate
bubble forming sample volume or
 Take note: wag sa bubbles quantitative analysis
ng sample kukuha (if because it does not have
blood) markings or graduated
lines (walang
 Volume of micropipet - less measurement)
than 100uL (maximum)
 Its purpose is to simply
 To change the volume, i-i- to transfer or deliver
ikot ang operating button samples measured by
sa taas. drops; technically it is
just a dropper in
laboratories
TRANSFER PIPETS  Has no exact volume;
has no graduation lines
 These pipets are designed to dispense one or markings
volume without further subdivisions.
 Ostwald-Folin pipets
AUTOMATIC PIPET (FIXED/VARIABLE)  The piston does not come in contact in the
liquid
 Most routinely used pipet in today’s clinical  In positive displacement, it does not require to
chemistry laboratory. Automatic and change tip every use.
semiautomatic pipets have many advantages:  Disadvantage: prone to carry-over because it
o Safety repeatedly used the same tip.
o Stability  To clean and avoid carry-over, remove the
o Ease of use pipet tip and wash in running water. Blot the
o Increased precision tip or wipe to remove the liquid residues, use
o The ability to save time gauze pad or tissue
o Less cleaning required
o tips used are disposable o Dispensers and dilutor/dispensers are
 It may be a fully automated, self-operating, automatic pipets that obtain the liquid from
semi-automatic, or completely manually a common reservoir and dispense it
operated device. There are three general types repeatedly. The dispensing pipets may be
of automatic pipets: bottle-top, motorized, handheld, or attached
o Air-displacement pipet relies on a piston to a dilutor. The dilutor often combines
for suction creation to draw the sample into sampling and dispensing functions.
a disposable tip that must be changed after
each use. The piston does not come in
contact with the liquid.
o Positive-displacement pipet operates by
moving the piston in the pipet tip or barrel,
much like a hypodermic syringe. It does not
require a different tip for each use. Because
of carry-over concerns, rinsing and blotting
between samples may be required
Note:
 Automated pipet need not to be cleaned anymore
 The standard for automatic pipet: Pipet tips are
disposable
 Note: automatic pipets are calibrated because it is Note:
automated. Who will calibrate? Engineers calibrate
automatic pipet, assess the pipet, ire-recalibrate;  It delivers a specific, exact same volume at
gagawa ng PMS (Preventive Maintenance Report the same time in different tubes
and Calibration Report)  Used when madaming pina-process, like
serial dilution
 The blue colored liquid is the dilutor
 It has a dilutor and dispenser
 Dispenser and dilutor/dispensers are used
when obtaining a liquid from the common
reservoir (yong dilutor)
 Micropipette and Macropipette - bestie sa
lab
 Paano i-adjust and volume
 Rotate the operating button in
micropipette
 For pasteur pipettes - droplets; commonly
used in kits

Major difference:
Body: Air displacement has tip ejector button, press this
to eject the pipet tip
If using positive displacement, remove the pipet tip using
the tip ejector button sa taas ng volumeter display

Note:
 Air displacement uses disposable tips, so it Answer: By rinsing and blotting
must be changed every after use
Different brands can be used for one particular LESSON 6
pipet but they do not necessarily perform in an QUALITY CONTROL
identical manner. Plastic burrs may be present in Quality Control in the laboratory involves the
the interior of the tip that cannot always be systematic monitoring of analytic processes in
detected by the naked eye. order to detect analytic errors that occur during
analysis and to ultimately prevent the reporting
A method using a 0.1% solution of phenol red in of incorrect patient test results.
distilled water has been used to compare the Note: The sole purpose of QC is to prevent
reproducibility of different brands of pipet tips. incorrect reporting of patient test result.
Although gravimetric validation is the most
desirable method, pipet calibration may also be Factors of QUALITY CONTROL
accomplished by using photometric methods,
 Performance monitoring
particularly for automatic pipetting devices.  Automated machines (semi or fully) are the one
being monitored. Problems and malfunctions
When a spectrophotometer is used, the molar can happen and so it must be calibrated. HINDI
extinction coefficient of a compound, such as MACHINE LANG ANG GUMAGAWA. As a
potassium dichromate, is obtained. After an aliquot medtech, you must know how to control your
of diluent is pipetted, the change in concentration machine, and know if there’s something wrong,
will reflect the volume of the pipet. and when to calibrate.
 QC material (lyophilized) assay
 Lyophilized assay –
powder form; solute
 Control 1 and Control 2 for
pathogenic (abnormal)
A quick, daily check for many larger-volume control and non-pathogenic
automatic pipetting devices involves the use of (normal) control – provided
volumetric flasks. by the supplier of your
machine
For example, a bottle-top dispenser that routinely
delivers 2.5 mL of reagent may be checked by  Control limits
dispensing four aliquots of the reagent into a 10-
 Target values
mL Class A volumetric flask.
 Aliquot preparation
 Daily logging of control results
The bottom of the meniscus should meet with the
 Record of every day control results is one of the
calibration line on the volumetric flask requirements inspected/checked by the DOH
 Reconstitution of QC material

 Note: Diluent/solvent used to dissolve the


control: distilled water
 Ihalo ang control sa Distilled Water,
Shake, invert ->solution
 avoid bubble formation

 QC monitoring charts
 One of the most common QC chart is the Levey-
Jennings chart (LJ chart)
 also a requirement by DOH being inspected
yearly
 Analytic methods
c. Reanalyzing control and patient
Quality Control Charts
data.
 Control charts graphically represent the Analytic methods are considered in control if a
observed values of a control material over time symmetrical distribution of control values about
in the context of the upper and lower control the mean are seen, and few values outside the
limits. 2SDs (2s) control limits are observed.
 Points falling outside the control limits may
suggest that problems may be developing. Some laboratories define a method out of control if
 Kung ikaw ang medtech, nakita mong a control value exceeds the 2s limits.
may lampas sa upper or lower limit,
dapat alam mong may problema either Other laboratories use the 2s limit as a warning
sa machine or sa reagent, at alam mo limit and the 3s limit as an error limit.
kung anong dapat at tamang gawin.
 Control charts can detect errors in accuracy In this particular case, a control point between 2s
and imprecision over time. and 3s would alert the technologist to a potential
problem, while a point greater than 3s would
Random Error require a corrective action.
 The underlying rationale for running repeated
assays is to detect random errors that affect Multirules Rule
precision.
 Random errors may be caused by variations in The “multirule” procedure was developed by
technique. Westgard & Groth to further judge whether
 presence of outliers (lampas sa control results indicate out-of-control situations.
controlled/allowed limits) These rules established a criterion for judging
 not precise; not accurate whether an analytic process is out of control.

Systematic Error
 May be due to several factors:
 poorly made standards
 reagents instrumentation problems
 poorly written procedures
 consistent error, problem is either with the
reagent or with the machine
 precise but not accurate
 will form/create a trend (upward/downward)

The terms that describe the performance of any


detector are the frequency of true alarms and
frequency of false alarms; for QC procedures, these
characteristics are called the probability for error
detection and the probability for false rejection,
resp.
 Probability for error detection, P ed, is the
probability of rejecting an analytical run having
an error occurring in addition to the stable
imprecision of the measurement procedure.
Ideally, Ped should be 1.00, which means there
would be a 100% chance of detecting an error.
Operation of Quality Control System A practical design objective is a Ped of 0.90,
The QC program can be thought of as a three-stage which means there would be a 90% chance of
process: detecting an analytical problem.
1. Establishing allowable statistical limits of  Probability for false rejection, Pfr, is the
variation for each analytic method. probability of rejecting an analytical run when
2. Using these limits as criteria for evaluating there is no error except for the stable
the QC data generated for each test. imprecision of the measurement procedure.
3. Taking action to remedy errors when Ideally, Pfr should be 0.00 to provide a 0.00%
indicated. chance of false rejection. In practice, a practical
a. Finding the cause(s) of error design objective is a Pfr of 0.05 or less, which
b. Taking corrective action
means there would be only a 5% or less chance  The laboratory must incorporate
of false rejection. proficiency testing into its routine
workflow as much as possible.
 The test values/samples must not
shared with other laboratories at any
time during the testing cycle.
 Proficiency samples are tested by bench
technical staff who normally conduct
patient testing; there can be no
unnecessary repeats or actions outside
of how a patient sample would be tested
and reported.
 Testing should be completed within the
usual time it would take for routine
patient testing.
 The proficiency testing samples also can serve
as valuable troubleshooting aids when
Approach for Improving QC Performance investigating problem analytes.
 Proficiency tests can also be beneficial in
 Eliminate those control rules that have a high validating the laboratory’s measurement
probability of false rejection. method, technical training, and uncertainty
 Select a combination of rules having at least budgets for new tests.
one rule responsive to random error and one to
systematic error.
 Assess the probabilities of rejection for that
combination of rules.
 Choose the total number of control
measurements (N) to provide the desired
probability of error detection.

Proficiency Testing

Inspirasyon niyo sa pagre-review 

Quality Management

 Acceptable performance in proficiency testing


programs is required by CAP, CLIA, and TJC to
maintain laboratory accreditation.
 A series of unknown samples are sent to the
laboratory from the program offering this
analysis. Note:
 The samples are analyzed in the same manner  Quality assurance – refers to the end-users
as patient specimens, and the results are (patient) who must benefit from this top
reported to the proficiency program. The quality patient care
program then compiles the results from all of  Quality improvement – improvement in the
the laboratories participating in the survey and process; e.g. fast turnaround time, high quality
sends a performance report back to each reagents, calibrated machines, double checking
participating laboratory. of results before releasing
 When a laboratory performs proficiency testing,
there are strict requirements as follows:
Quality Improvement: Lean Six Sigma
 Lean Six Sigma provides a culture,
infrastructure, methodology, and metric for
quality improvement.
 Lean Six Sigma begins with a belief in the
relentless pursuit of continuous improvement
toward excellence in products or services,
processes, and people.
 An infrastructure must support the Lean Six
Sigma initiative through a coalition of senior
members from the organization. These
organizational leaders select and assign quality
improvement projects to teams.
 Project coaches/leaders (Black Belts)
 Project team members (Green Belts)
 Project sponsors (Blue Belts)
 Lean Six Sigma uses a problem-cause-solution
methodology to improve any process through
waste elimination and variation reduction.

What is DMAIC?
 The DMAIC (Define, Measure, Analyze,
Improve, and Control) methodology is more
than a series of phases and steps, or tools and
techniques. It is the belief that quality
improvement requires sound problem
solving.

A process sigma represents the capability of a


process to meet (or exceed) the process
specifications (requirements). It reflects the
number of defects (errors) per million opportunities
(DPMO).

A Six Sigma process test has a narrow process SD


(i.e., it is very precise) and produces only three
errors for every million tests.
n of Phleb checking of disposal
kit your
 Patient previous
request, patient test
doctor’s result
order
 Specimen *Anything you
order do when
 Patient analysing
history specimen is part
of the analytical
process*

METRICS – Lean and Six Sigma


Six Sigma Metrics can be plotted graphically using
an operational process specification “OPSpecs”
chart.

Sensitivity vs. Specificity

PRE-ANALYTICAL, ANALYTICAL, POST-


ANALYTICAL PHASES

Note:
 Pre-aalytical – before mag-analyze/mag-process Sensitivity Specificity
 Analytical – the actual analysis Probability of a positive Probability of a
 Post-analytical – results test among patient with negative test among
Pre-Analytical Analytical Post-Analytical condition patient without
Patient  Reconstituti  Printing condition
Preparation on of QC results Tests with high Tests with high
 Specimen  Reagent  Logging of sensitivity are used to specificity are used to
labelling results rule out condition rule in condition
preparation
 Preparatio  Centrifugation  Interpretati (“snout=rule out”) (“spin=rule in”)
n of tubes  Delta on of results True positive – help True negatives – help
 Preparatio checking –  Waste identify patients with identify patients without
condition condition Lesson 7
LABORATORY MATHEMATICS

Significant Figures
 Minimum number of digits needed to express a
particular value in scientific notation without
loss of accuracy.

Examples:
0.0000800910 – 6 sig figs
1.0032 – 5 sig figs
899, 000 – 3 sig figs
645.010 – 6 sig figs
502.99000 – 8 sig figs

Logarithms
The logarithm of a number, which is written in
decimal format, consists of two parts: the character
or characteristic, and the mantissa

CHARACTERISTIC – number to the left of the


decimal point in the log and is derived from the
exponent.
1.0093

MANTISSA – portion of the logarithm to the right


of the decimal point and is derived from the
number itself.

Rulings in Logarithm
1. Log (1) = 0
2. If the BASE of the Log and the EXPONENT is
the same, the answer is 1
Example:
Log6 6 = 1
3. If there’s no base given, it’s always assumed as
Antilogarithms
10
 Log 12 = y (log base 10 of 12)  To determine the original number from a log
value, the process is done in reverse.
4. If the exponent is in FRACTION form, the
answer will be NEGATIVE
Negative Logarithms
 Log3 = y
X – NEGATIVE exponent base 10 expressed
= -y
without the minus sign
N – decimal portion of the scientific notations
5. If the base of the Log is a larger number than
expression
the exponent, answer will be in FRACTION
form.
 Log81 9 = y
= Sample problem:
1. Find pH of a solution that has a H+
6. If the given number has TRAILING ZEROS, the concentration of 0.00065.
answer will depend on the total number of Given: N = 0.00065
zeros.
Find: pH
 Log (100) = 2
Solution:
 Log (10,000) = 4
pH = X – log N (note: walang given for X value)
7. If the given number has LEADING ZEROS, the = - log N
answer will depend on the total number of = -log (0.00065)
zeros. pH = 2.1871 2.2 (note: 2 sig figs from 0.00065)
 note: if encountered decimal, automatic
the answer will be negative (because 2. Given: [H+] = 5.4 x 10-6
decimal can be written in fraction form) X = 6 (derived from the exponent)
 Log (0.1) = -1
N = 5.4
 Log (0.00001) = -5
Find: pH
8. If the WHOLE NUMBER is the same with the Solution:
BASE of the Log, simply cancel out and the pH = X – log N
answer will be the EXPONENT value. = 6 – log (5.4)
 6 log 6 12 = y = 5.2676
 6 log 6 12 = 12 pH = 5.3
(note: when working with pH, reporting of
answer should be 1 whole number and 1
decimal)

Concentration
Percent Solution
 determined in the same manner regardless of
whether weight/weight, volume/volume, or
weight/volume units are used.
 Percent implies “parts per 100”, which is
represented as percent (%) and is independent
of the molecular weight of a substance.

w/w
Make up 100 g of a 5% aqueous solution of HCl
(using 12M HCl).

5% 0.05
(note: converting % to decimal form is always the first
step)

0.05 X 100 g = 5 g of aqueous solution of 12M HCl


 Normality either equals or is greater than the
w/v molarity
Make up 1,000 mL of a 10% solution of NaOH. Step 1: Units needed?
10% 0.10 Step 2: Units you have?
Step 3: Rearrange the equation so that like terms
0.10 X 1,000 = 10 g can be cancelled out.
v/v H2SO4 gmw = 98 g
Make up 50mL of a 20% concentrated HCl V=2
20% 0.20 Solution: N = = 49 eq/L
0.20 X 50 mL = 10 mL
Sample Problem:
1. What is the N of a 500mL solution that
Molarity contains 7 g of H2SO4 ?
 Routinely expressed in units of moles per loter Find: N of 500mL solution with 7g H2SO4
(mol/L) or sometimes millimoles per millilitre Given: gmw H2SO4 = 98 g
(mmol/mL). V=2
 Remember that 1 mol of a substance is equal N of H2SO4 = 49 g/L
to the gram molecular weight (gmw) of that
substance. Solution:
 When trying to determine the amount of x x = 0.2857 or 0.3 eq/L
substance needed to yield a particular
concentration, initially decide what final
concentration units are needed.
Specific Gravity
Step 1: Which unit is needed in the final answer?  Density is expressed as mass per unit
Step 2: Assess other mass/volume terms used in volume. The specific gravity is the ratio of the
the problem. density of a material when compared to the
Step 3: Cancel out like units. density of water at a given temperature.
 The units for specific gravity are grams per
Sample Problem
milliliter.
1. How many grams are needed to make 1L of a
 Specific gravity is often used with very
2M solution of HCL? (gmw HCL = 36.5g/mol)
concentrated materials.
Find: grams
 The actual concentration is equal to the specific
Solution:
gravity multiplied by the assay or percent
x = 73 g/L of HCl purity value (expressed as a decimal) stated on
the label of the container.
2. A solution of NaOH is contained within a Class  In some instances, a chemistry laboratory may
A 1L filled to the calibration mark. Content report a given analyte using two different
label reads 24 g of NaOH. Determine the concentration units.
molarity. (gmw=40g)
Find: M (mol/L) Note:
Solution:  do not report SP 1.000; in such case increment
x = 0.6 mol/L or 0.6 M 1, meaning, add 0.0005 kasi possible na faulty
ang reagent strip
 memorize the conversion factors of different
Normality reagent
 Normality (N) is expressed as the number of
equivalent weights per liter (Eq/L) or
milliequivalents per milliliter (mEq/mL).
 Equivalent weight is equal to gmw divided by
the valence (V).
 Normality has often been used in acidbase
calculations because an equivalent weight of a
substance is also equal to its combining weight.
 When the valence of a substance is 1, the
molarity will equal the normality.
SERIAL DILUTIIONS
 Defined as multiple progressive dilutions
ranging from more-concentrated solutions to
less-concentrated solutions.
 Serial dilutions are extremely useful when the
volume of concentrate or diluent is in short
supply and needs to be minimized or a number
of dilutions are required.
 The serial dilution is initially made in the same
manner as a simple dilution. Subsequent
dilutions will then be made from each
preceding dilution.

Dilutions
 Note that the dilution factor indicates the parts
per total amount; however, in making the
dilution, the sum of the amount of the stock
material plus the amount of the diluent must
equal the total volume or dilution fraction  To establish the dilution factor needed for
denominator. subsequent dilutions, it is helpful to solve the
 The dilution factor may be correctly written as following equation for (x):
either a fraction or a ratio.
 A ratio is always expressed using a colon; a
dilution can be expressed as either a fraction or
a ratio.
 Sample dilutions should be made using either
reagent grade water, saline, or method-specific
diluent using Class A glassware.

SIMPLE DILUTIIONS
 The laboratory scientist must decide on the
total volume desired and the amount of stock
to be used.
Graphing and Beer’s Law

 mathematically establishes the relationship


between concentration and absorbance in
many photometric determinations.
 Beer’s Law is expressed as
A = abc
A = absorbance
a = absorptivity constant for a particular
compound at a given wavelength under
specified conditions
b = length of the light path
c = concentration
 If a method follows Beer’s law, then absorbance
is proportional to concentration as long as the
length of the light path and the absorptivity of
the absorbing species remains unaltered during
the analysis.
 Assays measuring absorbance generally obtain
the concentration results by using a Beer’s law
graph, known as a standard graph or curve

Enzyme Calculations
 When calculating enzyme results, the rate of
absorbance change is often monitored
continuously during the reaction to give the
difference in absorbance, known as the delta
absorbance, or A.
 The Enzyme Commission of the International
Union of Biochemistry recommended using one
unit, the international unit (IU), for reporting
enzyme activity.
Additional readings:
From Germanna Academic Center for Excellence
https://germanna.edu/sites/default/files/2022-
03/Significant%20Figure%20Rules.pdf

Courtesy of Ar-Rayan Edres:


For Molarity computation

Mass = g
or mg

Molar Mole =
mass = mol or
g/mol or
mmol
mg/mmol

Mole = mol
or mmol

Molarity = Volume =
M L or mL
Note:
Lesson 8 o wavelength – horizontal distance between
peaks of two frequency (waves)
ANALYTICAL TECHNIQUES o frequency – number of waves; measured by
Hertz
SUMMARY OF CHEMISTRY ANALYZER o Visible light – visible in naked eye, 400 to
OPERATIONS 700 nanometer; between ultraviolet and
Identification and Preparation infrared light
1. Sample  Usually done by reading  Electromagnetic radiation is described as
identification the bar code photons of energy traveling in waves.
 Information can be  For a ray of electromagnetic radiation to be
entered manually observed, it must have the same frequency as a
2. Determine  LIS communicates to rotational frequency in the atom or molecule
test(s) to the analyzer which that it strikes. It depends upon the solution or
perform test(s) have been analyte na gagamitin sa spectro
ordered.  Absorption or emission of energy by atoms
Chemical Reaction results in a line spectrum.
3. Reagent systems  One or more reagents
and delivery can be dispensed into Beer’s Law
the reaction cuvet.  states that the concentration of a substance
4. Specimen  Small aliquot of the is directly proportional to the amount of
measurement sample is introduced light absorbed or inversely proportional to
and delivery into the reaction cuvet. the logarithm of the transmitted light
5. Chemical  The sample and
reaction phase reagents are mixed and
incubated.
Data Collection and Analysis
6. Measurement  Optical readings may be
phase initiated before or after
all reagents have been
added.
7. Signal  The analyte
processing and concentration is
data handling estimated from a
calibration curve that is
stored in the analyzer.
8. Send result(s) to  The analyzer
LIS communicates results
for the ordered tests to
the LIS.
Operations generally occur in the order listed from 1 to 8.
However, there may be slight variations in the order. Some steps
may be deleted or duplicated. Most analyzers have the capability  all light absorbed or blocked results in 0% T
to dilute the sample and repeat the testing process if the analyte  no light absorbed = 100% T
concentration exceeds the linear range of the assay.
Note: o 2 factors: absorbance and concentration
4 Basic Disciplines in Analytic Chemistry  ↑ absorbance rate = ↑ concentration of
1. Spectrometry – commonly used in lab solution
2. Luminiscence  relationship between absorbance and
3. Electro analytic methods concentration = standard curve or
4. Chromatography graph

Spectrometry transmitted light


1. Atomic Absorption Spectrophotometer
2. Flame Photometry
3. Fluorometry
incident light
SPECTROPHOTOMETER
 measure light intensity without consideration
of wavelength
thermal paper
incubator

sample holder

digital display
 Absorbance (A) – amount of light absorbed. It
cannot be measured directly by a Note:
spectrophotometer but rather is  Spectrophotometer has incubation period/time.
mathematically derived from %T.  Typical incubation period: 5 minutes
 Absorptivity depends on molecular structure  After incubation, saka i-process.
and the way in which the absorbing molecules  the colored green screen is the digital display.
react with different energies.  We run samples only when the spectrophotometer is
in its optimal temperature.
 For any particular molecular type, absorptivity
 FBS, Lipid Profile and BUA are the tests that
changes as wavelength of radiation changes. require incubation period prior to processing in the
The amount of light absorbed at a particular spectrophotometer.
wavelength depends on the molecular and ion  thermal paper – where results can be printed, can be
types present and may vary with bought commercially
concentration, pH, or temperature  each number in the keys has corresponding command
(environmental changes that can affect your depending on the list of commands/test in the
solution). spectrophotometer. (ex: 4 is for BUA)
Note: Spectrophotometer has incubation  This semi-automated stat fax cannot run batch
 Optimal temp for incubation of Spectro - 37°C testing. Only fully-automated machines can run batch
testing.
 Absorbance is directly proportional to
concentration.
Spectrophotmeter Quality Assurance
Spectrophotometric Instruments  Wavelength accuracy means that the
wavelength indicated on the control dial is
the actual wavelength of light passed by the
monochromator. It is most commonly checked
using standard absorbing solutions or filters
with absorbance maxima of known wavelength.
 Stray light refers to any wavelengths outside
the band transmitted by the monochromator.
The most common causes of stray light are
Note: reflection of light from scratches on optical
 Incandescent lamp / Tungsten Iodide lamp – surfaces or from dust particles anywhere in
light source used in Spectrophotometer the light path and higher-order spectra
 Monochromator – separate/differentiate the produced by diffraction gratings.
wavelengths o Stray light is unwanted light that causes
interference/variation in the result.
 PM tube – photomultiplier tube
 Linearity is demonstrated when a change in
 A/D – Analog-digital converter concentration results in a straight-line
o converts the result digitally and displays
calibration curve.
the result
Atomic Absorption Spectrophotometer
 is used to measure concentration by detecting
absorption of electromagnetic radiation by
atoms rather than by molecules.
 The analyzed sample must contain the reduced
metal in the atomic vaporized state.
 The amount of light absorbed is proportional to
the concentration.
 When a ground-state atom absorbs light
energy, an excited atom is produced. The
excited atom then returns to the ground state,
emitting light of the same energy as it
absorbed. The flame sample thus contains a
dynamic population of ground-state and
excited atoms, both absorbing and emitting
radiant energy.
 Atomic absorption spectrophotometry is
sensitive and precise. It is routinely used to
measure concentration of trace metals that are
not easily excited.
 Flameless atomic absorption requires an
instrument modification that uses an electric
furnace to break chemical bonds
(electrothermal atomization).
 The presence of an intense static magnetic field
will cause the wavelength of the emitted
radiation to split into several components. This
shift in wavelength is the Zeeman effect.

 Fluorescence concentration measurements


are related to molar absorptivity of the
compound, intensity of the incident radiation,
quantum efficiency of the energy emitted per
quantum absorbed, and length of the light
path.
 In dilute solutions with instrument parameters
held constant, fluorescence is directly
proportional to concentration.
o The higher the fluorescence, the higher the
concentration.
 In fluorescence polarization, radiant energy is
Fluorometry polarized in a single plane.
 Each molecular type possesses a series of
electronic energy levels and can pass from a
lower energy level to a higher level only by
absorbing an integral unit (quantum) of light
that is equal in energy to the difference
between the two energy states.
 Because the molecules are excited by
absorption of radiant energy and lose energy by
multiple interactions, the radiant energy
emitted is less than the absorbed energy.
 The difference between the maximum
wavelengths, excitation, and emitted
fluorescence is called Stokes shift.
 Filter fluorometers measure the concentrations
of solutions that contain fluorescing molecules.

3 Sequential Steps in Fluorescence mechanism:


1. Absorption
2. Non-radiative dissipation
3. Emission

How is fluorescence used in PCR?


 Detection of PCR products in real-time can
be accomplished by using fluorescent dyes or
probes. Fluorescently-labeled probes detect the
amount of specific double-stranded DNA  Chemiluminescence reactions are oxidation
sequences while fluorescent dyes detect only reactions of luminol, acridinium esters, and
the amount of double-stranded DNA. dioxetanes characterized by a rapid increase in
 FLUOROPHORES - organic dyes such as: intensity of emitted light followed by a gradual
decay.
 Enhanced chemiluminescence techniques
increase the chemiluminescence efficiency by
Note: including an enhancer system in the reaction of
 Polymerase chain reaction – analytical technique a chemiluminescent agent with an enzyme.
where fluorescence/fluorometry is utilized.
 RT PCR - gold standard/confirmatory testing for Applications of Chemiluminescence
COVID testing. It is not only used for COVID. 1. Protein Blotting
DNA is being collected/obtained.  Western Blot (detects Lyme disease,
 Fluorescently-labelled probes detects the DNA HIV infection, hepatitis C
sequence infection,syphilis)
 Fluorescent dyes/coloring agents – detects the  The principles of Western blotting
amount of DNA. are equal loading of proteins,
separation of proteins by molecular
Advantages and Disadvantages of Fluorometry weight, electrophoretic transfer to a
 Fluorometry has two advantages over suitable membrane, and probing of
conventional spectrophotometry: specificity antibodies.
and sensitivity. Fluorometry increases  Western blot is commonly used.
specificity by selecting the optimal wavelength
for both absorption and fluorescence, rather
than just the absorption wavelength seen with
spectrophotometry.
 Fluorometry is approximately 1,000 times
more sensitive than most spectrophotometric
methods. One reason is because emitted
radiation is measured directly; it can be
increased simply by increasing the intensity of
the exciting radiant energy.
 The biggest disadvantage is that fluorescence
is very sensitive to environmental changes. Notes:
 Changes in pH affect availability of electrons,  Step 1 – get patient sample. From the sample, you
and temperature changes the probability of need to extract the protein
loss of energy by collision rather than  Step 2 – electrophoresis – also an analytic technique;
fluorescence. separation of proteins based on size of proteins
 Contaminating chemicals or a change of present in the sample
solvents may change the structure.  Step 3 – blot transfer – transferring proteins from gel
 UV light used for excitation can cause onto blotting membrane
 Step 4 – immunostaining – proteins present in
photochemical changes.
electrophoretic step are different kinds of proteins.
 Any decrease in fluorescence resulting from Use antibodies to detect the type of proteins.
any of these possibilities is known as  Step 5 – gel imaging – using a digital imager, target
quenching. protein bands are visualized and analysed using the
o Quenching effect – any decrease in principle of chemiluminescence. You will be abl to
fluorescence resulting from contaminants, recognize the type of protein based on its pattern of
changes in pH/temp. separation.

Chemiluminescence 2. Immunoassay - biochemical test that


measures the presence or concentration of
 Part of the chemical energy generated produces a macromolecule or a small molecule in a
excited intermediates that decay to a ground solution through the use of an antibody or
state with the emission of photons. an antigen; detects disease and works on
 Chemiluminescence is different than the principle of antigen-antibody reaction.
fluorescence in that no excitation radiation is  immuno – there should be an antigen-
required and no monochromators are needed antibody reaction or antigen-antibody
because the chemiluminescence arises from complex
one species.
measured for 10 seconds), ease of use (most
assays are one-step procedures), and simple
instrumentation.
 The main disadvantage is that impurities can
cause a background signal that degrades
sensitivity and specificity.

Turbidimetric
 Turbidimetric measurements are made with a
spectrophotometer to determine
concentration of particulate matter in a
sample.
 The amount of light blocked by a suspension
of particles depends not only on concentration
but also on size. Because particles tend to
aggregate and settle out of suspension, sample
Notes: handling becomes critical.
 LFIA – Lateral flow immunoassay  Instrument operation is the same as for any
 Example: Dengue blot test – screening test –
spectrophotometer.
qualitative test – no exact value of results
o sample flows from sample pad by capillary flow
o control line and testing line Turbidimetry Principle
o Dengue blot – IgG and IgM  The more lights blocked, it is MORE TURBID.
o IgG – always the past/chronic infections;  Turbidity is INVERSELY PROPORTIONAL to
meaning patients has past dengue infection Transmittance
o IgM – present/acute infection o the more turbid the solution, the lesser the
transmittance of incident light
 Specimen concentration is DIRECTLY
PROPORTIONAL to the amount of light blocked.
o the more turbid the sample is, the greater the
amount of light blocked

Application of Turbidimetry
1. Detection of bacterial growth and
bacterial culture.
2. Coagulation studies - If the instrument
detects a turbidity, that indicates a clotting
time or Prothrombin time.
Note:
Notes:
 Turbidimetry is not only used in
 factors to consider in immunoassay: antigen and
chemistry, but also in hematology
antibody
 Antigen-antibody complex is formed when antibody 3. Protein concentration in urine - Using of
bind with antigen Sulfosalicylic Acid (SSA) to confirm
 Antibody levels of body depend on the presence of presence of Proteins in urine; Turbidity is
disease to fight off the antigen. graded 1+ to 4+
o IgE – elevated level during parasitic or Note:
allergic reaction  macroscopic indication for high protein –
 Antibody – defense mechanism of the body; immunity foamy, dark urine
complex; produced by B cells antibodies.  microscopic – SSA testing – confirmatory
 5 Antibodies of the Body testing for protein in urine
1. IgA 2. IgE 3. IgM i. drop 1 to 2 drops of SSA
4. IgD 5. IgG ii. if the tube becomes cloudy or
turbid (sulfosalicylic acid reacted
with proteins in urine), thus it is
3. Pharmacological testing positive for protein
4. Toxicological testing
Nephelometry
Advantages and Disadvantages of  Light scattered by the small particles is
Chemiluminescence measured at an angle to the beam incident on
 Advantages of chemiluminescence assays the cuvet.
include sub-picomolar detection limits,  Light scattering depends on wavelength and
speed (with flash-type reactions, light is only particle size.
 Charged particles migrate toward the
Electrochemistry opposite charged electrode.
1. Potentiometry  The velocity of migration is controlled by the
2. Amperometry net charge of the particle, the size and shape of
3. Coulometry the particle, the strength of the electric field,
4. Polarography chemical and physical properties of the
supporting medium, and the electrophoretic
Galvanic and Electrolytic Cells temperature.
 In a galvanic cell, as the electrodes are
connected, there is spontaneous flow of
electrons from the electrode with the lower
electron affinity (oxidation; e.g., silver). These
electrons pass through the external meter to
the cathode (reduction), where OH ions are
liberated.
 This reaction continues until one of the
chemical components is depleted, at which
point, the cell is “dead” and cannot produce
electrical energy to the external meter.
 Current may be forced to flow through the dead
cell only by applying an external electromotive
force E. This is called an electrolytic cell.
 In short, a galvanic cell can be built from an
electrolytic cell.

Note:
 Ethidium bromide – commonly used to stain DNA
fragments; to enhance the contrast for easier
identification
o not biologically safe for the user (lab workers)

Electrophoresis Support Materials (Gel)


1. Cellulose Acetate
Note: 2. Agarose Gel
 Anode – negative electrode 3. Polyacrylamide Gel
 Cathode – positive electrode 4. Starch Gel
 In electrically charged electrodes using
electrochemical cell, we observe redox reactions
Gel Electrophoresis
 Oxidation – lose electrode
 Reduction – gain electrode

Electrophoresis
 Electrophoresis is the migration of charged
solutes or particles in an electrical field.
 Iontophoresis refers to the migration of small
ions, whereas zone electrophoresis is the
migration of charged macromolecules in a
porous support medium such as paper,
cellulose acetate, or agarose gel film.
 In a clinical laboratory, the macromolecules of
interest are proteins in serum, urine,
cerebrospinal fluid (CSF), and other biologic
body fluids and erythrocytes and tissue. Note:
 Electrophoresis consists of five components:  bp – base pair – unit for molecular size/length of DNA
fragment
the driving force (electrical power), the support
medium, the buffer, the sample, and the
detecting system.
Chromatographic Procedures
1. High-Performance Liquid Chromatography
2. Gas Chromatography

Column

High Performance Liquid Chromatography, or


HPLC
Chromatography o The gold standard method for hemoglobin
A1c testing.
 It refers to the group of techniques used to o A specific testing for glucose;
separate complex mixtures on the basis of o Hemoglobin A1c used in testing glucose that do not
different physical interactions between the require fasting
individual compounds and the stationary phase o requested by physicians to track the glucose
of the system. intake for past 3 months
Note: Gas Chromatography
 chromatography – separation by migration but does  Used to separate mixtures of compounds that
not need electrical power as source; just migrate are volatile or can be made volatile.
automatically
 The setup is similar to HPLC, except that the
mobile phase is a gas and samples are
partitioned between a gaseous mobile phase
and a liquid stationary phase.
Mass Spectrometry
 Definitive identification of samples eluting from
GC or HPLC columns is possible when an MS is
used as a detector.
 The coupled techniques, GC/MS and LC/MS,
have powerful analytic capabilities with
widespread clinical applications.
MS/MS
 Tandem MSs (GC/MS/MS and LC/MS/MS)
can be used for greater selectivity and lower
detection limits.
Chromatography Applications
 Biophysical technique that enables the
separation, identification, and purification
of the components of a mixture for qualitative
and quantitative analysis.
 separate along the stationary and mobile phase
1. Protein purification – purified protein

Modes of Separation
1. Absorption
2. Partition
3. Steric Exclusion or SXC – solution
separates according to sizes
GC/MS  The H2O2 is coupled to peroxidase to produce a
 Widely used for measuring drugs of abuse in color whose intensity is measured as a function
urine toxicology confirmations. of concentration and measured using
o common drugs – Methampethamine or Meth (shabu) reflectance photometry.
& Tetrahydrocannabinol or THC (marijuana)  Biosensors - A biosensor couples a specific
o gas chromatography tandem with mass biodetector, such as an enzyme, antibody, or
spectrometry – gold standard for drugs of abuse
nucleic acid probe, to a transducer for the
o drug testing is composed of screening testing (quali,
positive or negative), confirmatory testing (if donor direct measurement of a target analyte without
tested positive in either of the meth or THC testing), the need to separate it from the matrix.
o note: those na nagpapadrug test sa lab is called
DONOR (board exam must know)
o Confirmatory testing of drug of abuse is not
performed by medtech, but by the REGISTERED
CHEMISTS. Medtech only perform SCREENING
TESTING
 Drugs and metabolites must be extracted from
body fluids and typically reacted with
derivatizing reagents to form compounds that
are more volatile for the GC process.

LC/MS
 LC requires b and derivatization is rarely used,
saving time and expense.
 LC/MS also has great potential for measuring
low-level and mixed-polarity analytes such as
vitamin D, testosterone, and
immunosuppressant drugs due to its superior
sensitivity and specificity over immunoassays.

Osmometry
 An osmometer is used to measure the
concentration of solute particles in a solution.
The mathematic definition is:

where is osmotic coefficient, n is number of


dissociable particles (ions) per molecule in the
solution, and C is concentration in moles per
kilogram of solvent.
 to detect dehydration

Analytic Techniques for


Point-of-Care-Testing
 The major attraction of POCT is the reduced
turnaround time needed to deliver results.
 POCT relies on the same analytic techniques as
laboratory-based instrumentation:
spectrometry, electroanalytic techniques, and
chromatography.

Note: screening tests are analytic techniques considered


as POCT.

Point-of-Care-Testing
 Glucose monitoring - The first-generation
devices use a photometric approach, whereby
glucose produces hydrogen peroxide (H2O2)
with glucose oxidase immobilized onto test
strips.
AMINOACIDOPATHIES

Phenylketonuria Tyrosinemia Alkaptonuria Maple Syrup Isovaleric Homocystinuria Citrullinemia Cystinuria


Disease Acidemia
Enzyme Phenylalanine Type fumarylacetoacetate Homogentisate Ketoacid Isovaleryl-CoA Cystathionine Typ Argininosuccin *Defect in amino
absent/defi hydroxylase (PAH) I hydrolase oxidase (HGA) decarboxylase dehydrogenase synthetase eI -ic acid acid transport
cient/low synthetase system rather than
level Type tyrosine Typ Cells are enzyme deficiency*
II aminotransferase e II prevented
Type Hydroxyphenylpyru from making
III v-ate dioxygenase citrin

Characteri  Mental Type Most severe  Ochronosis  Sweet odor of  Failure to  Mental Typ  lack of  Cystine
stics retardation I  Arthritis-like urine, breath thrive retardation eI appetite precipitates out
 Microcephaly degeneration, and skin  Lethargy  Osteoporosis  failure to of the urine
Type Mental retardation thrive and forms
dark spots on  Lethargy  Multisystemic
II Photophobia the sclera  Failure to disorder of the  if left stones in the
untreated,
thrive connective kidneys,
can lead to
 Stupor tissue severe
ureters, or
 Mental  Thrombosis brain bladders
retardation (cardiovascular damage
Type risk) Typ  Japanese
III e II populatio
n
Tests  Guthrie test Confirmatory test: Urinalysis:  Modified  MS/MS  Guthrie test Urine:
o inhibitor: Beta-2- elevated level of the  Ferric chloride Guthrie test o inhibitor: L-  addition of
thienylalanine abnormal metabolite (+) Alkoptonuria
o o inhibitor: 4- methionine cyanide
o bacteria: will result in
Bacillus subtilis
succinylacetone azaleucine sulfoximine nitroprusside –
black color
o sensitive enough  Microfluoromet  MS/MS producing red-
change in urine
to detect serum
ric assay  HPLC purple color
phenylalanine (+)
o confirmatory
levels of 180-240  MS/MS
mol/L (3-4 o > 2 mg/dL (+)
 Pre-natal:
mg/dL)  LC-MS/MS
 Microfluoromet decarboxylase o elevated urinary
ric assay enzyme total
o direct concentration homocysteine
measurement of
phenylalanine in in amniotic
dried blood filter fluid
disks. This method
yields quantitative
results, more
adaptable to
automation.
 HPLC
o reference method
 MS/MS
o greater sensitivity
o phenylalanine to
tyrosine
Drug  Kuvan Nitisinone (NBTC) High-dose vitamin Oral administration High dose of  Arginine  consistent high
treatment (sapropterin C of glycine and vitamin B6 supplementatio fluid intake
dihydrochloride) carnitine n  Penicillamine
supplementation
 Administration
of sodium
benzoate and
sodium
phenylacetate
Urine “Cabbage-like” odor Black/brown-black Burnt sugar/sweet Sweaty feet odor
color odor
AMINO ACIDS AND PROTEINS  random (midstream clean catch) -
qualitative
Overview  quantitation – 24 hour urine preserved
AMINO ACIDS with thymol
 building blocks of proteins. Testing
 growth, repair, and maintenance of all cells are  TLC – method of choice
dependent on amino acids  Two-dimensional chromatography
 The chemical properties of the amino acids of o amino acids migrate along one solvent front and
proteins determine the biologic activity of the then the chromatogram is rotated 90 degrees
protein. and a second solvent migration occurs
 Proteins catalyze almost all of the reactions in o Solvents:
living cells, controlling virtually all cellular  Butanol
processes.  Acetic acid
BASIC STRUCTURE  Water and ethanol
 Ammonia
 One amino functional group and one carboxylic
acid functional group  Chromatogram – stained with ninhydrin, mostly
giving a blue color
 Polypeptide – chain of amino acids
 MS/MS
 Protein – large polypeptide
Proteins
 Collagens are the most abundant protein in the
human body.
 A typical protein contains 200–300 amino acids,
but some are much smaller (peptides) and some
are much larger (titin, in muscle).
Protein Synthesis
Metabolism  Most plasma proteins are synthesized in the liver
and secreted by the hepatocyte into the
 Nutritionally essential amino acids must be
circulation.
supplied by the diet in the form of proteins.
 Transcription: The double-stranded DNA unfolds
 10 Essential Amino Acids
in the nucleus, and one strand is used as a
o Arginine (Arg) o Methionine (Met)
template for the formation of a complementary
o Histidine (His) o Phenylalanine (Phe)
strand of messenger RNA (mRNA).
o Isoloecine (Ile) o Threonine (Thr)
 Translation: The process of synthesizing a
o Leucine (Leu) o Tryptophan (Trp)
protein from an mRNA template. Protein synthesis
o Lysine (Lys) o Valine (Val)
occurs at the rate of approximately two to six
 The primary purpose of amino acids is for the
peptide bonds per second.
synthesis of body proteins, including plasma,
 The hormones that assist in controlling protein
intracellular, and structural proteins.
synthesis are (TGIT) thyroxine, growth
 Humans do not have all the enzymes required for
hormone,
the biosynthesis of all of the amino acids. Under
insulin, and
normal circumstances, proteolytic enzymes, such
testosterone.
as pepsin and trypsin, completely digest dietary
 The hormones
proteins into their constituent amino acids.
that assist in
Amino acids are then rapidly absorbed from the
controlling
intestine into the blood and subsequently become
protein
part of the body’s pool of amino acids.
catabolism
 Aminoacidopathies – an enzyme defect that
are glucagon
inhibits the body’s ability to metabolize certain
and cortisol.
amino acids.

Amino Acid Analysis


Specimen Collection
 Fasting: 6 to 8 hours
 Specimen:
o Plasma – Green top (Heparin)
 plasma separated from cells through
centrifugation
o Urine
 screening test
 Typical blood panel provide the following
Classification of Proteins by Functions measurements: TPAG
Enzymes Catalyze chemical reactions  total protein  globulin
Hormones Chemical messenger that control the  albumin  albumin/globulin ratio
actions of specific cells or organs
Transport Transport movement of ions, small Albumin
Proteins molecules, or macromolecules
Immuno- Produced by B-cells (lymphocytes) or  Synthesized in the liver
globulins plasma cells in the bone marrow that
 Highest concentration in the plasma
mediate the humoral immune response  Responsible for nearly 80% of the colloid osmotic
to identify and neutralize foreign objects pressure of the intravascular fluid (fluid balance)
 IgA, IgG, IgM, IgD, IgE  Negative APR
Structural Connective tissue
 Binding characteristic is also exhibited with
proteins certain dyes (method for the quantitation)
Storage Reserves of metal ions and amino acids  Decreased concentrations of serum albumin may
proteins that can be released and used later be caused by the following: inadequate source,
without harm occurring to cells during liver disease, protein-losing enteropathy, kidney
the time of storage loss, gene mutations, hypothyroidism
Osmotic Maintenance of water distribution
force between cells and tissue, interstitial Globulin
compartments, and the vascular system
 The globulin group of proteins consists of 1, 2, ,
of the body.
and fractions. Each fraction consists of a number
of different proteins with different functions.

Alpha-1-Antitrypsin (AAT)
 Synthesized in the liver
 Also an APR
 FUNCTION:
o Inhibition of the protease neutrophil
elastase; NE is released to fight off
infection but can destroy alveoli of the
lungs leading to Emphysema.
 Deficiency: Mutations in the SERPINA1 gene
 INCREASED in: inflammatory rxns, pregnancy,
contraceptive use
 METHODS OF ANALYSIS: Radial
Immunodiffusion, Immunonephelometric assays
Methods of Analysis
 Specimen collection Alpha-1-Fetoprotein (AFP)
 serum (red top – plain tube)  Synthesized in the liver and developing fetus and
 Interferences embryo
 Lipemia – milky white or cloudy serum  FUNCTION:
 Hemolysis o Binds estradiol in normal fetuses and
If such interferences occur, reject the sample and protects the fetus from immunologic
recollect. attack by the mother; no known function
 Reference interval: 6.5 – 8.3 g/dL (65 – 83 g/L) in normal adults
 INCREASED in: Spina Bifida, NTDs, anencephaly,
fetal distress
 DECREASED in: LOW levels in maternal AFP
indicates Down syndrome and Trisomy 18
 AFP SCREENING: done between 15-20 weeks
gestational age; affected by materna weight, race
and DM
 METHODS OF ANALYSIS: fractionated by Affinity
electrophoresis into 3 ISOFORMS (L1, L2, L3)
based on their reactivity with Lectin Lens
Plasma Proteins Culinaris Agglutin (LCA), RIA, EIA
2 Major Groups: ALBUMINS & GLOBULINS  MATERNAL SCREENING TEST: QUADRUPLE
Blood Panel TEST using these 4 substances: AFP, hCG,
unconjugated estriol, inhibin A
Note:
 APR – acute phase reactants – first to elevate during infection or inflammation
 hCG – human chorionic gonadotropin – produced by placenta during pregnancy
 Multiples of the Median (MoM) - calculated by
dividing the patient’s AFP value by the median
 reference value for that gestational age. Ceruloplasmin
 0.5 MoM - 2.0 MoM Note:
 MoM – multiples of the median  Synthesized in the liver
 Also an APR
Alpha-1-Acid Glycoproein (Orosomucoid)
 COPPER-CONTAINING
 Synthesized in the liver  INCREASED in: inflammation, severe infections,
 Also an APR tissue damage, pregnancy, use of oral
 INCREASED in: stress, inflammation, tissue contraceptives and medications such as:
damage, acute M.I, trauma, pregnancy, cancer, Carbamazepine, phenobarbital, valproic acid
pneumonia, R.A  90% of serum copper is found in Ceruloplasmin
 METHODS OF ANALYSIS: RID, Immunoturbidity, while the 10% is bound to albumin
Nephelometry  DECREASED in: malnutrition, malabsorption,
 NORMAL RANGE: 50-120 mg/dL severe liver disease, nephrotic syndrome, Menkes
syndrome (kinky hair
Alpha-1-Acid Antichymotripsin disease)
 PATHOGENESIS:
 Synthesized in the liver DECREASED levels in
 Also an APR Wilson’s disease
 Function: (KAYSER-FLEISCHER
o Inhibits enzymatic activities of Cathepsin RINGS)
G, pancreatic elastase, mast cell chymase,
chymotrypsin  METHODS OF
 INCREASED in: inflammation ANALYSIS: RID,
 DECREASED in: liver disease Nephelometry, Copper
 Pathologic findings: Parkinson’s disease, COPD, oxidase assay
Alzheimer’s disease
Alpha 2-Macroglobulin
GC-Globulin (Group Specific Component)
 Synthesized in the liver
 Synthesized in the liver  Function:
 Function: o Inhibits proteases such as trypsin,
o major carrier of VITAMIN D and its thrombin, kallikrein and plasmin
metabolites, co-chemotactic factor in  INCREASED in: Nephrosis
chemotaxis of neutrophils and monocytes,  METHODS OF ANALYSIS: RID,
binds actin released from cells upon injury Immunonephelometry, ELISA, LIA
 The resulting decrease in concentration is used as
a prognostic indicator of a significant tissue injury
Transferrin
after trauma
 INCREASED in: 3rd trimester of pregnancy,  Synthesized in the liver
estrogen oral contraceptives  NEGATIVE APR
 METHODS OF ANALYSIS: Immunonephelometry  Most important iron pool
 Major component of the Beta-Globulin fraction
Haptoglobin that appears as a distinct band on electrophoresis
 FUNCTION:
 Synthesized in the liver o Transport of iron and prevention of loss of
 Also an APR iron through kidneys
 This is a tetramer molecule consisting of 2 alpha  Used to determine cause of anemia, gauge iron
chains and 2 beta chains metabolism, and iron carrying capacity of blood
 INCREASED in: ulcerative colitis, acute  DECREASED in: liver disease, malnutrition,
Rheumatic disease, heart attack, severe infections excessive loss through kidneys, infection,
 FUNCTION malignancies
o Binds free Hgb to prevent loss of Hgb  INCREASED in: Iron Deficiency Anemia (IDA)
(Free Hgb is not contained inside  METHODS OF ANALYSIS: Immunodiffusion,
RBCs) Immunonephelometry
 HAPTOGLOBIN-HEMOGLOBIN COMPLEX: HP
and Hgb attaches to each other and this
Hemopexin
attachment/complex is removed by the
reticuloendothelial cells (spleen), with iron and  Synthesized in the liver
amino aids being recycled while HP is destroyed.  Also an APR
 PATHOGENESIS: Hemolytic Anemia,  FUNCTION: scavenge the heme released or lost in
Intravascular hemolysis METHODS OF the blood stream, protecting the body from
ANALYSIS: RID, Immunonephelometry
oxidative damage caused by the free heme
(Highest affinity to heme)
 INCREASED in: inflammation, DM, Duchenne-
type muscular dystrophy, melanomas
 DECREASED in: Hemolytic anemia
 METHODS OF ANALYSIS: RID

Beta 2-Microglobulin
 The light chain component of the Major
Histocompatibility Complex (HLA)
 Found mainly on the surface of most nucleated
cells
 Mostly are reabsorbed by the PCT in kidneys
(>99%)
 INCREASED in: R.A, SLE, HIV
 METHODS OF ANALYSIS: Immunoassays

C-Reactive Protein
 Synthesized in the liver
 One of the first APRs to rise during inflammations
(not specific but rather serve as general
indicator) Troponin
 CRP bound o bacteria and fungi promotes binding  FUNCTION: GOLD STANDARD in the diagnosis of
of Complement facilitating the uptake of Acute Coronary Syndrome (ACS) due to its
phagocytes (OPSONIZATION) specificity for myocardial damage
 INCREASED in: tissue necrosis, inflammatory  Compared to Myoglobin, cTn is elevated for a
diseases, Atherosclerosis (chronic inflammatory longer period of time
process), bacterial infections, acute Rheumatic  SPECIMEN COLLECTION: Serum or heparinized
fever, R.A, gout, viral infections plasma
 Most infections and inflammations result in CRP  METHODS OF ANALYSIS: ELISA,
above 10mg/dL immunoenzymometric assays
 METHODS OF ANALYSIS: Nephelometry, EIA  REFERENCE INTERVAL: 0.1-3.1 ng/mL

High-Sensitivity CRP Brain Natriuretic Peptide (BNP)


 Detects low levels of CRP (below 1 mg/L)  TYPES: Atrial Natriuretic peptide (ANP), B type
 Monoclonal antibody-based test methodologies (brain), C type (CNP), Dendroaspis Natriuretic
 Function: peptide (DNP)
o Determine risk of cardiovascular risk,  Marker for CHF
future cardiovascular events, and  METHODS OF ANALYSIS: Immunoradiometric
recurrent coronary events assay, microparticle enzyme immunoassay, ECLIA

Other Proteins of Importance Fibronectin


 FUNCTIONS: cell adhesion, tissue differentiation,
Myoglobin growth and wound healing, serves as a
 FUNCTION: primary oxygen-carrying protein NUTRITIONAL MARKER
found in skeletal and cardiac muscles, serves as a  FETAL FIBRONECTIN (fFN): predicts short-term
CARDIAC BIOMARKER (when striated muscles risk of premature delivery; maintains adherence
are damaged, Myoglobin is released) of placenta to the uterus
 Requires a very low oxygen tension to release
bound oxygen; mostly are dissolved in the Adiponectin
cytoplasm
 Recent studies have shown an inverse
 AMI: increased levels are seen 2-3hrs of onset,
relationship between BMI and adiponectin values
peak concentration in 8-12hrs, return to normal
 LOW levels correlate with an increased risk of
in 18-30hrs after AMI (speed of appearance and
heart disease, type 2 DM, and obesity
clearance of Myoglobin)
 INCRESED in: progressive muscular dystrophy,
crushing injury Beta-Trace Protein
 METHODS OF ANALYSIS: Latex agglutination,  Prostaglandin D Synthase
ELISA, Immunonephelometry, ECLIA,  Serves as an accurate marker of CSF LEAKAGE
Fluoroimmunoassays
 FUNCTION: Detects renal impairment, diagnosis SERUM PROTEIN ELECTROPHORESIS
of perilymphatic fluid fistulas

Cystatin C
 Produced and destroyed at a constant rate
 FUNCTION: sensitive endogenous marker for
GFR, kidney dysfunction monitoring
 Cystatin C levels are NOT affected by muscle
mass, gender, age, race, nor drugs
 May be used as an alternative to creatinine and
Creatinine Clearance
 METHODS OF ANALYSIS: Particle-enhanced
immunoturbidimetry, immunonephelometric
methods

Amyloid
 Fibrous protein aggregates formed due to an
alteration in Beta pleated sheets
 Stains with CONGO RED
 PATHOGENESIS: AMYLOIDOSIS, abnormal
deposits to organs/tissues

Total Protein Abnormalities


Hyperproteinemia Hypoproteinemia
 Dehydration - When  Negative Nitrogen
excess water is lost balance occurs
from the vascular  Plasma proteins can
HIGH RESOLUTION ELECTROPHORESIS
system, the proteins, be lost by excretion in
because of their size, the urine in renal
remain within the disease; leakage into
blood vessels. the gastrointestinal
 Excessive production tract in inflammation
of gamma globulins
of the digestive
system; and the loss
of blood in open
wounds, internal
bleeding, or extensive
burns
NONPROTEIN NITROGEN COMPOUNDS
Non-protein nitrogen - used to monitor renal function

UREA
 highest concentration
 major secretory product of protein metabolism
 Urea concentration:
 renal function
 protein content in the diet
 rate of protein catabolism
 Clinical applications:
 hydration states
 nitrogen balance
 renal disease diagnosis
 adequacy of dialysis
 Urea nitrogen/creatinine ratio:
 Normal values - 10:1 to 20:1
 Azotemia
 Uremia/Uremic syndrome
o very high plasma concentration of urea with URIC ACID
renal failure  Catabolism of PURINE nucleic acids
o treatment: Dialysis/Transplant  Reabsorbed and reused (98-100% by the PCT)
o Causes:  Insoluble in plasma and with high concentrations, can
o Pre-renal – reduced blood flow be deposited in joints causing inflammation
 CHF, Shock, dehydration,  Present in plasma as MONOSODIUM URATE
haemorrhage
 In humans, uric acid is the final breakdown product of
o Renal – acute renal failure, glomerular PURINE METABOLISM.
nephritis, tubular necrosis
 Some other mammals, they have the ability to
o Post-renal – obstruction of urine flow
catabolize purines to ALLANTOIN.
 Methods of Analysis:
 Clinical application: Gout
 Ammonium is measured by color change
 Methods of Analysis:
associated with pH indicator (Dry Reagent
o Caraway Method – oxidation of UA in a
Strips)
protein-free filtrate; not specific
 Coupled method of urease reaction + glutamate
o Uricase/Uricate Oxidase - Catalyzes oxidation
dehydrogenase (GLDH) (Kinetic Method)
of UA to Allantoin; more specific; measures at
 Specimen:
293nm the differential absorption of UA and
o Plasma Note: DO NOT USE SODIUM CITRATE and/or
Allantoin (SPECTRO)
o Serum SODIUM FLUORIDE because it inhibits urease
o Coupled Enzymatic Methods- Measurement of
o Urine
Hydrogen Peroxide provided when UA is
 Originally, it is performed on a PROTEIN-FREE
converted to Allantoin catalyzed by
FILTRATE of whole blood and based on the
PEROXIDASE or CATALASE - Color produced
amount of NITORGEN in the sample.
is DIRECTLY PROPORTIONAL to UA
 Urea nitrogen concentration = Urea x 2.14
concentration.
o unit: mg/dL
 Wet and Dry chem analyzers
 Reference Method: ISOTOPE-DILUTION
 Interferences: Bilirubin & Ascorbic
MASS SPECTROMETERY (IDMS)
Acid destroy peroxide
o Specimen: plasma, serum, urine
o Reference Method: ISOTOPE-DILUTION
MASS SPECTROMETERY (IDMS)
 Pathophysiology:
o Lesch-Nyhan Syndrome (Males) - Complete
deficiency of Hypoxanthine Guanine
Phosphoribosyltransfe rase (HGPRT) base of
this enyme prevents the neutralization of
purine bases.
o Hyperuricemia (Gout) - often develop renal  interferences: alpha ketoacids,
calculi (stones); in severe cases, deposits of cephalosphorins
crystalline UA (TOPHI) form in tissues. o Coupled Enzymatic Methods (Dry Slide
 Treatment: Allopurinol – inhibis Analyzer)
xanthine oxidase  use of creatininase, creatinase,
o Hypouricemia sarcosine oxidase, peroxidase
 Fanconi syndrome – defective tubular o Specimen: plasma, serum, urine
reabsorption o Reference Method: ISOTOPE-DILUTION
MASS SPECTROMETERY (IDMS)

CREATININE
 Excreted into the plasma at CONSTANT RATE related
To muscle mass
 INVERSELY PROPORTIONAL to GFR
 PLASMA CREATININE CONCENTRATION:
o Relative muscle mass
o Rate of creatine turnover
o Renal function
 CLINICAL APPLICATIONS
o Sufficiency of kidney function
o Severity of kidney damage
 Urinary constituents may be expressed as a ratio to
creatinine quantity
 CREATININE CLEARANCE (GFR)
o Measures the amount of creatinine eliminated
from the blood by the kidneys CREATINE
 Methods of Analysis:  Plasma creatine is an insensitive marker and may not
o Jaffe be measurable increased until renal function has
 creatinine + picric acid = red-orange deteriorated more than 50%
chromogen  Creatine is synthesized primarily in the liver from
 interferences: acetoacetate, acetone, arginine, glycine, and methionine
ascorbate, glucose, pyruvate  Plasma creatine & urinary creatine are increased in:
o Kinetic Jaffe Method 1. muscular dystrophy
 serum + alkaline picrate 2. poliomyelitis
 rate of change in A is measured 3. hyperthyroidism
4. trauma
5. Methods of Analysis: LIPIDS
 Endpoint Jaffe Method Role of Lipids (fats)
 Creatine Creatinine  store excess calories
heating
 integral part of cell membranes
 Difference between the 2  structural role in cells
sample measurements is the Fatty Acids
creatinine concentration  May exist as FREE or UNESTERIFIED form (bound to
 HPLC – creatinine plasma ALBUMIN)
concentration is inversely proportional  Majority are found as constituent of Triglycerides or
to Creatinine clearance Phospholipids C
 Specimen: plasma, serum, urine  LASSIFIED AS:
1. short chain (4-6 carbon)
AMMONIA 2. medium chain (8-12 carbon)
3. long chain (>12 carbon)
 Deamination of Amino acids during protein
metabolism.
 Free Ammonia is toxic but low concentrations are CHOLESTEROL
present in the blood stream  amphipatic lipid found on the surface of lipid layers
 Bacterial metabolism occurs in the lumen of the  not readily catabolized by most cells
intestine  does not serve as body’s source of fuel
 Anaerobic metabolism in skeletal muscles Cholesterol Measurement:
 Clinical applications:  Routine laboratory:
1. Reye’s syndrome - seen in children; acute 1. The use of Enzymatic reagents - reasonably
metabolic disorder of liver that shows severe accurate quantitation WITHOUT the necessity
fatty infiltration of that organ. for any pretreatments, mild and better suited
2. Liver disease for automated chemistry analyzers
 PATHOPHYSIOLOGY:  Early analytic methods
1. High concentration of NH3 is NEUROTOXIC 1. Use of strong acids together with the other
associated with encephalopathy chemicals to produce a measurable color (Non
2. HYPERAMMONEMIA is associated with specific)
inherited deficiencies of urea cycle  Reference method:
 Methods of Analysis: 1. Uses HEXANE extraction after hydrolysis with
1. Microdiffusion chamber – separation of the alcoholic KOH followed by reaction with
compound by its vitality LIEBERMANN-BURCHARD color reagent
2. Enzymatic method – using of Glutamate (sulfuric acid, acetic acid, acetic anhydride)
dehydrogenase (chem analyzer)  Definitive method: IDMS
3. Thin film colorimetric assay (spectro) - NH3  Specimen collection: Serum (red top)
reacts with an indicator to produce a colored  Interferences: vitamin C, bilirubin
compound (blue dye) 1. Both are reducing agents that could interfere
4. Specimen: heparinized plasma, EDTA with the peroxidase-catalyzed color reaction
5. Patient preparation: Cigarette smoking by the
patient should be AVOIDED as this can
contaminate the sample.
6. Ammonia contamination is a potential problem
in the lab

1. Cholesteryl ester hydrolase cleaves fatty acid residue


from cholesteryl esters converting them to unesterified or
free cholesterol
2. Free cholesterol reacted by the 2nd enzyme (cholesterol
oxidase) producing hydrogen peroxide
*H2O2 is a substrate common for enzymatic color reaction
using horseradish peroxidase
3. colored compound
Note: intensity of resulting color is proportional to amount of
cholesterol
TRIGLYCERIDES
 3 fatty acid molecules attached to 1 molecule of glycerol
by ESTER BONDS
 HYDROPHOBIC, making it water insoluble
 no charge, making it a NEUTRAL lipid
 Triglycerides containing SATURATED fatty acids:
1. pack together more closely
2. SOLID at room temp
 Triglycerides containing UNSATURATED fatty acids:
forms oils at room temp LIPOPROTEINS
Triglyceride Measurement:  AMPHIPATIC HELIX - ability of apolipoproteins to
 Enzymatic Triglyceride rxns react with any bind to lipids
ENDOGENOUS FREE GLYCEROL (interference)  protein segments arranged in coils
 Serum can be a significant source of interference  HYDROPHOBIC AA - interact with lipids
wherein most endogenous free glycerol contributes a  HYDROPHILIC AA - faces toward the aqueous
10-20 mg/dL OVERESTIMATION of Triglycerides. environment
 UV endpoint  Spherical in shape ranging from 10-1200 nm in size
1. Freed glycerol participates in several enzymatic  Amphipathic cholesterol and phospholipid molecules -
sequences SURFACE
2. The use of GLYCEROL KINASE and  Triglycerides and cholesteryl ester molecules -
PYRUVATE KINASE converting NADH to hydrophobic; CENTER/CORE
NAD+  Separation technique: ULTRACENTRIFUGATION
3. DISADVANTAGES: susceptible to
 Apolipoproteins are located on the surface of
interference and side reactions
lipoprotein molecules - structural integrity; ligands for
 Common correction: cell receptors; activators/inhibitors of various enzymes
1. the use of DOUBLE-CUVET BLANK:
 LIPOPROTEIN CALSSIFICATION SYSTEM BY
 accomplished with a second parallel
DENSITY FRACTIONS:
measurement using Triglyceride rgt
1. Chylomicrons
WITHOUT the lipase enzyme to
2. VLDL
quantify only the free glycerol blank.
3. LDL
 BLANK-CORRECTED
4. HDL
TRIGLYCERIDE RESULT: Total
 Apolipoprotein (APO) A-1
glycerol measurement - Glycerol blank
o Major protein of HDL
measurement
o antiatherogenic
2. SINGLE-CUVET BLANK
 Apolipoprotein B
1. Lipase-free reagent
o large protein (500kD)
2. incubation
o principal protein on LDL, VLDL, chylomicrons
3. blank reading: measures only the
o 2 forms:
endogenous free glycerol
 APO B – 100
4. addition of lipase enzyme
 APO B-48 – Chylomicrons
5. incubation
 Apolipoprotein E – LDL, chylomicrons
6. final reading
o 3 mahor isoforms: APO E2, E3, E4
3. Designated calibration blanking
 Another convenient and cost-efficient
Lipoprotein Methods
method
 Reference method: Ultracentrifugation – lipoprotein
 Adjusting the calibrator set points t net
quantification: classically defined in terms of hydrated
or blank corrected values,
density
compensating for the average free
1. Ultracentrifugation
glycerol content of specimens.
o fractionation by density
4. Reference method (used by CDC)
2. Elctrophoretic separation
 Alkaline hydrolysis
o differences in CHARGE and SIZE
 solvent extraction
o detecting unusual or variant patterns
 color reaction with chromotropic acid
o uses agarose gel as most common medium
qualitative analysis
3. Chemical precipitation method
o depends on PARTICLE SIZE, CHARGE and
differences on APOLIPOPROTEIN CONTENT
o specific o Reference method: Beta quantiiificaiton
o usually with polyanions together with divalent  beta designation refers to electrophoretic term
cations, most common for HDL for LDL
4. Chromatographic method  combines ultracentrifugation and chemical
o size differences in MOLECULAR SIEVING precipitation
method  Lipoprotein (a)
5. Immunochemical methods o LDL-like particles that contain 1 molecule of apo
o for research and routine (a) linked to apo B-100 by disulphide bonds
o using antibodies specific to epitopes on the o Increased risk for premature CHD and stroke
apolipoproteins o High level of homology with plasminogen:
6. Direct Homogenous reagents competes with plasminogen for binding sites,
o fully automated chemistry analyzers promoting CLOTTING which is a key contributor
o uses smaller scale separations using tabletop to both MYOCARDIAL INFARCTION and
ultracentrifuges STROKE

CHYLOMICRONS High Density Lipoproteins (VLDL)


 Produced by the intestines  Smallest and most dense
 Largest (1200nm) and least dense  synthesized by the liver and intestine
 reflect light and account for the TURBIDITY of  FORMS:
postprandial plasma 1. Spherical
 Because of their lightness, they readily FLOAT ON 2. Discoidal - 2 molecules of apo A I;
TOP of stored plasma and form a CREAMT LAYER  nascent or newly secreted HDL;
 Once they enter the circulation, Triglycerides and  MOST ACTIVE FORM in removing excess
cholesteryl esters in chylomicrons are rapidly cholesterol from peripheral cells
hydrolyzed by lipases and are transformed into  Transformation of discoidal HDL to spherical
chylomicron remnant particles. HDL: When discoidal HDL has acquired
 They are recognized by the proteoglycans and remnant additional lipid, cholesteryl esters and Trigly
receptors in the LIVER, faacilitating their uptake. form a core region between its phospholipid
bilayer
Very Low Density Lipoproteins (VLDL)
 Antiatherogenic: Reverse cholesterol transport
 Produced by the liver  HDL Methods
 MAJOR CARRIERS of endogenous (Hepatic derived) o Chemical precipitation – two step procedure with
Triglycerides and transfer Trigly from the liver to the manual pre-treatment
peripheral tissues.  serum/plasma + precipitation reagent
 Also reflect light and account for most turbidity in  centrifugation
hyperlipidemic specimens.  HDL is quantified as cholesterol in supernate
 interference: elevated triglyceride levels
Low Density Lipoproteins (VLDL)  Low density of aggregated lipoproteins
 more cholesterol-rich than other apolipoproteins may cause floating during
 They form as a consequence of the lipolysis of VLDL centrifugation.
 It is readily taken up by cells via the LDL RECEPTOR  This incomplete sedimentation is
in the liver and peripheral cells indicated by cloudiness, turbidity, r
Note: particulate matter floating in the
o Atherosclerosis supernate.
 can infiltrate into the extracellular space of vessel  This will result in overestimation of
walls where they can be oxidized and taken up by HDL cholesterol
MACROPHAGES.  SOLUTION: HIGH SPEED
 Macrophages that taken up too much lipid become CENTRIFUGATION
filled with intracellular lipid drops and turn into  Reagents: Dextran sulphate (synthetic
FOAM CELLS. heparin)
 Foam cells are the predominant cell type of fatty o Homogenous assays
streaks, an early precursor of  highly precise and accurate
ATHEROSCLEROTIC PLAQUE.  have replaced pretreatment methods in
 LDL Methods: routine laboratory
o Chemical precipitation  Reference method (three-step procedure)
 used to separate HDL either from the serum 1. Ultracentrifugation – removes VLDL
or infranate obtained from ultracentrifugation 2. Heparin – manganese precipitation –
 Cholesterol is quantified removes LDL
3. Analysis of supernatant cholesterol –
Abell-Kendall assay
 Specimen collection
o Serum (red top)
o Serum (yellow top)
 Need for correction/dilution (NSS): lipemic samples
 Fasting: 12 – 14 hours

LIPID PANEL (C-T-H-L-V)


 Cholesterol
 Triglycerides
 HDL
 LDL
 VLDL

Friedewald Equation

You might also like