Clinical Chemistry 1 LAB_First Semester
Clinical Chemistry 1 LAB_First Semester
Clinical Chemistry 1 LAB_First Semester
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
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.
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
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.
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
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)
Proficiency Testing
Quality Management
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.
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
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)
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
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
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
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.
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
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
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:
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
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.
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
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
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
Friedewald Equation