CC2 Laboratory (Finals)

1 Phlebotomy Clinical Chemistry 2 (LAB)
Phlebotomy: A Review Sources and Types of Blood Specimens

Definition of Terms Source of Blood Specimens
Phlebotomy ● Arterial Blood
● invasive procedure that involves puncturing o Composition is generally uniform.
blood vessel (artery/vein) using a hypodermic o When it comes to collection, it is
needle-syringe set up in order to draw blood inherently difficult and hazardous as
● General definition – in order for us to draw this type of specimen is usually only
bodily fluids; in this case, when we are focused reserved for blood gas evaluation or
on our profession, it would be focused on analysis.
drawing blood. ▪ When it comes to collection,
Professional this is usually only done by a
licensed medical physician
● It is an important attribute of a phlebotomist
or rather a physician or a
wherein they must project a professional image
trained personnel.
that involves appearance, attitude,
● Venous Blood
communication, skills, and bedside manners
o Composition is generally affected
towards our patients.
by the metabolic activity of the
Patient Consent
individual.
● It is also very important for us to have patient
o It is also inherently lower or has lower
consent be obtained before initiating any
O2 levels compared to that of
medical procedures. In this case, it would be
arterial blood because veins carry
venipuncture or phlebotomy.
deoxygenated blood which is in this
● There are different types of patient consent:
case venous blood.
o Informed consent – implies voluntary
o Chloride, glucose, pH, CO2, lactic
permission for a medical procedure, test, or
acid, and even ammonia may also
medication will be given
differ from venous blood compared
o Expressed consent – may be given verbally
to arterial blood.
or in writing
o If venous blood flow is impaired, this
o Implied consent – does not require verbal
may affect several analytes.
expression of consent but rather actions
Examples of these would be protein
that implies consent. Example: A patient
and certain electrolytes.
holds out an arm. after being told a blood
● Capillary Blood
specimen is going to be collected
o Contains both your arterial blood,
o HIV consent – these are laws specify exactly
venous blood and even the
what type of information must be given to
surrounding tissue fluid that
information or to inform the client properly
surrounds the capillary network.
o Consent for minors – parent or guardian
o It has inherently high glucose levels
consent is required, especially, for
compared to that of your venous
healthcare personnel who do not obtained
blood.
it, are liable for assault or battery
o It has lower levels of calcium,
o Refusal of consent – an individual right or
potassium, and total protein.
constitutional right to refuse a medical
o However, potassium may increase in
procedure such as venipuncture
capillary blood when in collection, if the
Infection Control collection procedure is or has a squeezing
● It is a standard precautions that are regularly motion when collecting capillary blood.
followed which must be taken with every Types of Blood Specimens
patient to prevent the spread of infection
● SERUM
● These precautions are the
o Liquid portion of blood which is
o Use of personal protective equipment
clear, and pale yellow liquid in
(PPEs) such as laboratory gowns, gloves etc.
coloration
o Employ proper hand hygiene
o Separated from clotted blood via
o Transmission-based precautions such as centrifugation
isolation. o If centrifugation is not possible,
gravity may be used.
TRANSFORMERS 1
o Majority of chemistry tests usually we need to add an additive which is an

utilize this type of specimen anticoagulant
● PLASMA e.g. EDTA (Ethylenediamine Tetraacetic) Acid
o Somewhat similar to that of serum Citrates, heparin, and oxalates.
because it is also the liquid portion Antiglycolytic Agents
of blood - These are additives that prevent cellular
o Clear to slightly hazy, pale yellow uptake of blood glucose. Meaning that this
fluid prevents your blood cellular elements from
o However, it can only be seen in consuming the glucose in the specimen. So,
anticoagulated blood specimens this prevents your Erythrocytes, Leukocytes
o Plasma contains fibrinogen which from consuming glucose.
serum does not - Preserves 3 days, but glucose can still
o Many chemistry test can be decrease by 10 mg/dL.
performed using plasma particularly - Has antibacterial properties.
in cases of emergency and other - Used in blood alcohol analysis, due to its
situations ability to prevent the fermentation of
● WHOLE BLOOD glucose in order to increase alcohol by
o This type of specimen contains the inhibiting bacterial growth.
cellular elements of blood and the - Used in conjunction with potassium oxalate.
liquid portion of blood e.g. Sodium fluoride- Used in conjunction
o Contains blood cells and plasma with potassium oxalate.
o Only seen in anticoagulated blood Note:
specimens - Sodium fluoride- ADDITIVE
o Usually used in hematology, but can Potassium oxalate- ANTICOAGULANT
also be used in clinical chemistry Clot Activators
particularly in POCTs ( for acute care ● These are additives that encourage clot
or STAT situations/emergencies) formation
● Provide additional surface area for platelet
Phlebotomy Equipment activation
Tube Additives ● Examples:
● An additive functions as a solutions that o Thrombin
keeps the specimen in optimum condition o Glass (silica) particles
prior to analysis or even after analysis Inert clay (diatomite or diatomaceous earth or
● Tube additives are somewhat preservatives, celite)
they preserve the integrity of the specimen Thixotropic Gels
● An additive functions optimally when the ● Are inert substances contained in or near
tube is filled to its intended volume and the bottom of certain tubes
gently inverted immediately after collection o Inert substance – does not affect
o Underfilled or overfilled tubes may the blood specimen in any way
not function properly or the additive Function: during centrifugation, the gel lodges
may not function properly to between the cellular and fluid layers
preserve the specimen Trace Element-Free Tubes
Specimen quality can be affected if the tube is not ● Technically, this is not an additive. Instead, it
filled and handled properly. is a type of tube, however, this is included in
Anticoagulants the additives portion of our discussion
- These are the most commonly encountered
additives in the laboratory. ● These are tubes that are contamination-free
- These are additives that prevent the clotting as much as possible
behavior of collected blood, because once
blood is collected, this activates ● Used in collecting specimens for trace
coagulation cascades. So, for us to prevent elements, toxicology, nutrients, and other
this coagulation cascade from triggering, tests that detect minute concentration of
analytes
TRANSFORMERS 2
● May contain EDTA, heparin, or no additive tubes. That’s the reason why it is behind your blood
at all culture.
o EDTA and heparin may be present in Order of Draw:
order to preserve the collected Glass non additive tubes
specimen Tube Stopper Color:
Stopper Color Additive Department/s Red
Light Blue Sodium Citrate Hematology Rationale:
(Coagulation Prevents contamination by additives in other tubes
Red (Glass) None CC, IS/BB Order of Draw:
Red (Plastic) Clot Activator CC Plastic clot activator tubes
Red/Light Gray None CC or Discard Serum separator tubes (SSTs)
(Plastic) Tube Tube Stopper Color:
Red/Black Clot Activator CC Red
(Tiger) Gold with gel Red and gray
Green/Gray Lithium Heparin CC Gold Plastic
Light Green and gel Rationale:
Green Lithium or CC Filled after coagulation tests (or your glass
Sodium nonadditives) because silica particles activate
Heparin clotting and affect coagulation tests. That’s the
reason why it is always behind your coagulation
Lavender/Pink EDTA Hematology
tubes that may carry-over and ruin your
Gray Sodium fluoride CC
anticoagulant or your coagulation studies (carry-
and Potassium
over of silica into subsequent tubes can be
oxalate
overridden by anticoagulant in them).
Tan (glass) Sodium heparin CC
Order of Draw: Plasma Separator Tubes (PSTs) and
Tan (plastic) EDTA CC
Heparin/Heparinized Tubes
Yellow Sodium Microbiology Tube Stopper Color: green and Gray Rubber. Light
polyethanol Green Plastic
sulfonate (SPS) Rationale: Heparin affects coagulation studies
Yellow Acid citrate IS/BB we’ve mentioned and interferes in the collection of
dextrose (ACD) serum specimens. It causes the least interference in
tests other than coagulation tests
Order of Draw Order of Draw: EDTA Tubes
● Order of draw is very crucial in order for us to Tube Stopper Color: Lavender / pink
have no contamination between the different Rationale: the reason why the EDTA tubes are
tubes. placed in the latter part of the order of draw, is
● It depends on the order of draw, the tube because majority of the times. They are the one
color, and the rationale for collecting order. that have the most problems with carryover. EDTA
Order of Draw: elevated sodium and potassium levels. The reason
Blood cultures (sterile collections) behind this is because of the concoction or the
Tube Stopper Color: composition of how edta is prepared, because we
Yellow SPS have disodium edta and potassium edta. That is
Sterile media bottle the reason why edta elevated sodium and
Rationale: potassium. It also chelates and decreases calcium
Minimizes the chance of microbial contamination and iron levels and also elevates pt and ptt results
from other tubes and the like. because it is an anticoagulant and results
Order of Draw: particularly for sodium fluoride and potassium
Coagulation tubes oxalate affect sodium and potassium respectively.
Tube Stopper Color: After hematology tubes because oxalates
Light blue damage cell membranes and cause abnormal rbc
Rationale: morphology. Because for edta, we primarily use for
The first additive tube in the order because all other hematology,
additives affect coagulation tests such as EDTA,
heparin, oxalate, etc. will affect coagulation study
TRANSFORMERS 3
Order of Draw: 1. Plasma preparation Tubes (PPTs) o Patient’s physician should be

2. Oxalate fluoride tubes consulted before drawing from an
Tube Stopper Color: 1. Pearl Top 2. Gray arm on the same side as the
Rationale: oxalates and fluoride should be at the mastectomy, lymph nodes removal
end of the order of draw. Because lole what we’ve typically a part of the procedure
mentioned, they interfere with the cellular integrity can cause lymphostasis which is the
or the morphology of red blood cells. And then stoppage of lymph flow which
oxalates also interferes with enzymatic reactions. makes the arm susceptible to
swelling and even infection.
Pre-Analytical Conditions Procedural Error Risks
Problematic Sites ● Hematoma Formation
● Burn, scars, and tattoos o This is the rapid swelling at or near
o Should be avoided as they may be the venipuncture site, which is due
difficult to palpate and draw from, to blood leaking into the tissues.
and these areas have impaired o Venipuncture must discontinue and
circulation that can affect the test pressure should be applied.
results. ● Iatrogenic Anemia
o Recently burned or tattooed areas o An anemia as a result of treatment
are susceptible to infection, tattoos (e.g. frequent blood draws or
contain dyes that can interfere in removing large quantities at a time)
testing and areas with dyes should o Only minimum amounts of blood
be avoided unless no other site is should be drawn, particularly from
available. infants.
● Damage veins ● Inadvertent arterial puncture
o Sclerosed or which we call o This is the accidental sticking of an
hardened or thrombosed clotted artery, often the result of deep or
veins are obstructed, they feel hard blind probing, or attempting to draw
and cord-like lacking resiliency, they from a basilic vein.
have difficulty puncture, have o Venipuncture must discontinue and
impaired blood flow, that leads to pressure should be applied for at
erroneous test results and should be least 5 minutes.
avoided. o Identify specimen as “arterial blood’,
● Edema if submitted for testing.
o Edematous or edema swelling ● Infection of the site
caused by an abnormal o These are adverse effects of
accumulation of fluid in the tissues bacterial multiplication.
makes veins harder to locate, o Infection can be minimized by using
specimens collected from aseptic technique, including,
edematous areas may yield cleaning the site properly and not
erroneous test results because touching it again before needle
swelling alters a blood composition, insertion – which is a very common
so choose another site if it is possible. mistake by phlebotomists.
● Hematoma ● Nerve injury
o Is a swelling or mass of blood that o This results from poor site selection,
escaped from a vein during or inserting the needle too deeply or
following venipuncture so, never too quickly, in which you will be
draw blood from hematoma puncturing a nerve.
because it is painful and leads to o May be due to patient movement,
inaccurate test results if there is no needle insertion, excessive lateral
other site is suitable, draw the needle redirection, or even simply
specimen distal to the hematoma so blind probing.
that free flowing blood is collected. o Extreme pain, burning, or electric
● Mastectomy shock sensation of the arm, and
even numbness, and pain radiating
TRANSFORMERS 4
up or down the arm are all signs of Excessive Bleeding

nerve involvement. ● For example, those patients who are taking up
▪ This requires immediate anticoagulant therapy such as warfarin take
removal of the needle. longer than normal to stop bleeding.
● Reflux ● Kindly apply pressure to the site until the
o This is the backflow of blood from bleeding stops.
the tube into the patient’s vein that ● If it continues beyond 5 minutes, kindly notify an
can occur if blood in the tube is in appropriate professional to handle the situation
contact with the needle during a Syncope or fainting
blood draw. ● This is very much common particularly those
o To prevent reflux, the patient’s arm individuals who have phobias to needles or
must be in a downward position too, from the hospital setting itself
so that the collection tubes fill from ● Warning signs that a person may faint include
the bottom up. o Perspiration beads on the forehead
● Vein Damage o Hyperventilation
o These are scar build-ups that can o Loss of color (namumutla)
result from many venipunctures in ● Vasovagal syncope: fainting due to abrupt
the same area for an extended pain or trauma comes on suddenly without
period of time, improper redirection warnings
of the needle, or even blind Nausea or vomiting
probing. ● Reassure a nauseous patient and provide a
o Vein damage can impair vein container of some sort to hold as a precaution
patency and make it difficult to ● Ask the patient to breathe slowly and apply a
perform subsequent or succeeding cold to his/ her forehead
venipunctures. ● If the patient vomits, terminate the procedure
Patient Conditions/Complications and notify first aid personnel or appropriate
● Allergies to supplies and equipment personnel
● Excessive bleeding Obese Patients
● Syncope or fainting ● If there is no easily palpable vein in the
● Nausea or vomiting antecubital area which is very common, ask
● Obese patients the patient what site have been successful for
● Pain past blood draws
● Petechiae ● One area to focus on is the cephalic vein,
● Seizures/convulsions which is more easily located by rotating the
Allergies to supplies and equipment patient’s arm. In doing so, the weight of the
● Patients can be allergic to antiseptics such excess tissue often pulls downward making the
as iodine, adhesives on glue and vein easier to see or palpate
bandages; and even latex which can Pain
cause a life-threatening reaction in those - General pain that your patient is suffering,
who are severely allergic to latex. aside a slight amount of pain is expected
Particularly to latex gloves in items such as during a routine venipuncture or capillary
gloves and even tourniquet use an puncture, so a stinging sensation is very
alternative antiseptic if required if the much automatic to your patient. This can
patient is allergic to iodine and then use be avoided by allowing the alcohol to dry
paper tapes place over folded gauze or completely after the cleaning site or to
self-adhesive bandages material can be perform the venipuncture properly.
used in place of adhesive bandages. And - Severe pain or a burning or electric shock
then like what you've mentioned if a sensation numbness or a pain radiating up
patient is allergic to latex never use latex or down the arm like what we've
items to your patient because like what we mentioned ago with nerve damage, kindly
mentioned they are sensitive. If the patient terminate the procedure and kindly notify
did not mention that they are allergic to immediate or appropriate personnel to
latex immediate response should be handle the case
attended too.
TRANSFORMERS 5
Petechiae
- This is when a tourniquet is applied to
certain individuals, tiny red spots called
petechiae appear on the arm below it.
- These spots are actually minute amounts of
blood that escaped from the capillaries
and come to the surface of the skin as a
result of platelet abnormalities or a defect
in the capillary wall or even might be that
the tourniquet is tight or is tied too tightly so
they do not indicate that the phlebotomist
has done anything wrong.
Seizures/convulsions
- These are to continue blood collection
immediately if a patient has a seizure or
goes into convulsions, hold pressure over
the side if possible, without limiting the
patient's movement and immediately notify
appropriate personnel to handle the
situation.
TRANSFORMERS 6
1 Liver Function Clinical Chemistry 2 (LAB)
LIVER FUNCTION o Serum is the preferred sample.

TOPIC OUTLINE: ● Fasting sample – lipemia increases bilirubin
o Fasting is required because lipemia would
increase bilirubin concentrations
1. Bilirubin
● Avoid hemolysis – decrease the reaction of
2. Urobilinogen in Urine and Feces
bilirubin
3. Enzymes
o Hemolysis should be avoided because it
4. Autoimmune markers
decreases the reaction of bilirubin with
5. Tests measuring hepatic synthetic ability
the reagent.
6. Tests measuring Nitrogen metabolism
● PROTECT SAMPLE FROM LIGHT
7. Laboratory Demonstration for Bilirubin
o Upon collection of the sample, we must
LIVER FUNCTION TESTS protect the sample from the light
because bilirubin is photosensitive. Once
● Bilirubinometry in Newborns
it is exposed to light, it would falsely
● Sample Collection and Precautions
decrease the bilirubin and the actual
● Bilirubin determination
sample.
● Urobilinogen in urine and feces
o If you notice in the picture, this is a
● Liver Enzymes
bilirubin test; it is not covered by light.
● Autoimmune markers
Usually, in more developed countries, it is
● Test measuring hepatic synthetic ability
an amber-colored container or here in
● Test measuring nitrogen metabolism
the Philippines, we will cover the syringe
BILIRUBINOMETRY (TRANSCUTANEOUS)
with either the paper, or the syringe and
● This is commonly used in newborns.
the test tube.
o While extracting, we must protect our

sample from light.
● If you look at the picture, there is a portable
device and it is noninvasive so because it is Bilirubin Determination
noninvasive, it means that there is no need to The bilirubin determination is based on the
●
collect blood samples from the newborn. Diazo reaction.
● This applicable for newborns because they do ● The Diazo reagent contains sulfanilic acid,
not have any food intake yet. Why? Because hydrochloric acid and sodium nitrite on
food intake may actually interfere with measuring bilirubin
Transcutaneous bilirubinometry. ● In performing bilirubin tests in the laboratory, it
● The interference from food are usually from is important to take note that there are 3
carotenoid compounds fractions of Bilirubin.
● PRINCIPLE: It measures reflected light from the o These 3 react in all laboratory analysis. They
skin using two wavelengths that provide a also make up the total bilirubin
numerical index based on spectral reflectance. Three (3) Fractions of Bilirubin
● For newer generations, they use 1. Unconjugated (Indirect) bilirubin
microspectrophotometers to determine optical o This does not readily react with the
density of bilirubin diazo reagent. It will only react to it
Clinical Significance in the presence of an accelerator.
● What is the clinical significance if there is Therefore, there must be an
increased bilirubin in newborns? It is the accelerator for the unconjugated
Jaundice of the Newborn. bilirubin
o It is caused by the inactive enzyme 2. Conjugated (Direct) bilirubin
uridyl diphosphate glucuronyl o It will directly or readily react with
transferase (UDPGT). the diazo reagent. Meaning, it does
o Unconjugated bilirubin is the type of not need the presence of an
bilirubin that is increased in this accelerator.
condition because the UDPGT enzyme is 3. Delta bilirubin
the one responsible for the conjugation o It is described as a conjugated
of bilirubin and since the newborn there bilirubin bound to an albumin.
is no UDPGT yet and it is not active, o Since it is a conjugated bilirubin, it
unconjugated bilirubin is the one that reacts similarly to the direct bilirubin,
increases. where there is an immediate
Specimen Collection and Storage reaction to the diazo reagent.
Why do we have delta bilirubin?
● Serum
TRANSFORMERS 1
This actually happens when there is too @ 600 nm and would then give the
much or increased conjugated bilirubin in concentration of CONJUGATED BILIRUBIN.
the body. That’s why it starts to attach to
albumin
Different Types of Bilirubin Determination
1. Malloy-Evelyn Method
2. Jendrassik-Grof Method
Malloy-Evelyn Procedure Second Aliquot
● The bilirubin in PATIENT’S SAMPLE (BILIRUBIN)
enmixed with an ACCELERATOR
(Caffeine-Benzoate, this would then solubilized
our UNCONJUGATED BILIRUBIN). And then it
Patient Sample would react with DIAZO REAGENT + ASCORBIC
ACID (would stop the reaction)+ALKALINE
TARTRATE SOLUTION (alkalinize the solution).
FINAL PRODUCT = BLUE PRODUCT (read
spectrophotometrically @600nm, and this
● May also be called the Evelyn-Malloy would give us the TOTAL BILIRUBIN. Because of
Procedure the addition of our ACCELERATOR
● It is based on the Diazo Reaction (Conjugated + Unconjugated = Total Bilirubin).
● In this procedure, we have the patient sample
which was protected from light and was aliquot
into 2.
o Aliquot means you get a portion from a
larger whole
● For the first aliquot in the sample is mixed with
the Diazo reagent and would yield an
Azobilirubin, which is now red ● At the end of performing both the procedure
spectrophotometrically at 560 nm. with both aliquots, we now have the
o With the addition of the Diazo reagent, conjugated and total bilirubin.
this would give us just the Direct
Bilirubin/Conjugated Bilirubin. To get the concentration of UNCONJUGATED
● For the second sample (Aliquot 2), it is mixed BILIRUBIN
with the Diazo reagent but with the addition of ● Subtract Conjugated bilirubin from total
the Methanol that would act as an accelerator bilirubin.
and it would also give us Azobilirubin which is
also be read at 560 nm.
o The addition of the accelerator,
Methanol, would then give us Total
Bilirubin. Reference Ranges
● How do we get the Unconjugated Bilirubin Taken from Bishop (2013). Reference ranges may
Concentration? vary among laboratories, this also depends on
o For the Unconjugated Bilirubin, we just what lab methods are used, but for this course, we
subtract conjugated bilirubin from total will use this table as our basis for the reference
bilirubin in the test. ranges for bilirubin
Jendrassik-Grof Method
● Similar to Malloy-Evelyn method.
● We have the sample and then we will
ALIQUOT INTO TWO.
First Aliquot
● The first one we have the addition and
reaction with the PATIENT’s SAMPLE Adults
(BILIRUBIN)+DIAZO REAGENT+ASCORBIC ACID Conjugated bilirubin 0.0-0.2 mg/dL(0-3 μmol/L)
(would then terminate the reaction) Unconjugated 0.2-0.8 mg/dL
+ALKALINE TARTRATE (alkalinize the solution). bilirubin (3-14 μmol/L)
Which will then make the AZOBILIRUBIN a Total bilirubin (TB) 0.2-1.0 mg/dL
more intense BLUE COLOR. This also lessens the (3-17 μmol/L)
interferences in the sample. FINAL PRODUCT = Premature infants
BLUE PRODUCT (read spectrophotometrically TB at 24 hours 1-6 mg/dL
(17-103 μmol/L)
TRANSFORMERS 2
TB at 48 hours 6-8 mg/dL

(103-137 μmol/L)
TB at 3-5 days 10-12 mg/dL
(171-205 μmol/L)
Full-term infants
(34-103 μmol/L)
(103-120 μmol/L)
TB at 3-5 days 4-6 mg/dL ● Determination of Total Bilirubin
(68-103 μmol/L)
Test Product (Bilirubin Jendrassik-Grof Test: DIASYS)
o We use the four reagents which are the

reagents 1,2,3, and 4.
An example of test kit that utilizes Jendrassik-Grof
o We have to prepare both the sample
method and this is by the brand DiaSys (Diagnostic
blank and the sample
Systems)
o For reagent 2 we add 50 µL followed by
● Required sample is SERUM
reagent 1, 200 µL then the reagent 3, 1000
o Serum specially for bilirubin assay must
µL and for the sample 200 µL then mix and
be protected from light, because it is
allow it to stand for 10 to 60 minutes at 15
photosensitive
to 25 degree Celsius then add reagent 4
● Test kit includes: 4 reagents
which is 1000 µL. Mix all of them together
o Reagent 1 – Sulfanilic acid solution
and after 5 to 30 minutes measure the
o Reagent 2 - Sodium nitrite solution
absorbance sample against the sample
o Reagent 3 – Accelerator (Caffeine
blank.
benzoate)
o Prepare for the sample blank (red square
o Reagent 4 – Alkaline Fehling’s solution
of the pic above), and read using
▪ This is to alkalinize the solution
Spectrophotometer at 578 nm.
● To get the Unconjugated / indirect bilirubin
just simply subtract the total bilirubin and
Conjugated / Direct bilirubin
o Total BILI – Conjugated
Reference Range
Bilirubin total
Neonates 24h <8.8 <150
mg/dL μmol/L
2 day
nd
1.3 – 11.3 22 – 193
mg/dL μmol/L
3RD day 0.7 – 12.7 12 – 217
● To measure the direct (conjugated) mg/dL μmol/L
bilirubin, we make use of reagents 1 & 2 4th-6th day 0.1 – 12.6 1.7 – 216
and a NaCl solution. mg/dL μmol/L
● For the sample preparation we add 50uL of Children >1 month 0.2 – 1.0 3.4 – 17
reagent 2, then we add 200uL of reagent 1, mg/dL μmol/L
to 2000uL of NaCl solution, with 200ul of the Adults 0.1 – 1.2 1.7 – 21
sample. mg/dL μmol/L
● We will then mix immediately and allow
Bilirubin direct
standing of the sample preparation at 15 to
Adults and children ≤ 0.2 3.4 μmol/L
25 deg C for exactly 5 minutes.
mg/dL
● Measure the absorbance against the sample
Each laboratory should check if the reference
blank, this is used with Spectrophotometer.
ranges are transferable to its own patient
● For the measurement of direct bilirubin by
population and determine own reference ranges if
DIASYS we make used of the wavelength Hg
necessary.
546 nm
CLINICAL SIGNIFICANCE
● The reagent blank it is similar to the
preparation of the sample blank just follow the Unconjugated ● Crigler – Najjar syndrome
steps (Red square of the pic below) Bilirubin ● Gilbert’s syndrome
● Jaundice of the Newborn
TRANSFORMERS 3
● Pre-hepatic jaundice semi-quantitative determination methods

● Hepatic jaundice for fecal urobilinogen and this is the same
(Metabolism) principle used in urine urobilinogen
o Abnormality in the determination which is also the ehrlich
metabolism of bilirubin reaction. So the test is done using an
Conjugated ● Dubin – Johnson aqueous extract of fresh feces and then
Bilirubin syndrome urobilin is present in the feces Is reduced
● Rotor syndrome urobilinogen by the alkaline ferrous
● Increased bilirubin hydroxide then the ehrlich reagent is then
in blood added and the end product would be a
● Post-hepatic jaundice red color.
● Due to Reference Range
obstructions or ● 75 to 275 Ehrlich unit per 100 grams of fresh
tumors which may feces
lead to increased ● 75 to 400 Ehrlich units per 24-hour specimen
bilirubin LIVER ENZYMES
● Hepatic (excretion) Hepatocellular (functional) vs Obstructive
● The conjugated (mechanical) Liver Disease
bilirubin is also
increased in blood - Injury to the liver would cause the release of
enzymes into the circulation of an individual. There
UROBILINOGEN IN URINE AND FECES is an increase in this enzymes in the blood which we
Can also aid in diagnosis of liver disease are testing in the CC lab. This means that there is a
UROBILINOGEN liver damage or liver injury. So the liver enzymes
● It is a colorless end product of bilirubin that are used would tell us to differentiate whether
metabolism which when oxidized it give us it is hepatocellular or functional liver disease from
urobilin. other obstructive or mechanical liver disease or
● Urobilin- gives the brown color of the stool hepatocellular or functional liver disease.
● Urobilinogen that is reabsorbed by the systemic Aminotransferases (AST and ALT)
circulation is secreted by the kidneys through ● Damaged or necrotic hepatocytes
the urine ● These are the enzymes that are most useful
● Increased urobilinogen in the urine = excessive in detecting hepatocellular damage to the
bilirubin being metabolized by the body liver.
o Hemolytic disease Alanine aminotransferase (ALT)
o Defective liver cell function ● Found mainly in the liver
● Absence/Decreased urobilinogen in urine or ● More specific Liver enzyme
stool ● If there are elevated AST and ALT levels, this
o Complete Biliary obstruction is due to leakage of these enzymes from the
- the conjugated bilirubin damaged or necrotic hepatocytes.
does not reach the Aspartate aminotransferase (ASP)
intestines, that is why no ● This is distributed in other tissues other than
urobilinogen detected in the liver such as the heart and skeletal
urine or stool samples. muscle.
Determination of Urine Urobilinogen Phosphatases (ALP)
● Sample: 2 – hour urine specimen ● Extrahepatic biliary obstruction
● Urobilinogen in urine + Ehrlich’s reagent → red ● Aid in diagnosing liver conditions
color (spectro) Alkaline Phosphatase (ALP)
● The laboratory method employs the ● It has high activity in the liver, bone,
principle of Ehrlich’s reaction where in intestine, kidney and placenta.
urobilinogen in urine reacts with ● For liver function, ALP is used in
p-dimethylaminobenzaldehyde (Ehrlich’s differentiating hepatobiliary disease from
reagent) to form a red color that is osteogenic bone diseases.
measured spectrophotometrically. ● In the liver, the enzyme is found in the bile
Reference Range canaliculi, making it a marker of
● 0.1 to 1.0 Ehrlich unit every 2 hours Extrahepatic biliary obstruction.
● 0.5 to 4.0 Ehrlich units per day ● An example is if there is a stone or tumor in
Determination of Fecal Urobilinogen the common bile duct.
● Visual inspection ● Very high concentrations of ALP are
● Semi-quantitative determination observed in Extrahepatic biliary obstruction
- Aqueous extract fresh while only slight to moderate are observed
feces in other hepatocellular disorders.
Phosphatases (5’ – nuleotidase)
● Usually, visual inspection is enough to ● Also called as 5-nt
detect decrease or absence of
urobilinogen but there are also
TRANSFORMERS 4
● Used to differentiate ALP elevations due to liver TESTS MEASURING HEPATIC SYNTHETIC ABILITY
problems from other conditions such as bone “All proteins are synthesized by the liver.”
diseases. ● The measurement of serum proteins are
■ This is because 5-nt has NO bone source. useful for quantifying the severity of hepatic
■ In liver diseases, both ALP and 5-nt are dysfunction
ELEVATED. ● Serum albumin
■ In primary bone diseases, only the ALP is o If it is decreased, it may be due to
ELEVATED, while the 5-nt is NORMAL or can decreased liver protein synthesis
only be SLIGHTLY INCREASED. ● Serum alpha globulins
Phosphatases (GGT) o If it is decreased, it may be due to
● Also known as gamma-glutamyl transferase chronic liver disease
● Differentiates ALP elevations ● Serum gamma globulins
● Can also aid to determine the cause of o Increased in acute and chronic liver
elevated ALP. disease, chronic active hepatitis
■ This is because high levels of GGT is also and post necrotic cirrhosis
seen in biliary obstruction. ▪ IgG and IgM in chronic
■ VERY HIGH ALP levels are observed in active hepatitis
extrahepatic biliary obstruction, so if ▪ IgM in biliary cirrhosis
partnered with HIGH GGT levels, it is highly ▪ IgA in alcoholic cirrhosis
suggestive of BILIARY OBSTRUCTION. ● Prothrombin Time
● Also INCREASED in chronic alcohol and drug o Measures clotting factors
ingestion. o Increased/Prolonged also in liver
Liver Enzymes disease
CONDITIO ALP 5-NT GTT ▪ This may be due to
N inadequate production of
NOT liver High Normal Normal clotting factors or disruption
disease of bile flow
Biliary High High High o Used as serial measurements to follow the
Obstructio progression of liver disease (tested in
n hematology section)
Chronic Normal / Normal High TEST MEASURING NITROGEN METABOLISM
alcohol / slightly ● Measures the ability of the liver to convert
drug high ammonia to urea.
intake ⮚ This would reflect the ability of the
● NOT liver disease liver to convert ammonia to urea.
o Could be other conditions that involve high ⮚ The liver is exclusively responsible for
ALP conc., but it doesn’t necessarily mean removing ammonia from the blood,
that it is a liver disease. This is because ALP is by converting it to urea.
also found in other organs of the body. ● Advanced liver disease and hepatic coma
⮚ Ammonia and other toxins increase
in the blood, and this can cause
AUTOIMMUNE MARKERS
hepatic coma.
● Method: ELISA
● Plasma ammonia (arterial blood- PREFERRED
● Primary Biliary Cirrhosis
SPECIMEN) EDTA, heparin, potassium
o the most common autoimmune liver
oxalate (on ice)
disease
⮚ On the other hand, if you make use
o it causes fibrosis or scarring of bile
of arterial blood, the following test
canaliculi
tube should contain EDTA, Lithium
o most patients with this condition is
heparin, Potassium oxalate (on ice).
positive for the anti-mitochondrial
So we must place the sample from
antibody (AMA)
extraction on ice, until we are able
▪ this antibody is directed to
to separate the cells from plasma.
the antigens found in the
Hence, it should remain on ice until
inner mitochondrial
separation of cells from plasma
membrane known as M2
occurs. In addition, we must also
▪ AMA with anti-M2 specificity
take extra precaution that hemolysis
is 100% specific for primary
would be avoided because this
biliary cirrhosis
would increase ammonia
● Primary Sclerosing Cholangitis
concentration.
o It is an autoimmune disease which is
associated with destruction of both
● Venipuncture- avoid tourniquet, fist
intrahepatic and extra hepatic bile
clenching and relaxing
ducts
⮚ Sometimes, if it’s hard to extract,
o >80% patients with this condition is
venous blood may be used, but it is
positive for anti-neutrophil
not really recommended. But, if you
cytoplasmic antibodies marker
are to use venous blood through
TRANSFORMERS 5
venipuncture, we have to avoid the

use of tourniquets and fist clenching
are to be avoided, and see to it that
the patient is relaxed.
● Avoid Hemolysis (Increased ammonia)
TESTS MEASURING NITROGEN METABOLISM

❖ The most common laboratory
determination of ammonia concentrations
is based on the following reaction:
NH4 + alpha keto glutaric acid + NADH -----------🡪

glutamic acid + NAD+
❖ To measure ammonia, one of the methods

that is used is by enzymatic assay, so we
make use of glutamate dehydrogenase. So,
this catalyzes the reaction of alpha keto
glutaric acid and ammonia to form
glutamate and then there is oxidation of
NADH to NAD, which is the indicator.
❖ Resulting decrease in absorbance at 340
nm is measured and proportional to
ammonia concentration.
AMMONIA
ADULT PLASMA 19-60 11-35
ug/dL umol/L 2. A serum sample
URINE, 24H 140- 1,500 10-107
mg N/d mmolN/d
CHILD (10 PLASMA 68-136 40-80
DAYS TO 2 ug/dL umol/L
YEARS
OLD)
(Bishop, 2013)
● What is the clinical significance of
increased ammonia in blood?
⮚ This my be suggested of Reye’s
syndrome, which is an acute 3. Total bilirubin working reagent
metabolic disorder of the liver
wherein there is fatty infiltration of
the liver which is common among
children and salicilates were used
for the viral infection.
LABORATORY DEMONSTRATION
Overview
● Bilirubin in serum can either be direct or
indirect. Both types can be distinguished
and quantitated through the Diazo 4. Direct bilirubin working reagent
Reaction.
● Direct bilirubin consists of conjugated and
water soluble derivatives that may react in
the absence of an accelerating reagent.
● On the other hand, indirect bilirubin consists
of unconjugated bilirubin bound to serum
albumin which only reacts in the presence
of an accelerator.
Materials and Equipment 5. Distilled water
6. Clean test tubes
1. Micropipette and disposable tips
7. Spectrophotometer
**always take note that bilirubin is a photosensitive

analyte. Specimen container should always be
TRANSFORMERS 6
covered with an opaque material such as carbon

paper. If a specimen is directly exposed to sunlight,
it may lose up to 50% of the total bilirubin level.
Procedure
1. Pipette 1mL of the direct bilirubin working
reagent to a test tube.
2. Mix 50 uL of the sample to the same tube

and allow it to stand at room temperature
for 5 minutes.
3. Pipette 1mL of the total bilirubin working

reagent to another test tube
4. Mix 50 uL of the sample to the same tube

and allow it to stand at room temperature
for 5 minutes.
5. After incubation, lid the tube in the

spectrophotometer and measure the
absorbance at a wavelength of 555 nm.
TRANSFORMERS 7
3 Clinically Significant Enzymes Clinical Chemistry 2 (LAB)
CLINICALLY SIGNIFICANT ENZYMES ▪ All of them are related to the bones

Topic Outline: • ALP is also increased to other hepatic
Phosphatases conditions or diseases that involve the liver;
Transaminases/Transferases these include the:
Digestive enzymes o Obstructive jaundice
Other major enzymes o Hepatitis
Other minor enzymes o Cirrhosis
PHOSPHATASES • Aside from these conditions, it also has a
Alkaline Phosphatases special mention that is Sprue
Acid Phosphatases o It is an immune reaction to gluten
o Hence, if the patient has sprue, they have
ALKALINE PHOSPHATASE
an increased ALP level.
• ALP (3.1.3.1)
• Hypophosphatasia
o 3.1.3.1 – E.C. number or Enzyme
o It is an inborn deficiency, but it decreases
Commission number associated with ALP
the ALP levels
• “Alkaline Orthophosphoric Monoester
• ALP also decreases in some cases of
Phosphohydrolase” – another name of ALP
malnutrition
• Major Tissue Sources:
MAJOR ISOENZYMES
o Liver
o Bone We have 4 major isoenzymes of ALP. If you have to
o Placenta focus on one part or one aspect of ALP, these
o Renal Organs (sp. Kidneys) would involve the major isoenzymes because ALP is
• It is considered as the non-specific enzyme a non-specific enzyme capable of reacting to the
because it is capable of reacting to various various substrates. ALP has isoenzymes which could
substrates help us determine what is the source or specific/
o Enzymes have different specificity, the ALP pathological increase that has caused the
is a non-specific enzyme because based elevations of the ALP.
on the major tissue sources, it can be seen REMARKS
on the bones, placenta, and renal organs LIVER Obstructive Jaundice; increased
o Hence, even though there is an increase in fraction
the ALP, we have to utilize other laboratory BONE Immunohistochemical techniques
tools or analytes that we can use in order to Paget’s Disease / Osteitis Deformans
determine what causes the increase of the PLACENTAL Normal Pregnancy; 16-20th Week
ALP INTESTINAL Blood Group, Secretor Status Type B
• The primary reaction involved in ALP is that it is & O: Fatty Meals
a non-specific enzyme that induces or ALP ISOENZYMES
facilitates the Hydrolysis of organic phosphate LIVER • Often increased in cases of
esters to produce alcohol and phosphate ions ISOENZYME obstructive jaundice
at an ALKALINE PH • Is inhibited by levamisole and
o It is the reason why it is named Alkaline urea
Phosphatase because it occurs at an BONE • Commonly measured using
ALKALINE pH ISOENZYME immunohistochemical
Reference Range techniques
Male 90-190 u/L • Highest elevation of this enzyme
Female 85-165 u/L is observed in Paget’s
Clinical and Diagnostic Significance disease/Osteitis deformans
• Bone isoenzymes elevated in children • Inhibited by urea and levamisole
o ALP (bone isoenzyme) is high in children PLACENTAL • Commonly observed in
because bone isoenzyme increases due to pregnant women
osteoblastic activity. • Placental ALP activity can be
o Normally, children or growing children, their detected during the 16th-20th
levels of bone isoenzyme for ALP are usually week of gestation
increased during their growth period • Is inhibited by the phenylalanine
o Aside from children, ALP is also increased reagent
among the geriatric population, those INTESTINAL • Is affected by the blood group
individuals belonging to the age groups of and secretor status
50 and above. - In the blood bank, we have
• Increased ALP are also observed on the the different blood group
following conditions: systems (Type ABO)
o Osteitis deformans - If you are a secretor, your
o Osteoblastic bone tumors ABO antigens can also be
o Osteomalacia found in other body fluids
o Rickets (saliva)
o Bone cancer - If you are secretor positive,
TRANSFORMERS 1
your blood type can also be

determined using your saliva
• The presence of intestinal ALP in
the serum is dependent on the
blood group and secretor status
of the individual
o For instance, intestinal ALP of
individuals with blood type B
& O becomes elevated upon
the consumption of fatty • BLUE = GEL
meal • BOXES = MICROWELLS
Inhibited by phenylalanine reagent • Imagine electrophoresis gel as a flat layer of
ISOENZYME DIFFERENTIATION gelatin. On the left portion of the gel, near the
• As mentioned, ALP isoenzymes are not normally cathode, are the microwells (boxes).
seen • Microwells are the location where the MT will
• When ALP isoenzymes are elevated, it doesn’t embed the sample using micropipette
have that much clinical significance, as the
increase in ALP may be a result from a variety
of isoenzymes
• Therefore, differentiation of isoenzymes should
be done
• Common techniques used for isoenzyme
differentiation are:
o Electrophoresis
o Heat fractionation test
o Urea incubation test • MT will collect the sample and embed the
ELECTROPHORESIS samples into the microwells, in a way that it will
• Distinguish the ALP isoenzymes qualitatively enter the microwells BUT WILL NOT pass through
• Differentiates the isoenzymes of ALP the gel underneath (para hindi mabutas yung
qualitatively gel sa ilalim). Parang pag nagveni, papasok
Parts and components of Electrophoresis machine: yung needle sa vein, pero hindi tatagos sa vein
1. Anode (red) – positively charged electrode; (hindi magthrough and through)
is usually color red
2. Cathode (black) – negatively charged
electrode; often color-coded electrode
• Next, the electrophoresis machine will be

turned on, and the electric current will pass
3. Electrophoresis gel – there are 2 gels that
through from the cathode up until the anode
are commonly used for electrophoresis gel
• The difference in the electrical charge and
➢ Polyacrylamide gel
molecular weight of the ALP isoenzymes will
➢ Agarose gel
cause them to migrate
4. Microwells – boxes where the samples are
• Clinical significance: it would allow us to see
placed
the different isoenzymes
• For electrolytes, the cations are the positively
charged ions, which migrates to the cathode
(negatively charged)
• Anions are negatively charged ions which
migrates to the anode (positively charged)
Virtual demonstration
• Intestinal ALP – most cathodal

• Placental ALP – second most cathodal
• Bone ALP – second most anodal
TRANSFORMERS 2
• Liver ALP – is the most anodal in MAJOR isoenzymes

Clinical significance of electrophoresis (Alp • Reagan: most heat stable in
isoenzymes) MINOR isoenzymes
Inhibited by Phenylalanine reagent
Inactivation: Heat at 65 C for 30
minutes
Both Reagan & Nagao are referred

as carcino-placental alkaline
phosphatase
they are called as such since they
are similar to the placental ACP
• Most cathodal would be the intestinal ALP, however, they are often observed
followed by placental, bone, and liver ALP. among cancer patients
• Liver: most anodal ALP isoenzyme NAGAO “Pancreas ALP”
• Bone: 2nd most anodal ALP ALP Variant of Regan
• Placental: least cathodal ALP ALP
STABLE - It is often observed in
Heat at 56 C for 15 minutes adenocarcinoma of the
• to inactivate ALP pancreas, adenocarcinoma
• 90% of total ALP is already of the bile duct, and
inactivated if heating is done adenocarcinoma of the
Bone: 50% | Liver: 100% pleural cancer
• In heat fractionation test, ALP - It is inhibited by the
is measured before and after phenylalanine reagent. Aside
the test from this, it is also inhibited by
• If residual ALP is <20% = bone L-leucine
isoenzyme elevations SPECIMEN CONSIDERATION
• If residual ALP is >20% = liver - Hemolysis, Fatty Meals, Low temperatures
Heat isoenzyme elevations (<4°C) = FALSE ELEVATION/INCREASE OF
Fractionatio • The residual ALP activity after ALP
n Test heating is recorded - Room Temperature = DECREASE IN ALP due
Heat Denaturation Test to the loss of CO2
• performed for placental ALP - Inhibitor: P Activator: Mg, Mn
• Same procedure but instead - Specimen of choice: Serum, Plasma
of heating it at 56 C, it is (heparinized)
heated at 60 C for 10 OWERS AND MCCOMB METHOD FOR ALP
minutes DETERMINATION
Expected results of Heat Reference method in determining ALP;
Fractionation Test Szasz Modification IFCC recommended method
• Placental: most heat STABLE Continuous monitoring technique
• Bone: most heat LABILE pH: 10.15 Wavelength: 405nm
•
Placental→Intestinal→Liver→Bone *IFCC- International Federation of Clinical
(heat stable to heat labile) Chemistry and Laboratory Medicine
Urea AKA Urea inhibition test
Incubation Used with electrophoresis Para-nitrophenylphosphate, in the presence of
Test • since results of ALP, will be hydrolyzed to para-nitrophenol and
electrophoresis are in a phosphate ion p-nitrophenylphosphate  ALP → p-
qualitative manner nitrophenol + phosphate ion
2-3M Urea= reagent SUMMARY OF METHODS FOR ALP DETERMINATION
Bone and Liver ALP
METHOD SUBSTRATE
• inactivated by the test
Bodansky
(significance of test)
Shinowara
MINOR ISOENZYMES
Jones b-glycerophosphate
REMARKS
Reinhart
REGAN ALP “Lung ALP”
King and Armstrong Phenylphosphate
Migrates with Bone ALP
• during electrophoresis Bessey, Lowry, Brock p-nitrophenylphosphate
Most Heat Stable ALP Bowers and McComb
• Reagan ALP is more heat Huggins and Talalay Phenolphthalein
stable in comparison to diphosphate
placental ALP Moss α-naphtholphosphate
• Placental: most heat stable Klein, Babson and Buffered Phenolphthalein
TRANSFORMERS 3
Read phosphate diagnosis, and management of Prostatic

END PRODUCTS Carcinoma (Prostate Cancer) alongside Prostate
Bodansky Specific Antigen.
Shinowara • Prostatic Carcinoma: Acid phosphatase
Jones (ACP) & Prostate Specific Antigen (PSA)
can be utilized to monitor the recurrence of
Reinhart
prostate cancer. However, PSA is more
King and Armstrong
sensitive in comparison to Prostatic ACP
Bessey, Lowry, Brock
specifically, if we are determining the stage
Bowers and McComb of prostate cancer.
Huggins and Talalay Phenolphthalein Red • Osteoporosis, Multiple Myeloma, Paget’s
Moss α-naphthol Disease, Gaucher’s Disease, Niemann Pick
Klein, Babson and Free Phenolphthalein Disease, Thrombocytopenia
Read o Increased levels of ACP
REMARKS *Decreased ACP levels are usually not significant.
Bodansky SPX CONSIDERATION FOR ACP DETERMINATION
Shinowara o Hemolysis
Jones o False increase in ACP values
Reinhart *Kapag merong hemolysis or hemolyzed sample,
King and Armstrong the presence of hemoglobin is released sa sample.
Bessey, Lowry, Brock The presence of hemoglobin and bilirubin interferes
Bowers and McComb with the chemical reaction or enzymatic reaction
when we measure ACP levels.
Huggins and Talalay Utilizes Phenolphthalein
• Fluoride Anticoagulants
diphosphate as its
o False decrease in ACP values
substrate and the end
o Should be avoided
product would be
Phenolphthalein Red.
• Serum ACP decreases if left at room
Moss Utilizes α-
temperature for more than 1-2 hours.
naphtholphosphate as its
• Citrate Buffered
substrate and the end
o The anticoagulant of choice for
product would be α-
ACP determination.
naphthol.
o We maintain it at 6.2 – 6.6 pH level
Klein, Babson and Utilizes Buffered • Specimen of choice is plasma.
Read Phenolphthalein • Freezing temperature
phosphate as its substrate *If there is a delay in the sample measurement,
and the end product ano yung gagawin ni Medical Technologist? We
would be Free store it at a freezing temperature specifically at -
Phenolphthalein 20°C. If we’re going to measure it, we’re going to
thaw the sample. However, kapag repeated
ACID PHOSPHATASE 3.1.3.2 (ACP) freezing and thawing of sample, this would result to
• Other name is “Acid Orthophosphoric enzyme denaturation which will significantly
Monoester Phosphorylase” decrease the values of enzymes.
• Major Tissue Sources: Prostate, Semen, RBC, COMMONLY USED SUBSTRATES FOR ACP
PLT, Liver, Spleen, Kidney, and Bone Marrow DETERMINATION
(BM) REMARKS
o All of these release ACP. However,
PARA NITROPHENYL • Yellow color in Alkaline
yung pinaka mataas is from the PHOSPHATE Solution
prostate source. That’s why your • pH: 10.0
ACP is useful for Forensic Chemistry • Primary disadvantage:
or Forensic Science. Sample size around 0.1
• Forensic Chemistry: In cases of rape victims,
mL
we can utilize Vaginal Washings in order to • Incubation time: 30
determine Acid Phosphatase minutes
REFERENCE RANGE • Substrate of Choice or
0.5-1.9 U/L THYMOLPHTHALEIN the measurement of Acid
CLINICAL AND DIAGNOSTIC SIGNIFICANCE MONOPHOSPHATE Phosphatases
• Vaginal Washings: >50 IU/L – indicates the o Primarily detects the
presence of seminal fluid in the sample. prostatic ACP, so
o How long can we detect ACP yung mga prostatic
activity on vaginal washings enzymes ng ALP
samples? Up to 4 days (max.) natin: bones, liver,
*Aside from vaginal washings in forensic chemistry, intestinal, placental.
we can also utilize ACP for the identification, Pagdating naman sa
TRANSFORMERS 4
ACP which we will Rietz and Guibalt This method is a

further dicuss later, fluorescent/ fluorometric
we have prostatic method, since it utilizes
ACPand non- fluorometry as its
prostatic ACP. primary principle, Rietz
o It ideal because if and Guibalt method
detects the prostatic offers increase
ACP. sensitivity of our assay.
• Reaction is carried out in ACP ISOENZYME TECHNIQUES
a citrate buffer at pH 6.0, REMARKS
for 30 minutes and read Chemical Inhibition • Bulky in nature,
at 410-450 nm. means
• Interference: hemoglobin complicated
and bilirubin procedure
o There will be an • It is also non-specific
interference in the Electrophoresis • Not easily
measurement of reproduced
Acid Phosphatase o If you remember in
because it also ALP, we have lots
measured at 410- of procedure,
450nm. which involves
ALPHA- • Continuous monitoring highly trained
NAPHTHYLPHOSPH o Method of choice individuals in order
ATE if we are to perform
employing or electrophoresis.
utilizing a • The materials for
Continuous electrophoresis are
Monitoring. often too
SUMMARY OF METHODS FOR ACP DETERMINATION complicated for
METHOD SUBSTRATE routine clinical use.
Gutman and Gutman Phenylphosphate Recommended
King Armstrong Phenylphosphate Immunoassay approach for ACP
Shinowara p-nitrophenylphosphate Determination
Hudson p-nitrophenylphosphate TARTRATE INHIBITION
Babson, Read, and a-naphthylphosphate TRAP: Tartrate Resistant Acid Phosphatase
Phillips Prostatic ACP: Non-TRAP
Roy and Hillman Thymolphthalein RBC ACP: TRAP
monophosphate Cu (cupric ion)+ Formaldehyde: Inhibits RBC and
(react with prostatic ACP
ACP) RBC ACP PROSTATIC ACP
Rietz and Guibalt 4-methylumbelliferylone Copper Inhibited Not Inhibited
phosphate Tartrate Ion Not Inhibited Inhibited
END PRODUCTS Prostatic ACP = Total ACP ― TRAP
Gutman and Gutman Inorganic phosphate
King Armstrong Inorganic phosphate Example 1:
Shinowara p-nitrophenol Total ACP: 20
Hudson p-nitrophenol TRAP: 10
20 - 10 = 10
Babson, Read, and a-naphthol
Phillips Example 2:
Total ACP: 15
Roy and Hillman Free thymolphthalein
TRAP: 5
Rietz and Guibalt Fluorescence
15 - 5 = 10
REMARKS Example 3:
Gutman and Gutman Nonspecific methods Prostatic ACP: 7
King Armstrong Nonspecific methods TRAP: 7
Shinowara Total ACP = 7? (diba 14)
Hudson
Babson, Read, and A complicated
Phillips procedure and it has Transaminase/Transferases
less sensitivity ASPARTATE AMINOTRANSFERASE 2.6.1.1 (AST)
compared to the other Old name: Serum Glutamic Oxaloacetic
methods. Transaminase
Roy and Hillman Major Tissue Sources:
TRANSFORMERS 5
Cardiac muscle, Cardiac tissue, Liver, Skeletal ▪ It will eventually decrease or

Muscles normalize after 120 hours or 5 days.
Minor Tissue Sources:
Kidney, Pancreas, RBC Condition wherein makikita yung pinakamataas na
elevations ng bone isoenzyme ng ALT
▪ That would be your Paget’s disease (other
SGOT: Serum Glutamic Oxaloacetic Transaminase name: Ostitis deformans)
o Pag dating naman kay AST, the highest
Transfer of amino group in aspartic acid for an a- elevations of AST are observed in Acute
keto group in a-ketoglutaric acid forming hepatitis.
oxaloacetate and glutamate o Tumataas din po yung AST in conditions of
hepatocellular disorders and skeletal
o Your AST is involved in the transfer of amino muscle disorders.
group in aspartic acid. So, yung amino Kapag ang diseases involves hepatocellular and
group sa aspartic acid, pinapalitan nya ito skeletal muscles, tumataas yung AST levels natin.
ng a-keto group galling kay a-keto glutaric
acid and it forms oxaloacetate & AST levels are also released to a certain degree in
Glutamate. chronic disorders such as a chronic disorder of the
liver. So kapag meron na tayong mahahabang
Products: Oxaloacetate & Glutamate diseases associated with the liver and it also results
to increase in the AST levels.
Major Isoenzymes:
Cytoplasmic AST and Mitochondrial AST Other increase pa ng AST,
AMI
So, ireview natin yung Isoenzymes natin so far. Hepatocellular disorders
▪ ALT isoenzyme: Disorders involving skeletal muscles.
Bone, liver, intestinal, placenta,
Minor isoenzyme: we have ligand, we have Uremia: Decrease AST levels
nagao.
▪ For acid phosphatase, ano yung Specimen consideration:
isoenzymes natin, prostatic ACP, non- o Hemolysis
prostatic ACP. o Icteric- mataas yung bilirubin; it appears
▪ For AST naman, our enzymes are yellow
Cytoplasmic AST and Mitochondrial AST. o Lipemic- mataas yung lipid contents ng
Now, the Cytoplasmic AST is the predominant samples natin. It appears milky.
isoenzyme. Around 80% cytoplasmic activity pero
only 20% yung mitochondrial AST. o Hemolysis, Icteric, Lipemic = falsely increase
levels of AST specifically hemolysis.
Cytoplasmic AST
- 80% cytoplasmic acitvity o Kapag gumamit ka ng hemolyzed sample
- Makikita sa serum. for AST determination, it may increase as
- Cytoplasmic activity na nakikita sa serum much as 10x the base line value.
REFERENCE RANGE
5-37 U/L o Aside from this, the specimen of choice for
<55 U/L AST is serum or plasma. Either of the two
can be utilized for AST determination.
The reference values for AST varies from different METHODS FOR DETERMINING AST LEVELS
references. So meron tayong kay Bishop, and other KARMEN method
references. Some books indicate that normal • Coupled Enzymatic reaction: NADH, MD
reference value is around 5-37 U/L. While some • Gumagamit tayo ng 2 enzymes: NADH
references indicate that a value of <55 U/L of AST is (Nicatonamide Adenine Dinucleotide and
already normal. Malate Dehydrogenase
CLINICAL AND DIAGNOSTIC SIGNIFICANCE • Your MD or malate dehydrogenase is your
Myocardial Infarction (6-8, 24, 120 hours) indicator enzyme. We utilized it for the
Hepatocellular Disorders, Skeletal Muscle conversion of oxaloacetate to malate.
Chronic Disorders of Liver
Muscular Dystrophy, Acute Pancreatitis Principle:
Uremia We determine the change in the absorbance.
Specifically yung pagbaba ng degree of
Myocardial Infarction absorbance and we set our monochromators at
o AMI (acute myocardial infarction) 340nm at a pH of slightly alkaline of 7.5
o Myocardial infarction pattern is as follows: Remarks
▪ It increases/ rises at 6-8 hours. Coupled Enzymatic Reaction: NADH, MD
▪ Magppeak sya sa 24 hours. Change in Absorbance at 340nm
TRANSFORMERS 6
pH 7.5 Decreased ALT levels: Not clinically significant

Aspartate + a-ketoglutarate  AST → Oxaloacetate METHODS FOR DETERMINING ALT LEVELS
+ Glutamate Oxaloacetate + NADH + H+  MD → REMARKS
Malate + NAD+ Coupled - The enzyme used here are
Principle: Enzyme similar with those of Karmen
- Aspartate + a-ketoglutarate in the method
presence of AST will form Oxaloacetate - NADH is used but the indicator
and Glutamate. enzyme is different which is the
- The oxaloacetate will be used in the Lactate dehydrogenase (LD)
second part of reaction, wherein it will - The change in Absorbance is
react with NADH, in the presence of malate being monitored specifically,
dehydrogenase this will result to the like in Karmen method, the
formation of your malate and NAD+ DECREASE in absorbance is
Reitman and Frankel Method being monitored.
- Also known as DNPH method - Coupled Enzymatic Reaction:
- Primary reaction: Reaction with 2,4-DNPH NADH, LD
(505nm) - Change in Absorbance is
- 2,4- DNPH stands for 2,4 measured at 340nm at pH 7.5
Dinitrophenylhydrazine L-
- This reaction is being performed at a Principle:
wavelength of 505nm. - L- alanine in the presence of
Ketoacids + 2,4-DNPH = Ketoacidhydrazones + a-ketoglutarate and ALT will
NaOH (blue) form oxaloacetate and
- The reaction of the DNPH method produces Glutamate.
an intense blue color, which is brought - The oxaloacetate will then
about NaOH. react with the Pyruvate, NADH
The primary disadvantage of this method is it lacks and H+, with the presence of,
Specificity. LD, it will form Lactate and
Diazonium Salt Coupling NAD+
- Coupling with Diazonium Salt Alanine + a-ketoglutarate  ALT →
- It utilizes Diazonium Salt wherein it will be Pyruvate + Glutamate Pyruvate +
coupled together with the target enzyme, NADH + H+  LD → Lactate +
AST. NAD+
Ketoacid + Diazo Compound = Diazonium Diazonium • This reaction similar to those of
Derivative Salt AST, it involves the coupling
The end product of this reaction is Diazonium Coupling with Diazonium Salt, so as in in
derivatives the reaction below, it would
Babson Method be ketoacid plus Diazo
- Violet Color compound would for a
ALANINE AMINOTRANSFERASE 2.6.1.2 (ALT) Diazonium Derivative.
Major Tissue Sources: Liver
- More liver specific Coupling with diazonium salts
SGPT: Serum Glutamic Pyruvic Transaminase Ketoacid + Diazo Compound →
- Transfer of amino group in aspartic acid for Diazonium Derivative
an a-keto group in a-ketoglutaric acid CORRELATION OF ALT AND AST
forming oxaloacetate and glutamate • Physicians usually use these tests in
- Similar with AST, the difference is that the conjunction.
products of ALT are Pyruvate and • The clinical significance of ALT and AST are
Glutamate. often increased when compared to one
- Remember: ALT is beneficial because it another.
doesn’t have isoenzymes. • When transaminases are mentioned, they
- Other enzymes w/o isoenzymes: ALT, are referring to AST and ALT
Lipase, GGT (Gamma glutamyl Transferase), Coenzyme: Pyridoxal phosphate (Vitamin B6)
5’ nucleosidase Aminotransferases are present in human plasma,
Note: AST= SGOT while AST=SGPT, OTPT= both SGOT and SGPT
AST is more liver sensitive kasi mas unang tumataas si AST
bile, CSF, and saliva
kapag liver diseases pero ang ALT ay mas specific sa liver. Children: ALT __<__ AST Adult: ALT __>___ AST
REFERENCE VALUES: o Children have greater AST than that of
6-37 U/L 5-35 U/L adults, while Adults have higher ALT
CLINICAL AND DIAGNOSTIC SIGNIFICANCE compared to that of children.
Increased ALT levels: Hepatic parenchymal FACTORS INVOLVED IN THE MEASUREMENT OF ALT
disease, viral hepatitis, obstructive jaundice, Reye’s AND AST
syndrome, heart failure, AMI, Infectious Both enzymes are affected by Hemolysis, but not
mononucleosis, muscular dystrophy as much as LDH
TRANSFORMERS 7
Both enzymes may be elevated in Heparin amylase is smallest, normal amount of

Therapy, How high? 3x the normal value. amylase is usually present in the urine.
Both enzymes are significantly lower in Renal failure • 15% higher value in women
More significant diurnal variation in ALT than AST REFERENCE VALUES
ALT 60-180 SU/dL (Somogyi units) 95-290 U/L
Major Organ Liver ISOENZYMES
Affected • P3 isoenzyme is the most predominant
Substrate Alanine a-ketoglutaric acid pancreatic amylase variant.
End Products Glutamic acid + Pyruvic o most predominant out of all pancreatic
acid isoenzymes of amylase.
Color Developer 2,4-DNPH o first to increase in the onset of acute
Color Intensifier 0.4N NaOH pancreatitis.
AST • Pancreatic amylase – most predominant in
Major Organ Heart general.
Affected SALIVARY TYPE PANCREATIC
Substrate Aspartic a-ketoglutaric acid TYPE
End Products Glutamic Acid + OTHER NAME S type or P type or
Oxaloacetic acid Ptyalin Amylopsin
Color Developer 2,4- DNPH MIGRATION Anodal Cathodal
Color Intensifier 0.4N NaOH PATHOLOGIC Parotitis Pancreatitis
DE RITIS RATIO INCREASE
SPECIMEN CONSIDERATION
Ratio between ALT and AST; Diagnostic tools; used
to determine the cause of liver damage • Sample Dilution with NaCl to prevent
Severe viral hepatitis or toxic hepatitis patients may inactivation of amylase specially when the
produce 20x the normal limits sample has high amylase level.
Highest elevations: Acute hepatitis • Endogenous inhibitors are present in serum that
Moderate elevation: Chronic hepatitis, Hepatic can interfere with amylase determination. This
cancer and Infectious Mononucleosis (IM) includes wheat germ lectin and triglycerides.
Slight increased: hepatic cirrhosis, alcoholic • Presence of Citrate, EDTA, Oxalate as
hepatitis, and obstructive jaundice anticoagulant may interfere with the chemical
or enzymatic reaction.
DE RITIS RATIO (ALT/AST) REMARKS
• Primary Substrate in all methods in the
>2 Alcoholic Hepatitis,
measurement of amylase is: Starch
Hepatocellular Injury,
METHODS FOR DETERMINING AMYLASE LEVELS
Hepatocellular
carcinoma PRINICPLE
1-2 Acute Hepatitis, Cirrhosis Measures the amount of
reducing sugar that are
<1 Viral Hepatitis
SACCHAROGENIC produced from the
hydrolyzed starch during
chemical reaction.
DIGESTIVE ENZYMES
AMYLOCLASTIC • Measures the
AMYLASE 3.2.1.1 (AMS)
decrease in
• Enzyme Commission number of 3.2.1.1 substrate
• A.K.A “α-1,4-Glucan-4-Glucohydrolase” concentration
• Major Tissue Sources: Pancreas and Salivary (degradation of
cells/glands starch).
o Salivary cells specifically they release • The decrease in
amylase specifically the acinar cells. substrate
• Minor Tissue Sources: Adipocytes (fat concentration is
containing cells), fallopian tubes, small proportional to the
intestines, skeletal muscles. levels of amylase. It
• Primary purpose of amylase is for the also used colored
breakdown of starch starch.
• Amylase is also known as the smallest enzyme • Decrease in colored
in terms of size starch = increase in
o It is small enzyme in a way that the amylase levels.
presence of amylase or certain amount
CHROMOGENIC • Measures increase in
of amylase present in the urine is usually
color intensity
normal.
• Utilize soluble dye
o Other enzymes often increase wherein they
substance which will
have high molecular weight and sizes, in
be used as primary
a way that it is already pathologic when
substrate.
this enzyme seen in the urine. Since
• Increase in color
TRANSFORMERS 8
intensity = increase colored starch. Ibig

in amylase levels sabihin, nagagamit si
COUPLED-ENZYME Uses α-glucosidase and substrate natin.
hexokinase • In amylosclastic, we
REMARKS measure the amount
Saccharogenic • Reference Method of the SUBSTRATE,
• Expressed in Somogyi specifically ung
Principle: Measures Units (SU; mg of substrate na nawala,
reducing sugar Glucose processed ung COLORED
produced from in 30 minutes) substrate na
hydrolyzed starch • Amount of Reduced NAWALA.
Sugar = Amount of • The lesser the
Amylase Activity substrate, which is
• Measure the amount the colored starch,
of reducing sugars results to an increase
formed in your amylase.
• To simplify, we have • Pag mas mababa
our enzyme which is ung substrate natin,
the amylase, and the mas mataas ung
substrate which is the amylase. Bakit mas
starch. So amylase mababa? Ibig
plus starch, when it sabihin nagamit ung
undergoes hydrolysis starch ng amylase.
will result to the So kung nagamit
formation of ung starch, ibig
reducing sugars. sabihin mataas ung
• So, we measure the amylase.
amount of products • The DECREASE in the
which is your amount of colored
reducing sugars. starch, or colored
So how do we interpret substrate, is equal to
the results? Pag mas the INCREASE in the
mataas si PRODUCT, amylase
mas mataas si AMYLASE. concentration.
Mas mataas si reducing • Time of
sugars, mas mataas si Decolorization =
amylase Amount of Amylase
Activity
Chromogenic • In chromogenic,
tandaan natin ung
Amyloclastic • Determine the Principle: main formula natin,
degradation of Measures increase in enzyme + substrate =
Principle: starch (which is the color intensity product. But as
Measures the decrease substrate) mentioned a while
in substrate • In the amyloclastic ago, we utilize a
concentration method, the soluble dye substrate
substrate that we use solution, in this case,
is COLORED starch. the substrate will
Kapag dumadami react together with
ung reducing sugars, another dye, this
bumababa ung forms an enzyme-
TRANSFORMERS 9
substrate complex amylase, result into

wherein the product maltotriose and
formed will have a maltose. Then the
color too. maltotriose will bind
• Kung kanina in the together with
amyloclastic, ung maltose, in the
substrate lang ung presence of alpha-
colored, in glucosidase will yield
chromogenic, ung glucose (5-Glucose).
mismong product So the end product
ung magkakakulay, of the initial/
so since PRIMARY reaction
magkakaroon ng would be glucose.
kulay ung product • The value of glucose
natin. will then be
• An INCREASE in the measured through
value of your routine glucose
products, specifically methods. The
your colored reference method
products, will result to for glucose
an INCREASE level of determination is
amylase. glucose hexokinase.
• So since mas marami • So gagamitin natin
ung naform na dye, dito si hexokinase. So
tapos ung dye again in the initial
nagrerect lang siya part of the reaction,
sa enzyme and we will measure
substrate, ibig kung gaano karami
sabihin pag mas si amylase by
matas ung dye na determining the
nameasure natin, glucose.
mas mataas ung • In the second part of
levels ng amylase the reaction we use
natin. the HEXOKINASE
Amount of Starch Dye METHOD, wherein si
destroyed = Intensity of glucose, will bind to
Color = Amount of ATP, in the presence
Amylase Activity of hexokinase, will
yield 5-Gluc-6Phos
and 5 NAD, then we
will measure here
ung amount ng
glucose levels natin,
so nalaman na natin
kung gaano karami
etong glucose na
ito, so what we will
determine here is
that, mas maraming
glucose, mas
• We utilize amylase mataas si amylase.
and alpha- So DIRECT ung
glucosidase, and the proportion nila
assay being used is a • So kapag couple
CONTINOUS enzyme sa amylase,
Coupled enzyme MONITORING ASSAY. una nating gagawin
Since coupled is we will determine
(Coupled enzymatic enzyme tayo, the amount of
reaction) gumagamit tayo ng amylase through the
TWO methods. measurement of
Principle: • First is the AMYLASE your glucose, und
Uses a-glucosidase and METHOD. Wherein second part naman,
hexokinase maltopentose, in the iqu-quantify natin
presence of ung glucose na
TRANSFORMERS 10
naproduce from • Earliest marker to increase during

amylase. pancreatic inflammation (often increase 2-
The INCREASE in the 12 hours after the onset of pancreatitis,
amount of glucose peaks after 24 hours and, decrease after
formed is equal to the 72-120 hours or 3 to 5 days)
INCREASE of the • If a patient has acute pancreatitis in 2 hours
amount of amylase amylase increases
• Increased AMS blood levels = AMS urinary
secretion
o It is normal to see subtle amount of
amylase in the urine since it is a
small enzyme capable of passing
through kidneys, however if there is
an increase blood amylase level
there will also increase level of
amylase in the urine.
OTHER METHODS • During acute pancreatitis the A:C Ratio is 4-
PRINCIPLE 15% MACROAMYLASEMIA; it increases
beyond the high base line value
Electrophoresis 6 Bands produced
(lumalagpas sya ng 1-5%)
o The first 3 bands include:
• MACROAMYLASEMIA
S-TYPE or salivary
o Macroenzymes – enzymes bound to
o The last 3 bands include:
an immunoglobulin. Example is the
P-TYPE or pancreatic
MACROAMYLASE is an amylase
Inhibition Test Triticum vulgaris (a wheat
bound to an IgA. If amylase bind
germ lectin) will inhibit the S-
with IgA it can no longer pass to the
type amylase
renal filters of our kidneys.
Monoclonal An immunological and o Elevated Serum AMS = Low urine
Antibodies serological method AMS; Increased in amylase level in
Utilization of monoclonal
blood but not in urine because it
antibodies that targets
can’t pass through the kidneys it
specific isoenzymes bound to antibodies.
Pwede tayong gumamit ng o AMS bounded to an Ab
antibodies that are directed • Increase in AMS level is often observed in
for s-type or those that are
Renal Failure and Parotitis (inflammation of
directed for p-type. This is
parotid gland may be due to mumps;
similar sa paggamit ng
mumps especially for male are at risk of
antisera, which are used for infertility)
blood typing (anti-A, anti-B, • Three-fold AMS increase; PEG precipitation
anti-D). o If there is an increase in threefold
CLINICAL AND DIAGNOSTIC SIGNIFICANCE amylase level within 24 hours the MT
• Amylase/Creatinine (A:C) Ratio must repeat sample collection and
When we are determining this ratio we will perform PEG precipitation
need to determine the: (Polyethylene Glycol)
o Urinary Amylase • Increased AMS level may also observed to
o Blood Amylase the following condition Ectopic Pregnancy,
o Urinary Creatinine Peptic Ulcers, Alcoholism, Mumps, Diabetic
o Blood Creatinine acidosis, severe burns, recovery from
Meron tayong 4 values na kailangan in order to thoracic surgery
determine the amylase to creatinine ratio. • Decreased in AMS is observed in Pancreatic
Kailangan natin mameasure ung amylase sa urine insufficiency
and blood of the patient as well as the creatinine Drugs that might interfere with AMS level
level in the blood and urine of the patient. determination Codeine, Morphine,
• Normal A:C Ratio: 1-5% Glucocorticoids, dexamethasone, oral
• Formula: contraceptives, x-ray contrast dye
LIPASE 3.1.1.3 (LPS)
• Major Tissue Source: PANCREAS
You need to memorize the formula and will simply • Most specific pancreatic marker because
substitute the values that will be given. The purpose only produce in pancreas
of this is that you know how to apply or paano • Other name “Triaglycerol Acylhydrolase”
madetermine ung amylase to creatinine ratio • Advantage in regards to pancreatic
natin. function: not affected by other renal
disorders not unlike other enzyme; if lipase
ACUTE PANCREATITIS level increases it is attributed to disease
TRANSFORMERS 11
related to pancreas. Olive oil as their primary substrate

• Primary function: Hydrolysis of ester linkages • Your olive oil functions as the substrate
of fats to produce alcohol and fatty acid because other esterases can hydrolyze
(end products of lipase reaction) triglycerides and synthetic diglycerides
• Nomal Values: 0-1.0 U/mL • A substitute for olive is Triolein, it is a more
• Concentrations are normal in conditions of pure form of triglyceride in comparison to
salivary gland involvement olive oil, although Triolein is expensive that is
why substrate of choice parin si Olive oil
ACUTE PANCREATITIS • Colipase are proteins secreted by the
Superior diagnostic tool (Trypsinogen-2 & Elastase- pancreas. Bile salts and Colipase may be
1) added to the enzymatic reaction in order
6 Hours, 1-7 Days, 8-14 Days to increase ↑ the sensitivity and specificity of
CHRONIC PANCREATITIS: our assay by adding bile salts and colipase
Acinar cell degradation = loss of AMS and LPS • Colipase also prevents the inactivation of
lipase
Notes: • Presence or utilization of a Hemolyzed
• Lipase is a superior diagnostic tool because sample inhibitis the activity of your lipase.
it is only secreted by the pancreas Kapag si hemoglobin narelease through a
• So, kapag mayroon tayong increase in hemolyzed sample, this interferes with the
levels of Lipase sa circulation natin, it is Lipase measurement which results to a false
often attributed to a damage, disorder, or decrease↓ lipase values
disease associated to the pancreas • Bacterial contamination= false ↑increase in
• Trypsinogen 2 and Elastase 1 are also good lipase values
diagnostic tool. So, Trypsinogen 2, Elastase Hemolysis (Hemoglobin)= false ↓decrease in lipase
1, and Lipase are all superior markers or values
diagnostic tools for Acute Pancreatitis 3 methods for Lipase REMARKS
• Lipase increases 6 hours after the onset of determination
acute pancreatitis, remains to be elevated CHERRY CRANDAL Reference Method for
for up to 1 week, and will begin to decline and lipase measurement
normalize 8-14 days after the onset of your -Hydrolysis of Olive oil,
infection. incubation at 24 hours,
• In Chronic pancreatitis the presence of acinar 37°C
cell degradation is already evident so since -Titration of FA using
mababa or nawawala or nasisira yung mga NAOH
-TAG+Water←LPS→2-
acinar cells natin, nawawala na yung
Monoglyceride+FA
nagproproduce ng amylase pati lipase
PEROXIDASE COUPLING Most Commonly used
• In Acute Pancreatitis= Amylase and Lipase ↑
method
increase
TURBIDIMETRIC -Olive oil and Triolein
In Chronic Pancreatitis since there is already -Decrease in
degradation of acinar cells this will result to a loss↓ of Turbidimetry=↑LPS
amylase or lipase resulting to their decrease kapag Notes:
Chronic Pancreatitis • Cherry Crandal is considered the reference
REFERENCE VALUES method for Lipase measurement
0-1.0 U/mL • Key facts: we must incubate our sample at
CLINICAL SIGNIFICANCE 24 hours, at 37°C
Increased LPS Values: Acute pancreatitis, • Primary principle of Cherry Crandal is we
Pancreatic cyst / pseudocyst, obstructive jaundice, titrate the fatty acids using the NaOH or
peritonitis, intestinal obstruction Sodium Hydroxide
Decreased LPS Values: Pancreatic insufficiency • Principle: Triglyceride in the presence of
METHODS FOR LIPASE MEASUREMENT water ihyhydrolyze niya in the presence of
your lipase into 2 monoglyceride and fatty
Substrate: Olive Oil
acids
Alternative Substrate: Triolein
• The 24 hours and 37 °C was a previous
Increases Sensitivity and Specificity: Bile salts and
question in the board exam
Colipase
• Although Cherry crandal is the reference
Inhibitor: Hemoglobin (Hemolyzed sample)=False↓
method, Peroxidase Coupling is still the
of lipase values
most commonly used method, mas
Bacterial contamination can falsely increase LPS
ginagamit siya sa mga lab
values
• Peroxidase coupling method utilizes a
Notes:
colorimetric assay and the principle
• If sa Amylase measurement Starch yung
requires minimal amount of sample kaya
primary substrate
siya ginagamit in comparison to cherry
• All methods of Lipase measurement use
crandal mas konti yung sample na
TRANSFORMERS 12
kailangan kapag ginagamit natin si ISOENZYMES

peroxidase coupling PERCENTAGE
• Turbidemetric assay, turbidimetric LD1 17-27%
measurement of lipase involves the LD2 27-37%
utilization of olive oil and triolein as the LD3 18-25%
substrate
LD4 3-8%
• A decrease in ↓turbidity is equal to an
LD5 0-5%
increase in ↑lipase value
PRIMARY/MAJOR TISSUE SOURCES
We determine the decrease in turbidimetry or the
decrease in turbidity LD1 & LD2 Heart, RBC, Kidney
LD3 Lungs, Pancreas, Spleen
LD4 & LD5 Skin, Ilium, Skeletal muscle, Liver,
OTHER MAJOR ENZYMES Intestines
LACTATE DEHYDROGENASE 1.1.1.27 (LD) REMARKS
Major Tissue Sources: Heart, RBC, Kidneys, Lungs, LD1 &LD2 - Most anodal, heat stable,
Pancreas, Spleen, Skeletal Muscles, Liver, Intestine abundant
Interconversion of lactic and pyruvic acids -LD2: Major isoenzyme in Serum
COENZYME: (NAD) Nicotinamide Dinucleotide LD3 -Moderate in abundancy.
Tetrameric molecule: Four subunits of two possible -Also known as “intermediary
forms: H and M form Isoenzyme”
Clinically significant if separated into its Isoenzymes LD4 & LD5 -Least anodal, heat labile, scarce
LD6 -A minor isoenzyme.
Notes: -Also known as “Alcohol
• LD stands for Lactate dehydrogenase, also dehydrogenase”
referred to as LDH -Appears on the 6th band in
• It has an EC number of 1.1.1.27 electrophoresis
• Heart, RBC, Kidneys, Lungs, Pancreas, -Often used in determining patients
Spleen, Skeletal Muscles, Liver, Intestine are with drug hepatotoxicity, obstructive
the major tissue sources of LDH so jaundice, arteriosclerotic failure.
maraming organs ang affected kapag CLINICAL SIGNIFICANCE
nagincrease ang LDH natin, this means that Normally, LD2 would have the highest levels
an increase or elevated level of LDH most followed by LD1 > LD3 > LD4 > LD5
often than not provides no clinical - Normal pattern in serum:
significance unless we have determined the o LD2 >LD1>LD3>LD4>LD5
different isoenzymes - Normal pattern in CSF sample
• Similar kay alkaline phosphatase na kapag o LD1>LD2>LD3>LD4>LD5>
tumaas siya, alam mo lang na may - Expected pattern for patients experiencing
something wrong with the patient acute myocardial infarction
• Similar kay Lactate dehydrogenase na o LD1>LD2>LD3>LD4>LD5>
kapag mataas si LD levels alam mo lang na - Expected pattern for patients experiencing
may sakit si patient natin seizure:
• We have to determine the specific cause o LD2 >LD1>LD3>LD4>LD5
of the increase in the LDH levels by
determining the different isoenzymes ACUTE MYOCARDIAL INFARCTION
• Facilitates the interconversion of lactic and - “Flipped Pattern” LD1 > LD2
pyruvic acids, this requires a hydrogen o LD1>LD2>LD3>LD4>LD5>
transfer enzyme that uses the coenzyme - LD1 increases after 12-24 hours of the onset,
NAD (Nicotinamide Dinucleotide) peaks at 48-72 hours, and gradually
• Our coenzyme in LDH is NAD declines and returns to normal after 10-14
• LDH is a tetrameric molecule with 2 forms, days
the H form and M form
• Tetrameric means 4 so meron siyang 4 2-3x Viral Hepatitis, Cirrhosis
subunits LD2/3/4: Cancer Markers
• 4 subunits, 2 forms (the H form and the M LD5: Hepatic Carcinoma, Toxic Hepatitis
form) Cardiac Muscle: AMI, CSF, Myocarditis
Normal values differs depending on the method
we are trying to analyze • Bacterial Meningitis – the normal
REFERENCE VALUES electrophoretic pattern of your lactate
Forward: 35-90 U/L dehydrogenase would be 5.4.3.2.1. (FLIP)
Reverse: 95-200 U/L • In normal serum and when the patient has
Note: 95 to 200 asa handout pero 95 to 100 asa seizures the normal electrophoretic mobility of
powerpoint and discussion, sabi ni sir claclarify lactate dehydrogenase would be 2.1.3.4.5
nalang non pero di ko alam ano niyan yung • Acute myocardial infection – the normal value
talaga would be 1.2.3.4.5. It has a characteristic leap
TRANSFORMERS 13
pattern and it is also observed kapag naman Wroblewski • MOST PREFERRED METHOD –
CSF sample naman ginamit natin mas mataas Ladue because it is twice as fast in
si 1 followed by 2.3.4.5. comparison to wacker/direct
• Other causes of lactate dehydrogenase method.
increase includes your: • Reverse method or Indirect
o Pernicious Anemia, Hemolytic Disorders or method
Hemolytic Anemia – The highest level of • Facilitates the conversion of
lactate dehydrogenase is observed kapag Pyruvate to Lactate at lightly
meron ng Pernicious Anemia. alakaline pH 7.2
o 10x in Hepatic Carcinoma and Toxic • Method of choice or Preferred
Hepatitis – It is usually presents with a 10 method for LD-5
times in increase. Increases the values of Pyruvate + NADH ←LDH→ Lactate
lactate dehydrogenase up to 10 times to +N AD
normal value. Heat LD1 is stable
o 2-3x Viral Hepatitis, Cirrhosis – Slightly Denaturatio
increase approximately 2 to 3 times the n Test
normal values Most heat labile isoenzyme – LD5 or LD-5
o LD2/3/4: Cancer Markers (Cancer CREATINE KINASE 2.7.3.2 (CK)
Biomarkers) – Kapag mataas ang LD2/3/4 • Major Tissue Sources:
ng patient ay they may be utilized for the ✓ Brain
detection of Acute Leukemia, Germ cell ✓ Smooth Muscle
tumors, Breast Cancers, and Lung Cancers. ✓ Skeletal Muscle
Pinaka significant as cancer marker is LD3. ✓ Cardiac Muscle
LD5: Hepatic Carcinoma, Toxic Hepatitis – LD5 • Unlike most enzymes CK cannot be found in the
increased kapag may Hepatic Carcinoma tsaka RBCs and Liver
Toxic Hepatitis • Reference Values Male: 15-160 U/L Female: 15-
o Cardiac Muscle: AMI, CHF (Congestive 130 U/L
heart failure), Myocarditis • CK facilitates or catalyzes the transfer of a
o Skeletal Muscle: Muscular dystrophy, phosphate group between creatinine
muscle trauma phosphate and adenosine diphosphate
o Hepatic Parenchymal Disease: Viral • Recall that in the muscular system, CK is
Hepatitis, Cirrhosis, obstructive jaundice, IM involved in the creatine phosphate formation
(Infectious mononucleosis) using ATP and their intercore(?) version of ATP
o Megaloblastic and Pernicious Anemia and ADP. Therefore, CK is involved in the
Decreased amounts of lactate dehydrogenase muscles.
are usually not significant. ATHLETES & TRAINED INDIVIDUALS: Often/Tend to
SPECIMEN CONSIDERATION have an increased baseline level of CK
Severely affected by hemolysis because your RBC USE OF INTRAMUSCULAR INJECTIONS: Results in an
contains increase amount of lactate increased CK level by up to 5 times
dehydrogenase BEDRIDDEN PATIENTS: Have dereased baseline
• Lactate dehydrogenase is being utilized to values since they are bedridden, wala masyado
Differentiate Transudates from Exudates. muscular activity
o Transudates – mababa or decrease lactate REFERENCE VALUES
dehydrogenase Male: 15-160 U/L
o Exudates - mataas or increase lactate Female: 15-130 U/L
dehydrogenase.
ISOENZYMES
o Normal ratio of Transudates to Exudates –
CK is involved in the storage of high energy
1:2 or lower
creatinine phosphate = two different monomers
Decrease values from frozen samples; 24-48 hours
o Creatinine Phosphate is one of the major
at room temperature (25 degree Celsius) -
sources of energy upon muscular activity
Decrease values of Lactate dehydrogenase are
o Composed of two monomers:
observed kapag yung samples natin are frozen.
✓ Monomer M
Therefore we must perform analysis within 24-48
✓ Monomer B
hours upon sample collection in room temperature.
•MINOR ISOENZYMES FOR CK:
PRINCIPLE
✓ Mitochondrial CK
Wacker • Forward method or Direct ✓ Macro-CK
method o Often observe when there are advanced
• Facilitates the conversion of malignancies
Lactate to Pyruvate at an
MAJOR ENZYMES OF CK
alkaline pH 8.8
ISOENZYME MAIN ORGAN
• Method of choice or Preferred
CK-BB Brain
Method for LD-1
Lactate + NAD ←LDH→ Pyruvate CK-MB Heart
+NADH CK-MM Miuscle
TRANSFORMERS 14
REMARKS these conditions

• Rarely • Also known as brain type CK RELATIVE INDEX (CKI)
seen in creatine kinase • Percentage of total CK that is attributed to CK-
Adult • Also known as CK-1 MB
Sera • Possible release of CK-MB from non-cardiac tissue
• Most • This computed in order to determine the
Anodal possible release of CK-MB from non-cardiac
CK tissues
• Most • For this, we only have to substitute the values of
Labile CK-MB and Total CK since they will be given.
• 20% of • Also known as hybrid type
the total creatine kinase
CK-MB is • Also known as CK-2
present • Significant amount of CK-MB
in the that is present in the circulation is SPECIMEN CONSIDERATIONS IN CK MEASUREMENTS
cardiac already indicative of myocardial • Activator: N-Acetyl-Cystine and Mg
tissue damage o Added in the enzymatic reaction in order
• >6% • Any elevation of CK-MB in the activate CK
Total CK serum circulation of a patient is • Hemolysis; Adenylate Kinase
= already indicative of myocardial o Hemolysis falsely increases the levels of CK
Myocar damage o Hemolyzed sample also releases another
dial • To be more specific >6% total CK enzyme which is the Adenylate Kinase. This
Damag is again indicative of myocardial enzyme will result to falsely increased levels
e damage of CK
• Not • Buffer: Imidazole
elevate • Inhibitor: Urate and Cystine
d in • Photosensitive
cases of o This marker (CK) is photosensitive meaning it
Angina falsely increases the values when exposed
• Cardiac • Also known as muscle type to light specifically UV light
and creatine kinase • Decrease after storage; Cleland’s Reagent +
Skeletal • Also known as CK-3 Glutathione
Muscle • Most abundant CK fraction in o CK level decreases after storage
the serum If this happens, we utilize the Cleland’s reagent
• Major • Major isoenzyme = >94% of CK in and add glutathione to remedy the loss of CK in
Isoenzy serum is attributed to CK-MM the sample
me in REMARKS
Serum, Tanzer- pH: 9.0 @ 340 nm
Lest Gilvarg
Anodal Creatine + ATP CPK
• M for Muscle Creatine phosphate + ADP
• B for Brain
CLINICAL AND DIAGNOSTIC SIGNIFICANCE ADP + Phosphoenolpyruate
• Acute Myocardial Infarction (AMI) (4-8, 12-24, PK Pyruvate + ATP
48-72 hours)
o Laging bumabalik AMI. This is because it is Pyruvate + NADH
one of the diseases that is often managed LD Lactate + NAD
using different enzymes Oliver- pH 9.0 @ 340 nm
o In AMI, Lactate dehydrogenase is Rosalki
considered as a sensitive indicator bec. CK- Creatine phosphate + ADP
MB will increase 4-8 hours after the on-set of CPK Creatine + ATP
AMI and will peak at 12-24 hours and will
decline at 48-72 hours later ATP + Glucose HK
• Duchenne Disorder or Duchenne Muscular ADP + Glucose-6-Phosphate
Dystrophy
o Highest Level of CK is observed in Glucose-6-Phosphate + NADP
Duchenne disorder G6PD
• Elevated after trauma 6-Phosphogluconate + NADPH
o CK is elevated after trauma such as Electrophor Reference method:
accidents esis • (-)mCK
• Hypothyroidism, Pulmonary Infarction, Reye’s • CKMM
Syndrome, Strenuous exercise, Rocky Mountain • MCK
Spotted Fever, CO Poisoning • CKMB
o Marked elevation of Total CK is observed in • CKBB(+)
TRANSFORMERS 15
NOTES: LD 8 72 hours 5-7 days

Tanzer-Gilvarg and Oliver-Rosalki -
• Just like in LDH, there is also a forward and 1
reverse method in CK measurement: 0
o Forward/direct: Tanzer Gilvarg
o Reverse/indirect: Oliver-Rosalki h
• Preferred method: Oliver-Rosalki r
o It provides a faster turn-around time s
(TAT). NOTES:
• Tanzer-Gilvarg is utilized by setting it at a pH • The table above is the summary of the
of 9.0 (Alkaline) and set at 340 nm cardiac profile of Acute Myocardial
• Oliver-Rosalki has an acidic pH of 6.8, set at Infarction (AMI)
340 nm. • Mnemonic: MY TROPiCAL
o MYoglobin
Electrophoresis o TROPonin I
• If electrophoresis is performed with CK o CK-MB
sample or if we intend to measure the o AST
different isoenzymes, this is how the normal o LD
values will appear: • Gold standard in the diagnosis of AMI:
o (-)mCK: the mitochondrial CK (mCK) Troponin I
will appear in the cathode (-) • First enzyme to increase during AMI: CK-MB
▪ Cathode (-) • First analyte to increase during AMI:
▪ Anode (+) Myoglobin
o CKMM • First two analytes in the table (Myoglobin
o MCK: Macro CK and Troponin I): PROTEINS
o CKMB Last three analytes in the table (CK-MB, AST, LD):
o CKBB(+) ENZYMES
• Normal isoenzymes: CKMM, CKMB, CKBB
(+)
Board exam questions: Do not include the minor OTHER MINOR ENZYMES
isoenzymes unless they are asked in the question or GAMMA GLUTAMYL TRANSFERASE 2.3.2.1 (GGT)
mentioned in the choices. • Main Tissue Source: Kidney, liver, pancreas,
CARDIAC PROFILE FOR AMI prostate, and the brain
• Transfer of gamma glutamyl group form of
MYOGLOBIN 4 12-49 hours 7-10 days gamma glutamyl peptide to another
- peptide or amino acid
1 • Canaliculi of the hepatic cells
0 o Other tissue source that releases
GGT
h o Particularly, the epithelial cell lining
r of the biliary ductules.
s REFERENCE RANGE
TROPONIN I 4 12-49 hours 7-10 days Male: 5-40 U/L
- Female: 5-25 U/L
1 CLINICAL AND DIAGNOSTIC SIGNIFICANCE
0 • Indicator of alcoholism
o Sensitive indicator of alcoholism
h o The most sensitive marker for
r alcoholic hepatitis
s • Useful for differentiating source of an
CK-MB 4 12-24 hours 1-2 days elevated ALP
- o Since ALP is non-specific, GGT can
6 be utilized to determine the source
of the ALP elevation.
h • Hepatobiliary disorders and biliary tract
r obstruction
s • Primary biliary cirrhosis
AST 6 48 hours 4-5 days o Highest elevations of GGT is seen in
- PBC
8 • Increased levels of GGT are observed on
the following conditions: Obstructive
h jaundice, Cirrhosis, Tumors, Infectious
r Mononucleosis (IM), Hepatotoxicity,
s
TRANSFORMERS 16
Chronic Alcoholism, Antiepileptic / Most widely used

Anticonvulsant drug administration, renal Ellman SUBSTRATE:
diseases Butyrylcholine
Hydrolyzed substrate
SZASZ ASSAY reacts with colorless
• Different from the Szazsz Modification 5,5’-dithios-2-
• Photometric Enzyme Assay for the nitrobenzoic acid
measurement of GGT Notes:
• Substrate: L-gamma-p-nitroanalide / L- • Michel
gamma-3-carboxy-nitroanilide o The change in pH is what we would
• Product: Yellow find to determine if there is a
• Indicator: production of p-nitroaniline or 5- change in cholinesterase levels
amino-2-nitrobenzoate • Ellman
• Monitored at 405-410 nm What you need to remember here is that its
TRUE CHOLINESTERASE 3.1.1.7 monochromator is set at 410 nm
PSEUDOCHOLINESTERASE 3.1.1.8 ALDOLASE 4.1.2.1
• It has 2 types of isoenzymes: True • Main Tissue Sources: Skeletal muscles, Heart
Cholinesterase and Pseudocholinesterase muscles, Liver, and RBC
• True Tissue Source: RBC, Lungs, Kidney, • Primary function: Splitting of fructose 1,6-
Liver, Pancreas, Prostate, Brain diphosphate to glyceraldehyde-3-
• Pseudo Tissue Source: Heart, Liver, phosphate and dihydroxyacetone
Pancreas, Brain phosphate
• Reference Range: 6,000-12,000 U/L • Other name: “Fructose-1,6-Diphosphate
• Other name: Aldolase”
o True: Acylcholine Acyhydrolase • Isoenzymes
o Pseudo: Acetylcholine • Aldolase A: Skeletal Muscles
Acetylhydrolase o Kapag mataas ang Aldolase A,
• Hydrolysis of esters with acetylcholine to more often than not, this is due to a
choline and acetic acid skeletal muscle
• Decreased levels of cholinesterase disorder/involvement
indicates disease. • Aldolase B: WBC, Liver, Kidney
CLINICAL AND DIAGNOSTIC SIGNIFICANCE • Aldolase C: Brain Tissue
• Assessment of parenchymal function • Increased: Progressive Muscular dystrophy,
• Insecticide poisoning, Metastatic inflammatory muscle disease, MI, Liver
Carcinoma, Parenchymatous disease disease, Liver necrosis, Pulmonary infarction,
• Expected results: Low or decreased malignancy, leukemia, hemolytic anemia
Cholinesterase levels Decreased: Not significant
ORNITHINE CARBAMOYL TRANSFERASE 2.1.1.3 (OCT)
• Main tissue source: Liver
METHODS IN DETERMINING GGT LEVELS • Catalyzes the reversible conversion of
• INHIBITOR: None but we have an indicator (refer Ornithine to Citrulline
to Szasz Assay: p-nitroaniline or 5-amino-2- o This is involved in the synthesis of urea
nitrobenzoate) • Laboratory Methods in the measurement of
REMARKS OCT levels
Electromagnetic o Reichard and Reichard / Isotopic
method that determines Microdiffusion
changes in pH • Colorimetric Assay
Change in UV • Increased: Acute Viral Hepatitis, Obstructive
absorption would jaundice, cirrhosis, metastatic carcinoma,
Michel indicate that there is heart failure, delirium tremens, cholecystitis
hydrolysis of • Decreased: Not significant
benzoycholine = LEUCINE AMINOPEPTIDASE 3.4.4.1
changes in pH • Main sources: Urine, Serum, Bile
DISADVANTAGE: • Primary function: Hydrolysis of N-terminal
specialized instrument residues from certain peptides and amides
Monometric Primary Principle: o This contains many amino acids
Liberation of CO2 from • Laboratory Method: Goldbarg and
acetic acid as Rutenberd (Fluorometric and Colorimetric
acetylcholine is Method)
hydrolyzed • Increased: Hepatobiliary disease (e.g.,
hepatitis, cirrhosis, obstructive jaundice,
Photometric/Colorimetri
metastatic carcinoma of liver and
c enzyme reaction at
pancreatitis
410 nm
TRANSFORMERS 17
Decreased: Not significant Dehydrogenase

5’ NUCLEOTIDASE 3.1.3.5 (5’N) ISOENZYMES
• A phosphoric monoester hydrolase that is AST Cytoplasmic
predominantly secreted from the liver Mitochondrial
• Marker for hepatobiliary disease and CK 1, 2, 2
infiltrative lesions of the liver (Increased) LDH 1, 2, 3, 4, 5, 6
• Laboratory Methods: Dixon and Purdon, ACP Prostatic
Bellfield and Goldberg, Campbell Non-prostatic
o If the enzyme discussed does not have a ALP Liver
specific instructions or remarks for the Bone
methods, all you have to remember is Placenta
which method is for the enzyme Intestines
ANGIOTENSIN-CONVERTING ENZYME 3.4.15.1 (ACE) AMS Pancreatic
• Main sources: Macrophages and Epithelioid Salivary
Cells A, R, C
• Primary function: Converts the Angiotensin I Cholinestera True
to Angiotensin II within the lungs se Pseudo
• Other name: “Peptidyldipeptidase A”
NON ALT
“Kininase II”
GGT
• Neuronal dysfunction (i.e., Alzheimer’s
LPS
Disease via the CSF analysis)
5’N
• Other causes of increase: Sarcoidosis,
Acute and Chronic Bronchitis, leprosy
CERULOPLASMIN 1.16.3.1 LABORATORY DEMONSTRATION
• Copper-carrying protein that also functions ALANINE AMINOTRANSFERASE (ALT)
as an enzyme Overview
• A known marker for Wilson’s Disease
For this laboratory demonstration, the Indirect or In-
(Increased)
vitro Quantitative Detection of Alanine
It is a hepatocellular disease that involves the liver
aminotransferase (ALT) in serum will be performed.
GLUCOSE-6-PHOSPHATE DEHYDROGENASE 1.1.1.49
(G-6-PD) Alanine Aminotransferase (ALT)
• Maintains the NADPH in the reduced form • It is widely distributed in tissues with the Highest
inside RBC Concentrations found in the Liver and the
• Newborn screening marker; Deficiency can Kidneys.
lead to drug-induced hemolytic anemia • It is Liver-specific enzyme in comparison to
o Especially if there is intake of Aspartate aminotransferase (AST)
primaquine which is an anti-malarial
drug Clinical Significance of ALT
• Main Tissue Source: adrenal cortex, spleen, • Increase enzymatic levels of ALT are often
RBC, lymph nodes observed in liver disease such as:
• Specimen of Choice: Red Cell Hemolysate, o Cirrhosis
Serum o Hepatitis
• MI, Megaloblastic Anemia o Metastatic Carcinoma
REFERENCE VALUES
10-15 U/g of Hemoglobin or 1,200-2,000 mU/mL of • Elevated levels of ALT are also observed on
Packed RBC patients diagnosed with:
SUMMARY o Infectious Mononucleosis (IM)
Acid o Muscular Dystrophy
RBC, Prostate o Dermatomyositis
Phosphatase
Alanine Material and Equipment
High Aminotransferas Liver 1. Serum Sample
Specificity e 2. ALT Working Reagent
Pancreas, 3. Distilled Water
Amylase
Salivary glands 4. Clean Test Tubes
Lipase Pancreas 5. Micropipettes
Aspartate Liver, Heart, 6. Disposable Micropipette Tips
Aminotransferas Skeletal 7. Spectrophotometer
Moderate
e Muscle
Specificity
Heart, Skeletal
Creatine Kinase
Muscle, Brain
Alkaline Liver, Bone,
Low
Phosphatase Kidney
Specificity
Lactate All Tissues
TRANSFORMERS 18
next 2 minutes
o Calculate for the mean absorbance in
order to obtain the result for ALT.
Procedure
1. Pipette 1mL of the ALT working reagent into
two tips:
o 1mL for the Blank
o 1mL for the Sample
2. Transfer 10uL of Distilled water to the blank

tube
3. Mix and incubate the sample at 37 degrees
Celsius for 1 minute.
o
4. Transfer 10uL of sample to the sample tube
5. Mix and incubate the sample at 37 degrees
Celsius for 1 minute.
o
6. After the incubation, feed the reagent blank
and the sample in the spectrophotometer
and measure the first absorbance at a
wavelength of 340nm
7. Repeat the readings every 1 minute for the
TRANSFORMERS 19
4 Clinically Significant Electrolytes Clinical Chemistry 2 (LAB)
CLINICALLY SIGNIFICANT ELECTROLYTES each side of the membrane into an electric

SODIUM potential.
Specimen of Choice ● For sodium measurements, most analyzers
with integrated ISE system contain a
● Serum/plasma/urine
o Glass ion-exchange membrane.
o Other specimen include whole
● The ISE method used 2 electrodes.
blood, sweat, feces and
o 1 electrode will serve as the
gastrointestinal fluid.
reference electrode. This reference
o Take note that when plasma is used
electrode has the constant
the following anticoagulants are
potential.
preferred or suitable for sodium
o While the other one is the (2)
measurement.
measuring electrode
● PLASMA
o The difference in potential between
o Lithium heparin, ammonium heparin
the two (reference and measuring
and lithium oxalate as
electrode) will be used to calculate
anticoagulant
the concentration of the ION.
o Remember also that the Red blood
● Activity of the ion is the one being
cell only contain small amount of
measured.
sodium. Hemolysis does not cause
significant changes on the serum or Types of ISE
plasma sodium level unless there is 1 Direct
marked hemolysis. 2 Indirect
● Marked hemolysis- decreased levels Direct
o When there is marked hemolysis the Undiluted sample interacts with ISE membrane
levels of sodium decreased. Indirect
● Urine specimen of choice: 24-hour urine Diluted sample is used
collection specimen. No significant difference between two results
Methods of Determination except when sample is hypoprotenimic or
● Chemical methods hyperlipidemic. This kind of samples may lead to
o Are one of the earliest methods that FALSE DECREASE of Na levels due to diluting of
were used to measure sodium. sample. Diluting is seen in indirect ISE. Due to
o However these methods are situations, direct ISE is considered more accurate.
considered obsolete, since they POTASSIUM
required large volume of sample Specimen of Choice
and the results are not precise. Serum/Plasma (preferred if high PLT ct. because K
o One of the obsolete method for from platelets will be released during clot formation
sodium determination as well is the causing FALSELY INCREASE result) /Urine/Whole
Colorimetric Method. Blood
● Colorimetric method (Albanese-Lein) Anticoagulant of Choice
o The colorimetric method for sodium Heparin
determination is known as Serum > plasma and WB
Albanese-Lein method. ✔K+ concentration in serum is higher than in
● Other Methods: plasma & WB
● FES (Flame emission spectrophotometry) Hemolysis – must be avoided (high K in RBCs)
● AAS(Atomic absorption spectrophotometry) ✔ Unlike Na+, it should be avoided at all
● ISEs(Ion selective electrode) times during K+ concentration measurement,
o Considered as the most routinely because RBCs has high K+ content
used or the commonly used method Urine specimen of choice: 24-hour urine
in the laboratory nowadays. Methods of Determination
Ion Selective Electrode 1 Colorimetric method (Lockhead-Purcell)
● ISE or Ion Selective Electrode makes use of: ✔ Obsolete test
● Selectively permeable or the 2 FES
semipermeable membrane to convert the
3 AAS
activity of different ion concentrations in
4 ISEs (ion selective electrode)
TRANSFORMERS 1
✔ Method of choice - If uncapped the CO2 may escape

✔ Use Valinomycin membrane – to and lead to DECREASED in CO2
selectively bind with Ca+ levels
Note: In Na measurement, use glass ion Methods of Determination
exchange 1 ISE
Colorimetric Method (Lockhead-Purcell) ● to measure total CO2 - acid reagent
FES Acid reagent is used to convert all forms of
AAS CO2 into CO2 GAS then it will be measured
ISEs (ion selective electrode) using another electrode.
● Method of choice 2 Enzymatic Method
● Valinomycin membrane ● Conversion to HCO3 - Alkalinized
▪ Selectively binds with potassium The sample will be alkalinized first and convert
CHLORIDE all forms of CO2 to bicarbonate.
Specimen of Choice MAGNESIUM
● Serum / plasma / urine / whole blood / sweat Specimen of Choice
● Lithium Heparin ● Non-hemolyzed serum or lithium heparin
■ Anticoagulant of choice plasma
● Hemolysis - Routine specimen and
■ Does not cause significant changes in the anticoagulant of choice
serum or plasma levels of chloride - Serum must be separated
■ Marked hemolysis - decreased levels immediately
Urine specimen of choice: 24-hour urine ● Oxalate, citrate, EDTA - unacceptable
Method of Determination - because these anticoagulant bind
1 ISEs (ion selective electrode) with magnesium
2 Amperometric-coulometric titration ● Hemolysis - should be avoided
3 Mercurimetric titration (Schales and Schales) - e.g. Potassium hemolysis should be
avoided when measuring
4 Colorimetry
magnesium levels
ISEs (ion selective electrode)
- Why? there is greater concentration
● Ion-exchange membrane
of magnesium inside the RBCs that
● tri-n-octyl-propylammonium chloride decanol
that of extracellular fluids
▪ Most commonly used membrane for
● Urine specimen of choice: 24-hour urine
chloride determination
(acidified with HCl)
Amperometric-coulometric titration Acidification with HCl is to avoid precipitation
● Uses Silver (Ag2+ ions) that will combine with Methods of Determination
chloride to quantitate the concentration of
1 Colorimetric methods:
chloride
1. Calmagite
● Cotlove chloridometer
- Mg + Calmagite ions =
▪ One of the devices that makes use of this
reddish-violet complex
principle
- Can be read at 532 nm
Mercurimetric titration (Schales and Schales) 2. Formazan dye
Colorimetry - Mg + Formazan dye = colored
BICARBONATE complex
Specimen of Choice - Can be read at 660 nm
● Serum / Plasma 3. Methylthymol blue
- Testing for total CO2 Mg + chromogen = Colored complex
● Serum or Lithium Heparin Plasma 2 Dye Lake method
- Anticoagulant of choice - Make used of titan yellow
● Capped samples 3 Reference method: ASS
- Always be capped until the serum Calmagite
or plasma is separated and the ● Binds to magnesium ions
sample should be tested Forms reddish-violet complex at 532 nm
immediately
Formazan dye
TRANSFORMERS 2
● Binds to magnesium ions ● The anticoagulant of choice for plasma is

● Colored complex at 660 nm LITHIUM HEPARIN
Methylthymol blue
● Mg + chromogen = colored complex TAKE NOTE:
CALCIUM The anticoagulants to be avoided are EDTA,
Specimen of Choice CITRATE and OXALATE, because they may interfere
● Serum or lithium heparin plasma with measurement. Also remember that hemolysis
o Routine specimen: Serum or plasma should also be avoided at all times as well when
o Other specimen: Urine measuring for phosphorus because of its higher
o Anticoagulant of choice: Lithium heparin concentration inside the red blood cells.
● Dry heparin vs liquid heparin ● Phosphorus is also known to reach high levels in
o Liquid heparin should not be used because the morning and low levels at night.
some heparin anticoagulants bind to calcium, The urine specimen of choice to be used is a
thereby lowering its concentration (Dry 24-hour urine specimen.
heparin eliminates the aforementioned Method of Determination
interference) 1 ● Current methods for the determination of
● EDTA and oxalate – unacceptable phosphorus involve the formation of an
o They bind with calcium ammonium phosphomolybdate complex.
● For urine specimens: This method is also known as
● Must be accurately timed urine acidified FISKE-SUBBAROW METHOD.
with HCl to avoid precipitation This complex that will be formed is known to be
Methods of Determination colorless and can be measured by the
1 Colorimetric methods: (Most common) ultraviolet absorption at 340 nm or it can be
2 Precipitation and Redox titration reduced to molybdenum blue which can be
read between 600-700 nm. But the unreduced
3 ISE (Liquid membrane)
complex at 340 nm is considered as the most
4 EDTA titration, FES
accurate measurement of inorganic
5 Reference method: AAS
phosphorus.
Colorimetric methods
● Orthocresolphthalein complexone (CPC)
● Arsenazo III dye
Laboratory Demonstration
Precipitation and Redox titration
ALBANESE & LEIN METHOD FOR IN-VITRO
● Clark Collip
DETERMINATION FOR SODIUM
o End product: oxalic acid
Sodium
● Ferro Ham – Chloranilic Acid
• Major extracellular cation in the human body
End product: chloranilic acid
• Responsible for determining the plasma
ISE (liquid membrane)
osmolality levels
- a method for calcium determination
• Sodium concentration in the body will
and it makes use of liquid
depend on various factors such as the intake
membrane
and excretion of water and blood volume
EDTA TITTRATION, FES, and AAS status
AAS • Hyponatremia: Decreased plasma sodium
- considered as the Reference levels
Method for calcium o One of the most common electrolyte
PHOSPHATE/PHOSPHORUS disorders among individuals
Specimen of Choice Material and Equipment
● SERUM or LITHIUM HEPARIN PLASMA 1 Sodium standard reagent
2 Serum sample
In testing for phosphorus, the specimens that may 3 Sodium working reagent
be used are serum or plasma. Other specimen 4 Sodium color developer
such as URINE may also be used.
5 Micropipette (50 uL, 1500 uL)
6 Distilled water
7 Disposable tips
TRANSFORMERS 3
8 Clean test tubes

9 Spectrophotometer
Procedure
1. Pipette 50 uL of the sodium standard into a tube
labeled as standard.
2. Pipette 50 uL of the serum sample into a tube

labeled as sample
5. Allow the tubes to stand at room
temperature for 1 minute.
3. Add 1.5 mL of Sodium working reagent on each

tube.
6. After incubation, read the tubes in the

spectrophotometer and measure the
absorbance at a wavelength of 550 nm.
4. Add 50 uL of sodium color developer

reagent on each tube. **(50uL sinabi ni sir
pero sa video 1.5uL nakattach).
TRANSFORMERS 4
5 Blood Gas Analysis Clinical Chemistry 2 (LAB)
BLOOD GAS ANALYSIS ● Although, we can undergo training, but

● Blood gas analysis (BGA) is used for laboratory generally, we are not the ones who collect
testing that relates to acid-base balance and arterial blood
oxygenation status of a patient ● Most of the time, this is collected by
● used as a diagnostic tool to evaluate partial physicians or doctors
pressure of gas and blood in acid-base
contents or balance
● understanding the use of BGA enable our
doctors to interpret respiratory, circulatory, and
metabolic disorders of our body
Specimen Collection
Specimen of Choice: ARTERIAL BLOOD
● sample type required for correct assessment
of gas exchange, functions of the lungs
SPECIMEN COLLECTION
such as that of PO2 and PCO2 measures,
● Syringe and needle must be
specifically our oxygenation status
pre-heparinized by drawing up heparin into
● Composition: uniform and not dependent
the syringe to wet its interior
on changes in systemic or local circulation;
● Any excess should be expelled
hence, it is the best spx of choice for BGA
● Most of the time, MTs are the ones who will
Anticoagulant: 0.05 mL LITHIUM HEPARIN per mL of
perform pre-heparinization of the syringe,
blood
then prepared syringe will be given to the
● Recall item in board exams (Lithium
doctor
heparin)
VIDEO OF PRE-HEPARINIZATION
● 2 kinds of heparin (sodium and lithium) but
● Draw 1 cc of 1000 units per cc heparin into
the one used for BGA is lithium heparin
3 or 5 cc syringe
● Sodium heparin – can alter the sodium
● After removing the needle from the heparin,
concentrations of the blood because of the
pull the plunger all the way back to coat
presence of sodium
the syringe, and expel all the heparin,
● If asked in the board exams and only
leaving only the amount trapped in the hub
heparin is the choice then, heparin is
● Change the needle to the one that will be
acceptable
used for puncture
Usual Sites for arterial puncture are the radial,
SPECIMEN COLLECTION
brachial, and femoral artery
● Butterfly infusion sets is NOT recommended
● Radial artery – most commonly used site for
for arterial puncture
BGA
- Syringe and needle is always the best
● Brachial artery - may be preferred for larger
● Any air trapped in the syringe during
volumes of arterial blood
collection should be immediately expelled
- More difficult to puncture because of its
at the completion of draw
deeper location between the muscles
- Presence of gas/air bubbles in the
and connective tissues
syringe should be removed by gently
● Femoral artery - large vessel that is easily
tapping the side of the syringe
palpated and punctured but is rarely used
- We are testing for gasses, therefore,
in clinical practice due to poor collateral
presence of trapped bubbles in the
circulation and increased chance of
syringe can greatly affect the test results
infections and hematoma
● Following air-bubbles expulsion,
Again, these are the usual sites of arterial puncture,
homogenization of the sample with the
the radial, brachial, and femoral artery, wherein
anticoagulant should be done to avoid clot
the most commonly used is the radial artery
formation
● But take note, medical technologists, in
- Same with blood collection tubes, roll
general, are not trained to perform arterial
the syringe between the palms or invert
puncture
it vertically, or mix it in a figure of 8
- Sample should be properly mixed so as
to prevent clot formation
TRANSFORMERS 1
● The best method for blood gas collection in the ● Syringe and needle or the device of collection
newborn is by indwelling umbilical artery should be pre-heparinized
catheter. ● Once the doctor or a trained [certified]
medical technologist, the capillary tube or
syringe should be capped and remove any air
bubbles that is present by tapping the sides of
the sample
● Be sure to label the sample
● To prevent clot formation, mix the tube by
rolling it between your hands or palm for ten
times [10x] or mix it in a figure of 8 for at least 20
seconds.
● Blood gas analysis has a time requirement
wherein in a certain time period, the sample
has already been tested.
[refer to this image] ● Sample analysis should be performed as soon
● They will do a catheterization on the umbilical as possible when it is received in the laboratory.
cord of the newborn. ● After the draw, blood gas [especially from
● Arterial puncture or from radial artery may not arterial blood] can only be tested within 30
be advisable for them since they have a small minutes. After that, it should be rejected and
body. request for specimen collection if not tested
● A capillary blood sample can be considered Specimen Consideration
as an adequate substitute for the arterial blood Effect
in neonatal and pediatric units. Prolonged standing of pH and pO2 decreases
● A capillary blood can replace an arterial blood the specimen pCO2 increases
sample only when arterialization technique is Specimen should be This will prevent oxygen
used. Although, entirely reliable pO2 chilled during transport consumption by the RBC
measurement can only be achieved only when and release of acidic
an arterial blood sample is used. metabolites, thereby
● Skin puncture is also applicable for adults altering the pH
patients with severe burns, tendencies to Glycolysis May lead to a decrease
thrombosis, obese, and geriatric patients. in blood pH
Avoid excess heparin- May cause a downward
In summary… most common source of shifting of blood pH
● Indwelling umbilical artery catheter – Best preanalytic error
method of collection
Lower temperature Increased oxygen
● Capillary blood sample – Alternative Method of
solubility
collection
Left shift in the
oxyhemoglobin curve
Exposure to room air pH and PO2 will
increase (HI PHO)
pCO2 will decrease (LO
CO)
Explanation:
● Heparin- anticoagulant of choice
● Most of the time during the preparation of
syringe and needle when you pre
heparinized, sometimes the medical
technologist input excess heparin in the
syringe which may alter the result which
[refer to the image above]
may cause downward shifting of the pH in
the blood
Summary of the specimen collection for blood gas
analysis
TRANSFORMERS 2
● Recall from the board exam: When we

expose our arterial blood to room air, the
possible effect would be the increase in pH
and PO2, while there is a decrease pCO2 in
the specimen
Common Errors in Specimen Collection & Handling
1. Form and concentration of anticoagulant
2. Speed of syringe filling
3. Maintenance of anaerobiosis B. Natelson
- When you expose our arterial blood in room air
this can happen in HI PHO and LO CO, and that is
also one of the most common errors
4. Mixing of samples
- you can mix the sample by rolling it in your palms
or making a figure of eight
5. Collection device transport and storage time
before analysis
- the time of analysis should be within 30 minutes
only, after that you should reject the sample and
● This is the instrument under Natelson,
request for a repeat collection
and again it is very vintage
Methods for Blood Gas Analysis ● Natelson is composed of 4
Gasometer Method: (4 Different Compositions under Natelson):
Has 2 Methods but is no longer included in the 1. Mercury
modern books ● Produces the vacuum
A. Van Slyke 2. Caprylic alcohol ★
● Liberated and isolated “the desired gas ● Antifoam reagent
contained in a known volume of ● Recall question in board exam before
solution, and recording in the millimeters ● Gasometer methods were recall
of mercury in the pressure of the gas at question in board exam
a known fixed volume” 3. Lactic acid
Maam, bakit po wala na to sa mga recent 4. NaOH and NaHSO3 (Sodium Hydroxide and
textbooks natin? Sodium Bisulfide)
Like Van Slyke, this method is also an obsolete
As you can see in the illustration mapapansin niyo method.
na parang vintage and itsura ng machine Spectrophotometric Determination of Oxygen
Saturation (CO-Oximetry)
Actually the Van Slyke method under the ● the actual percent oxyhemoglobin (O2Hb)
gasometer is an obsolete method. can be determined spectrophotometrically
using a CO-oximeter designed to directly
When I was researching this method for the blood measure the various hemoglobin species
gas analysis, the researches that I got were around ● At a minimum, instruments should have four
the 1950s, 1960s, so during this time ito yung mga wavelengths for measurements of the HHb,
unang lumabas na methods for the blood gas O2Hb, and the two most common
analysis but not used anymore since we have dyshemoglobins, COHb and MetHb
modern blood gas analyzers which can run our
samples within a short amount of time. Take note that when it comes to CO-Oximetry, we
are actually measuring the hemoglobin.
Hemoglobin will carry the oxygen. Hence, using the
measurement of hemoglobin, we can know the
oxygenation status of the patient. Take note again
hemoglobin is the one that is being directly
measured not the oxygenation.
TRANSFORMERS 3
● At a minimum, instruments should have four ● And of course when it comes to bacterial
wavelengths for measurements of contamination again if present, our bacteria
(deoxyhemoglobin) HHb, O2Hb, and the present in the sample can consume the oxygen
two most common dyshemoglobins, hence it can lower the pO2 values of our
Carboxyhemoglobin (COHb) and sample
Methemoglobin (MetHb).
Blood Gas Analysis: pH, pCO2, and pO2 Blood Gas Analyzers: Measurement of pH
● Blood gas analyzers use electrodes ● Principle: Potentiometry
(macroelectrochemical or o pH measurement is potentiometrics so
microelectrochemical sensors) as sensing that is its principle in which change in
devices to directly measure pO2, pCO2, voltage indicates the activity of each
and pH. analyte
● The blood gas analyzer can also calculate ● Electrodes:
several additional parameters: ✔ Ag-Ag Chloride electrode - reference
bicarbonate, total CO2, base excess, and electrode
SO2. ✔ Calomel electrode (Hg2Cl2) - reference
*These parameters are calculated maybe electrode
depending on some type of formula like the ✔ Glass electrode - most commonly used for pH
Henderson-Hasselbalch equation. o To measure pH, a glass membrane
sensitive to hydrogen is placed around
Blood Gas Analyzers: Measurement of pO2 an internal Ag-AG Chloride to form a
● Principle: Polarography-Amperometry measuring electrode. Potential
*The reduction of oxygen produces a current that is develops at a glass membrane as a
proportional to the amount of oxygen present in result of hydrogen diffusion from the
the sample. unknown solution into the membrane
● Electrode: Clark electrode surface
● Factors to consider: ● Again measurement of pH, principle is
o pO2 test is affected by buildup of Potentiometry
proteins on the surface of the ● Electrode used here, we have 3:
membrane. 1. Ag-Ag Chloride electrode which can be
o Bacterial contamination used a reference electrode in
Bacterial contamination, if present, will consume potentiometry
oxygen and cause a low value of pO2. 2. Calomel electrode (Hg2Cl2) is a
reference electrode as well
Again just to summarize the measurement for pO2 3. Glass electrode is the most commonly
● Principle again is Polarography-Amperometry used for the pH
● The electrode that you should use is Clark ● Take note that the Calomel electrode can be
electrode toxic because of the presence of mercuric
● Factors to consider is the pO2 test is affected by chloride or mercury in general. As we all know,
build up of proteins on the surface of the mercury can be a toxic substance so Calomel
membrane electrode is not actually routinely used
● Here is an example of the membrane. What if because of that reason so Glass electrode is
there is actually a build up of protein here? The the most commonly used followed by the
oxygen should pass our membrane is hindi siya Ag-Ag Chloride electrode.
agad-agad makakapasok kasi may mga
blockage or build up nga ng proteins
TRANSFORMERS 4
Blood Gas Analyzers: Measurement of pCO2

● Principle: Potentiometry
● Electrode: Severinghaus electrode
● It has an outer semipermeable membrane that
allows CO2 to diffuse and dissolve into an
internal layer of electrolyte, usually a
bicarbonate buffer.
https://www.youtube.com/watch?v=PSyR29yc-Tc&
feature=youtu.be
Kindly watch the video provided in the myclass for
the demonstration of how modern blood gas
analyzers are used in the laboratory
This is the illustration of Severinghaus electrode

wherein there is a presence of semipermeable
membrane that will allow our carbon dioxide to
diffuse and dissolve into an internal layer of
electrolyte, usually a bicarbonate buffer
Modern Blood Gas Analyzers Continuous Monitoring for pO2

● Routinely contain 3 electrodes ● Another method for the blood gas analysis
(“electrolytes” in the video) ● Uses transcutaneous (TC) electrodes placed
● Which provides a rapid, accurate, and directly on the skins of the patient
direct measurement of the three ● Commonly used for: neonates and infants
parameters: ● Non-invasive procedure
o pH In comparison with arterial blood sample
o pCO2 collection, that is an invasive method due to the
o pO2 use of a syringe. But for the TC electrodes it is not
The routinely used modern blood gas analyzers in invasive because it will only need to be placed or
the lab already have three electrodes that can attached to the skin of the patient so that we can
rapidly, directly, and accurately measure our pH, monitor for the pO2
pCO2, and pO2 Factors Affecting Blood Gases and pH
Measurement
1. Temperature
TRANSFORMERS 5
● (37 degrees Celsius +/- 0.1’ only) is HCO3 Bicarbonat 21-28 Actual
acceptable e; includes mmol/L bicarbona
● The temperature is the most IMPORTANT true serum; te is a
factor that can affect our blood gas and bicarbona 18-23 derived
pH measurement te, mmol/L measurem
● For every 1 degree Celsius increase in fever, bicarbona arterial ent
there may be: te , and calculated
o 3% INCREASED in pCO2 dissolved from the
o 7% DECREASED in pO2 free CO2. pH and
This is a recall question in the board exam pCO2 of
2. Elevated plasma protein concentration an
● pO2 parameter is mostly affected aerobically
● (1) Our PROTEINS may alter the diffusion of drawn
the gases and hinder electrode response arterial
● (2) Sensitivity of electrodes is related to the specimen.
thickness of the membrane Standard
o If the patient’s plasma protein is bicarbona
elevated, the proteins can block the te is
membrane, therefore the pO2 will derived
have a hard time diffusing in the from the
electrode Henderson
3. Bacterial contamination - pO2 is mostly affected -Hasselbac
● When bacteria are present, they can h equation
consume the oxygen. and
● Once they consume the oxygen, the pO2 indicates
levels may decrease the
● So the pO2 is mostly affected then, when it bicarbona
comes to bacterial contamination. te level in
4. Improper transport of blood specimen an
● Blood sample not transported on ice (or not oxygenate
chilled): pO2 changes rapidly than pH and d plasma
pCO2 specimen
○ In this case, when you do not at 98.6°F
transport our sample on ice during (37°C)
transportation in the laboratory, the and pCO2
most affected parameter is your
of 40 mm
pO2 as well.
Hg.
● Samples should be kept at room
sO2 Oxygen 95-100% Derived
temperature and analyzed immediately
saturation value
after blood collection (30 mins max; best is
of calculated
within 10 mins)
hemoglobi using sO2%
○ Note: you have 30 mins after blood
n. =
draw before you test the sample but
cO2Hb/(c
again, the 30 mins is the maximum
O2Hb +
range
cHHb) x
○ For best results, our sample should
100.
be tested within 10 mins only but 30
Calculate
minutes maximum is still acceptable.
d value
For best results, again, 10 mins is the
does not
recommended time of testing.
account
Calculated Parameters
for other
Calculated Description Reference Comments
hemoglobi
Parameters Range
ns or
TRANSFORMERS 6
actual so that we can compute the

pCO2. bicarbonate.
Oxyhemog ○ So this bicarbonate is not directly
lobin is measured, this is just calculated from
directly the parameters that you have
measured directly measured which is the pH,
using pCO2, and pO2 nga.
oximetry. ● Another calculate parameter is your sO2
P50 pO2 at Calculate ● You also have your P50 which is pO2 at
which d which hemoglobin is 50% saturated with
hemoglobi parameter oxygen.
n is 50% ● You also have your buffer base and the
saturated base excess.
with Take note that the base excess uses the values of
oxygen pH and pCO2.
Buffer base Total of all 44-48 Calculate Quality Control
anionic mmol/L d ● In any type of machine that you should use
buffers in parameter inside the laboratory, quality control is
the blood; ; should laging minemeasure at laging dapat
includes not be finofollow.
hemoglobi affected ● For the quality control for our blood gas
n, by analyzers, in general, we have minimum
bicarbona respiratory requirements for the QC or the quality
te, disorders. control.
inorganic ●
phosphate QC Minimum Requirements
, and ● 1 sample every 8 hours; and
proteins ○ We should test 1 sample every 8
with a hours
negative ● 3 levels of control (acidosis, normal,
charge. alkalosis) every 24 hours.
Base Number of Calculate ○ Very very important
excess millimoles d ○ You should have 3 levels of controls
of strong parameter to control or measure acidosis, the
acid ; should normal rangel and alkalosis which is
needed to not be performed at least once every 24
titrate a affected hours.
blood by
sample to respiratory
pH 7.4 at disorders.
pCO2 40
mm Hg.
Notes:
● For the calculated parameters, this can be
based on different types of equations such ● The 3 levels of controls are just like vials
as the Henderson-Hasselbach equation (picture), which have level 1, 2, 3 which
wherein another calculated parameter, we corresponds to acidosis (level 1), normal
have your bicarbonate, dun sya binase. (level 2), alkalosis (level 3). Wherein if we test
○ Kumbaga kasi, sabi nga dito, when it using the machine the levels of control
you calculate for the pH and pCO2, should be within the normal range or
makikita na natin sya kasi we are reference range.
directly measuring it. ● For instance, if it is acidosis (level 1) it should
○ And that value, we can use po in be measured by our machine in which it is
the Henderson-Hasselbach equation on its acidic range. While for normal (level
TRANSFORMERS 7
2) should be measured by our machine the National reference lab for CC or

wherein it is on normal range. Lastly for it should be on the acceptable
alkalosis (level 3) should be measured by range of the measured unknown
our machine wherein it is in alkaline range. sample.
Calibration ● The different sections of the laboratory such
● Every instrument used inside the laboratory as hematology, microbiology have different
should be regularly calibrated. National reference labs.
● Each analyzer depending on the Additional Notes
manufacturer has recommended ● Blood gas results are affected by the gas
calibration protocols that include specific mixture the patient is breathing and by the
calibration materials and frequency. patient's body temperature.
● Each analyzer has different manufacturers ● The total CO2 in arterial blood (Plasma or
and has different methods for calibration. serum) is equal to HCO3 in arterial blood.
● Normally, two different gas mixtures with ● Calculation of base excess uses pH and
known pCO2 and pO2 levels are used for pCO2 values
calibration. ● Blood gas results should be back to the
Proficiency Testing physician preferably within 10 minutes after
● Participating in external, interlaboratory draw to obtain maximum benefit from
surveys or proficiency testing programs is them.
another essential component of ensuring
the quality of blood gas measurements.
● Inside the laboratory we are performing
internal quality control that will reflect on
what is inside. Our test/results should go
inside with the other laboratory. Hence, you
should perform interlaboratory service or
you should participate in interlaboratory
surveys or proficiency testing programs.
● Ongoing comparisons of results through
proficiency testing help ensure that
systematic (accuracy) errors do not slowly
increase and go undetected by internal QC
procedures. There can be a possibility that
our internal QC which we run everyday,
nandun siya sa loob ng range ng control.
However, when you test it in comparison
with other laboratory baka magkaroon ng
deviation kaya dapat nichecheck natin
yung ganun para alam natin if quality
results yung nilalabas ng ating machine. So,
we can perform proficiency testing or our
laboratory can participate on proficiency
testing program.
● National reference lab for CC: Lung Center
of the Philippines
o What they do para malaman natin
or mag go inside yung ibang
laboratory is that magpapadala yan
ng unknown sample (without
measurement) at yung sample na
yun irurun natin sa mga machine
natin sa loob ng lab. At yung
magegenerate na result should go
inside with the reference range from
TRANSFORMERS 8
6 Clinically Significant Trace Elements Clinical Chemistry 2 (LAB)
Clinically Significant Trace Elements ➢ Ang ating mga enzymes ay

Overview of the Discussion importante, essential sila kapag
1) Introduction – will give the learners an tumutulong sila sa mga enzymes
overview of the module natin para mapabilis ang isang
2) Biologic functions of trace elements – chemical reaction.
important functions of trace elements in the ➢ And so we call this
body enzyme/metals/trace elements as a
3) Specimen collection and processing – co-factor.
provide knowledge to learners how a o Trace elements are found in such small
specimen is to be used, collected, and quantities in mg/L such as iron, copper, zinc.
stored most specifically for trace elements. ➢ Kapag sinabi nating iron, this is
4) Methods and Instrumentation – learners will involved in the regulation of our
be able to determine the important RBCs, specifically with the oxygen
laboratory instruments to evaluate the content that is found in the
presence of essential or toxic elements. hemoglobin.
• The following instruments will be ➢ Same as through with copper.
discussed: Atomic Emission Tutulong din ang copper sa
Spectroscopy, Atomic Absorption pagpaprocess ng ating iron
Spectroscopy, Inductively Coupled component na makikita sa RBCs that
Mass Spectroscopy is found also in the hemoglobin
5) Interferences – will equip the learners of the synthesis.
commonly encountered interferences in the ➢ Zinc. Lagi nating nakikita itong zinc
laboratory and what to do when these na to sa ating mga vitamins.
problems arises. Because one of the important
6) Elemental speciation – involves the function of our zinc is to boosts our
determination of the chemical forms of an immune system.
element in a particular matrix. ➢ So these are examples of essential
7) Essential and non-essential trace elements – trace elements.
will tackle the essential trace elements o Ultratrace elements: these are extremely
found in the body. small amount in our body less than
Introduction micrograms per liter.
Essential Trace Elements Non-Essential Trace Elements
o It is considered essential if a deficiency o Of medical interests because many of them
impairs biochemical or functional process are toxic.
and replacement of the element correct this ➢ Non-essential because they are of
impairment. medical importance. And many of
➢ Kapag nawala sila sa katawan natin, the non-essential trace elements,
magkakaroon tayo ng deficiency or once they enter our body, will
magkakaroon tayo ng sakit. become toxic or pwede itong
➢ Pwedeng masabi natin na essential magcause ng sakit sa atin.
tong bagay na ito kapag pinalitan or o Conditions that can result in deficiency of
by means of substitution, mawawala several trace elements might be because
itong ating sakit. of:
➢ So we call this element an essential • Decrease intake
trace elements. • Impaired absorption
o These are elements we need to have in our • Increased secretion
diet. • Genetic abnormalities
➢ Makikita rin natin ito sa pagkain. o Minsan nagkakaroon tayo ng trace element
These are important because they deficiencies dahil nakulangan ka sa diet. For
are also found in our diet. example there are some trace elements
o Associated with an enzyme or other protein that are found in meats pero paano kung for
as essential component or co-factor. example ikaw ay isang vegetarian. So
TRANSFORMERS 1
pwede kang magkaroon ng kakulangan sa ● Some metallic ions, such as iron and
trace elements na yun dahil decrease ang copper, participate in oxidation-
intake mo. It might also be because of reduction reactions in energy
problems like in impaired absorption. For metabolism
example, in the small intestine we have the ○ One of the characteristics of our
duodenum, jejunum, ileum. Paano kung metal is that they are protons,
halimbawa mayroon kang problem dito sa positively charged ions
iyong gastrointestinal tract. And therefore, it (cations). Therefore, they give
would lead to a deficiency in a particular electrons or donators of protons
trace element. Thus, there would be an or electron donors. Nonmetal,
impaired absorption. on the other hand, shares
electrons
Trace and Toxic Elements ○ One of the biological functions
● The essential elements are often associated of our trace elements is that
with an enzyme (metalloenzyme) or they participate in the redox
another protein (metalloprotein) as a reactions which are important
cofactor for energy metabolism and this
○ Most of our essential elements are is with regards with the
metals, when metals are bounded production of ATP
with enzymes, they are called 3 Role in Oxygen Transport
metalloenzymes and they give an ● Iron, is a constituent of hemoglobin and
important functions with regards to myoglobin, also plays a vital role in the
our chemical reactions transport of oxygen
○ Essential elements may be a protein, o Iron is very important because it is part
if important metals are included of the hemoglobin synthesis where it
with the presence of metal and the carries oxygen and it is found in the
protein, then our metal will act as a hemoglobin. Iron can also be seen in
cofactor or otherwise known as a myoglobin which is found in the muscles
metalloprotein and play a vital role in the distribution of
● Deficiencies typically impair one or more oxygen in the different parts of the
biochemical functions body.
● Excess concentration are associated with • Metal activated enzyme - when the metals
at least some degree of toxicity help increase the activity of the enzyme
Although trace elements, such as iron, copper, and
zinc are found in milligram per liter or parts per Distinction between the metalloenzymes and
million concentrations, ultratrace elements, such as metal activated enzymes
selenium chromium, and manganese, are found in
microgram per liter or parts per billion
concentrations
Biological Functions of Trace Elements
1 Catalysts
● Trace elements function primarily as
catalysts in enzyme systems
o When we talk about catalysts, they
speed up chemical reactions. Now,
what are the characteristics of trace
elements? They are what we call as
metalloenzymes and they help speed
up chemical reactions
2 Participate in the Oxidation-Reduction for • Definition
energy metabolism o Metalloenzymes are enzymes that are
tightly bound to the metal ion
TRANSFORMERS 2
Metal activated enzymes, the metals are

o leads to the creation of ATP in a
enzymes that have increased activity complete system named oxidative
because of the presence of the metal ions phosphorylation
• Attachment of the cofactor o It occurs in mitochondria in both
o A metalloenzyme is firmly bounded cellular respiration and
to a metal ion and this is the cofactor photosynthesis
o For the metal activated enzyme they o One of the most important function
have no firm bound to the metal ion of the trace elements, wherein they
or it is not firmly bounded to a metal are involved in the processing of
iron energy of the cells and that is called
• Requirements for the activity the electron transport chain
o For the metalloenzymes, it requires o The powerhouse of the cell is the
either one or two metal ions that is mitochondria and this is where we
bound to the specific region of the create our adenosine triphosphate.
enzyme. Kailangan naka bound The essential trace elements help in
talaga sila doon sa enzymes, ung creating and processing, to have an
mga metals enough energy for each cells in the
o For the metal activated enzyme it body.
requires high concentration of metal o The electrons come from breaking
ions around them. Hindi niya down organic molecules, and
kailangan na laging nakabound or energy is released.
nakaattach sa ating enzymes, ung o Iron is an important metal that
metal, kahit maraming contents lang participates in electron transport
• Examples chain
o Metalloenzymes – thermolysin, Specimen Collection and Processing
dioldehydrase, cytochrome c • Specimen collection
oxidase peroxidase, and arginase o Specimens for the analysis of trace
o Metal activated enzymes - elements must be collected with
pyruvate kinase and scrupulous attention to details such
phosphotransferases as anticoagulant, collection
apparatus, and specimen type, this
usually includes the (urine, serum,
plasma, or blood).
o Because of the low concentration in
biologic specimens, extraordinary
measures are required to prevent
contamination of the specimen.
o This includes using special (specific)
sampling and collection devices,
specially cleaned glassware, and
water and reagents of high purity.
Dito makikita natin na dapat
macontrol natin ung mga
environmental factors na pwedeng
magcause ng inaccurate results, as
we all know our specimens are
“trace” element, ibig sabihin sila ay
• The biological functions include also the: napakakonti or minute, na kapag
Electron transport chain merong mga uncontrolled variables
o is a series of four protein complexes na hindi maiiwasang makikita sa
that couple redox reactions creating environment such as the air, or dust
an electrochemical gradient that particles or ung mga iba na
TRANSFORMERS 3
pwedeng constituents ng ating mga ceiling tiles natin which can cause
test tubes ay pwedeng magcause inaccurate results
ng false or inaccurate results sa ating (3) Carefully controlled air flow to
mga specimens. Kaya dapat bigyan minimize particulate contamination
ng scrupulous attention etong mga – Some of the essential elements
ito. can be acquired airborne and
o The selection of needles, evacuated therefore we should control the
blood collection tubes, airflow
anticoagulants and other additives, (4) Disposable booties, worn over shoes
water and other reagents, pipettes, and particle monitoring equipment
and sample cups must be carefully should be done
evaluated for use in trace and Commonly Used Instrumentation for Trace Elements
ultratrace analyses. • Specimen for Trace Elements (TE) analysis
o It is also recommended that In the must be collected in a manner as to
laboratory environment, placing the minimize contamination of other TE that are
trace elements laboratory in a not found in the specimen
separate room incorporating • What we need here are the following:
rigorous contamination control ✓ Royal Blue EDTA Tube – Ideal in collecting
features (Napakaimportante na as the TE
much as possible kung pwede sana ✓ Royal Blue TE Serum Tube
ilagay sila sa separate room para ✓ Stainless needles
mas controlled natin ang
environment) , such control features
contains placing sticky mats at doors
(some of the special trace elements
maybe acquired through gas or air),
non-shedding ceiling tiles, carefully • TE specimen is collected FIRST - In collecting
controlled air flow to minimize specimens, pinaka unang kinukuha yung mga
particulate contamination, pangtrace element prior to the other tests
disposable booties worn over shoes
and particle monitoring equipment METHODS AND INSTRUMENTATION
should be done Atomic Absorption Spectroscopy (AAS)
• It is recommended that in the laboratory • The commonly used instrumentation for trace
environment that we: elemenlents and toxic metal analysis is the
o Place the trace elements laboratory in a Atomic Absorption Spectroscopy.
separate room incorporating rigorous • AAS is also known as Atomic Absorption
contamination control features spectrometer or Atomic Absorption photometer
▪ Importante na ilagay natin trace – They can either make use of a flame or without
elements sa separate rooms para mas a flame
controlled natin ang environment • Atomic Absorption Spectroscopy can either be:
natin. Such control features are: 1. Flame atomic absorption spectroscopy
(1) Placing a sticky mats at the doors – (FAAS) – Uses a flame
Some trace elements may be 2. Graphite furnace atomic absorption
acquired through glass and air. As spectroscopy (GFAAS) atomization –
much as possible macontrol natin Flameless
and ito and one way is placing Atomic Emission Spectroscopy (AES)
sticky mats at the doors • AKA Atomoc Emission Spectrophotometru
(2) Non-shedding ceiling tiles – Baka • Also alled Emission Flame Photometry
mahulog ibang trace elements na • Also useful for some elements
nasa paligid natin kagaya ng mga • Used together with inductively coupled plasma
nasa kisame natin or yung mga atomic emission spectroscopy (ICP-AES) –
Makikita ito sa tests or instruments that are used
TRANSFORMERS 4
in determining the presence of TE; Inductively Couples Plasma Mass Spectroscopy

pinagsasabay AES and Inductively coupled (ICP-MS)
plasma emission spectroscopy. These are for • More sensitive
atomization and excitation. • Can tests wide range of elements
• Madalas pinipair si AES kay ICP-AES • Relatively free from interferences
More preferred than the other tests
• Depende sa ating trace elements na
babasahin nya, pipiliin ng mochromator
kung sino lang yung gusto nating tignan na
trace element. From there mag seselect po
ang monochromator at isa lang po and
lalabas sa detector, the rest or other
elements that are not read will not be
detected and so therefore ang lalabas lang
po dito ay isang element lang.
• When a metallic salt is burned in a flame, the Three most important COMPONENTS of AES
heat energy that the atom absorbs drives one or
more of the electrons out of their usual orbits –
Pagna ignite using a flame, it will be excited. This
will make the electrons more excited thus they
will go out of their orbits
• As the excited electrons return to a lower or
ground electronic state, they emit
electromagnetic radiation → Electromagnetic
radiation is read
• The heat energy absorbed and the light emitted
are now the characteristics of the atom under
consideration – Kung ano gusto natin mabasa o
gusto iabsorb nung particular TE, yun yung
makikita natin under the detector
o Remember that each metal has their own
spectrum showing emission at E 24:05 – 30:05
characteristic wavelength
• Take a look at the picture shown above
o Pag yung metallic salt nag-undergo ng ● The three most important components of atomic
flame, it will excite the electrons emission spectroscopy (AES) are as follows:
o As they excite the electrons, they will going 1. Source - in which the sample is atomized at
to emit electrical charges then baba sa sufficient temperature to produce an excited-
ground state state species. Those species will emit radiation
o Nung naexcite electrons, they emitted upon relaxation back to the ground state.
electromagnetic radiation which is read at - When metallic salt is ignited the
the monochromator → Pipiliin ng electrons will be excited and bababa
monochromator yung gusto mong sila in a ground state. The important
mabasang TE. Monochromator will select thing here is the GAS.
one TE at isa lang ang lalabas sa detector ● Gas - source of the flame energy. It
o The other elements that are not read or the includes a mixture of hydrogen and
elements that are not needed will not be oxygen gas, natural gas, acetylene, and
detected → Isang element lang lalabas propane in conjunction with air and
then that is the corresponding oxygen. The flame temperature should be
characteristic wavelength that will be read held constant because it affects sensitivity
by our detector and response to flame photometers.
TRANSFORMERS 5
- Nebulizer - sample is introduced into the

2. Wavelength selecting device chamber using a nebulizer that converts
(Monochromator) - the spectral liquid into a fine spray.
dispensation of the radiation and separation - In AES, both atomic and ionic excited states
of the analytical line from other radiation. It can be produced (depending on the
isolates specific wavelengths of interest. element and the source), which leads to the
- Upon the excitation of electron and production of complicated emission
then they will be on ground state, spectra.
mag eemit ito ng electromagnetic - Thus kapag naka gawa na sya ng spectra,
radiation at mababasa ito ng eto po yung ating mga fine line naten.
monochromator, eto ngayong ang
pipili kung anong wavelength of
interest ang kukunin naten.
3. Detector - Measurement of radiation
intensity. detectors require water and
standards to establish thermal equilibrium
before measurements are taken.
- Makikita natin sa detector na isa lang
babasahin nya na nanggaling sa ating
monochromator.
Typical Fuel Gases Used in AES
● The most commonly used sources in AES are
flame and inductively coupled plasma (ICP).
● Flames are capable of producing temperatures
up to 3,000 K. - The “emission spectrum” of an element is
● Typical fuel gasses include following: composed of a series of very narrow peaks
- Hydrogen (sometimes known as “lines”) with each line
- Acetylene at a different wavelength and each line
- Oxidant gasses matched to a specific transition.
● Air - Example of emission spectra (picture above)
● Oxygen that are found our atomic emission
● Nitrous oxide spectroscopy
Mixing Chamber Burner for Flame Atomic - Each element has their own characteristics
Absorption emission spectrum.
- Example: Sodium can be detected by
tuning the monochromator to a wavelength
of 589
- Ideally, each emission line of given elements
would be distinct from all other emission lines
of other elements.
- However, there are many cases where
emission lines from distinct elements overlap
resulting in interference.
- The choice of interference free-wavelength
(atomic or ionic line) may be challenging
- While there are possible wavelengths for a
given element, wavelengths producing
suitable analytical performance, such as
- The gasses are combined in specially limit of quantification, freedom from
designed mixing chambers. interference, and robustness, are selected.
Interferences in AAS
TRANSFORMERS 6
● A common problem in Graphite Furnace 2. MASS ANALYZER: component that takes

Atomic absorption spectroscopy GFAAS is that ionized masses and separates them based
analyte volatility depends on the molecular on charge to mass ratios and outputs them
form of the analyte and the sample matrix. to the detector where they are detected
● To overcome these limitations, chemical and later converted to a digital output
modifiers (palladium nitrate, magnesium nitrate, 3. ION DETECTOR: measures the value of an
or a mixture of both) are frequently added to indicator quantity and thus provides data
samples, calibrators, and controls. for calculating the abundance of each ion
● Chemical Interferences present
- It occurs when flame cannot dissociate PROCESS
with the sample • The ARGON PLASMA induced by
● Ionization interferences commercial ICP instruments (both ICP-AES
- It occurs when sample in flame become and ICP-MS) generates temperatures
excited ranging from 6,000 to 10,000 K (Kelvin) and
● Matrix Interferences serves several purposes.
- it occurs when light absorption is enhanced • First, it dries and then vaporizes the droplets
by organic substances produced by the nebulizer.
- As well as when the sample is evaporated in • This step is followed by atomization of any
the flame and produces solid particles. molecular species.
Inductively Coupled Plasma Mass Spectrometry • Finally, atoms are thermally ionized, at
(ICP) which point they are ready for introduction
• ICP-MS: state-of-the-art analytical technique into the mass spectrometer.
for elemental analysis
• the term “plasma” in ICP refers to an IONIZED
GAS (Typically argon), in which a certain
proportion of electrons are free
• measures the MASS-TO-CHARGE RATIO
o molecular mass divided by ionic charge
[m/z] of selected analyte ions
o Where: m means charge; z means ions
o m/z is the unit used for ICP-MS
(Simplified schematic of ICP-MS instrumentation.

ICP, inductively coupled plasma; RF,
radiofrequency; MS, mass spectrometer)
• Nearly, all ICP torches consist of three

concentric quartz tubes surrounded by a
coil carrying radiofrequency (RF) power.
• The middle tube of the torch carries the
argon (Ar) that forms the plasma.
• Quantitative analysis for clinical samples is
(picture of ICP-MS) best performed with the use of an internal
standard.
COMPONENTS OF ICP-MS • All patient samples, calibrators, and
1. ION SOURCE: part of mass spectrometer controls are diluted with an internal
that ionizes the material under analysis (the standard, usually a solution of an
analyte) uncommon element such as YTTRIUM.
a. the ions are then transported by Quadruple Mass Spectrometry
magnetic or electric fields to the
mass analyzer
TRANSFORMERS 7
• The typical mass spectrometer most • The magnetic field is adjusted to allow only
commonly used for ICP-MS is a quadrupole ions of a selected m/z to reach the
mass spectrometer. detection system at any given point in time.
• The analyzer consists of four parallel • A second device known as an electrostatic
conducting rods arranged ion a square analyzer corrects for certain nonideal
array. effects, allowing the instrument to achieve
➢ Applying for the RF (radio high resolution.
frequency) and constant (DC) • Commercially available high resolution ICP-
direct current voltages to the rods, MS instruments are capable of a resolution
the instrument can be tuned so that of 10,000 (10% valley)
only ions of specific would be • However, magnetic sector instruments are
determined. not able to resolve elemental isobaric
➢ m/z ratio can pass through the interferences which would require solution
device to reach the detector. much higher than 10,000
Where: m (mass) means charge,
and z means ions.
➢ This type of instrument tends to be
relatively simple to use and
maintain, but the resolution (the
ability to discriminate between
closely spaced m/z values) is
limited, being able to well resolve
peaks separated by one m/z unit
but not able to resolve peaks
separated by a small fraction of an
m/z unit.
Relative Advantage and Disadvantage of Main

Techniques for Elemental Analysis
Flame GFAA ICP-AES ICP-MS
AA
Sensitivity Moderate Excellent Moderate Excellent
Selectivity Excellent Good Poor Good
Elemental Moderate Good Good Excellent
coverage
Speed of Fast Slow Fast Fast
High Resolution Mass Spectrometry one
• Other ICP-MS instruments incorporate high- analyte
resolution mass spectrometers. Multi- No No Yes Yes
• These are usually “double facing sector element
capabiliti
field” instruments. es
• Such instruments separate ions of different Initial cost Low Moderate Moderate High
m/z values via deflection in a magnetic of
field, with ions of greater m/z being instrument
deflected to a lesser degree than those of Cost of Very Low Very High Low Moderate
Consuma
lower m/z. bles
Ease of Excellent Poor Moderate Moderate
Operation
TRANSFORMERS 8
Interferences information, to mathematically correct for

1 Are factors that hinder or impedes a interferences.
procedure. • Another approach interposes a reaction
2 In general, the interferences in the element cell or collision cell between the main ion
analysis are either classified as spectroscopic lenses and the mass analyzer.
and nonspectroscopic. • A small amount of a gas such as helium or
Spectroscopic: Spectral Overlap ammonia introduced into the cell removes
Spectral interferences generally result from a interferences, either by chemical reaction
spectral overlap with the spectrum of the target or by an energy filtering process, using the
analyte. fact that polyatomic species, with their
larger collisional cross sections, lose energy
faster than atomic ions.
• High-resolution mass spectrometers provide
a way to remove interferences.
Spectroscopic: Isotopes of Nearby Elements
• For example, in AAS certain molecular

species may have broad absorption spectra
that may overlap the line spectra of the
elements of interest, leading to false
elevations of the target concentrations.
• A much less common occurrence would be
for the absorption spectrum of one element
to overlap with that of another.
Now, paano po natin ma dedeal ung mga • Kung titingnan po natin yung example
ganitong interference? natin dito, andyan po yung tin at tsaka si
• Various strategies are used to deal with cadmium.
spectral interferences in AAS. • Makikita mo yung isotope po ng tin natin is
• A continuum source background corrector 114 same with the isotope of our cadmium
may be included in the instrument design at which is 114.
the cost of some instrument complication. • So pwede po silang magoverlap ulit.
• Another alternative is the Zeeman • This will again become an interference.
background correction, which relies on
shifting the atomic spectral line by the A second source of spectral interferences in ICP-MS
application of magnetic field. arises from nearby elements in the periodic table.
• For example, tin (Sn) and cadmium (Cd)
Spectroscopic: Polyatomic ions
both have isotopes at 114 Da (atomic mass
Several approaches are used to deal with
unit), so they could potentially interfere with
polyatomic interferences in ICP-MS.
each other if the instrument is set to
• One applies algebraic equations, together
measure 114 m/z.
with relative isotopic abundance
TRANSFORMERS 9
o Pwede po silang mag-interfere sa o Anything that could interfere with

bawat isa with regards to their charge atomization of the sample could be
ion ratio. classified as a nonsprectral interference.
• This can usually be handled by using a o For example
different isotope for the analysis. o In AAS (atomic absorption
o Tingin tayo ng isotope na wala silang spectrometry), a flame may not be hot
pareho para maiwasan ang mga enough for efficient atomization.
interferences. o Kung hindi enough yung flame, hindi
• For example, cadmium also has an isotope magiging gas particles, hindi magiging
at 111 Da that is free from isobaric atom yung mga sample, therefore, it
elemental interferences. would interfere with the reading of the
Non-spectroscopic: Matrix Interferences result.
o One of the most common non- o Difference in sample viscosity between
spectroscopic interferences is the matrix standards and unknown samples,
interference. resulting in differing rates of sample
Matric interferences involve the bulk physical o AES, anything that would prevent the
properties of the sample to be analyzed. efficient excitation or emission of
o Pag dating sa matrix, ito yung attributes sppectral lines used for the analysis
or qualities ng ating specimen. would constitute a nonspectral
o Vicious ba siya, diluted ba sya, o interference
watery. Elemental Speciation
o Itong mga to ang kino-consider natin ⚫ The toxixity of elements may depend on their
that can cause interferences habang chemical forms
kinukuha natin ang trace element. ⚫ There are elements that has a toxic and
o The aqueous samples may behave nontoxic form
differently than organic and biological ⚫ Ex.
specimens, depending upon the ◼ Arsenic
technology used and the analyte of - arsenobetaine (nontoxic)
interest. - methylated forms of arsenic are intermediate
o The properties of significance as in toxicity, and inorganic arsenic, such as As(V)
viscosity, presence of easily ionized and As(III) are highly toxic
elements, and presence of carbon, ◼ Medical evaluation of patients
o Matrix matching of the calibrators, - can be important to know whether an
controls, and specimens helps to elevated arsenic level is due to relatively
overcome matrix interferences. innocuous forms (arsenobetaine) perhaps from
o Kung alam natin na magkaiba pala a seafood meal ingested up to 3 days before
yung ating viscosity sa gagamitin na the specimen collection, or by dangerous
control or standard, then mas forms such as inorganic arsenic
maganda na pareho sila. ◼ Concentrations of methylated forms
o Kaya tinatawag sila na matrix - may beuseful information for monitoring recovery
matching. from toxic exposure
o In order to avoid interfering with the Hyphenated Techniques: LC-ICP-MS
physical attributes or physical properties Liquid chromatography, inductively coupled mass
of our specimen, mas maganda na spectroscopy
pareho sila para mas maging accurate ⚫ Allow for speciation determinations
and makuha nating result. ⚫ Combination of two or more complementary
o Dilution of the specimens helps minimize analytical techniques used to measure the
matrix effect, but it is only applicable to specific form of an analyte
certain analytical techniques and to ⚫ Classic example is liquid chromatography-ICP-
the determination of analytes with MS (LS-ICP-MS)
higher concentrations. ◼ The sample is injected into a liquid
chromatograph which separates the
TRANSFORMERS 10
different chemical forms of the analyte

producing a characteristic retention time
◼ The eluting sample is continuously analyzed
by a mass spectrometer
◼ The retention time partially identifies the
analytes, and the mass spectrometer
further identifies the element
Hyphenated Techniques: AAS
⚫ Atomic Absorption Spectroscopy
◼ May be a sample for mass spectroscopy in
elemental speciation schemes ● Voltammetric methods, such as anodic stripping
⚫ Methods for elemental speciation are voltammetry (ASV) and adsorptive stripping
becoming more common, esp in Europe voltammetry, can be used in determination of
⚫ Despite the chemical matrices being among selected metals and are the basis for some point-
the most difficult for speciation, several of-care devices.
applications are reported; some among them ○ Point of care devices are new generation of
are as follows: more compact instruments that are more
◼ Arsenic speciation in urine by LC-ICP-MS automated and user friendly.
and HG (hybrid generation) - GFAAS ○ They are available for bedsite testing,
◼ Copper in urine by size exclusion screening projects, wellness centers,
chromatography (SEC)-ICP-MS emergency room, operating rooms and clinics
◼ Copper in red blood cells (RBCs) by SEC- ○ Most POC chemistry analyzers uses samples
ICP-MS that are less than 50 microliters in volume with
◼ Lead in blood by gas chromatography turn around time in less than 10 minutes.
(GC)-GFAAS
◼ Selenium in serum by SEC-GFAAS ● Example of AAT (Ion Chromatography):
◼ Zinc in urine by anion exchange
chromatography ICP-MS
○ Copper in urine by size exclusion
chromatography (SEC)-ICP-MS
○ Copper in red blood cells (RBCs) by SEC-ICP-MS
○ Lead in blood by gas chromatography (GC)-
Graphic Furnace Atomic Absorption Spectroscopy
(GFAAS)
○ Selenium in serum by SEC-GFAAS
○ Zinc in urine by anion exchange
chromatography ICP-MS
*Majority of the Hyphenated techniques makes use

of a spectroscopy and coupled with that of a ● Ion chromatography can be used for the
chromatography; usually liquid chromatography determination of copper, iron, and zinc in blood,
and gas chromatography, including the size serum, and plasma and for the determination of
exclusion chromatography. Together with another zinc in urine.
instrument, either atomic absorption or mass ● Gas chromatography–mass spectrometry (GC-
spectroscopy (MS). MS) is capable of determination of trace elements
Alternative Analytic Techniques such as cadmium, chromium, cobalt, copper,
lead, and selenium in urine, copper in serum, and
● Example of AAT (Anodic stripping voltammetry):
lead in blood.
● The methods accommodating direct analysis of
solid samples, for instance, laser ablation –
Inductively Coupled Plasma – Mass Spectroscopy
(LA-ICPMS), are gaining recognition for selected
clinical applications.
TRANSFORMERS 11
• Example of Glass 3. Mercury

Chromatography Definition of Terms
• Essential parts: Copper
✓ Carrier Gas Tank o Sequestration – removal or isolate from
✓ Pressure Regulator the rest
✓ Flow Controller o Ceruloplasmin
✓ Sample Injection Port o An alpha 2-globulin that binds to 95% of
✓ Column Copper
✓ Detector o It is a ferroxidase enzyme that in
✓ Column Oven – contains the Column humans is encoded by the CP gene
✓ Outlet o Major copper-carrying protein in blood
✓ Processor Signal o Plays a role in Fe metabolism
Essential Trace Elements o Transporter and nagdadala ng Cu from
These are all important to the body because it is different organs of body
needed/essential for processes such as: o 2 trace elements ang tinatransport and
• Enzyme process hatid sa different organs (mostly Cu
• Production of O2 for Hgb & Fe in the RBCs followed by Fe
• Responsible in helping the mitochondria in
electron transport chain Sources of foods:
o Oysters and other shellfish, whole grains,
COPPER: beans, nuts, potatoes, and organ meats
- Is an essential trace element present in all (kidneys & liver) are good sources of Cu
body tissues. Dark leafy greens, dried fruits such as prunes,
- Small amount is needed for the body to cocoa, black pepper, and yeast – sources of
function, but the body cannot make its own copper in the diet
copper.
- It is important to get this from food. o Most abundant trace element in the
human body.
IRON: o It participates in cellular respiration
- Is considered an essential mineral because o Helps in processing the most important
it is needed to make Hgb in certain part of energy which is the Adenosine
blood cells. Triphosphate.
SELENIUM: o DNA and RNA reproduction
- Is an essential component of various o Maintenance of cell membrane integrity
enzymes and proteins, called o Sequestration of radicals
Selenoproteins, that help to make DNA and o Tinatago ang free radicals
protect against cell damage and infections. o Free radicals- cells that lacks electron
o Since free radicals lack electrons, it will
ZINC: get electrons from normal cells, as a
- Trace mineral – body only needs small result the normal cell will have
amounts, and yet it is necessary for almost incomplete electrons and will undergo
100 enzymes to carry out vital chemical oxidation.
reactions. o When cells oxidize, it will result stress on
- Major player: creation of DNA, growth of the cells which will cause different
cells, building proteins, healing damaged disorders in the body, aging and even
tissue, and supporting a healthy immune cancer formation
system (it is stronger if Zinc is combined w/ o Exposure to different environmental
Ascorbic acid). factors could result to different disorders.
Vitamin C is much stronger if combined with Zinc o Copper is distributed in the liver, wherein
Non-Essential Trace Elements it has the highest concentration.
1. Arsenic o The copper released from the liver is
2. Lead attached mostly to Ceruloplasmin.
TRANSFORMERS 12
Ceruloplasmin
o - The plasma that the instrument is
Transport protein
o pertaining to is the argon
Acts as mobilization of iron in the storage
o • Quantitative analysis for clinical samples is
sites. best performed with the use of an internal
o It functions as peroxidase enzyme during standard
the ferrous- ferric conversion of iron. • All patient samples, calibrators, and controls
CLINOCO PATHOLOGIC CORRELATION are diluted with an internal standard
- Internal standard – usually a solution of
Copper deficiency an uncommon element such as yttrium
o It I observed in premature infants and - Yttrium – is the frequently used calibrator
undernourished children in the quality control of the instrument
o It is a contributing factor in Osteoporosis QUADRUPLE MASS SPECTROMETER
and Cardiovascular diseases. • The typical mass spectrometer used for ICP-
Menkes Disease MS is a quadrupole mass spectrometer
• The analyzer consists of 4 parallel
conducting rods arranged in a square array
- Applying radio frequency (RF) and
constant (DC) or direct current voltages
to the rods, the instrument can be turned
so that only ions of a specific will be
determined
o “Kinky hair syndrome” - Pwedeng galawin yung radio frequency
o There is a propound decrease in and current voltage para kung ano lang
ceruloplasmin levels and diminished yung gusto natin kunin na trace
concentration of copper in hair. element, yun lang yung kukunin nya
o Presence of truncal hypotonia (loss of • m/z ratio can pass through the device to
the muscle tone *refer to the picture above) reach the detector, where;
o The one responsible for generating - m means mass (charge nakalagay sa
Menkes protein is the ATP7A gene. ppt)
o Menkes Disease is also described as - z means ions
rapid brain atrophy predisposing to - this is frequently used lalong lalo na
subdural hematoma. pagdating sa mass spectrometer
o Spares kinky hair (unevenly distributed) • this type of instrument tends to be relatively
simple to use and maintain,but the resolution
Wilson’s Disease (the ability to discriminate between closely
spaed m/z values) is limited, being able to
well resolve peaks separated by one m/z
unit but not able to resolve peaks separated
by a small fraction of an m/z unit
- relatively simple to use and maintain –
kasi nagagalaw yung radio frequency
and direct current voltage doon sa
gusto nating madetermine na specific
o Copper toxicity ion
o Increased tissue and serum levels of - limitation: the ability to discriminate
copper. between closely spaced m/z values or
o Acute copper poisoning may be caused charge and ion, is limited.
by ingestion of excess copper, ➢ Pagdating sa resolution, medyo
fungicides containing copper sulfate or limited yung mass spectrometer
exposure to industrial sources. Nahihirapan syang mag-discriminate closely kapag
• The middle tube of the torch carries the sobrang dikit na ng charge and ion values,
argon (Ar) that forms the plasma nahihirapan na yung quadruple mass spectrometer
TRANSFORMERS 13
• with ions of greater m/z being deflected to

a lesser degree than those of lower m/z.
• The magnetic field is adjusted to allow only
ions of a selected m/z to reach the
detection system at any given point in time
• A second device known as an electrostatic
analyzer corrects certain nonideal effects,
allowing the instrument to achieve high
resolution
• Commercially available high resolution ICP-
MS instruments are capable of a resolution
of 10,000 (10% valley)
• So yung maganda dito, double focusing
sya, unlike doon sa quadruple mass
spectrometer natin. Ito, pwede din syang
magalaw, meron din syang pang-turn doon
sa gusto nating makita, at the same time,
meron din syang isa pa na tinatawag na
second device, which is known as
electrostatic analyzer
Diagram:
Iron
• It has a source, it has a non-resonant ions,
and we also have the resonant ions, o It is a measurement of great value in detecting
followed by the detector iron deficiency anemia because it occurs early
• Yung importante na napapalitan/naadjust in its development.
natin are yung dc and ac voltages or yung o Increased serum iron ferritin is observed in the
radio frequency and direct current voltages. following conditions:
Importante na na-aadjust ito based on the • Fever
desired trace element that we want to get • Acute infection
• Rheumatoid arthritis
HIGH RESOLUTION MASS SPECTROMETRY • Viral hepatitis
o As shown in the picture, pag tinatago natin si
iron nandyan si ferritin. Ngayon pag gusto
naman I distribute yung iron palabas gagamitin
natin ay si transferrin.
o Transferrin – is a transport protein that will
transfer/move the iron into the circulation.
SOURCES OF FOODS
• nuts
• dried fruit
• pasta and bread
• iron fortified bread and breakfast cereal
• legumes (mixed beans, baked beans, lentils,
chickpeas)
• dark leafy green vegetables (spinach, silver
beet, broccoli)
• Other ICP-MS instruments incorporate high- • oats
resolution mass spectrometers • tofu
• These are usually “double focusing sector • spinach
field” instruments (double facing sector field • raisins
instruments sinabi ni maam) • apricot
• Such instruments separate ions of different • prunes
m/z values via deflection in a magnetic field • meat
TRANSFORMERS 14
• chicken o Dietary iron has to be in ferrous form (Fe+2) in

order to be absorbed.
INTRODUCTION o Ascorbic acid, the acid pH in the stomach,
o Iron is the most abundant metal in the human along with reducing substances enhances iron
body with a concentration of approximately 40- absorption.
50 mg iron per body weight o Once absorbed, iron is transported by plasma
o Iron containing proteins are required in the transferrin.
metabolism of collagen, tyrosinase, and o Ferritin –provides accessible reserve of iron for
catecholamines the synthesis of iron containing compounds.
o It has multiple effect in the cell mediated
immunity such as: CLINICO PATHOLOGIC CORRELATIONS
• by modulating the propagation and Iron deficiency
differentiation of lymphocyte subsets o there is a deficit in total body iron.
• by affecting the immune potential of I. Iron depletion is the earliest stage of
the macrophages iron deficiency.
II. Iron deficiency without anemia- this
DISTRIBUTION denotes additional decrease in iron
Iron is distributed in the following: storage that may limit heme
o RBC containing hemoglobin production, but without frank
o Ferritin and hemosiderin as iron stores anemia.
• Ferritin keeps the excess iron III. Iron deficiency anemia is the most
o Body tissues containing iron (muscles) in the form advanced stage iron deficiency
of myoglobin and non heme enzymes anemia and the most common
o Iron bound to transferrin cause of anemia in USA and
worldwide.
Causes of Iron deficiency include:
o Blood loss due to GI bleeding, accident
o Chronic drug ingestion
o Hookworms and helminth infection
o Impaired iron absorption (if the iron doesn’t
reduce into ferrous state or if the
environment of the GI system has
inadequate acidic pH)
o Renal failure
Iron overload
o Denotes excess total body iron resulting
from supply that exceeds iron requirements.
o It is seen in the following:
• Hereditary hemochromatosis – the most
common form of iron overload
• Hemosiderosis
*Refer to the picture
• Myoglobin – found in muscle Hemochromatosis
• Ferritin- found in liver o Iron overload are collectively referred to as
• Total iron binding capacity- the test that hemochromatosis, whether or not tissue
determine the number of transferrin that the damage is present.
iron will accommodate which is bounded to o Primary Fe overload is most frequently
transferrin. associated with hereditary
• Hemoglobin- presence of iron found in the hemochromatosis (HH).
RBCs • It is hereditary because it is a single-
gene homozygous recessive disorder
METABOLISM AND REGULATION
TRANSFORMERS 15
leading to abnormally high Fe ‘yon, and so therefore, ganon din

absorption, culminating in Fe overload. ‘pag hinawakan ka na ng chelator,
• HH causes tissue accumulation of iron, ‘yung ating mga toxic o ‘yung
affects liver function, and often leads to sumobra na mga elements natin,
hyperpigmentation of the skin. hihilain nya yan palabas sa
➢ Refer to the image below: katawan.
Sometimes, when we see our liver in Hemosiderosis
HH, it has traces of iron. You can see o Secondary Fe overload may result from
the yellow color; these are iron excessive dietary, medicinal, or
deposits that may be seen in the transfusional Fe intake or due to metabolic
liver. dysfunction.
o Hemosiderosis has been used to specifically
designate a condition of iron overload as
demonstrated by an increased serum iron
and low total iron binding capacity (TIBC) in
the absence of demonstrable tissue
damage.
Analytic methods
o Direct measurements yield quantitative,
specific, and sensitive determination of
tissue or body iron stores but yield invasive
procedures.
• Quantitative phlebotomy
• Bone marrow aspiration and biopsy
• Liver biopsy with quantitative
measurements of non heme iron
concentration
•
Some conditions associated with severe o Indirect measurements are easy and
hemochromatosis include diabetes convenient but they lack specificity,
mellitus, arthritis, cardiac arrythmia or sensitivity, or both.
failure, cirrhosis, hypothyroidism, o Serum iron is measured by:
impotence, and liver cancer. 1. Colorimetric method – it involves
➢ Cirrhosis – ‘yung liver palagi, lalong dissociation, reduction, and reaction of
lalo na dyan naka-store ‘yung iron. iron with chromogen (pangkulay) to be
Pano kung sumobra siya? So pwede measured spectrophotometrically at
syang magkaroon ng iron deposits specific wavelength.
• Treatment may include therapeutic ➢ Banthophenanthroline and ferrozine
phlebotomy or administration of are the 2 most widely used
chelators, such as deferoxamine. chromogens.
➢ Chelators are like magnet; they will 2. Atomic Absorption Spectroscopy (AAS)
remove our trace elements that are ➢ So, kung titignan natin, na-discuss
too much and they will excrete natin kanina sa atomic absorption
them through feces or urine. spectroscopy the light source and
➢ I-a-attract nila ‘yon, hindi na nila sya then the presence of the atomizer
bibitawan, and then isasama na ito or the nebulizer which will excite,
sa excretion. and the after the excitement of the
➢ Para din daw ang mga chelators, particles, they will be in a ground
kung idedescribe natin, parang state and they will emit
yung paghawak ng crab; kung electromagnetic radiation; thus, this
iimagine-in mo yung crab, diba will be read by the monochromator
sobrang lakas ng grip ng crab na which, sabi natin kanina, ang
TRANSFORMERS 16
monochromator, depende doon sa 4. Chemiluminescence Assay

gagamitin nating wavelength, kung
ano man ang trace element na • Zinc protoporphyrin
hahanapin natin. o The zinc for iron substitution that
➢ In this case, we have the iron, so si occurs during the periods of iron
iron lang ang ilalabas nya para deficiency will lead to the increased
madetect ng ating detector. zinc protoporphyrin formation
o This substitution process occurs
predominantly in the bone marrow
o The zinc protoporphyrin
(ZnPP/heme) ratio in the
erythrocytes reflects the iron status
in the bone marrow
▪ This test determines yung
• (refer to the photo above): In here, you will nangyayare sa loob ng
see the specific colors (yellow circle), those bone marrow natin
are the different chromogens to be used in Selenium
colorimetry. • Selenium (Se) is a naturally occurring
• The important parts of the colorimeter are metalloid with many chemical and physical
the tungsten lamp, collimator, entrance slit, properties similar to those of sulfur.
prism (monochromator), exit slit, sample, • Most processed Selenium is used in the
detector, and read in the meter. electronics industry; however, other uses
• Serum Transferrin is measured: include nutritional supplements, pigments,
1. Immunoassay pesticides, rubber production, anti-dandruff
2. TIBC (Total Iron Binding Capacity) – it is shampoos, and fungicides.
the maximum amount of iron that can • It is found naturally in foods or as a
bind to serum transferrin. This test supplement. Selenium is an essential
determines how well that protein component of various enzymes and
(specifically, transferrin), can carry iron proteins, called selenoproteins.
in the blood. o Selenoproteins
➢ Kung gusto nating malaman - help make DNA and protect
gaano karami ba yung iron na against cell damage and
makakapag-bind sa transferrin. infections
➢ Diba sabi kanina, si transferrin, - also involved in reproduction
kaya niyang humawak ng and metabolism of thyroid
dalawang atoms, two iron atoms hormones.
3. Transferrin Ratio – is the ratio of the - have an important role in
plasma iron to the TIBC. skeletal muscle
• Serum Ferritin is measured by: regeneration, cell
▪ Kung gusto naman nating maintenance, oxidative and
idetermine kung gaano calcium homeostasis, thyroid
karami ang na-store na iron, hormone metabolism, and
if we are to describe ferritin, immune responses.
parang nakatago yung mga Selenium – Absorption, Transport, and Excretion
iron sa boxes. Kapag • Selenium is well absorbed from the
kakailanganin natin, gastrointestinal tract (~50%). Its exposure
bibigyan tayo ng ferritin ng occurs primarily from food but can be
enough supply of iron found in drinking water, usually in the form
1. Immunoradiometric Assay (RIA) of inorganic sodium selenite or sodium
2. Enzyme-linked Immunosorbent Assay selenite.
(ELISA) • Selenium homeostasis is largely achieved
3. Immunofluorometric Assay by excretion via urine and feces.
TRANSFORMERS 17
• Other routes of elimination include: sweat, o Is a gene-environment interaction

and, at very high intakes, exhalation of disease.
volatile forms of selenium. o Symptoms include:
▪ Dizziness
• Other routes of elimination include sweat, ▪ Malaise
and at very high intakes, exhalation, of ▪ Loss of appetite
volatile forms of selenium ▪ Nausea
o But it is largely excreted in the urine ▪ Chills
and feces. ▪ Abnormal
SELENIUM – Health Effects, Deficiency and Toxicity electrocardiograms
Health Effects ▪ Cardiogenic shock
• In 1930s selenium is considered to be a toxic ▪ Cardiac enlargements
element ▪ Congestive heart failure.
• In 1940s, it is considered to be carcinogenic o Common symptoms manifested by
• In 1950s, it was declared as essential KD are heart related
element; and since 1960s, especially 1970s it o Selenium supplementation have
has been viewed as an anti-carcinogen. been shown to effectively control
• Glutathione peroxidase (in the form of KD.
selenocysteine) is part of the cellular ▪ Ang naging problem s
antioxidant defense mechanism against Keshan Disease ay kulang
free radicals. Selenium is also involved in the siya ng selenium sa pagkain
metabolism of thyroid hormones. (e.g., kaya kung bibigyan natin ng
deiodinase enzymes and thioredoxin selenium or selenium
reductase) supplement yung kanyang
o Selenium belongs to antioxidant diet, then it will improve the
property in combating the free disease condition.
radicals. • Kashin-Beck Disease
• Selenium deficiency has been associated o An endemic osteoarthritis that
with cardiomyopathy, skeletal muscle occurs during adolescent and
weakness, and osteoarthritis. preadolescent years, is another
A significant negative correlation was observed disease linked to low selenium status
between selenium intakes and the rate of cancer in northern China, North Korea, and
of the large intestines, rectum, prostate, breast, eastern Siberia.
ovary, and lungs and leukemia. o The etiology of KBD remains elusive.
Selenium Deficiency o 4 Factors have been convincing
• Keshan Disease associated with the disease;
o An endemic cardiomyopathy that selenium deficiency, iodine
affects mostly children and women deficiency, grin contamination with
in childbearing age in certain areas mycotoxin-producing fungi, and
in China water pollution with organic
o has been associated with selenium material and fulvic acid.
deficiency. • Acute oral exposure to extremely high levels
o Characterized by a dilated of selenium may produce gastrointestinal
cardiomyopathy closely related symptoms (nausea, vomiting, and diarrhea)
with a diet deficient in the mineral and cardiovascular symptoms such as
selenium. tachycardia or there is increased in heart
▪ Source of food ay kulang sa rate of a person, (since madalas affected
selenium. ang puso dito)
o It is named for the northeastern • Chronic exposure to very high levels can
Chinese country Keshan, where the cause dermal effects, including diseased
disease prevalence is high because nails and skin and hair loss, as well as
of selenium-deficient soil.
TRANSFORMERS 18
neurologic problems such as unsteady gait • Factors increasing zinc absorption include
or paralysis the presence of animal proteins and amino
o Madalas apektado ang balat acids in a meal, intake of calcium, and
• One specific form of selenium, which is unsaturated fatty acids.
selenium sulfide, is a probable human • Conversely, factors decreasing zinc
carcinogen absorption include the intake of iron, taking
• Selenium sulfide is a very different chemical zinc on empty stomach, presence of copper
from the organic and inorganic selenium at high levels, and age.
compounds found in foods and in the o Kumbaga, inversely proportional
environment ang ating zinc and copper, so
• in Hubei Province (China), during 1961 kanina diniscuss natin yung copper,
through 1964, almost half of the population pwe’pwede gamitin si zinc
of many villagers died from chronic selenosis protoporphyrin, so similarly dito rin,
(prolonged eating of food rich in selenium or nagpapababa ng absorption ng
acquisition through high levels of exposure) ating zinc si copper lalo na at high
• The most common signs of selenium levels
poisoning were loss of hair and nails, skin o Also yung ating age, and when
lesions, tooth decay and abnormalities of taking zinc in an empty stomach,
the nervous system and sabi nga na mas maganda
itake ang zinc when accompanied
LABORATORY TEST FOR SELENIUM by amino acid in a meal or animal
• Selenium is most often determined by ICP- proteins in our meal and taking zinc
MS (inductively coupled plasma – Mass in empty stomach will decrease the
spectroscopy) or GFAAS (Graphic Furnace – absorption
Atomic absorption spectroscopy) • In blood, the absorbed zinc is distributed
• The determination of urinary and blood between RBCs (80%), plasma (17%) and
selenium is an useful measure of selenium white blood cells (3%)
status • In normal dietary circumstances, about 90%
Zinc of zinc is excreted as feces.
• Zinc (Zn) is a bluish white, lustrous metal that
is stable in dry air and becomes covered Zinc- health effects, deficiency, and toxicity
with a white coating when exposed to • Zinc is second only to iron in importance as an
moisture. essential trace element.
• Zinc is used in a production of alloys, • The main biochemical role of zinc is seen in its
especially brass (with copper), in galvanizing influence on the activity of more than 300
steel, in die casting, in paints, in skin lotions, enzymes in classes such as oxidoreductases,
as treatment for Wilson’s disease and in transferases, hydrolases, leases, isomerases, and
many over-the-counter medications. lipases.
• Zinc is an essential trace element and o So, kasama po si zinc sa mga napapabilis
deficiency is common throughout life, ng chemical reaction ng atin mga specific
especially in individuals that DO NOT ingest enzymes
meat. • As a result of the importance of zinc for the
ZINC – ABSORPTION, TRANSPORT AND EXCRETION structure, regulation, and catalytic action of
• The body content in a normal individual various enzymes, zinc is indirectly involved in the
varies substantially with age and is synthesis and metabolism of DNA and RNA, the
predominantly distributed in the muscle synthesis and metabolism of proteins, the
(60%) and skeleton (30%). The remaining metabolism of glucose and cholesterol,
10% is distributed in various other tissues with membrane structure maintenance, insulin
highest concentration found in the eyes, function, and growth factor affects.
prostate, and hair o Besides na tumutulong ang ating mga
• Absorption of zinc mainly occurs in the small enzymes na magspeed-up ng chemical
intestine and especially in the jejunum. reactions, zinc has a lot of important uses,
TRANSFORMERS 19
and one of them is the synthesis and o The acquired form of this disorder generates
metabolism of DNA and RNA. Kaya nga similar symptoms.
napaka-importante ng ating mga o One transient form can result from failure of
important biological functions such as the the mother to secrete zinc into her breast
glucose metabolism and cholesterol, insulin milk.
function, and also for the protein function. o Other acquired forms of AE sometimes result
• Chronic oral zinc supplementation interferes after surgery to bypass some of the upper
with copper absorption and may cause copper intestine or from special intravenous
deficiency, forming the basis for using zinc to nutritional programs that are prepared
treat Wilson’s disease. without the appropriate amount of zinc.
• Zinc deficiency causes growth retardation, ▪ So, Acrodermatitis Enteropathica has
slows skeletal maturation, causes testicular two forms: inborn (congenital) form
atrophy, and reduces taste perception. and two acquired forms.
• Old age, pregnancy, lactation, and alcoholism ▪ For inborn, it is a genetic disorder and
are also associated with poor zinc nutrition. the problem is the intestine because it
• Zinc deficiency in adolescents is manifested by cannot absorb the zinc.
slow growth or weight loss, altered taste, ▪ For acquired forms, the first is when the
delayed puberty, dwarfism, impaired dark mother cannot secrete zinc into her
adaptation, alopecia, emotional instability, and breast milk and the second is when a
tremors. In severe cases, lymphopenia patient undergone surgery or bypass in
(decreased lymphocytes) and death can result which the upper portion of the intestine
from an overwhelming infection. (absorption of zince) is affected or by
• This is an example of Zinc deficiency, in which giving nutritional program or total
the child starts by having rashes in their face. parenteral nutrition, where there is a
lack of zinc nutrients giving to the
patient.
o Supplemental zinc usually eliminates the
symptoms.
▪ Dahil nga kulang si zinc, kapag
binigyan ng zinc mag-iimprove ang
kaniyang condition.
o Exposure to Zinc Oxide fumes and dust may
• Zinc deficiency cause “zinc fume fever,” with symptoms
o Infants with acrodermatitis enteropathica including chemically induced pneumonia,
(zinc malabsorption) first develop a severe pulmonary inflammation, fever,
characteristic facial and diaper rash. hyperpnea, coughing, pains in legs and
o If untreated, symptoms progress and chest, and vomiting.
include growth retardation, diarrhea, ▪ If palagi tayong na-eexpose sa zinc, sa
impaired T-cell immunity, insufficient wound usok or dust na meron, magkakaroon
healing, infections due to decreased in T- tayo ng "zinc fume fever" and the
cell, delayed testicular development in symptoms mentioned above might
adolescence, and early death. happen. Dahil nga nalanghap natin,
o Acrodermatitis enteropathica (AE) is a most often signs and symptoms na
disorder of zinc metabolism that occurs in pinapakita ay mostly respiratory signs
one of three forms: an inborn (congenital) and symptoms.
form and two acquired forms.
o The inborn (congenital) form of Zinc – Health effects, deficiency, and toxicity
Acrodermatitis Enteropathica is a rare • Exposure to Zinc Oxide fumes and dust may
genetic disorder characterized by intestinal cause “Zinc fume fever,” with symptoms
abnormalities that lead to the inability to including chemically-induced pneumonia,
absorb zinc from the intestine. server pulmonary inflammation, fever,
TRANSFORMERS 20
hyperpnea, coughing, pains in legs and Inorganic Arsenic

chest, and vomiting. • Refers to the pure metallic form or the
• Signs and symptoms are more inclined to arsenic that has binded to non-carbon
the respiratory system. elements. They do not contain carbon
atoms. Examples include arsenite and
Zinc – Laboratory Evaluation arsenate. They are highly toxic.
• Zinc is measured by GFAAS, ICP-AES, or ICP-
MS Methylation
o Measured by making use of the • It is a biological mechanism for the removal
Graphite Furnace Atomic of toxic metal(loid)s by converting them to
Absorption Sprectrometry (GFAAS), methyl derivatives that are subsequently
Induced Coupled Plasma-Atomic removed by volatilization. Methylation
Emission Spectrometry (ICP-AES), or allows the conversion of aqueous or solid
the Induced Coupled Plasma-Mass associated inorganic arsenic into gaseous
Spectrometry (ICP-MS). arsines and removes them from the living
• Lower urine zinc levels in the presence of medium, which is usually regarded as
low serum zinc levels usually confirm zinc detoxification. Methylated forms of arsenic
deficiency. are volatile and conveniently released into
o Low urine zinc levels + Low serum the surroundings.
zinc levels = zinc deficiency o Methylation is the process of
• Normal serum zinc cannot be interpreted as removing the toxic elements in the
evidence of normal zinc stores. Zinc body. Kapag nag add na tayo ng
concentration in RBCs in approximately 10 important component, like methyl,
times in that serum. mage-evaporate ang mga ‘to. Thus
o This is because the majority of the it will be removed from the body.
zinc concentration is found in the This is now regarded as
RBCs. detoxification.
o If normal serum zinc is observed, it Lavage
does not necessarily mean that the
zinc levels are normal. Therefore, it is
important to check the zinc RBC
concentration since the RBCs
contain approximately 10x than that
of the concetration found in the
serum.
• Copper status should be monitored in
patients undergoing long-term zinc therapy.
o Copper and Zinc have an inversely
proportional relationship. If copper Gastric lavage involves placing a tube through the
levels are elevated, zinc levels mouth (orogastric)or through the nose
decline, and vice versa. (nasogastric) into the stomach.
NON-ESSENTIAL TRACE ELEMENTS • Toxicants are removed by flushing saline
Arsenic solutions into the stomach, following by
Definition of Terms: suction of gastric contents.
o Eto yung example na pinapakain
Organic Arsenic nalang yung patients by means of
• Refers to the organic compounds having tubo or NGT (Nasogastric tube).
covalently bonded arsenic atoms. They o May iba’t-ibang functions itong
contain carbon atoms. Examples include NGT:
arsenobetaine and arsenocholine. These ▪ Lavage – Labas
are less toxic.
TRANSFORMERS 21
• Linalabas; To remove pumasok po ito sa katawan natin, pwede itong

ingested toxic maglead ng sakit satin. So these are some things
elements/toxicants we could do to counteract the effect of non-
▪ Gavage – Give essential elements or the toxic element.
• Gastric lavage must be performed soon o Arsenic- is abundant element displaying both
after ingestion to be at all effective in metallic and non-metallic properties.
removing drugs from the stomach. o Arsenic is a well-known wood preservative
o Must be one as soon as possible to o It is relatively tasteless.
immediately suction the ingested o Used as “rat poison”
poison or toxicant in the body. o Kapag narinig arsenic agad na po papasok sa
• For this reason, many clinicians do not isip natin na pamatay sa daga, pwede rin po
lavage patients who have overdosed is tayo mamatay dito, could lead to death lalo nap
more than 1 hour has elapsed since ag marami na pumasok sa katawan natin
ingestion. o For most people, Food is the largest source of
o It is possible that the poison ingested arsenic exposure (about 25 to 50ug/day), with
is already absorbed in the lower amounts coming from drinking water and
circulation if the patient has air.
overdosed for more than 1 hour. sources of arsenic na pwede po natin makuha sa
o Therefore, it is recommended that a environment:
patient undergo lavage within or o Pesticides, ginagamit natin si arsenic sa
less than an hour. pesticides. In fields in the south and central
America, they have high levels of arsenic due to
o Siyempre kung pwede pa alisin kung may oras pa pesticide use.
aalisin natin itong toxic chemical na to, o Also used to the rice fields,(water used to flood
ipapalavage (labas) natin the rice fields) itong rice fields na to dahil minsan
nasa tubig siya, pwede mabigyan ang ating rice
o Gastric lavage must be performed soon after field and as a result pag napunta sa pagkain
ingestion to be at all effective in removing drugs natin mabibigyan rin tayo ng food poison.
from the stomach o The American rice grows in flooded areas
o Mas maaga mas better, kase mabilis natin allowing arsenic uptake from the soil and water.
makukuha, masusuction yung pumasok na Ang mga sources po ng arsenic natin pwede po
poison sa katawan siya manggaling sa Industrialization companies
o For this reason, many clinicians do not lavage and so therefore kapag contaminated ang
patients who have overdosed if more than 1 hour water maapektuhan yung mga kakanin natin.
has elapsed since ingestion Ganun din po yung soil ang water content natin.
o So kung halimbawa lagpas na ng 1 hour, di na o Antibiotics and Fertilizers, the drugs containing
po natin gagawin ang lavage kase possible po arsenic are used in livestock for a variety of
na naabsorb na ng circulation natin so sayang reasons. Fertilizers produced from these animal’s
lang manure contains concentrated amounts of
o At least as much as a possible we perform lavage arsenic and is in distributed to crops.
if it is less than 1 hour o Natural occurrence some soils and water sources
naturally have higher arsenic levels than others.
o Activated Charcoal- also known as Activated o Genes certain rice varieties absorb more arsenic
Carbon, medication used for treating poisonings o So these are the sources of arsenic na pwede po
that occurred by mouth. natin makuha sa environment.
o To be effective it must be used within a short time
of the poisoning occurring, typically an hour. o Green wallpaper, chemists and paint makers
o Kung ayaw po natin gamitin ang lavage, pwede introduced arsenic to other colors as well such as
po natin gamitin ang activated charcoal, again canary yellow to create vibrant new hues.
it should be given in less than an hour. o Despite its vivid and eye-catching nature, doctors
o Bakit kailangan po natin pagusapan ito? Again eventually discovered that arsenical wallpaper
these are non-essential elements so kapag could kill.
TRANSFORMERS 22
o When we talk about arsenic, meron din po siyang o Lung

association sa mga wall papers noong unang o Bladder
panahon. Ang nangyare ang ganda ng mga o Kidney and liver cancers
wall paper maraming bumile and eventually ito • Nerve damage
po ay nalulusaw o nag disintegrate yung • Circulatory problems in skin
wallpaper so what happens pumapasok na po
sa respiratory tract itong arsenic and siyempre For chronic arsenic exposure, signs and symptoms
would lead to complication and several diseases may include:
have develop, kase once nasira wallpaper
nahahalo na sa environment and so therefore
pag pumasok sa katawan natin it would lead to
a possible diseases.
Arsenic – Health effects and toxicity
The relation of clinical signs and symptoms to

arsenic exposure depends on the duration and
extent of the exposure to inorganic and
methylated species of arsenic, as well as, the
underlying clinical status of the patient.
For acute arsenic exposure, the symptoms may

include:
• Gastrointestinal (nausea, emesis, adominal
pain, and rice water diarrhea)
• Bone marrow (pancoytopenia, anemia,
and basophilic stippling)
• Cardiovascular (ECG changes)
• Central Nervous System (encephalopathy • Dermatologic (Mees’ lines [lines on the
and polyneuropathy) nails], hyperkeratosis, hyperpigmentation,
• Renal (renal insufficiency and renal failure) and alopecia [hair loss])
• Hepatic (hepatitis) • Hepatic (cirrhosis and hepatomegaly)
• Cardiovascular (hypertension and
peripheral vascular disease [PVD])
• Central nervous system ( “socks and glove”
neuropathy and tremor)
o Numbness of hands and feet -
Laging naffeel na nagpa-pata ang
kamay at paa.
• Malignancies (squamous cell,
hepatocellular, skin, bladder, lung, and
renal carcinomas)
Other acute arsenic poisoning symptoms (refer to

the image above):
• Skin damage
o Pigment changes (Pigmentation)
o Hyperkeratosis (scaling skin)
• Increased cancer risk
TRANSFORMERS 23
• Chronic arsenic exposure has been shown • The methylated inorganic forms are
to cause blackfoot disease, a severe form considered less toxic than As(3+) and As
of PVD, which leads to gangrenous (5+); however they are eliminated slowly
changes. (1-3 weeks). The Biological Exposure Index
established by the American Conference of
Governmental Industrial Hygienists for the
sum of inorganic and methylated
metabolites of arsenic in urine is 35 ug/L.
However, clinical symptoms may not
evident at 35 ug/L.
o Methylated arsenic are less toxic.
However, if it stays longer in the
body, it can be converted into fully
• The white powder of arsenic trioxide is pledged organic arsenic, which is
odorless, tasteless, and one of the most highly toxic.
common poisons in human history.
• Doses of 0.01 to 05 g produce toxic Arsenic – Laboratory Evaluation
symptoms. • Arsenic is primarily measured using ICP-MS,
• The lethal dose is reported to be between GFAAS.
0.12 and 0.3 g. • In most cases, arsenic is best detected by
• Immediate treatment of expected urine due to the short half-life of arsenic in
exposure consists of lavage and use of blood
activated charcoal to reduce arsenic o Half-life – time it takes for half of the the
absorption. substance or element to be excreted out of
• The most effective antidotes for arsenic the body.
poisoning are the following chelating Lead
agents: • Metallic lead (Pb) is soft, bluish white, highly
o Dimercaprol (aka British Anti- malleable, and ductile.
Lewisite[BAL]) • It is a poor conductor of electricity and
o Penicillamine heat and is resistant to corrosion.
o Succimer (Dimecaptosuccinic acid) • Lead is used in the production of storage
batteries ammunition, solder and foils.
Note: Chelating agents serve as magnets when • Metallic lead (Pb) is soft, bluish white, highly
they come in contact with harmful chemicals, such malleable, and ductile.
as arsenic, it will deliver these substances out of the • It is a poor conductor of electricity and heat
body through the feces and/or urine. and is resistant to corrosion.
Arsenic – Absorption, transport, and excretion • Lead is used in the production of storage
batteries, ammunition, solder, and foils.
• Main routes of exposure are ingestion of • Toxic concentration of lead can be found in
arsenic-containing foods, water, ad areas adjacent to homes painted with lead-
beverages or inhalation of contaminated based paints and around highways where it
air. has accumulated from the past use of
• Organic forms of arsenic, such as, leaded gasoline.
arsenocholine and arsenobetaine are • In recent years, there have been massive
commonly found in fish and seafood, are recalls of toys and costume jewelry
considered relatively non-toxic, and are produced in China, due to concerns over
cleared or excreted within 1-2 days. elevated lead content.
• Inorganic species of arsenic are highly o Lead is not needed by our body and
toxic and occur naturally in rocks, soil, and is non-essential. Therefore, it is toxic to
groundwater. the body and may cause several
• They are found in many synthetic products, diseases.
poisons, and industrial processes.
TRANSFORMERS 24
• Lead plays no known role in normal human

physiology.
Lead-absorption, transport, and excretion
• The red blood cells will retain aggregates of

ribosomal RNA that causes the stippling of
the cells.
o Sa lead poisoning, dumadaan at
pumapasok sila sa ating mga RBCs.
• Exposure to lead is primarily respiratory or • Picture:
gastrointestinal. o Lower IQ from lead poisoning
• Inhalation results in 30% to 40% of absorption o Blood – anemia
efficiency. o Kidneys – impairment or damage
• Gut absorption depends on a variety of o Reproductive organs – lower sperm count or
factors, including age and nutritional status, damage sperm
with enhanced gastrointestinal absorption o Nervous system – damage
occurring in children younger than 6 years of o Body – decreased bone and muscular
age. growth
o Toddlers and children are the first to o Brain – reduce IQ, learning disabilities,
be affected by lead poisoning since behavioral problems, and hearing loss
their gut absorption is fast. • The clinical presentation of lead toxicity is
• Certain substances, such as iron, calcium, variable.
magnesium, alcohol, and fat, may weaken • In children, obvious symptoms are usually seen
lead absorption while low dietary zinc, at blood levels of 60 µg/dL or higher with 45
ascorbic acid, and citric acid can enhance µg/dL as the typical threshold for acute, clinical
the absorption of lead. intervention.
• About 99% of absorbed lead is taken up by • IQ declines are seen in children with blood lead
erythrocytes where it interferes with heme levels (BLLs) of 10 µg/ dL or higher.
synthesis. • Other central nervous system symptoms of lead
• Lead distributes to soft tissues, such as liver, toxicity in children may include clumsiness, gait
kidneys, and brain, with the skeletal lead abnormalities, headache, behavioral changes,
concentrations containing greater than 90% seizures, and severe cognitive and behavioral
of the body burden of lead. problems.
• Absorbed lead is exreted primarily in urine • Gastrointestinal symptoms include abdominal
(76%) and feces (16%), and the remaining pain, constipation, and colic.
8% is excreted in hair, sweat, nails, and
others.
Lead – Health effects and toxicity
TRANSFORMERS 25
• Lead exposure may primarily arise in two

settings:
o Childhood exposure; usually through
paint chips (likes to play and tend to
touch things which may contain
lead content, which can they eat
and can cause lead poisoning)
o Adult occupational exposure, in the
smelting, mining, ammunitions,
soldering , plumbing, ceramic
glazing and construction industries.
• Lead-laboratory evaluation
o The most common specimen type is
whole venous blood, the result of
which is commonly referred to as
the Blood Lead Levels(BLL).
o This is preferred over plasma and
serum as circulating lead is
predominantly associated with
• Picture: RBCs.
o Brain – memory loss, lack of concentration, o Elevated lead levels in capillary
headaches, irritability, depression should be confirmed with a venous
o Digestive system – constipation, nausea specimen to avoid the potential
and poor appetite contribution of external
o Nervous system – damage including contamination.
numbness and pain in the extremities o Urine lead may be useful for
o Body – fatigue, joint and muscle pain detecting recent exposure to lead
o Cardiovascular – high blood pressure or to monitor chelation therapy.
o Kidneys – abnormal function and damage o Other testing, such as aminolevulinic
o Reproductive system acid, whole blood zinc
▪ Men: decreased sex drive and sperm protoporphyrin, and free
count, and sperm abnormalities. erythrocyte protophorphyrins, may
▪ Women: spontaneous miscarriage be useful for screening in
• In adults, the following symptoms may be occupational exposures.
observed: o Noninvasive measurement of lead in
o Peripheral neuropathies, motor weakness, bone may be available
chronic renal insufficiency and systolic radiographically.
hypertension, and anemia. o Removal of further lead exposure
• Lead exposure primarily arises in two settings: and parental education are
o Childhood exposure, usually through paint essential parts to the management
chips for patients with elevated lead
o Adult occupational exposure, in the levels (BLLs).
smelting, mining, ammunitions, soldering, o ICP-MS (Inducely coupled plasma
plumbing, ceramic glazing, and mass spectroscopy) is a preferred
construction industries. method of analysis, although ICP-
• In adults , the following symptoms may be AES and GFAAS are also used.
observed: Mercury
o (Includes the CNS), peripheral • Half-life- the amount of time it takes for the
neuropathies, motor weakness, body to eliminate half of the specific
chronic renal insufficiency(kidney) substance is called half-life. Toxic heavy
and systolic hypertension(heart) and metal such as cadmium, mercury, and lead
anemia (blood cells). can accumulate over a lifetime and have
TRANSFORMERS 26
a long half-lives in different organs and eye drops, and nasal sprays.
tissues.
• Mercury (Hg) is also called quicksilver, is a • Mercury is widely used in the production of
heavy , silver metal. eye cosmetics, especially mascara.
• Along with bromine, mercury is one of only Mercury- absorption, transport and excretion
two elements that are liquid at room
temperature and pressure. Routes of exposure include
• There are three naturally occurring (1) inhalation, primarily as elemental mercury vapor
oxidation states of mercury: mercury zero but occasionally as dimethyl mercury;
Hg(0),mercury one positive Hg (1+), and (2) ingestion of HgCl2 and mercury-containing
mercury two positive Hg(2+). foods such as predatory fish species;
• Mercury is used in dental amalgams, (3) cutaneous absorption of methyl mercury (MeHg)
electronic switches, germicides, fungicides through the skin and even through latex gloves;
and fluorescent light bulbs. • Also we have cutaneous absorption so, lalo
• The use of mercury in medicine has greatly na halimbawa nabasag yung mga
declined in all respects; however, mercury thermometer nung unang panahon tapos
compounds are found in some over-the- nahawakan natin, pinaglalaruan natin yung
counter drugs, including topical antiseptics, mercury, so this would lead to the route of
stimulant laxatives, diaper-rash ointment, exposure na papasok satin yung mercury,
eye-drops and nasal sprays. specifically the methyl mercury may pass
through latex gloves.
Definition of terms
(4) injection of relatively inert liquid mercury and
Half life mercury-containing tattoo pigments; and
• The amount of time it takes for the body to (5) dental amalgams. Inhaled mercury vapor is
eliminate half of a specific substance is retained in the lungs to about 80%, whereas liquid
called half-life. Toxic heavy metals such as metallic mercury passes through the gastrointestinal
cadmium, mercury, and lead can tract and are largely unabsorbed.
accumulate over a lifetime and have long • So saan po madalas nagsstay at reretain an
half-lives in different organs and tissues gating mercury? → lungs.
• Mercury (Hg), also called quicksilver, is a • 80% ay nagsstay sila sa ating baga or lungs.
heavy, silvery metal. Pag dating naman sa gastrointestinal
• Along with bromine, mercury is one of only system, hindi nya ito naaabsorb so hindi ito
two elements that are yung site of absorbtion ng ating GIT.
liquid at room temperature and pressure. • Usually, this remains unabsorbed whereas, sa
lungs natin marami ang absorption ng
• There are three naturally occurring oxidation mercury.
states of mercury:
• Hg(0)
• Hg(1+)
• Hg(2+).
• Mercury is used in dental amalgams,

electronic switches, germicides, fungicides,
and fluorescent light bulbs.
• The use of mercury in medicine has greatly

declined in all respects; however, mercury
compounds are found in some over-the-
counter drugs, including topical antiseptics,
stimulant laxatives, diaper-rash ointment,
TRANSFORMERS 27
Mercury enters the food chain primarily by volcanic urine for chronic exposure to inorganic
activity and manmade sources such as coal mercury.
combustion and smelting. • So pwede po ang half life po nito pwede
• Kung titignan po natin ang drawing po syang 5 days in the blood para sa
natin, we have the antropogenic Hg so phenylmercury. Pwede rin poi tong 90 days
nakuha po natin yan sa industries, local and in the urine para sa chronic exposure to
regional deposition → atmospheric Hg kung inorganic mercury.
saan madedeposit and mahuhulog sila sa
ating tubig so syempre magkakaroon ng • Normally, the highest concentration/
methylated Hg sa water system. accumulation of mercury is in the kidney,
• Global deposition ay mahuhulog sa ating liver, spleen, and brain.
soil or land natin and also remitted • Mercury can accumulate in pituitary and
anthropogenic and natural Hg syempre thyroid glands, the pancreas, and the
mageevaporate yan, maguundergo ng reproductive organs.
evaporation, condensation, precipitation. • The bulk of mercury accumulated in the
• So babalik ulit, iikot ulit. Ang distribution ng body is eliminated in approximately 60 days
Hg po ay from volcanic activity to o Again, the bulk accumulated in the
anthropogenic Hg activity, and then babalik body is supposed to be eliminated at
ulit satin and the cycle continues. approximately 60 days.
• Most of the dietary intake comes from o So matagal bago poi to tuluyan
consumption of meat and fish products, with maeliminate sa katawan natin.
estimates of dietary intake varying based
upon geographical location and dietary AB 15:05 – 21:05
sources.
• Kagaya ditto, syempre kung malapit yung • Liquid elemental mercury is poorly absorbed
volcano sa lake, ganun din kung malapit and relatively nontoxic but elemental mercury
yung industrial company sa lake, then vapor is highly absorbed and is highly toxic.
pwede po tayo mahawa or mabigyan kase Inorganic, ionized forms of mercury are toxic.
nga contaminated yung water therefore • Further bioconversion to an alkyl mercury, such
pati yung mga fish na nanjaan or yung mga as MeHg (Methyl Mercury), yields a very toxic
domesticated na animals na anjaan, species of mercury that is highly selective for
pwede po tayo mahawa. lipid-rich mediums such as the brain.
o The mercury is attracted to the brain
kaya umaakyat siya kaagad sa brain
• The kidney is the major storage organ after natin. The methyl mercury is very toxic
elemental or inorganic mercury exposure. and it is highly selective o gustong gusto
• Methyl mercury (MeHg) is efficiently niya pumunta sa lipid-rich medium
absorbed from the gastrointestinal tract, which is our brain.
and distribution to tissues, including the • Mercury intoxication can manifest in many signs
brain, appears complete in 48 hours. and symptoms that affect several organ
• Movement of MeHg across the blood–brain systems, including headache, tremor, impaired
barrier appears to be dependent on coordination, abdominal cramps, diarrhea,
coupling with the amino acid cysteine. dermatitis, polyneuropathy, proteinuria, and
• So, ditto sinasabi na pweding magpass sa hepatic dysfunction.
brain yung ating mercury kapag nakapag • Because many of these are relatively
bound sya sa cysteine. Movement of MeHg nonspecific signs and symptoms, laboratory
across the blood–brain barrier may be testing provides a key role in assessing mercury
dependent when it was able to bound to intoxication
cysteine. o Kung nakita natin ang signs & symptoms
• Half-lives vary according to the route of na pinakita sa mercury such as
exposure and form of mercury, from 5 days headache, tremor, impaired
in blood for phenylmercury to 90 days in coordination, marami ding mga sakit
TRANSFORMERS 28
kumbaga pare-pareho na pwede nila

mafeel o ma-experience yung
headache, tremor, impaired
coordination, diarrhea. Ang daming
sakit kung saan yan din ang signs &
symptoms na nararamdaman nila kaya
kailangan talaga magpa-assess ng
mercury intoxication para masigurado
natin na ang mga signs & symptoms na
nararamdaman natin is related to the
poison and that is your mercury. kasi nga
pare-pareho itong signs & symptoms,
hindi sila clearly stated kasi halos general
yung minamanifest na signs & symptoms
kaya importante na magpatest tayo
that we have a mercury testing.
Ipapatest natin ang ating dugo at urine
in the laboratory for further analysis.
Mercury-laboratory evaluation
• Mercury is usually determined as total mercury
levels in blood and urine without regard to
chemical form.
Analytical methods include ICP-MS (Mass

spectroscopy) and cold vapor AAS (Atomic
Absorption Spectroscopy)
TRANSFORMERS 29
7 Drug Testing Clinical Chemistry 2 (LAB)
DRUG TESTING ALCOHOL

Topical Outline: • Most common toxic substance is encountered
• Principles of Toxicology everyday
• Toxic Agents Affecting Patient’s Health • Causes:
• Laboratory Methods o Disorientation
DEFINITION OF TERMS o Confusion
Toxicology o Euphoria – well-being or elation
• It is the study of toxic substances and how they
affect the human body. • In excess, it can progress to:
• It is the study of adverse effects of xenobiotics in o Unconsciousness
humans o Paralysis
o Death
Xenobiotics
• Xenobiotics are chemicals that are not normally
𝑨𝒍𝒄𝒐𝒉𝒐𝒍 → 𝑨𝒍𝒅𝒆𝒉𝒚𝒅𝒆 → 𝑨𝒄𝒊𝒅
found or produced in the body.
Legend:
Poisons • ADH: Alcohol Dehydrogenase
• Chemical agents that have an adverse effect • ALDH: Aldehyde Dehydrogenase
on a biological system.
Toxins Rationale:
• Substances synthesized in living cells or • This is the common way on how the body
organisms. metabolize the alcohol
• Example: Botulinum Toxin
DOSE-RESPONSE RELATIONSHIP Procedure:
• It is an essential concept in toxicology as it 1. The alcohol is degraded into simple aldehyde
correlates the exposure with toxic substances with the help of the enzyme ADH [Alcohol
with changes in body functions or health Dehydrogenase].
• The relationship between the dose and the 2. The aldehyde will be further converted into
response is direct acid by the help of the Hepatic ALDH [Hepatic
• Directly Proportional Aldehyde Dehydrogenase]
• If the dosage of the toxic substance is increased, Types of Alcohol
then the toxic response in the body will also 1 Ethanol
increase. 2 Methanol (Common Solvent)
• The dose makes the poison 3 Isopropanol (Rubbing Alcohol)
• Everyday a person encounters tiny amounts of 4 Ethylene Glycol (1,2-Ethanediol)
toxic substances in the environment, but for it to
Ethanol
generate an evident physiological response, its
• In long term, it may cause toxic hepatitis and
concentration in the body should be sufficiently
liver cirrhosis
high.
• Liver cirrhosis is the condition wherein the liver
develops scarring
↑ 𝑫𝒐𝒔𝒆 = ↑ 𝑹𝒆𝒔𝒑𝒐𝒏𝒔𝒆
Methanol (Common Solvent)
TOXICOLOGY OF SPECIFIC AGENTS • End product is formic acid which may cause
1 Alcohol severe acidosis and blindness
2 Carbon Monoxide • Formic acid is a very toxic substance or acid.
3 Cyanide Isopropanol (Rubbing Alcohol)
4 Arsenic • Metabolized in the body into simple acid called
5 Cadmium acetone which is a strong central nervous
6 Lead system (CNS) depressant.
7 Mercury Ethylene Glycol (1,2-Ethanediol)
8 Pesticides • It is converted to oxalic acid and glycolic acid
in the body
• May cause severe metabolic acidosis
TRANSFORMERS 1
• Also, one of the product of the degradation • The carboxyhemoglobin causes a left shift in
products of this alcohol is the calcium oxalate, oxygen-hemoglobin dissociation curve causing
which can be found in the body as crystals in Hypoxia.
the urine which may cause renal tubular • Hypoxia is a condition where there is a decrease
damage concentrations of oxygen in the body
Determination of Alcohol Methods of Carbon Monoxide Determination
Specimen of Choice • Serum 1 Spot Test
• Plasma 2 Gas Chromatography
• Whole Blood 3 Spectrophotometric
Considerations: Spot Test
• Clean the venipuncture site with alcohol-free • In performing the spot test, the NaOH is added
disinfectant as the analyte to be measured is to aqueous solution of whole blood
alcohol. Using an alcohol product during • The positive result is a pink solution
venipuncture may interfere with the test causing Gas Chromatography
a falsely increased alcohol levels.
• It is uses Potassium ferricyanide
• For the alcohol determination, iodine is utilized.
• Carbon monoxide is detected through thermal
• Specimen must be tightly sealed and capped at
conductivity after the addition of potassium
all times to avoid evaporation since alcohol is a ferricyanide
volatile substance
Spectrophotometric
• Sodium fluoride as additive, this will also inhibit
• The difference in the spectral absorbency
the bacterial growth
curves is used to measure carbon monoxide
Methods of Alcohol Determination
1 Osmometric Method
CYANIDE
2 Gas Chromatography
• Considered as a super toxic agent because
3 Enzymatic Method
even in tiny amounts, it can produce an adverse
Osmometric Method effect in the body
• Measurement of alcohol through the difference • Found in insecticides or rodenticides, and even
in the osmolar gap in burning plastic
• It is only a screening test • It manifests its effect by binding to heme iron
Gas Chromatography causing:
• Determine the concentration of alcohol through o Headache
the comparison of the internal standard o Dizziness
• N-propanol is the typical internal standard used o Seizure
Enzymatic Method o Coma
• The ADH enzyme is added Methods of Cyanide Determination
• Upon the addition of ADH, the NADH is 1 Urinary Thiocyanate Measurement
produced and is measured at 340nm. 2 Ion-Specific Electrode
• The NADH is equivalent to the levels of alcohol 3 Photometric Analysis
• NADH = Alcohol levels Urinary Thiocyanate Measurement
• Cyanide may be detected in the urine through
CARBON MONOXIDE the measurement of urinary thiocyanate
• Colorless, odorless, and tasteless gas • Thiocyanate is a degradation of cyanide in the
• It is formed from the incomplete combustion of body
carbon-containing substances such as: Ion-Specific Electrode
o Gas engines
• The activity of a specific ion dissolved in a
o Wood solution is converted into an electrical potential
o Plastic fires • Electrical potential is equivalent to Cyanide
• Once it enters the body, it will bind with
Photometric Analysis
hemoglobin causing carboxyhemoglobin
• This is utilized following a two-well microdiffusion
TRANSFORMERS 2
ARSENIC • It manifests its toxic effects by binding to proteins

• It is a primary constituent of many organic and and cellular constituents which leads to renal
inorganic compounds damage
• There are several types of arsenic, but the arsine • “Itai-Itai” is a condition due to a long-term
gas is the most potent exposure to cadmium contaminated rice
• Arsenic can be found on seafoods such as:
o Clams
o Oysters
o Scallops
o Crabs
o Lobsters
• Arsenic manifests its toxic effects by binding to
the thiol group of proteins
• Mees’ Lines is a condition wherein there is
distinctive white lines in the fingernails caused
by the deposition of arsenic in fingernails
• It is manifested by Osteomalacia and

Osteoporosis
Method of Cadmium Determination
1 Atomic Absorption Spectrophotometry
Atomic Absorption Spectrophotometry
• It uses light wavelength to measure the intensity
of specific elements, particularly in this case is
the cadmium
Determination of Arsenic LEAD

Specimen of Choice • Blood • It is commonly found in household paints and
• Urine gasoline
• Hair • This affects the vitamin D metabolism and heme
• Fingernails synthetic pathway
Methods of Arsenic Determination • If the vitamin D metabolism is affected, then the
1 Atomic Absorption Spectrophotometry bones are affected as well
Atomic Absorption Spectrophotometry • If the heme synthetic pathway is affected, then
• It is utilized for the measurement of the arsenic the red blood cells are affected causing difficult
with urine as its specimen of choice. in the delivery of oxygen
• Lead manifests its toxic effects by binding to soft
CADMIUM tissues and bones
• It is most commonly found in: • Long-term exposure to lead may lead to
o Electroplating encephalopathy characterized by edema and
o Galvanizing ischemia
o Cigarette smoking • One of the effects of the lead is the decrease in
• It is also reportedly found in: IQ level or Intelligence Quotient
o Organ meats • One mark of lead in the blood that is observed
o Lettuce in the peripheral blood smear (PBS) is the
o Spinach basophilic stippling in the erythrocytes
o Potatoes
o Soybean
o Sunflower seeds
**but is mostly caused by contamination.
TRANSFORMERS 3
PESTICIDES
• It is commonly acquired due to food
contamination
• It manifests its toxic effects by the inhibition of
acetylcholinesterase
• Acetylcholinesterase is important neuro-
muscular transmitter
• The absence of the acetylcholinesterase may
lead to increase:
o Salivation
o Lacrimation
Methods of Lead Determination o Involuntary urination
1 Inductively Coupled Plasma Mass Spectrometry o Involuntary defecation
Inductively Coupled Plasma Mass Spectrometry • Pesticides can be detected by the decreased
(ICP-MS) erythrocytic acetylcholinesterase activity
• This method atomizes the sample and creates • Normally, the erythrocytic acetylcholinesterase
an atomic and small polyatomic ions, which are activity is increased, However, in cases of
then detected. pesticide poisoning, it decreases
3 Types of Pesticides
MERCURY 1 Organophosphates
• It is commonly acquired in industrial setting 2 Carbamates
• It manifests its toxic effects by binding to protein 3 Halogenated Hydrocarbons
which results in a change of structure and Rationale:
function • Among all the types of pesticides, the
• Ingestion of mercury may lead to bloody organophosphates are the one associated with
diarrhea food contamination
• It may also cause: Methods of Pesticides Determination
o Tachycardia 1 Serum Pseudocholinesterase (SChE) Activity
o Tremors Serum Pseudocholinesterase (SChE) Activity
o Thyroiditis • The increased in Organophosphates will
o Loss of Renal Function decrease the Serum Pseudocholinesterase
Determination of Mercury (SChE) Activity
Specimen of Choice 24-hour urine specimen
Methods of Mercury Determination TOXICOLOGY OF DRUG ABUSE
1 Atomic Absorption Spectrophotometry 1 Amphetamines/Methamphetamines
2 Stripping Anodal Volametry 2 Anabolic steroids
Atomic Absorption Spectrophotometry 3 Cannabinoids
• Method of choice 4 Cocaine
Stripping Anodal Volametry 5 Opiates
It is composed of two (2) steps: 6 Phencyclidine
1. Deposition 7 Sedative-hypnotics
2. Stripping AMPHETAMINES/METHAMPHETAMINES
• Wherein the analyte of interest is electroplated
• It is commonly used to treat narcolepsy and
on the working electrode during a deposition
attention deficit disorder
step and oxidized from the electrode during the
stripping step. Biological Effect:
• The current is measured during the stripping o It blocks dopamine receptors in the brain
step.
causing increased mental and physical
activity
TRANSFORMERS 4
Adverse Effect: • The major urinary metabolite of the

o Restlessness cannabinoids is11-nor-tetrahydrocannabinol-9-
o Irritation carboxylic acid (THC-COOH) that stays in the
o Psychosis urine for 3-5 days
Methods of Cannabinoids Determination
End Product of its Metabolism: 1 Immunoassay
• Benzoic Acid 2 Gas Chromatography
3 Mass Spectrophotometry
**Another type of this drug is Ecstasy which is also
referred to as Methylenedioxymethylamphetamine COCAINE
Methods of Amphetamine/Methamphetamine • It is referred to as the “Rich man’s drug” since it
Determination is expensive
1 Immunoassay • Sometimes, it is used as a local anesthetic
2 Liquid Chromatography
3 Gas Chromatography Biological Effects:
Immunoassay o Excitement
• The antibody is used to detect the analyte o Euphoria
• It is used as a presumptive test
Adverse Effects:
ANABOLIC STEROIDS o Hypertension
• It is originally used to treat male hypogonadism o Arrythmia
o Seizure
Biological Effects: o Myocardial Infarction
o Increases muscle mass
o Increases athletic activity • Primary metabolic product is Benzoylecgonine
Methods of Cocaine Determination
• But since it increases the muscle mass and 1 Immunoassay
athletic performance, some athletes used this to 2 Gas Chromatography
increase their physical activity specially in 3 Mass Spectrophotometry
professional and competitive sports
OPIATES
Adverse Effects: • These are used as analgesic, sedation, and
o Toxic Hepatitis anesthesia
o Atherosclerosis • This comes from opium poppy
o Stroke
o Myocardial Infarction Adverse Effects:
o Respiratory Acidosis
• Testing involves the detection of parent drug o Myoglobinuria
Methods of Anabolic Steroids Determination o Cardiac damage
1 Chromatography
• There will be an increased serum Troponin and
CANNABINOIDS CK-MB due to cardiac damage
• Are psychoactive compounds found in 3 Types of Opiates
marijuana 1 Opium
2 Morphine
Biological Effects: 3 Codeine
o Increases sense of well-being Methods of Opiates Determination
o Euphoria
1 Immunoassay
2 Gas Chromatography
• It is a lipophilic substance, which it attaches to
3 Mass Spectrophotometry
brain and fats
TRANSFORMERS 5
Phencyclidine LABORATORY DEMONSTRATION

• It has a depressant, anesthetic, and Overview
hallucinogenic properties • According to the dangerous drugs board, as of
2019, Methamphetamine Hydrochloride
Adverse Effects: commonly referred to as the Shabu is still the
o Agitation Philippines’ main drug of abuse comprising of
o Hostility more than 90% of total drug surrenderers.
o Paranoia • Statistics also show that Cannabis or Marijuana
and Contact Cement or Rugby follows next in
• It is a lipophilic drug line, respectively.
Methods of Phencyclidine Determination Effects of Methamphetamine
1 Immunoassay • Physiologic Effects:
2 Gas Chromatography o Its effects persist for 2-4 hours with a half-life
3 Mass Spectrophotometry of 9-24 hours
• Majority of the Methamphetamine is excreted
SEDATIVE HYPNOTICS as Amphetamine and Other Reaction
• This are central nervous system (CNS) Derivatives after deamination and oxidation
depressants • Around 10-20% of Methamphetamine is
excreted in the urine in an unchanged form,
Adverse Effects: and it can be detected for up to 3-5 days
o Lethargy • Detecting Methamphetamine in the urine is
o Slurred Speech indicative of Methamphetamine use.
o Coma Effects of Cannabinoids
• Active ingredient: Tetrahydrocannabinol
• Respiratory depression is the most serious toxic • Physiologic Effects:
effect o Its effects peak at 20-30 minutes and can
2 Types of Sedative Hypnotics persist up to 90-120 minutes
1 Barbiturates • THC metabolites such as Delta-9-THC-COOH are
o Secobarbital detected in the urine within hours after use or
o Pentobarbital exposure
o Phenobarbital • It will remain detectable in the urine for 3-10 days
2 Benzodiazepines Principle of the Test
o Diazepam Principle:
o Lorazepam o Lateral Flow – Immunochromatographic Assay
Methods of Sedative Hypnotics Determination o The test kit utilizes this principle for the
1 Immunoassay simultaneous detection of MET and THC
2 Gas Chromatography Metabolites
o It relies on the competition for the antibody
3 Liquid Chromatography
binding between the drug-protein conjugates
and the possible presence of the
aforementioned drugs in the urine sample
Materials Needed
1 Urine Sample
2 MET/THC Test Kit
Rationale:
MET/THC test kit
• A single test strip includes the following parts:
o Disposable urine dropper
o Sample Pad that contains:
1. Gold conjugates
2. Mouse Monoclonal anti-MET
TRANSFORMERS 6
3. Mouse Monoclonal anti-THC
• The first test line which contains the:

o MET-BSA Conjugate
• The second test line which contains the:
o THC-BSA Conjugate
• The Control Line which contains the:
o Goat Anti-Mouse IgG
STEP 3
**Take note that a special form which indicates the
Chain of Custody is required in processing Drug test
• Hold the dropper above the test device
Chain of Custody specifies all individuals involved in
• Dispense 3-4 drops of sample in the sample
the drug testing procedure starting from the
well labeled as “S”
specimen collection up until to the person who will
• As the test proceeds, a purple band that is
release the results
moving across the result window will then be
**Another form that a drug analyst or a DTA must
observed
have been the consent form of the individual that is
being tested.
Pre-Analytical Analysis
Check the Expiry Date
o Make sure that the test kit is not pass the expiry
date
Procedure
STEP 4
• Immediately interpret the results after 5

minutes
• Results interpreted longer than the specified
amount of time are considered as invalid
STEP 1
Interpretation
• Remove the test device from the foil pouch

• That should be performed immediately
after removing the test kit from the foil
pouch as the kit both sensitive to heat and
humidity
• Place the kit on a dry and flat surface
[Negative]
STEP 2
Rationale:
o This indicates that the drug concentrations of
MET/THC are below the detectable levels
• Obtain a urine sample using the provided

dropper
TRANSFORMERS 7
o This indicates that the concentrations of the MET

and THC is at or above the detectable levels
[MET-Positive]
Rationale:
o Lines at the THC and Control
o This indicates that the concentrations of
Methamphetamine in the sample is at or above
the detectable level
[Faint Lines]
Rationale:
o Faint bands are still considered in most test kits
[THC-Positive]
Rationale:
o Lines at the MET and Control
o This indicates that the concentrations of the THC
metabolites in the sample is at or above the
detectable levels
[MET-THC Positive]
Rationale:
[Invalid Result]
o Line at the Control
TRANSFORMERS 8
Rationale:
o The absence of the colored band in the control
regardless of the result obtain from the first and
second band is indicative of invalid result
o The test must be repeated with a new test kit
• The drug test analyst must preserve the sample

in the event of positive screening test result as it
will be subjected for a confirmatory testing
through GCMS or Gas Chromatography Mass
Spectrophotometry at East Avenue Medical
Center which is the Reference laboratory for
toxicology and drugs of abuse
TRANSFORMERS 9
Introduction to Clinical Endocrinology, Pituitary,
8 Adrenal, and Gonadal
Clinical Chemistry 2 (LAB)
PITUITARY, ADRENAL, & GONADAL FUNCTION na lang “idiopathic” or hindi known yung
PITUITARY TUMORS dahilan bakit siya nagkaroon ng pituitarism
1 Prolactin-secreting tumors Panhypopituitarism
2 Nonfunctioning or null cell tumors ● Complete loss of function
3 GH, Gonadotropins, ACTH, TSH tumors ● Meaning yung ating pituitary gland ay hindi na
talaga gumagana at all
Rationale:
● Treatment: We can only do replacement
● So pag nagdedevelop ang mga cancers sa
therapy for the primary target organ failure.
pituitary gland napakaraming factors, but the
o If the primary target organ is the pituitary
three major or most common factors that could
gland, yung mga hormones na sinesecrete
lead to pituitary tumors
niya or yung mga specific hormones na
1. Prolactin-secreting tumors – most common
kailangan talaga such as thyroid, growth
2. Nonfunctioning or null cell tumors
hormones, etc., kailangan natin ng
3. GH (Growth Hormone), Gonadotropins,
replacement therapy dun sa patient
ACTH, TSH tumors – the specific tumors sa
o However, the problem here is napaka
mga specialized cells
gastos. This is a costly therapeutic method.
Hypopituitarism
Growth Hormone/Releasing Hormone Feedback
● There is a low secretion of hormones secreted
System
by the pituitary gland
● Most common or the Monotropic hormone
deficiency
o Meaning Monotropic – a single area or
tissue ang may problem, it has a specific
abnormality kaya nagkakaroon ng
hormone deficiency
● Examples:
1. Pituitary tumors
2. Parapituitary/hypothalamic tumors
3. Trauma
4. Radiation therapy/surgery – natamaan or
nadamay ang mga pituitary tissues
5. Infarction – hindi dumaloy yung dugo sa
pituitary gland
6. Infection – bacterial invasion doon sa area
7. Infiltrative disease – nagkaroon ng foreign
substances Growth Hormone/Somatotropin
8. Immunologic – autoimmune diseases or ● Increased: acromegaly, chronic malnutrition,
yung immune system mismo ng patient renal disease, cirrhosis, and sepsis
attacks yung sariling tissues niya sa pituitary ● Decreased: hyperglycemia, obesity,
gland hyperthyroidism
9. Familial – genetic ● Method: Chemiluminescent Immunoassay
10. Idiopathic – tinatamad yung diagnostician, ● Reference Values: <7 ng/mL (in the blood)
hindi niya alam yung diagnosis, sasabihin Growth Hormone Disorders
TRANSFORMERS 1
● Most common growth hormone disorders are ● Andre the giant is an example for
dwarfism, acromegaly and gigantism. acromegaly, wrestler nung 80s, and
1. GH deficiency (GHD) / Pituitary dwarfism mapapansin niyo na mas Malaki siya sa
a. Idiopathic growth hormone deficiency normal size ng human, and bone structure is
o Meaning hindi known pero usually it is different, his nose bridge is bigger, and
genetic or familial jawline niya. Hindi lang height affected,
o It is the most common cause of GH pati bone structure ng patient
deficiency in children
b. Pituitary adenoma
o Maari ring caused by tumor or a tumor
in the pituitary gland
o Most common diagnosis of dwarfism in
adult-onset GH deficiency ●
2. Acromegaly and Gigantism ● The problem sa acromegaly and gigantism
o Meaning nagkakaroon ng is due to the overproduction of growth
napakaraming growth hormone sa hormone, often times patients suffering this
circulation syndrome is napapaikli buhay nila kaya
o Due to overproduction of GH need talaga ng treatment.
o >50 ng/mL, masyadong mataas ang Gh Gonadotropin Feedback System
meaning matangkad yung patient,
hindi na normal yung paglaki niya
o Normal value: <7 ng/mL
Notes:
Growth hormone deficiency/ Pituitary Dwarfism,
yung stature ng ating pasyente is mas maliit kesa
sa average human
Kapag naman overproduction or mataas value ng

growth hormone (>50 ng/mL) mataas to since
normal value lang is <7 ng/mL, so ibig sabihin since
masyado mataas sa patient, masyado niyan
matangkad patient. Hindi na normal paglaki niya
pati bone structure niya. Gonadotropins
Increased FSH: clue in the diagnosis of premature
Examples: menopause
Increased FSH and LH post menopause, due to
● Dagul (Growth Hormone Deficiency) as you lack of estrogen
can see nagsusuffer siya dwarfism, although ● FSH: Follicle stimulating hormone
day to day life niya is normal lang. ● LH: Luteinizing hormone
● If mamanage mo syndrome na to Pituitary-Thyroid Axis Feedback System
(dwarfism and gigantism), day to life would
be normal parin.
TRANSFORMERS 2
Thyroid Stimulating Hormone

Adrenocorticotropic Hormone
● Blood levels may contribute in the
evaluation of infertility Increased: Addison’s disease, ectopic tumors,
Main problem with the thyroid is doon sa mismong protein rich meals
thyroid organ, so dito sa pituitary walang masyado Main problem is on the tissue itself, sa adrenal
ambag sa katawan, doon lang sa infertility, doon glands
lang siya nagbibigay ng kanyang contribution Prolactin
Adrenocorticotropic Feedback System Increased: pituitary adenoma, infertility,
amenorrhea, galactorrhea, acromegaly, renal
failure, PCOS, cirrhosis, and primary and secondary
hypothyroidism
● Method to analyze prolactin: immunometric
assay
● Reference values:
● Male: 1-20ng/mL
● Female: 1-25ng/mL
● Remember the normal values
● Prolactinoma (>150 ng/mL)
● Pag Prolactinoma, this is a tumor na, oma
eh
● Tumor that secretes prolactin
Anong nangyayare sa patient natin?
● Reduced libido and erectile dysfunction
(male)
TRANSFORMERS 3
● Alteration on menopause (female), irregular ● Normoglycemia - normal blood glucose

menopausal dates level
● Abrupt onset of breast discharge, kahit ● Polyuria with low sp. Gravity
walang stimulation nagkakaroon ng ● Polydipsia- excessive thirst or excess
sudden release sa kanyang mammary drinking
glands ng gats ● Polyphagia- excessive or extreme
hunger
Hyperprolactinemia (25-100ng/ml) o Diabetes insipidus vs. Diabetes Mellitus
● Pituitary stalk interruption ● Their main difference is the
● Pituitary stalk is the connection ng pituitary amount/value of specific gravity
gland sa kanyang hypothalamus ● Diabetes insipidus has low specific
● Therapy for this is: Use of dopaminergic gravity
antagonist medications Diabetes Mellitus has normal to high specific
Hyperprolactinemia is due to Primary thyroid failure, gravity
or Renal failure, or pwede rin lumitaw sa Polycystic
ovary syndrome (PCOS) ADH disorder
Oxytocin Major Types of Diabetes Insipidus
o Pitocin – synthetic oxytocin to induce labors
o It is easily distributed to the tissue a. True Diabetes Insipidus
o It is very rare for it to have pathology or (Hypothalamic/Neurogenic)
pathogenesis ● ADH deficiency with NORMAL ADH
*No significant clinical pathophysiology receptor
Anti-Diuretic Hormone/Vasopressin ● Failure of the pituitary gland to secrete
oIncreased: increased plasma osmolality ADH
(>295 mOsm/kg) b. Nephrogenic Diabetes Insipidus
o Rise in plasma osmolality stimulate the thirst ● NORMAL ADH with ABNORMAL ADH
center of brain receptor
● ↑ in osmolality = thirst ● Failure of the kidneys to respond to
o ↑ in osmolality – SHRINK - hypothalamic normal or elevated ADH levels
osmoreceptors End Goal: POLYURIA
o ↓ in osmolality – SWELL – hypothalamic
osmoreceptors
Reference values: serum: 0.5-2 pg/uL ADRENAL
Anti-Diuretic Hormone/Vasopressin Disorder Cortex Steroidogenesis
o Diabetes insipidus
● This diabetes has no correlation with
glucose because it has a
normoglycemia.
● ADH deficiency; results in severe
polyuria (≥3L of urine/day)
● 24-hour urine sample is used for
screening diabetes insipidus
o Clinical features include:
TRANSFORMERS 4
For the adrenal gland, always remember that we ● Yung dalawa may marked virilization,
have the cortex steroidogenesis. Meaning, yung ibigsabihin nagkakaroon ng male
hypothalamus na nag sesecrete ng Corticotropic characteristics yung female. Sino lang yung
Releasing Hormone (CRH) and then magiging wala si 17 α-Hydroxylase
Adrenocorticotropic Hormone (ACTH), ACTH can ● However dito sa 3β-Hydroxysteroid
produce cholesterol. Cholesterol will enter the dehydrogenase, remember the High Lab
mitochondria of the target cells and then release Value
pregnenolone (end product).
● Hypertension is the most common symptom RAAS (from HAPP discussion)
that we can associate with cholesterol.
Meron din neto sa medulla.
Aldosterone
● Also known as Aldo
● Method: RIA and chromatography
o RIA - Radioimmunoassay
Pag may certain problems sa isa or dalawang
● Increased: Hyperaldosteronism (Conn’s
enzymes dyan, magkakaroon ng abnormality.
disease)
● GFR (glomerulosa fasciculata reticularis)
o Hypernatremia, hypokalemia,
o Three zones of adrenal cortex
metabolic alkalosis
● Pregnenolone
o High sodium, Low potassium,
o Precursor sa halos lahat ng
metabolic alkalosis
hormones na nirerelease.
● Decreased: Hypoaldosteronism
Congenital Adrenal Hyperplasia
o Hyponatremia, hyperkalemia,
Group of clinical entities that arise from absent or metabolic acidosis
diminished activity of enzymes involved in o Low sodium, High potassium,
steroidogenesis. metabolic acidosis
● Maaaring may abnormality doon sa
Aldosterone Disorders
enzyme. Pag nangyari yun, magbabago
A. Primary hyperaldosteronism
yung chemical na normally na nagiging
● Caused by
hormone.
o APA - Aldosterone adenoma
● For example, meron kang problem sa
▪ Ang ibig sabihin nyan
3-beta (β)-hydroxysteroid dehydrogenase
Aldosterone adenoma. Saan
(HSD). Anong nangyayari? Magkakaroon
galling ang PA? Ibig sabihin
ng slight virilization (yung female,
Primary ALdosteronism. Pag
nageexhibit siya ng male characteristics –
trinanscribe niyo, hindi siya
e.x. body hairs).
exactly APA. Yung A dito yun
TRANSFORMERS 5
lang ichecheck niyo, ● Most common clinical picture when you have
aldosterone adenoma Primary hypoaldosteronism:
o IHA - Adrenal hyperplasia ■ HIGH plasma ALDOSTERONE
▪ Saan galling yung I? Yung I ◆ May problema yung ADRENAL GLAND
dito is yung characteristic ng -> Excessive release of aldosterone
inyong adrenal gland, yung ■ LOW plasma RENIN
histology niya ■ PA:PRA > 25 [plasma aldosterone and
o GPA - Glucocorticoid-remediable plasma renin activity ratio]
glucocorticoid receptor defects SECONDARY HYPERALDOSTERONISM
▪ Sa pangalan pa lang, alam ● Indirect yung PAG-INCREASE
niyo na yung may may ■ Angiotensinogen -> angiotensin (galing
problema is glucocorticoid kay RENIN)
receptors na matatagpuan ◆ Angiotensin
din sa adrenal glands ● mag-uutos sa adrenal glands to
o Adrenal carcinoma - tumors/cancer release aldosterone
in adrenal glands ● If renin has a problem,
● HIGH plasma ALDO, LOW plasma RENIN aldosterone will also have a
● PA: PRA >25 problem
o PA:PRA - Plasma aldosterone: ● MAIN PROBLEM: RENIN
Plasma renin activity ● Caused by EXCESSIVE production of RENIN
o If greater than 25, it means Primary ● HIGH plasma ALDOSTERONE and RENIN
hyperaldosteronism Due to the elevated / excessive production of
RENIN, madadamay si ALDOSTERONE.
Note: Ano ba ang main difference? Pag primary, HYPOALDOSTERONISM (Addison’s disease)
the main cause of hyperaldosteronism is yung ● Addison’s disease - hypoaldosteronism and
mismong tissue or yung specialized cell na hypocortisolism
nagsesecrete ng aldosterone. Whereas kapag ● Caused by destruction of adrenal glands and
secondary ibig sabihin indirect, hindi involved yung glucocorticoid deficiency
main tissue na nagsesecrete ng aldosterone. ● Same with hypocortisolism
Meron pang dahilan bakit tumaas yung values ng LOW plasma ALDOSTERONE
aldosterone. Kaya tatandaan kapag secondary Cortisol
ibig sabihin yung tissue mismo na nagsesecrete for Method: HPLC-MS (High Performance Liquid
example dito yung adrenal glands mismo okay Chromatography - Mass Spectroscopy)
lang siya walang problema, may indirect na
dahilan or may nag-uutos sa kanya kaya mas INCREASED: Hypercortisolism (Cushing’s disease)
tumataas yung value niya. ● Hyperglycemia
● Glucocorticoid-remediable glucocorticoid ● Hypertension
receptor defects ● Hypercholesterolemia
■ PROBLEM: Glucocorticoid receptors, which ● Leukopenia
are found in the adrenal glands. ● Obesity
● Adrenal carcinoma
■ Tumors / cancers (adrenal glands) DECREASED: Hypocortisolism (Addison’s disease)
● Hypotension
TRANSFORMERS 6
● Hyperkalemia o Secondary meaning adrenal gland is not

● Weight loss the one who causes problem anymore but
HYPERCORTISOLISM (Cushing’s syndrome) still cortisol levels will remain at low levels.
● Caused by excessive production of cortisol o This is caused by a loss of ACTH
and ACTH ● Loss of ACTH
■ PROBLEM: Adrenal Gland (cortisol) and o The problem is in the pituitary gland.
Pituitary Gland (ACTH) o Pituitary gland should produce ACTH
● May also be caused by corticosteroid overuse supposedly since it can detect low levels of
● MAIN FEATURE: “buffalo hump” cortisol.
● HIGH CORTISOL and ACTH o Pituitary gland is a master gland therefore it
LOW ALDOSTERONE and RENIN can send a signal (send utos) to the
. Primary Hypocortisolism adrenal glands to produce cortisol.
o In primary, remember that the one causing However, in cases like this hindi masyadong
the problem (low secretion of cortisol) is the nagrrelease or pwedeng totally walang
irrelease si pituitary gland na ACTH.
tissue itself or the specialized cell that
o There will still be low levels of cortisol even if
secretes the hormone.
there is no problem in adrenal glands as
● Caused by decreased cortisol products well as low levels in ACTH since dito mismo
o Primary = Decreased cortisol production yung problem (di kasi nagrerelease
● Hyperpigmentation pituitary gland)
o Patient will experience hyperpigmentation – o Low ACTH = Low cortisol
There will be change in skin color making it ● No Hyperpigmentation
more golden (gumiginto) or bronze LOW CORTISOL and ACTH
depending on the skin color of the patient Cortisol Disorders
● HIGH ACTH, LOW CORTISOL, ALDO, and RENIN
o Why is ACTH high?
▪ The pituitary gland will continuously
release ACTH (kasi nga walang
problem sa pituitary gland), it can still
detect that there is a low level in
cortisol. Because of this, it will
continuously release ACTH = HIGH This photo shows the main difference of cortisol
ACTH disorders in terms of their clinical features
▪ However, there is a problem in adrenal ● Cushing syndrome
glands. Due to this, ACTH that is o Moon face
continuously released will not be o Gynecomastia – In males, lumalaki
converted into cortisol. Cortisol will not breast/chest area
go to the target tissues or when it is o Buffalo hump – There is a fat
able to go to the target tissues, build-up in the neck area (sa
nag-iiba naman yung kanyang likod so di kita sa pic)
structure (hindi siya nagiging cortisol) ● Addison’s disease
▪ The problem here is the tissue itself o Bronze/gold pigmentation of the skin
which is the adrenal gland o Hypoglycemia
C. Secondary Hypocortisolism Super daming HYPO except sa potassium
TRANSFORMERS 7
Adrenal Androgens o They are rapidly cleared because their

● Weak androgens/ Adrenal androgens effect is also rapid
● Increased: virilization for women Another characteristic is that they are
o When there is an increase in the value of HYDROPHILIC, which is why they can easily travel all
these androgens, nagkakaroon ng throughout the body.
virilization for women Epinephrine/Adrenaline
o This happens when there is an enzyme ● Major metabolite:
abnormality such those that are previously VMA (vanillylmandelic acid)
mentioned like hydroxylases and DHEA Dopamine
o Virilization – Female develops a male ● Major metabolite:
characteristic such as yung sa hair (pag sa HVA (homovanillic acid)
buhok, specifically hirsutism siya however Adrenal Medulla Disorders
dito general siya), body structure becomes 1. Pheochromocytoma
prominent ● Tumor of adrenal medulla or sympathetic
o – General term where females acquire ganglia (chromaffin cells)
male characteristics o From the name itself, “chromocytoma”
● Increased: virilization for women this means that there is the formation of
o Aside from hair growth, the body tumor in the chromaffin cells, most
structures of women also change, their specifically in the chromaffin cells of
shoulders become prominent and may adrenal medulla or sympathetic
have increased build-up of muscles. ganglia
o When we say “virilization”, it is the ● Overproduction of catecholamines
general term used to describe when a o This results to always being in a fight or
female acquires male characteristics flight mode which results to:
There is the presence of facial ● Hypertension, tachycardia, headache,
hair and increased hair growth tightness of chest, and sweating, these are
in the navel area, and clinical the manifestations
increased hair in the groin area.
This is specifically called 2. Neuroblastoma
“Hirsutism” which is still a part of ● Fatal malignant condition in children
virilization. ● Overproduction of norepinephrine
o Kapag bata ka palang and
Noepinephrine/Noradrenaline
napakataas ng norepinephrine mo
● Major metabolites:
ang nangyayari sayo at same lang,
o MHPG
hypertension, tachycardia, etc. Pag
(3-methoxy-4-hydroxyphenylgycol)
sobrang taas ng norepinephrine mo
o VMA (vanillylmandelic acid)
mas higit pa don ang pedeng
o These metabolites can also be checked
mangyari. Kapag naging chronic na ito
in the clinical microscopy
maaring ikamatay ng pasyente kasi
o These catecholamines’ half-life in the
sobrang delikado neto kaya tinawag
circulation is only SECONDS up to TWO
na fatal malignant
(2) MINUTES
● High urinary excretion of HVA or VMA or
both, and dopamine
TRANSFORMERS 8
nag poproduce kaya hypogonadism kaya

increased FSH). Note: Normal ang LH and
GONADAL most common ng Hypergonadotropic
Causes of Infertility Hypogonadism is menopause
-Ovarian failure🡪 Increased FSH (Normal LH)
most common manifestation: menopause
2. Polycystic Ovary Syndrome (PCOS)

-Common sa mga females sa Pilipinas na
medyo nahihirapan mag kaanak.
-Infertility (kase meron anovulation o absence of
egg cell production), hirsutism, anovulation,
glucose intolerance, hyperlipidemia (ung iba, hindi
lahat ng may PCOS,ung may PCOS minsan ay
mataas ung lipid in the circulation kaya medyo
tumataba), and hypertension.
Testosterone
● This is the summary of causes of infertility from Method: Most Test for infertility
Bishop - And usually nasa CM which is semenalysis,
● Infertility – decreased value of the gonadal testosterone, FSH and LH.
hormones - Reference value for testosterone: 3.9-7.9
● As you can notice in the table, almost all of ng/mL
them have decreased levels of gonadal - Specimen: Serum
hormones
Pathology
These pathology is more focused on females Testicular Infertility
1. Hypogonadotropic Hypogonadism
o Decreased FSH and LH of the females 1. Pretesticular infertility (Secondary
o More commonly known as Runner’s hypogonadism)
ammennorhea. Minsan daw ung mga - Hindi mismo ung testes ung may problem
babae, pag masyadong tumatakbo and - Ang may problem: Hypothalamus and
biglang sumasakit ung puson area, hindi Pituitary gland
ung gilid gilid. - Normal to low: testosterone, FSH and LH
1. Hypergonadotropic Hypogonadism
-Hyper ang FSH 2. Testicular infertility (Primary hypogonadism)
-Ang problem, nasa OVARY (d sya makapg - Ung testes mismo may problem
produce ng egg cells as well as estrogen - This is usually congenital (bata palang may
and progesterone. Dahil matitriger ang problem na testes)
pituitary gland na mag release ng FSH - Low: Testosterone
(para mag produce ng estrogen and - High: FSH and LH
progesterone, may ovarian failure d parin
TRANSFORMERS 9
o The problem is with the pituitary or

3. Post-testicular infertility the hypothalamus (hypothalamic or
- Walang problem sa endocrine gland, pituitary)
endocrine system. Ang problema nasa The function of the ovaries is normal but since there
mismong sperm. is a problem with the pituitary, FSH, LH, and
Normal: Testosterone, FSH and LH estradiol are low
Estrogen Progesterone
Decreased: Irregular and incomplete development ● Decreased: failure of embryo implantation
of ENDOMETRIUM (eto ung shineshed ng mga
females pag mens). Notes:
● If the progesterone of a female is low, this
Forms of Estrogen can cause a failure of embryo implantation
Estrogen Significance Endocrine Also leads to infertility
gland
Estrone (E1) Post-menopau Ovaries Human Chorionic Gonadotropin
sal women ● Female: pregnancy marker
Estradiol (E2) Pre-menopaus Ovaries ● Male: testicular carcinoma marker
al women, ● Method: Immunometric (sandwich)
major
estrogen Notes:
Estriol (E3) Major Placenta ● For the females, HCG is the pregnancy
estrogen (Precursor of marker found in the pregnancy tests.
during (E2) ● For the males, males also produce HCG
pregnancy even though they do not get pregnant.
From estradiol Males produce HCG if they have testicular
galling sa carcinoma. HCG is used as a testicular
ovaries carcinoma marker for the males.
pupunta ng ● The method used to detect the HCG is the
palcenta para immunometric or the sandwich ELISA.
maging estriol Inhibin A
Female Infertility ● 4th Down Syndrome marker – Quadruple test
1. Primary deficiency of female sex hormone for Down Syndrome (Trisomy 21):
o LOW E2, HIGH FSH and LH
o The problem is with the tissue or the Trisomy 21 Marker Down Syndrome Result
ovary itself. HCG Increased
o There is low estradiol but there is Urine E3 Decreased
high FSH and LH Alpha fetoprotein Decreased
2. Secondary deficiency of female sex Inhibin A Increased
hormone Notes:
o LOW E2, FSH, and LH ● The inhibin A is the 4th down syndrome
marker
1
TRANSFORMERS
0
● We have this test called the quadruple test 4. Aldosterone

for down syndrome or the quadruple test 5. Medullary Hormones
for trisomy 21. 6. Estrogens
● The four tests include: Summary of Hormonal Assays
o HCG These are the questions that you might ask yourself
o Urine Estriol while we’re going through this lecture so let’s
o AFP or the Alpha fetoprotein answer them now:
o Inhibin A 1. Why are we not discussing ALL hormone
● Increased: HCG and Inhibin A diagnostic lab procedures?
● Decreased: Estriol and Alpha fetoprotein o Because not all of the hormones are
● The results will be normal if the child is actually significant, meaning, they
normal. are not diagnostically significant.
● Sir, kung down syndrome result yan, ano o Certain hormones such as oxytocin
naman dapat yung maging result nya sa and prolactin and certain weak
normal na bata? androgens are actually just
o Pag normal yung bata, shempre, contributing factors to a certain
normal lahat ng result nya. Hindi mo disease, meaning, they are not the
babaligtarin. Normal lahat yan. main factor for a certain disease
Remember, in the quadruple test for down that’s why they are rarely tested
syndrome, we have inhibin A, and HCG and rarely conducted in the
and they are both increased. Estriol and laboratory procedures.
AFP are decreased. 2. Will we ever see these tests in the actual
clinical laboratory?
● Maybe, because almost all of these tests re
DIAGNOSTIC PROCEDURES not routinely done. So basically, you won’t
Objectives: find them in a routine clinical chemistry
● At the end of this presentation, the students laboratory.
shall: ● They are usually found on different sections
o Be able to recall the different endocrine of laboratory usually, and outside
functions of pituitary, adrenal, and laboratory too. You may find them in
gonadal hormones immunology section, special chemistry
o Be able to understand the principles section. Some tests are conducted by
behind the different laboratory clinical pharmacists, and some tests are
diagnostic procedures usually inside the nuclear medicine section.
Be able to correlate the different hormonal ● In your internship, limit lang yung makikita
laboratory values to their respective effects niyong tests for endocrinology.
Table of Contents
● Significant Diagnostic Laboratory 3. Why would it be relevant to know these
Procedures: procedures?
1. Growth Hormone ● Lalabaas sa boards
2. Anti-Diuretic Hormone
3. Cortisol and ACTH
1
TRANSFORMERS
1
● If you want to pursue a career in Growth Hormone

endocrinology when you become a Reference value: 7 ng/mL
physician or a clinical laboratory scientist.
● There are also opportunities abroad. GH Deficiency
o These tests are rarely done in the ● GH value of lower than 7 ng/mL
Philippines, but in abroad, medyo ● Insulin Growth Factor-1 (IGF-1) - low
common naman. However, you ● Physical Activity Test - screening
need a special training. o Procedure: Exercise test
▪ Example of growth hormone
stimulus
o Normal result: elevated GH serum
o If GH fails to increase, confirmation
must be made
▪ Because there are a lot of
factors that may be involved
to the failure of growth
hormone
● Insulin Tolerance Test - confirmatory
o Insulin Tolerance Test and Arginine
These are the common machines and instruments stimulation test
used when conducting endocrinology. Both pharmacologic tests
● Siemens machine (upper left) INSULIN TOLERANCE TEST & Arginine stimulation test
● Beckman coulter (lower left) (Confirmatory test)
● Cobas machine (lower right) ● INSULIN: inhibitor for GH release
o These 3 were usually found in CC o administration of insulin causes
laboratory decrease in GH; However, this
o They utilized the principles of decrease is only up until a certain
immunology, chemistry, as well as level because we have the
the immunochemistry threshold
● Exercise test (upper middle) o Negative feedback system occurs
o It has treadmill para magrelease ng when there’s already too much
certain hormones insulin (disruption in homeostasis)
● IV drip (upper right) ● ARGININE: a stimulus which increases GH at
o There are a lot of pharmacologic only a certain level
test that involves the hormone. PROCEDURE 24-hr/night time monitoring of
Pharmacologic tests involve GH
stimulation or inhibition of certain RESULT elevated serum GH until 5
hormones to assess its threshold ng/mL (adult) & 10 ng/mL
value, and to check for the increase (child)
or decrease in value. INTERPRETATION If GH fails to increase →
o In regards to that, you can confirm CONFIRMED GHD
a certain disease. ACROMEGALY
1
TRANSFORMERS
2
● has HIGH IGH-1 Anti-Diuretic Hormone

● Overnight Water Deprivation Test
SOMATOMEDIN C or insulin-like growth factor-1 (Concentration Test) – for anti-diuretic hormone
(a direct/screening test for IGF-1) o This is a confirmatory test para madistinguish
PROCEDURE Extract fasting serum mo ‘yung difference between a neurogenic
NORMAL RESULT IGF-1 is normal diabetes insipidus and nephrogenic
INTERPRETATION INCREASED → acromegaly diabetes insipidus.
DECREASED → GHD o ‘Yung screening test dito was yung sa CM,
GLUCOSE SUPPRESSION TEST or OGTT (75g glucose) ‘yung test for urine osmolality.
(Confirmatory test for acromegaly) o Procedure: You need a fasting serum and
PROCEDURE Extract blood every 30 mins. for the patient should not take any fluids at all
2 hrs. (patient should fast prior) ⮚ Ito ‘yung very very strict non per orem
RESULT Suppression of GH <1 ng/mL test procedure
(until 0.4 ng/mL) o Result: The urine osmolality does not rise
INTERPRETATION above 300 mOsm/kg serum ADH – normal
⮚ So, basically, ito ‘yung sa CM,
GH fails to decline (+ acromegaly
malamang walang DI ‘yung patient mo.
increased IGF-1)
⮚ And if the serum ADH is normal, very
GH fails to suppress below acromegaly
likely that your patient has no diabetes
0.3 ng/mL w/ ELEVATED
insipidus.
IGF-1
⮚ However, if your patient has increased
GH suppressed below 0.3 NOT acromegaly
mOsm or ‘yung kanyang urine osmolality
ng/mL w/ NORMAL IGH-1
(increased urine osmolality), it should be
GH suppressed but Other factors
a diabetes insipidus.
ELEVATED IGF-1
o Interpretation: Neurogenic DI – Low ADH;
Nephrogenic DI – Normal to high ADH
● If the GH is suppressed but elevated IGF-1 →
⮚ How are we going to
other factors
differentiate/distinguish between the
o If the GH is suppressed, so normal ‘yung
two DIs?
kanyang negative feedback mechanism,
✔ If your patient has increased urine
but the IGF-1 is elevated, meaning, there
osmolality pero low ADH, so this is
should be other factors and it is not
neurogenic. Once again, the
acromegaly – ito yung importansya ng
problem is in the hypothalamus or
confirmatory tests, so that you could
the pituitary gland.
distinguish acromegaly from other factors
✔ If your patient has a normal to
that could contribute to the elevated IGF-1
increased ADH, ibig sabihin, this is a
of your patient.
nephrogenic diabetes insipidus and
o So ano ‘yung other factors na yon? It may
with that, you could also conduct a
be liver problems, such as liver cirrhosis,
more confirmatory test.
pwedeng ‘yung tissue mismo may
✔ This is your gold standard – Overnight
problema, pwedeng ‘yung receptor inside
Water Deprivation Test. Pero you
that tissue may be abnormal, or maaaring
kaunti lang ‘yung kanyang amount.
1
TRANSFORMERS
3
could also conduct the ADH The next one is the 17-Ketogenic steroids or the
administration. Zimmermann reaction (method). The result should
o ADH administration: Neurogenic DI – be reddish purple.
Kidneys will compensate; Nephrogenic DI – Cushing’s Syndrome (AKA hypercortisolism)
No renal water reabsorption ● Expected result: Elevated cortisol levels
⮚ Ibig sabihin, you would induce/inject ● Normal value of cortisol: <0.5 ug/dL
you patient the hormone itself. Increased: >0.5 ug/dL (expect Cushing’s
⮚ We have the neurogenic DI, wherein the syndrome)
kidneys will compensate for the 3 Screening test:
problems in your hypothalamus. ● 24-hour urinary free cortisol test (direct
If you have nephrogenic DI, there is no renal water test*)
reabsorption, ibig sabihin, your ADH receptors ● Overnight dexamethasone suppression test
found in your tubules tyaka don sa loop of Henle, (Significant test**)
yung mga receptors, may problem. That is a o Dexamethasone – pharmacologic
conducive or conclusive of your nephrogenic DI. agent (stimulus) which could
Cortisol and ACTH stimulate the release of cortisol up
● For the next tests, we have for the cortisol and until a certain level --- Because in a
ACTH, and it involves a lot of tests kasi nga normal person, the negative
we’re trying to detect a disorder na very feedback system would proceed to
significant, which is ‘yung Cushing’s, as well as, suppress the increase in cortisol level
‘yung hypocortisolism. ● Midnight salivary cortisol test (direct test*)
● Urinary metabolites: o *Direct – it means you assess the
o 17-hydroxycorticosteroid saliva or the urine of the patient,
⮚ Method: Porter-Silber then directly detect cortisol levels
⮚ Result: Yellow color Results (screening test):
o 17-Ketogenic steroids ● Increased: rule in Cushing’s Syndrome but
⮚ Zimmermann Reaction cannot confirm it yet
⮚ Result: Reddish purple
3 Confirmatory test:
Note: ● Low-dose dexamethasone suppression test
o The first one, are the urinary metabolites. o Same as Overnight dexamethasone
We have the 17-hydroxycorticosteroid and suppression test, the only difference
17-Ketogenic steroids. is that you are giving your patient a
o So, ang gagawin mo lang dito are actually low dose (15g of dexamethasone)
organic chemistry tests, ibig sabihin, there then check for cortisol levels after 30
will be a coloration/colorimetry, so this is a minutes. This is to conduct for 3 days
colorimetric test. ● Midnight plasma cortisol (direct)
o So, ‘yung method for the ● Corticotrophin- releasing hormone (CHR)
17-hydroxycorticosteroid, we have the stimulation test (direct)
Porter-Silber and the result should be yellow, Results (confirmatory test)
if present ‘yung metabolite na ‘yon. ● >0.5 ug/dL = confirmed case of Cushing’s
syndrome
1
TRANSFORMERS
4
● Oral sodium loading test

Hypocortisolism (low cortisol) - Addison’s disease ● Fludrocortisone suppression test
● Detection and differentiation of primary to Captopril challenge test
secondary O 28:00 – 34:16
● ACTH stimulation test – screening test Fludrocortisone suppression test and the Captopril
● Insulin Tolerance test – confirmatory test challenge test.
*2 mentioned test are only for detection Medullary Hormones
and differentiation For the medullary hormones or yung mga nasa
adrenal medulla. Ito yung mga tests.
*Before proceeding to ACTH stimulation test, the ● Urinary metabolites:
aforementioned cortisol test (in Cushing’s o MHPG, VMA, Normetanephrines,
syndrome) can be used to detect if your patient Metanephrines, and HVA (lecture
has low cortisol values or serum level. discussion)
● After only a decreased result should you
proceed with ACTH stimulation test. For the detection of Phechromocytoma:
● Clonidine Test
● Primary Hypocortisolism: ● Glucagon Stimulation Test
o ACTH stimulation test and insulin
tolerance test expected result: **for this test once again this are the stimulation
▪ Decreased cortisol test. So ganun din, glucagon is actually a stimulus
▪ Decreased aldosterone of your norepinephrine and epinephrine. So
▪ Decreased renin basically same principle lang ng other stimulation
▪ Increased ACTH test. Kapag instimulate nya, you should expect na
● Secondary Hypocortisolism: increase pero at a certain level lang. Merong
o Delayed response in the cortisol threshold. And Kapag elevated siya, pwede mong
o ACTH stimulation test and insulin sabihin na disease Pheochromocytoma
tolerance test expected result: (Phechromocytoma sa ppt).
▪ Decreased cortisol Estrogens
▪ Decreased ACTH Kober reaction
Aldosterone ● For the analysis of different estrogens
Conn’s disease (hyperaldosteronism) – main ● Involves the utilization of hydroquinone and
significant/ relevant disease hot concentrated sulfuric acid (H2SO4).
● Plasma aldo concentration: plasma renin ● Positive result: Pink color
activity (PAC/PRA ratio) – screening test ● Specific on certain estrogen present
o Normal ratio – 1:1 ● Have certain concentration or different
o (+) result - >30 ratio – suggestive of levels of reagent to detect/differentiate the
primary hyperaldo three (3) estrogens.
o (+) result - >50 ratio – suggestive of Summary of Hormonal Assays
secondary hyperaldo SAMPLES
● Whole blood
Confirmatory test: (direct tests) o LH, testosterone
● Saline suppression test ● Plasma
1
TRANSFORMERS
5
o EDTA – ACTH, ADH

o Heparin – catecholamines, cortisol,
dopamine, FSH
● Serum
⮚ Aldo, weak androgens, estrogen,
FSH, GH, HCG, progesterone
● Urine
⮚ Estriol and urine metabolites
OTHER TESTS
● Classic Assay
⮚ Bioassays
⮚ research laboratory
⮚ Use other animals and study
their level of hormones using
this.
⮚ Competitive Protein Binding (CPB)
⮚ for insoluble hormones
⮚ in need of transport proteins
⮚ examples: steroid hormones,
testosterone, androgens,
thyroid hormones
● Fluorescent Techniques
⮚ Fluorescence Polarization
Immunoassay (FPIA)
⮚ Immunology section
⮚ This is where you can see the
Ag-Ab binding complex of
hormones
⮚ Kung mapapansin niyo para
silang Ab kasi mayroong
hormone [Ag] and receptor
[Ab].
● High Performance Liquid Chromatography
⮚ Stationary phase and mobile phase
● Colorimetry
⮚ Previously discussed like quarter silver,
Zimmerman, and Kober reaction.
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TRANSFORMERS
6
9 Thyroid, Pancreatic, and Gastrointestinal Clinical Chemistry 2 (LAB)
Thyroid, Pancreatic and Gastrointestinal Functions ● Thickened Skin

THYROID GLAND ● Cardiac Complications
Disorders of the Thyroid
Hypothyroidism
● Caused by insufficient amounts of thyroid
hormone
⮚ Low free T4 level, normal or high TSH
● One of the most common disorders of the
thyroid gland
CAUSES OF HYPOTHYROIDISM
Organ Conditions
Involved
Primary Thyroid Gland ⮚ Destruction of
the thyroid
gland itself
⮚ Subtotal
thyroidectomy
⮚ Hashimoto’s
Disease
⮚ Myxedema
● Hypothyroidism has signs and symptoms Second Pituitary ⮚ Hypopituitarism
like; ary Gland ⮚ Pituitary
⮚ Intolerance to the cold destruction
⮚ Receding hairline ⮚ Pituitary
⮚ Facial and Eyelid Edema adenoid
⮚ Dull-Blank Expression Tertiary Hypothalamu ⮚ Rare
⮚ Extreme Fatigue s
⮚ Thick Tongue- resulting to slow
speech ● Pituitary Gland- responsible for the
⮚ Anorexia production and the release of the Thyroid
⮚ Brittle nails and Hair Stimulating Hormone, which is the chief
⮚ Menstrual disturbances stimulant of the thyroid hormone secretions
⮚ Hair Loss ● In Secondary and Tertiary Hypothyroidism,
⮚ Apathy you will be able to see low T3 and T4 levels,
⮚ Lethargy as well as low TSH levels compared to your
⮚ Dry Skin (Coarse and Scaly) primary hypothyroidism.
⮚ Muscle Aches and Weakness ● For your thyroid gland to be able to
⮚ Constipation produce your T3 and T4.
● Late Clinical manifestation may include; ● In cases of hypothyroidism, there is a
● Subnormal Temperature decreased level of thyroid hormones in the
● Bradycardia (Slow pulse rate) circulation. In these cases, we have to find
● Weight Gain out what organ causes the decreased level
● Decreased Loss of Consciousness of T3 and T4, so we have to check on the
TRANSFORMERS 1
TSH as well. If the TSH is normal, and your T3 hypothyroidism and we call it
and T4 is decreased, most likely there is a myxedema.
problem with the pituitary gland, it means
that the pituitary gland is not adapting to
the need of the body to produce more T3
and T4. That is why it is still in its normal level,
or worse, it could be in a lower level, where
it is not able to adapt in the needs of the
body. If the TSH on the other hand, is
increased and your T3 and T4 is decreased, Thyrotoxicosis
it means that your TSH was able to identify ● Considered hyperthyroidism
the needs of the body to produce more T3 ● A constellation of findings that result when
and T4, and now it is the thyroid who is peripheral tissues are presented with, and
actually having the problem in producing respond to, an excess of thyroid hormone.
T3 and T4 despite having the presence of ● Can be the result of excessive thyroid
high levels of TSH. hormone ingestion (external factor),
Hashimoto’s Disease leakage of stored thyroid hormone from
● Increased TSH: since pituitary gland is storage in the thyroid follicles (that is why
capable of detecting the low levels of T3 there is a lot of T3 and T4 being released in
and T4, it will produce more TSH to tell your the circulation), or excessive thyroid gland
thyroid gland to produce more thyroid production of thyroid hormone.
hormones. o Thyrotoxicosis: If thyrotoxicosis is due
● But since there is this progressive to sources form the body itself
deterioration, of the thyroid gland, the o Thyrotoxicosis Factitia: T4 or T3
thyroid gland is not capable of doing or comes from an exogenous source
producing more T3 and T4 for that.
Myxedema
● A condition that occurs when your body
doesn’t produce enough thyroid hormone
● Considered to be hypothyroidism
● Result of having undiagnosed or untreated
severe hypothyroidism
● Also used to describe skin changes in
someone with severely advanced
hypothyroidism.
o Swelling of your face, which can
include your lips, eyelids, and
tongue
o Swelling and thickening of skin
anywhere on your body, especially SIGNS AND SYMPTOMS
in your lower legs is most likely the o Intolerance to o Weight loss
physical appearance of an heat
individual with severely advanced
TRANSFORMERS 2
o Fine, straight o Muscle ● Most common cause of increased T3

hair wasting and/or T4, with decreased/undetectable
o Bulging eyes o Localized TSH.
o Facial flushing edema o Since grave disease is more of an
o Enlarged o Finger autoimmune disorder that directly
erythroid clubbing attacks your thyroid gland, Pituitary
o Tachycardia (namaman gland will not produce TSH since
(high pulse as mga there are a lot of T3 and T4
rate) daliri) circulating in the body.
o Increased o Tremors o But because of the presence of TSH
systolic BP o Increased receptor antibody, your thyroid
o Breast diarrhea gland confuses it as your TSH and
enlargement o Menstrual now it keeps on producing your T3
changes and T4
(amenorrhe o Thus, you have increased level of
a) thyroid hormones coupled with
decreased or undetectable levels of
Grave’s Disease TSH
● Most common form of hyperthyroidism ● It occurs 5x more commonly in women than
● Stigmata of “Diffuse Toxic Goiter” in men
o Radioactive iodine uptake imaging ● Features:
(image) ✔ Exophthalmos (bulging eyes)
✔ Pretibial myxedema
● Diagnostic Test: TSH receptor antibody test
o Or the detection of your antibody
itself
● Ophthalmopathy
o Protrusion of the eyeballs
o Glowing butterfly like silhouette o Eyeball protrusion in severe
means that your thyroid is active condition stretches the OPTIC nerve
o All of the area which is glowing enough to damage vision.
signifies that particular area of the o The eyes are damaged because
thyroid is hyperactive leading to the eyelids do not close completely
production of more T3 and T4 than when the person blinks or sleep (will
usual eventually result to ulceration of
● An autoimmune disease in which cornea)
antibodies, TSH receptor antibody (TSHRAb), o So if there is this difficulty to close the
form against the TSH receptor (TSHR) in the lid during blinking or sleeping for
thyroid gland example, your eyes is exposed to
o This antibody binds to the receptor hair causing the dryness of the eyes
of TSH mimicking your TSH, signaling that could cause ulceration or
thyroid gland to produce more T3 scratches to cornea.
and T4
TRANSFORMERS 3
o As you can see here (image below),

radioactive iodine uptake scan will be able
to see multiple hot areas…
Toxic Adenomas and Multinodular Goiter
Toxic Adenomas
o This is hyperthyroidism with palpable
nodules.
▪ Palpable nodules – kapag kakapain
natin yung thyroid natin, we will be able
to see or feel irregularities on the thyroid
area; may mga bukol don sa thyroid
Drug-induced Thyroid Dysfunction
gland.
Amiodarone-Induced thyroid Disease
o In here, when we do your radioactive
Amiodarone-Induced Thyroid Disease
iodine uptake and have a scan for it, we
- Is one of the drug-induced thyroid
will be able to see a “hot scan”.
dysfunction known.
▪ Compared to your graves’ disease, the
- Amiodarone is a drug used to treat cardiac
whole thyroid area is actually glowing
arrhythmias (described as fat soluble with
because your thyroid gland itself is
long half life of around 50 days).
hyperactive.
- The problem with Amiodarone is that it has
▪ Here in toxic adenomas, only the
around 37% of Iodine and this is the
nodule is actually considered hot or
probable reason why there is this thyroid
active, so you will only see a circular
dysfunction.
glowing image here (image below),
- Wolf-Chaikoff effect (wherein its seen that if
which means that this nodule is the one
there is large dose of Iodine in the
responsible for the production of your T3
circulation, it acutely leads to the inhibition
and T4, or they are the ones more
of the thyroid hormones
metabolically active, producing T3s and
- Block T3 and T4 conversion
T4s, causing hyperthyroidism.
Note: This can lead to hypothyroidism and

hyperthyroidism. That’s why it is very important to
check on the patient’s medication in case that
there is thyroid related signs and symptoms.
Subacute Thyroiditis
● Post-partum Thyroiditis
● Exogenous hormone
Toxic Multinodular Goiter ● Ectopic thyroid tissue
o From the name itself, it causes an
appearance of goiter or inflammation of
the thyroid, but in reality, there are a lot of
nodules seen or attached in your thyroid
gland.
TRANSFORMERS 4
● Other tests other than the palpable

nodules, can used thyroid scans to see the
location of the presence of the toxic
adenoma/benign nodules.
TOXIC MULTINODULAR GOITER
● Foci of functional autonomy.
● There’s a presence of many nodule in the
thyroid. All of which are hyperactive giving
us a decrease TSH level, with high levels of
T3 and T4.
Here, you see the different pathogenic
● Increased RAIU
mechanism, the TSH levels, the RAIU, and other
● As seen on the thyroid scan, toxic
tests that can be used for the diagnosis of this
multinodular goiter lights up areas where all
particular conditions.
the nodules are actually present. These are
the hyperactive cells producing the T3 and
HYPERTHYROIDISM
T4.
GRAVE’S DISEASE
● Pathogenic mechanism of
TSH-SECRETING TUMOR
Thyroid-Stimulating Hormone Receptor
● Benign pituitary tumor
Antibody (TSHRAb), which binds to the TSH
● The problem here now is the pituitary gland.
receptor and acts as like your TSH causing
● Even though there is an increase levels of T3
increase levels of T3 and T4.
and T4, as we have a benign pituitary
● The TSH level here is decrease in radioactive
tumor, this tumor now acts as hyperactively
iodine uptake is actually increase.
and wouldn’t stop despite the presence of
● Other test that can be used is the TRAb or
increase levels of T3 and T4. Producing now
the TSHRAb tests, which will show a positive
more TSH and the normal.
result.
● The RAIU is also increased.
● Also the TSI/Thyroid Stimulating
● We can do pituitary MRI to see the location
Immunoglobulin.
of the tumor.
NONHYPERTHYROIDISM
TOXIC ADENOMA
PAINFUL THYROIDITIS HORMONE
● Pathogenic mechanism is the presence of
● It is due to leakage of thyroid hormone.
benign nodule in the thyroid.
● Due to the trauma, the thyroid keeps on
● TSH level is decrease.
releasing T3 and T4.
● Pituitary gland is normal
● Shows decrease levels of TSH and RAIU.
● Too much T3 and T4 in the circulation. So it
● Can check of thyroglobulin. If thyroglobulin
will stop producing TSH leading to the
is inappropriately high, meaning there is a
decrease levels of TSH.
leakage/damage in the follicle leading to
● The RAIU is also increase like in Grave’s
the leakage in the thyroglobulin, causing
disease. But as we’ve discussed earlier, they
the increase levels of T3 and T4.
have different imaging. For Grave’s disease,
the whole thyroid is actually flaring up.
POSTPARTUM THYROIDITIS
While in toxic adenoma, only the benign
● Same with painful thyroiditis, there is
nodule is seen in the scan.
leakage of thyroid hormone.
TRANSFORMERS 5
● Decrease levels of TSH and RAIU. o All assays are capable of

● Check on the presence of thyroid diagnosing primary hypothyroidism
peroxidase antibody which is seen usually with elevated levels of TSH.
high in this cases. o Second (2nd) Generation Assay
o Immunometric Assay
EXOGENOUS HORMONE o Derection limit = 0.1 mU/L
● Caused by medications. o Effectively screen for
● Decrease levels of TSH and RAIU. hyperthyroidism
rd
● Check on the medical history of the o Third (3 ) Generation Assay
patient. Or what medication they are o Chemiluminometric Assay
taking to see the correlation of the thyroid o Detection limit = 0.01 mU/L
levels to this medications especially the ▪ Higher detection
presence of amiodarone. limits
ECTOPIC THYROID TISSUE o More sensitive than 2nd
● Metastatic thyroid. generation
● It could be cancerous and has Struma o Accurately distinguish
ovarii which give us a levels of decrease TSH between euthyroidism
and RAIU. (normal state of the thyroid
Usually do thyroid scan to check on distant gland) and hyperthyroidism
metastasis for this particular case. (hyperactive thyroid gland).
TEST FOR THYROID FUNCTION o Preferred method for:
▪ Monitoring and
adjusting thyroid
hormone
replacement therapy
▪ Screening abnormal
thyroid hormone
production
Has the ability to detect “subclinical disease”.
Interpretation of Thyroid Test
Different test for thyroid function. We can do blood

tests or other tools for thyroid evaluation like
biopsies and scans.
Thyroid Stimulating Hormone Test
o Most useful test for assessing thyroid
function.
o There are three (3) generations of
assays used in measuring TSH. But
the book only focused on two (2) Low Free Normal High Free
generations which is the 2nd then the T4 Free T4 T4
3rd generation.
Secondar Subclinical
Hyperthyr
Low TSH y hyperthyr
oidism
oidism
TRANSFORMERS 6
hypothyroi Thyroglobulin
dism ● A protein synthesized and secreted
Severe Nonthyroi exclusively by thyroid follicular cells.
nonthyroi dal illness o It’s only the thyroid gland that is
dal illness capable of producing thyroglobulin.
Secondar So if there is a high levels of
y thyroglobulin in the circulation, it
Artifact
hypothyroi means that there is leakage coming
dism from the thyroid gland.
Pituitary ● A proof of the presence of thyroid tissue
hyperthyr o For example, those who have
Normal hyperthyroidism or Grave’s disease
Normal oidism
TSH which has surgical removal of
Severe Laborator
nonthyroi y draw thyroid, specifically the one that
dal illness within 6-9 requires the complete removal of
h of thyroid gland. Thyroglobulin are
thyroxine used or could be a good marker if
dose the surgery is successful. If there is still
Test thyroglobulin being detected in the
artifact circulation of the patient, that
Pituitary means that there are still thyroid
Primary Subclinical gland cells present in the body. This
hyperthyr
High TSH hypothyroi hypothyroi is usually done for patients who
oidism
dism dism have cancers of the thyroid.
Thyroid
o In cases of severe or late cases of
hormone
thyroid cancer, complete removal
resistance
of the thyroid gland is needed to
**Primary hypothyroidism: despite the increase
prevent the metastasis of the
levels of the TSH, the thyroid is not yet able to
cancer.
produce more T4, giving a low free T4 levels.
o To know if the thyroid gland is
Serum T3 and T4
completely removed, they check on
● Usually measured by radioimmunoassay
the thyroglobulin, as the thyroid
(RIA), chemiluminometric assay, or similar
gland is the only organ of the body
immunometric technique
that is capable of producing
● Serum T4: commonly measured by:
thyroglobulin.
o Competitive protein binding assay
● An ideal tumor marker, and thyroid cancer
(CPBA)
post treatment surveillance
o Radioimmunoassay (RIA)
● It is used in monitoring the course of
o ELISA technique
metastatic or recurrence of thyroid cancer.
● Serum T3:
● Increased levels:
o Radioimmunoassay
o Untreated and metastatic
Because serum T3 is not good and accurate to be
differentiated thyroid cancer
used for CPBA because T3 has a low affinity for PBG
o hyperthyroidism
compared to T4.
● Decreased levels:
TRANSFORMERS 7
o Infants with goitorous ● Thyrotropin binding inhibitory immunoglobulins

hypothyroidism (TBI)
o Thyrotoxicosis factitia o Determined by direct radioreceptor assay.
Thyroid Autoimmunity o The method assesses the capacity of Igs
Determination of Thyrotropin-Receptor Antibodies to inhibit the binding of radioisotope
(TRAb) labelled TSH to its receptors in human or
● Thyrotropin-receptor antibodies (TRAb) also animal thyroid membrane preparations.
known as “thyroid-stimulating hormone ▪ detergent-solubilized porcine TSH
receptor antibodies” wherein the receptors and 125 I-labelled TSH are
thyroid-stimulating hormone is also known as used.
thyrotropin, are group of related ▪ This TSH labelled with your iodine 125
immunoglobulins (Igs) that bind to thyroid cell is used for the detection, if there is a
membranes at or near the “TSH receptor” site. binding to the receptor or not.
● Note: ▪ The ability of a purified fraction of
o These antibodies show substantial serum Igs to displace 125 I-labelled
heterogeneity. TSH from the receptors is measured.
o Some cause thyroid stimulation. ▪ In here, we have this radio labelled
o Some may have no effect or decrease TSH that we will use as your reagent
thyroid secretion by blocking/inhibiting or a marker as well, then we’ll see
actions of TSH. now if it will bind. If it will bind now, if
o As your Thyrotropin-Receptor Antibodies there is the presence of your
(TRAb)could cause stimulation of the Thyrotropin binding inhibitory
thyroid or the blocking of the thyroid there immunoglobulins (TBI), it will have
are those antibodies that will bind to the the capacity to kick out this thyroid
receptor of your TSH receptor sites, that stimulating hormone, for them to be
will stimulate the production of more TSH able to bind to the TSH receptor
and there are antibodies that will bind to o Interpretation
your TSH receptor site, that will render no ▪ Normal immunoglobulin G (IgG)
effect or block now the production of TSH concentrates do not produce
or block the TSH itself from binding to the significant displacement and
TSH receptor, thus no TSH is being produces only less than 10 percent
produced, leading to the decreased inhibition.
levels of T3 and T4. It depends on the ▪ If your immunoglobulin G are normal
mechanism of the antibody found and these are not autoantibodies,
● Methodology: only 10% of your TSH receptors
o radioreceptor assays; would be inhibited
o bioassays ● The use of this method detects over 85% of
● Types of receptor antibodies: Two types have patients with Grave’s disease
been described:
o Thyrotropin binding inhibitory ● Thyroid stimulating immunoglobulins (TSIgs).
immunoglobulins (TBI) o In vitro bioassay utilized.
o Thyroid stimulating immunoglobulins o The method assesses the capacity of the
(TSIgs). Igs (antibodies) to stimulate a functional
activity of the thyroid gland such as
TRANSFORMERS 8
adenylcyclase stimulation leading to o Assay is rapid (only 2 hours

increase in cyclic-AMP formation. incubation period is required).
o Measurement of increase in cyclic-AMP ● RESULTS: the antibody concentration is
level can be done using human thyroid expressed as units/mL
slices, frozen human thyroid cells culture, ● INTERPRETATIONS
or a cloned line of thyroid follicular cells. o In NORMAL healthy persons: the
o Interpretations mean anti-TPo activity in serum is 69
▪ The effect of stimulation is expressed (+/-) 15 units/mL
as a percentage of basal activity. ● Detectable concentrations of TPo
▪ In normal: Range is 70 to 130 antibodies are observed on:
percent. o nearly all patients w/ HASHIMOTO’S
▪ Detects 95% of patients with THYROIDITIS
untreated Grave’s disease o spontaneous adult mixed edema
Determination of Antithyroid Peroxidase (idiopathic primary type)
● This is the antibody present in Hashimoto’s o majority of patients w/ GRAVE’S
disease and some subacute thyroiditis DISEASE
● Thyroid Peroxidase (Tpo) has been Frequency of detectable TPo autoantibodies found
identified and claimed as the main and in normal thyroid cases is actually similar
possibly the only autoimmune component Nuclear Medicine Evaluation
of microsomes. Your Tpo antibodies is RADIOACTIVE IODINE
considered as an antimicrosomal antibody, ● Useful in assessing the metabolic activity of
the only antimicrosomal antibody that has thyroid tissue and assisting in the evaluation
a direct effect to the thyroid gland. and treatment of thyroid cancer
● Its purification by using affinity o Orally administered. Hyperactive
chromatography and its production by thyroid gland will uptake it.
recombinant technology has led to the ● Radioactive iodine uptake (RAIU)
development of ELISA and RIA o HIGH uptake = metabolically active
(radioimmunoassay) methods for measuring o LOW uptake = metabolically
anti-TPo antibodies. inactive
● Principle: ● Important to interpret the scan in
o Immunometric assay is based on conjunction with an assessment of TSH levels
competitive inhibition of the binding o If TSH is decreased or undetectably
of radioiodinated TPo to an antiTPo low and there’s evidence of uptake
monoclonal antibody, coated onto of thyroid gland this means that
plastic tubes. despite the absence of TSH, thyroid
● Advantages: gland is working autonomously (w/o
o Easy to perform signals from TSH) in taking up its
o Provides greater sensitivity and iodine
specificity as compared to the TSH ● Can also be useful in the evaluation of
test and can be used for screening thyroid nodules in the presence of a low or
as a suitable immunometric assay undetectable TSH
has been developed for this o HOT nodules: areas are hyperactive
particular determination o COLD/warm nodules: areas are not
hyperactive
TRANSFORMERS 9
TRACERS (for RAIU)

131
I Most commonly used
● Short-lived isotopes (lower
half-life since tracers are
radioactive)
I123 & I132
● Preferred use in pediatric
practice, and in pregnant and
lactating women
99 ● Behaves like iodine
Tc
● Lower radiation dose
I: Iodine; Tc: Technetium
Fine-Needle Aspiration
THYROID FINE-NEEDLE ASPIRATION (FNA) BIOPSY
● Often the first step and most accurate tool
in the evaluation of thyroid nodules in the
absence of hyperthyroidism
o In cases of palpable nodules, there’s
no need for scans; But, if nodules
are NOT palpable, scans are
necessary Diseases of Pancreases
● Allows prompt identification and treatment ● 3 Diseases of the Pancreas
of thyroid malignancies and avoids ● Cystic Fibrosis
unnecessary surgery in most individuals with ● Pancreatic Carcinoma
benign thyroid lesions ● Pancreatitis
o MALIGNANT = need thyroidectomy ● Other than trauma, these 3 diseases cause
● Bethesda System for Reporting Thyroid more than 95% of medical attention
Cytopathology devoted to the pancreas
o We follow this in reporting thyroid ● If they affect the endocrine function of the
cytopathology pancreas, these diseases can result in the
o Has 6 diagnostic categories altered digestion in nutrient metabolism
Cystic Fibrosis
● AKA: “Fibrocystic disease of the pancreas”
and “Mucoviscidosis”
● An inherited autosomal recessive disorder
characterized by dysfunction of mucous
and exocrine glands throughout the body
Manifestations:
● Intestinal obstruction of the newborn
● Excessive pulmonary infections in
childhood
● Pancreatogenous malabsorption in adults
(common)
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TRANSFORMERS
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● Causes the small and large ducts and the ● The CFTR gene for Cystic Fibrosis occurs
acini to dilate and convert into small cyst commonly on Chromosome 7
which is filled with mucous Pancreatic Carcinoma
● This eventually results in the prevention of ● 4th most frequent form of fetal cancer and
pancreatic secretions to reach the causes about 38,000 death each year in
duodenum the US
● Depending on the age of the patient, for ● Represents about 7% of all from malignant
example, there is this plug that blocks the neoplasms deaths
lumen of the bowel leading to obstruction ● Commonly seen in males rather than the
● When the disease progresses, there is this females
increased destruction and fibrous scarring ● It is seen more frequently in African
of the pancreas and corresponding Americans than in whites
decrease in the function of it as well ● It arises as adenocarcinomas of the ductal
Cystic Fibrosis have varying manifestations: epithelium
● Excessively salty sweat ● Pain is a prominent feature of the disease
● Exocrine pancreatic insufficiency: the ● For pancreas, if the tumor grows on the
disability of the secretions of the pancreas head of the pancreas (there are a lot of
to reach the duodenum nerve endings in this area), pancreatic
● Fat-soluble vitamin malabsorption: one cancer seems to be painful and becomes
problem would be the absorption of B12 o the most prominent feature of the disease
● Cystic fibrosis-related diabetes ● Could be easily detected because of the
Cystic fibrosis or generally a problem of the pain that the patient will experience
pancreas could also lead to: ● If the tumor arises in the body or tail of the
● Osteoporosis pancreas, detection does not often occur
● Arthritis until an advanced stage of the disease
● Hypertrophic pulmonary osteoarthropathy because of its central location and the
For the lungs itself there will be: associated vague symptoms of pancreatic
● Small airway obstruction cancer specifically the tumors growing on
● Recurrent respiratory exacerbation the tail or body portion of the pancreas
● Pulmonary Infection- relatively occurring for compared to the ones on the head
childhood ● These are commonly the type of
pancreatic cancers that are difficult to cure
● Biliary cirrhosis as they are commonly detected at late
● gallstones stage already
● Hepatic steatosis ● Cancer of the head of the pancreas is
For Large intestine: usually detected earlier because of its
● Meconium ileus proximity to the common bile duct as well
● Distal Intestinal Obstruction Syndrome as the presence of a lot of nerve endings
Fibrosing colonopathy causing the pain that the patient will feel
for this particular type of cancer
● Transmitted as an autosomal recessive Signs and symptoms:
disorder with a high degree of penetrance ● Jaundice
● Dark urine and light stool
1
TRANSFORMERS
1
● Unintended weight loss ● It is ultimately cased by autodigestion of the

● Back pain pancreas as a result of reflux of bile or
● Upper abdominal pain duodenal contents into the pancreatic
● Anorexia duct
● Nausea ● Pathologic changes can include
● Pain after eating o Acute edema, with large amounts
● Cancer of the head of the pancreas is of fluid accumulating in the
usually detected earlier because of its retroperitoneal space and an
proximity to the common bile duct and as associated decrease in effective
well as the presence of a lot of nerve circulating blood volume
endings causing the pain for this particular o Cellular infiltration that could lead to
type of cancer the necrosis of the acinar cells with
● Signs and symptoms: hemorrhage as possible result of
o Jaundice necrotic blood vessels
o Dark urine and light stool o Intrahepatic and extrahepatic
o Unintended weight loss pancreatic fat necrosis
o Back pain ● It is a common cause of fat necrosis
o Upper abdomen pain observed in the pancreas. This fat necrosis is
o Nausea also described to have a chalky
o Pain after eating appearance.
● There is also these Islet cell tumors of the ● If you are going to see the pancreas gross,
pancreas that affects the endocrine you will be able to see these white spots
capability of the pancreas that looks like chalk. This is because of the
o Beta-cell tumor -> hyperinsulinism absorption of the calcium. Due to the
o Commonly you have your beta-cell affinity of the calcium to the triglycerides
tumor which causes your present in the pancreas, which also cause
hyperinsulinism that decreases the now the decrease of the calcium levels. This
circulating glucose in the body explains the hypocalcemic tendencies
● Gastrinomas when it comes to pancreatitis.
o Causes “Zollinger-Ellison syndrome” ● Often associated with:
and can be duodenal in origin. o Alcohol abuse – for chronic
Sometimes it doesn’t start in the alcoholic drinkers, have increased
pancreas but it starts from your risk for pancreatitis
duodenum, spreading through your o Biliary tract disease
pancreas ● Patients with hyperlipoproteinemia and
o Commonly associated with: those with hyperparathyroidism are also at
▪ Watery diarrhea a significantly increased risk for this disease
▪ Recurring peptic ulcer ● Pancreatitis is generally classified as:
▪ Significant gastric o Acute – there is no permanent
hypersecretion and damage to the pancreas done
hyperacidity during this type of pancreatitis. But
Pancreatitis once the pancreatitis advanced to
● Inflammation of the pancreas
1
TRANSFORMERS
2
the chronic stage, it becomes an hypocalcemia because of its affinity

irreversible injury. to the fatty acids found in your
o Chronic - irreversible injury pancreas or in your fat necrosis.
o Relapsing/ recurrent – it can be That’s why chalky appearance is
acute or chronic. It can go back: there.
acute pancreatitis, treated, then it Test of Pancreatic Function
goes back. It can manifest as both Secretin/CCK Test
acute or chronic pancreatitis the ● It is used to directly determine the exocrine
next time. Painful episodes can secretory capacities of the pancreas. Just like
occur intermittently, usually mentioned before, your pancreas has your
reaching a maximum of within exocrine and endocrine tissues.
minutes or hours of pain. There are ● To check the capacity of the pancreas
cases that lasts for several days or specifically its exocrine secretion
weeks and it is usually ● It involves intubation of the duodenum without
accompanied by nausea and the contamination of gastric fluid. This is very
vomiting. important that when we are going to intubate
● Laboratory findings: the duodenum, it will not be contaminated with
o Increased amylase gastric fluid because gastric fluid neutralizes
o Increased lipase your bicarbonate. Bicarbonate is the one that
o Increased triglycerides we actually check as well in secretin/cck test.
o Hypercalcemia, which is often ● Done after 6 hours of fasting or overnight
associated with underlying fasting
hyperparathyroidism ● For IV administration of secretin, its around
o Hyperproteinemia – attributed 2-3U/kg of body weight plus your CCK as well.
mainly to the notable loss of plasma ● But if you are only going to test secretin alone,
into the retroperitoneal spaces we increase the dosage.
that’s why there is this increase in ● In this test we get to measure the: pH, secretory
protein levels. rate, Enzyme activity and amount of
o Hypocalcemia may be found and Bicarbonate.
has been attributed to the sudden
removal of large amounts of INTERPRETATION:
calcium from the excess cellular ● IF there is Decrease pancreatic flow- it signifies
fluid because of impaired Pancreatic obstruction
mobilization or as a result of calcium ● If there is low concentration of bicarbonate and
fixation by the fatty acids liberated enzymes- it can be due to: Cystic fibrosis,
by increased lipase action on the Chronic pancreatitis, Pancreatic cysts,
triglycerides that’s why there is a Calcification, and Edema if the pancreas.
chalky appearance.
● Example: hyperparathyroidism
That is why it is very important that during the
intubation of the duodenum, gastric fluid will not
o There is tendency that your calcium
contaminate the areas, so that, no bicarbonate will be
will increase in your circulation, but
neutralized.
eventually, your body will consume
Fecal Fat Analysis
all of this calcium leading to the
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● These are derived from four (4) sources:

o Unabsorbed ingested lipid
o Lipids that were excreted into the
intestine (predominantly in the bile)
o Cells shed into the intestine
o Metabolism of intestinal bacteria
● Fat does not normally exceeded about 7 grams
in a 24-hour period, even though you had a
high lipid diet. But for patients with lipid free
diet, it is normal to see about 1 – 4 grams of
lipids in the feces in 24-HOUR PERIOD. Since
there are other sources of fecal lipids as well.
● For FREE FATTY ACIDS- It requires Heating with
● Normal fecal lipid is composed of about:
addition of 36% acetic acid. Without heating, it
o 60% fatty acids
won’t be stained by SUDAN III. By heating your
o 30% sterols, higher alcohols and
free fatty acids, you make it more vulnerable in
carotenoids.
mixing with stain. For slides or samples that has
o 10% triglycerides; and small amounts of
cool down already, your fatty acids shows like
cholesterol and phospholipids.
a crystallize out in long, colorless, needle-like
● Although significantly increased fecal fat can
sheaves.
be caused by biliary obstruction,
● We can also stain MEAT FIBERS with the use of
Severe steatorrhea usually associate with exocrine
SUDAN III. It should be mixed with 10% alcohol
pancreatic insufficiency or disease of the small
and solution of eosin stained for 3 minutes. Stain
intestine
as rectangular cross-striated fibers.
Qualitative Screening Test for Fecal Fat
● Splitting the samples in detecting neutral fat,
● We use FAT SOLUBLE STAINS LIKE:
the fatty acids and the undigested meat fibers
o Sudan III
can provide diagnostic information.
o Sudan IV
● Increases in fats and undigested meat fibers
o Oil Red O
are indicative of patients with steatorrhea of
o Nile blue sulfate
pancreatic origin. So, representative fecal
specimen is used to analyze
SUDAN III
● Normal feces could have up to 40 or 50 small.
● Neutral fats like your triglycerides is stained with
This means that it is 1-5 millimeters in size.
yellow-orange to red discoloration
● Neutral lipid droplets per HPO
● So steatorrhea is characterized by an increase
in the number of the size of stainable droplet.
So if there is an increase, the droplets are more
than 5 millimeters, this could be more often
characteristic of steatorrhea.
Quantitative Fecal Fat Analysis

● Definitive test for steatorrhea
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● 72-hour or 3 days stool collection is actually then extracted with other soluble lipids into
preferred. Sometimes the collection can last petroleum ether and ethanol.
long up until 5 days. o Fatty acid soaps, predominantly your
● Lipid rich diet for 2 days prior the collection. calcium and magnesium soaps of fatty
Need to eat 50 grams to 100 grams of lipid acids are converted to fatty acids
each day. followed by the extraction of most of
● Methods for fecal determination: the lipid into an organic solvent which is
o Traditional methods then evaporated so that the lipid
▪ Gravimetric method residue can be weighed.
▪ Titrimetric method ● After evaporation of the organic solvents,
o Infrared and nuclear magnetic the lipid residue is weighed. All feces for a
resonance spectroscopy 3-day period are collected in tared
Titrimetric Method containers.
● Uses lipids are saponified with hydroxide, ● Container:
and the fatty acid salts are converted to o Must be tared
free fatty acids using acid (acidic ▪ You have to take note that your
reagents). container should be tared, meaning
● The free fatty acids, along with various that the container is actually
unsaponified lipids, are then extracted with weighed because we would want
an organic solvent, and the fatty acids are to get the weight of the stools alone,
titrated with hydroxide after evaporation of not including the container.
the solvent and redissolving of the residue in o No wax coating
ethanol. ▪ Another consideration when you’re
going to choose your container is
Disadvantages: that the container should not be
● The titration methods obviously measure containing any wax as it could
only saponifiable fatty acids and, interfere or falsely increase the lipid
consequently, render results about 20% or fat present in the fecal sample.
lower than those from gravimetric methods. ● Sample must be refrigerated
● A further objection is that titrimetric o During the 3-day period of the
methods use an assumed average collection, you have to make sure that
molecular weight for fatty acids to convert the sample is refrigerated
moles of fatty acids to grams of lipid. ● Patients must not ingest castor oil, mineral
o There are still inconsistencies when it oil, or other oily laxatives and must not use
comes to conversion as we are only rectal suppositories containing oil or lipid for
assuming the average molecular 2 days before the test and during the test.
weight. Reference range: 1 to 7 g per 24 hours.
Gravimetric Method Sweat Electrolyte Determination
● The entire fecal specimen is emulsified with Sweat sodium and chloride concentration
water. ● Most useful test for the diagnosis of cystic
o You mix it with water and you emulsify it. fibrosis.
● An aliquot is acidified to convert all fatty o If you can still remember, one of the
acid soaps to free fatty acids, which are signs and symptoms of cystic fibrosis is
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having a salty or excessively salty-tasting o A more sensitive indicator of acute

sweat because of the presence of your pancreatitis.
sodium and chloride. ● But the thing with amylase is that you have
o Significantly elevated concentrations of your salivary amylase as well that we ingest
both ions occur in more than 99% of the
affected patients Renal clearance of amylase
o If there is 2-fold to 5-fold increases in ● Useful in detecting minor or intermittent
sweat sodium and chloride, these are increases in the serum concentration of this
diagnostic of cystic fibrosis in children enzyme.
already. ● Ratio of amylase clearance to creatinine
o Even in adults, there are no other known clearance can be computed through this
pathologic cause that could increase particular equation:
the levels of sodium and chloride in the
o
sweat of adults.
● Pilocarpine administration by iontophoresis
o An efficient method for sweat collection ▪ UA = Urine amylase
and stimulation. ▪ SA = Serum amylase
o Before, we have your manual methods ▪ SC = Serum creatinine
on how to stimulate and collect sweat ▪ UC = Urine creatinine
from the patient but this method ● Normal value = < 3.1%
requires skilled medical technologist to ● Renal clearance of amylase
perform it as this older method requires o Useful in detecting minor or intermittent
induction of sweat using the application increases in the serum concentration of this
of plastic bags or wrapping the patient enzyme.
in blankets. The problem with this o ratio of amylase clearance to creatinine
method is that it has a higher risk for clearance
dehydration, electrolyte disturbances,
and hyperpyrexia in the patient that’s ▪
why pilocarpine administration by
iontophoresis is done instead of these
▪ UA = Urine amylase
older methods.
▪ SA = Serum amylase
Serum Enzyme
▪ SC = Serum creatinine
Amylase
▪ UC = Urine creatinine
● One of the most common serum enzymes
o Normal value = < 3.1%
that we could use in assessing the pancreas
o Significant increase values averaging about
would be your amylase
8% to 9% occurred in pancreatitis but may
● Serum enzyme most commonly relied on for
also occurred at other conditions such as
detecting pancreatic disease.
burns, sepsis and diabetic ketoacidosis.
● Particularly useful in the diagnosis of acute
o The problem in amylase it has a shorter life
pancreatitis, in which significant increases in
in the circulation. Thus, some physicians uses
serum concentrations occur in about 75% of
lipase as persist longer in the circulation.
patients.
● Urine amylase
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● Lipase ▪ moderate pancreatic insufficiency =

o Improved analytic methods appear to 100 to 200 μg/g
indicate that lipase increases in serum severe pancreatic insufficiency = < 100 μg/g.
about as soon as amylase in acute Clinical Aspect of Gastric Function
pancreatitis and that increased levels ● Gastric analysis is used in clinical medicine
persist somewhat longer than those of mainly for the following purposes:
amylase o Was once widely used in clinical medicine
o Some physicians consider lipase more but has now been largely replaced by
sensitive than amylase as an indicator of fiberoptic endoscopy and improved
acute pancreatitis or other causes of radiologic procedures.
pancreatic necrosis. o Used clinically mainly to detect
hypersecretion characteristics of the
● Note: Zollinger-Ellison syndrome.
o Both amylase and lipase may be ● Pernicious Anemia
significantly increased in serum in many o Vitamin B12 deficiency
other conditions (e.g., opiate o Presence of gastric atrophy
administration, pancreatic carcinoma, ● Normal gastric fluid
intestinal infarction, obstruction or o Translucent
perforation, and pancreatic trauma). o pale gray
o Amylase levels are also frequently o slightly viscous
increased in: mumps, cholecystitis, hepatitis, o Has a faintly acrid odor.
cirrhosis, ruptured ectopic pregnancy, and o Residual volume should not exceed 75 mL.
macroamylasemia. ▪ Residual specimens occasionally
o Lipase levels are often significantly contain flecks of blood or are green,
increased in: bone fractures and in brown, or yellow from reflux of bile
association with fat embolism. during the intubation procedure.
o Using serum enzyme alone is not directly ▪ The presence of food particles is
diagnostic of any pancreatic problem. So it abnormal and indicates obstruction.
needs to test other to make sure that it is a Test of Gastric Function
pancreatic origin.
Fecal Elastase ● Gastric peptic ulcer is usually associated with
● Elastase-1 normal secretory volume and acid output.
o A chymotrypsin-like enzyme secreted by ● Duodenal peptic ulcer is usually associated
the pancreas. with increased secretory volume in both the
o Has been proposed as a sensitive test of basal and maximal secretory tests;
pancreatic function. considerable overlap occurs, nevertheless, with
o Performed on random stool samples and the normal range.
has the advantage of being noninvasive. o Sometimes it’s in normal range but there
o It has been shown to be useful in the are times that they are actually
diagnosis of cystic fibrosis in children. increased for duodenal peptic ulcers
o Reference range:
▪ fecal elastase for normal = > 200 μg/g, Measuring Gastric Acid
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● In measuring gastric acid, its stimulated ● invaluable in diagnosing Zollinger Ellison

secretion specimens shows the ability of the syndrome
stomach to secrete against a hydrogen o fasting levels > 1,000 pg /mL and can
gradient which is determined by measuring the reach 400,000 pg /mL,
pH of sample o normal range = 50 to 150 pg mL.
● The total acid output in a timed interval is ● not increased in simple peptic ulcer disease.
determined from a tiitratable acidities in Increased plasma gastrin levels → pernicious
volumes of the specimen anemia patients but decrease toward normal
● After intubation, the residual secretion is when hydrochloric acid is artificially instilled into the
aspirated and retained. stomach.
o Secretion for the subsequent 10 to 30 Clinicopathological Aspects of Intestinal Function
minutes is discarded to allow for
adjustment of the patient to the ● Clinical chemistry testing of intestinal function
intubation procedure.
→ evaluation of absorption and its
o Specimens are ordinarily obtained as 15
derangements in various disease states.
minute collections for a period of 1 hour.
o The gastrin response to intravenous ● As discussed in pancreatic function, disease of
secreting stimulation may be used to the exocrine pancreas and biliary tract may
investigate patients with mild elevated also cause malabsorption
gastrin levels ● Intestinal diseases that may cause the
● Pure porcine secretin is injected intravenously, malabsorption syndrome are highly varied in
and gastrin levels are collected at 5 minute
their etiology, pathogenesis and severity. These
intervals for the next 30 minutes.
include:
o Patients with Zollinger Ellison syndrome,
the gastrin level increases at least 100 o tropical and nontropical or celiac sprue
pg /mL over the basal o Whipple's disease
o Patients with ordinary peptic ulceration, o Crohn's disease
achlorhydria, or other conditions show a o primary intestinal lymphoma
slight decrease in gastrin concentration. o small intestinal resection
o Most healthy subjects secrete 0 to 6
o intestinal lymphangiectasia
mmol of acid in a total volume of 10 to
o Ischemia
100 mL.
▪ Maximal 1 hour test, using o Amyloidosis
histamine or pentagastrin as the o giardiasis.
stimulus, ● In addition to the malabsorption syndrome
⮚ Most men secrete 1 to 40 which ordinarily causes impaired absorption of
mmol of acid in a total
fats, proteins, carbohydrates and other
volume of 40 to 350 mL.
macromolecules, specific malabsorption state
⮚ Women and older
persons usually secrete also occurs like acquired deficiency of lactase
somewhat less acid than which prevents normal absorption of lactose
do young men. and Hartnup syndrome which is a genetic
Plasma Gastrin
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disorder which involve deficient intestinal unaltered in the proximal small

transport of phenylalanine and leucine intestine and do not require the
intervention of pancreatic lytic
enzymes
the ability to absorb D xylose is of value in
Test of Intestinal Function differentiating malabsorption of intestinal etiology
Lactose Tolerance Test from that of exocrine pancreatic insufficiency.
● Disaccharidases Notes: because only about ½ of orally administered
o Lactase and sucrase D-Xylose is metabolized or lose by action of
o Produced by the mucosal cells of intestinal bacteria, Significant amounts are
the small intestine. excreted unchanged in the urine during in the
o acquired deficiencies of lactase are 5hours following Ingestion of a 25 gram dose by a
commonly found in adults. fasting adult even with normal renal function false
▪ Abdominal discomfort positive and false negative results frequently occur.
▪ Cramps Blood levels measured one or more times after
▪ Diarrhea ingestion of D-xylose can be at 30 mins time
● The disaccharidases lactase which cleaves interval, 1 hour, 2 hours significantly improved the
lactose into glucose and galactose, and diagnostic reliability of the test. So, other than
sucrase which cleaves sucrose into glucose testing the basal and after 1 hour we do multiple
and fructose produced by the mucosal testing of the D-xylose not only in the urine but also
cells of the small intestine. in the blood samples.
● Congenital deficiencies of this enzyme is D-Xylose Test
actually rare but acquired deficiencies of ● After ingestion of a specified solution of
lactase are commonly found in adults that D-xylose, blood specimens (Potassium
can cause Abdominal discomfort, Cramps Oxalate) are obtained, and urine is
and Diarrhea upon intake of milk or milk collected for 5hour period to determine the
products and other dairy products as well extent of D-xylose absorption.
▪ ADULTS= 25g of D-xylose in 250mL of
Lactose tolerance testing water
● Was used to establish the diagnosis of ▪ CHILDREN= 0.5g/kg
lactose tolerance Notes: The patient will undergo overnight fasting in
● Subject to many false positive and false void prior the ingestion of the specified solution of
negative results. the D-xylose. For Adults, the recommended dose
Replaced by hydrogen breath testing. 25g of D-xylose in 250ml of water and for children
0.5g/kg.
D Xylose Absorption Test ● The concentration of D-xylose is determined
● D Xylose by heating protein free supernates of urine
o pentose sugar that is ordinarily not and plasma to convert Xylose to furfural,
present in the blood in any which is reacted with;
significant amount. ▪ Chromogen: p-bromoaniline
o As with other monosaccharides, (producing= pink)
pentose sugars are absorbed ▪ Absorbance: 250nm
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▪ Thiourea is added as an antioxidant The test DOES NOT distinguish among the various
to prepend formation of interfering etiologies of malabsorption.
chromogens while doing this test.
Notes: during the 5hrs period of testing the D-xylose
absorption the patient is NOT ALLOWED to drink any
fluid or to eat any food until the determination is
over.
Results of D-xylose test:
● Normal blood concentrations of D-xylose +
Decreased urine excretion
▪ Impairment of Renal function
▪ Incomplete urine collection
● Aspirin therapy= diminishes Renal excretion
of D-xylose
● Indomethacin= decreases intestinal
absorption. (taking this medication should
be noted when doing the D-xylose test)
● Healthy adults= at least 4g in the (urine)
5-hour period
Blood collection <25mg/dl at 2 hours should be
considered abnormal
Additional Test for Intestinal Function:
Serum Carotenoids
● Carotenoids are yellow to orange or purple
pigments that are widely distributed in
animal tissues.
● Mainly synthesized by many plants and
impart a yellow color to some vegetables
and fruits.
● Major Carotenoids in HUMAN SERUM:
▪ Lycopene
▪ Xanthophyll
▪ Beta Carotene (chief precursor of
vitamin A in humans)
Notes: Carotenoids are typically absorbs in Small
intestine in association with the LIPIDS
● Malabsorption of lipids typically results in a
serum concentration of Carotenoids lower
than the reference range of 50 to 250
mg/dl.
▪ Starvation, dietary idiosyncrasies
and fever
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1 Phlebotomy Clinical Chemistry 2 (LAB)

Phlebotomy: A Review Sources and Types of Blood Specimens

o Majority of chemistry tests usually we need to add an additive which is an

Order of Draw: 1. Plasma preparation Tubes (PPTs) o Patient’s physician should be

up or down the arm are all signs of Excessive Bleeding

LIVER FUNCTION o Serum is the preferred sample.

o While extracting, we must protect our

TB at 48 hours 6-8 mg/dL

Test Product (Bilirubin Jendrassik-Grof Test: DIASYS)

o We use the four reagents which are the

● Pre-hepatic jaundice semi-quantitative determination methods

venipuncture, we have to avoid the

TESTS MEASURING NITROGEN METABOLISM

NH4 + alpha keto glutaric acid + NADH -----------🡪

❖ To measure ammonia, one of the methods

**always take note that bilirubin is a photosensitive

covered with an opaque material such as carbon

2. Mix 50 uL of the sample to the same tube

3. Pipette 1mL of the total bilirubin working

4. Mix 50 uL of the sample to the same tube

5. After incubation, lid the tube in the

CLINICALLY SIGNIFICANT ENZYMES ▪ All of them are related to the bones

your blood type can also be

• Next, the electrophoresis machine will be

• Intestinal ALP – most cathodal

• Liver ALP – is the most anodal in MAJOR isoenzymes

Both Reagan & Nagao are referred

Read phosphate diagnosis, and management of Prostatic

ACP which we will Rietz and Guibalt This method is a

Cardiac muscle, Cardiac tissue, Liver, Skeletal ▪ It will eventually decrease or

pH 7.5 Decreased ALT levels: Not clinically significant

Both enzymes may be elevated in Heparin amylase is smallest, normal amount of

intensity = increase colored starch. Ibig

substrate complex amylase, result into

naproduce from • Earliest marker to increase during

related to pancreas. Olive oil as their primary substrate

kailangan kapag ginagamit natin si ISOENZYMES

REMARKS these conditions

NOTES: LD 8 72 hours 5-7 days

Chronic Alcoholism, Antiepileptic / Most widely used

Decreased: Not significant Dehydrogenase

2. Transfer 10uL of Distilled water to the blank

CLINICALLY SIGNIFICANT ELECTROLYTES each side of the membrane into an electric

✔ Method of choice - If uncapped the CO2 may escape

● Binds to magnesium ions ● The anticoagulant of choice for plasma is

8 Clean test tubes

2. Pipette 50 uL of the serum sample into a tube

3. Add 1.5 mL of Sodium working reagent on each

6. After incubation, read the tubes in the

4. Add 50 uL of sodium color developer

BLOOD GAS ANALYSIS ● Although, we can undergo training, but

● Recall from the board exam: When we

Blood Gas Analyzers: Measurement of pCO2

This is the illustration of Severinghaus electrode

Modern Blood Gas Analyzers Continuous Monitoring for pO2

actual so that we can compute the

2) should be measured by our machine the National reference lab for CC or

Clinically Significant Trace Elements ➢ Ang ating mga enzymes ay

Metal activated enzymes, the metals are

in determining the presence of TE; Inductively Couples Plasma Mass Spectroscopy

- Nebulizer - sample is introduced into the

● A common problem in Graphite Furnace 2. MASS ANALYZER: component that takes

(Simplified schematic of ICP-MS instrumentation.

• Nearly, all ICP torches consist of three