MEDICAL LABORATORY SCIENCE PRACTICE I

PHLEBOTOMY  adjusting excess body fluids (William

 ‘Phlebotomy’ comes from the Greek Harvey up to present) 
word phlebos, meaning veins, and Note: unknown of aseptic techniques 
tome, meaning incision.
 It is a invasive procedure that involves BLOOD LETTING TECHNIQUES
the body through cutting or puncture,
performed by phlebotomists. 1.Venesection – Came form Latin words vena
Two main phlebotomy procedures:  (vein) and secto (cutting).
(1) Venipuncture – used scalpel, lancets and fleams, 
(2) Capillary Puncture 2. Arteriotomy – was less frequently done
because it was a dangerous method, involves
Historical Perspective: the temporal artery vein.
 Historical evidence suggests 3. Cupping – involved a procedure that required
the possibility of blood letting for placing a suction cup over an area of skin.
therapeutic reasons may have begun Suction was applied to create a blister or
in EGYPT around 1400 B.C.  swollen area. 
 Tomb paintings from this time show Leeching – leeches were applied to the
the  patient’s skin on the affected area and allowed
application of a leech to a patient.  to fill themselves with blood. Leeches usually
fell off when they became full of blood. 
HIPPOCRATES (460-377 B.C.)-also known as the
father of modern science, was responsible for Phlebotomists- have been specifically trained in
early medical (HUMORAL) theory, which blood collection techniques and are employed
believed illness was caused by an imbalance in primarily to collect blood specimens. 
the body. 
The removal of this excess was thought Phlebotomy at Modern Times:
to restore this balance. Health was thought • Obtain blood for diagnostic purposes
to be restored by plugging, starving, • Blood Donation
vomiting, or blood letting.  • Treatment for Polycythemia Vera
• Venipuncture and Capillary Puncture 
GALEN –believed that blood was formed in the • Point of Care Testing (POCT)
liver and was brought from the intestines by the • Evacuated Blood Collection System –
portal vein.  manufactured by Hynson, Wescott, and 
ANDREAS VESALIUS – presence of valves in the Dunning 
veins.
REALDUS COLUMBUS – discovered pulmonary TRADITIONAL DUTIES OF THE PHLEBOTOMIST
circulation. 1. Correct identification and preparation of the
WILLIAM HARVEY – discovered patient before sample collection. 
blood circulation from observations on 2. Collection of the appropriate amount of
living animals and dissections; determined blood by venipuncture or dermal puncture for
the function of valve the specified tests.
3. Selection of the appropriate sample
Blood letting  containers for the specified tests.
 Removal of blood in the body. 4. Correct labeling of all samples with the
 Standard practice for releasing spirits required information.
(Egyptians) 5.Appropriate transportation of samples back to
 cleansing blood of the laboratory in a timely manner. 
impurities (Hippocrates)
MEDICAL LABORATORY SCIENCE PRACTICE I

6. Effective interaction with patients and advanced degree and several years of
hospital personnel. experience. Duties include overseeing all
7.Processing of samples for delivery to the operations involving physician and patient
appropriate laboratory departments.  services. 
8. Performance of computer operations and 3. Technical Supervisor (Section Head) –
record-keeping pertaining to phlebotomy.  responsible for the administration of an area
9. Observation of all safety regulations, quality and reports to the lab administrator .
control checks, and preventive maintenance 4. Medical Technologist/ Medical Laboratory
procedures. Scientist - performs all levels of testing in any
10 Attendance at continuing education area of the laboratory.
programs.  5. Phlebotomist – trained to collect blood
samples in the laboratory. Primary role is to
Two main categories of the Health Care collect blood by venipuncture or capillary
Delivery Systems: puncture.
1. Inpatient – hospitals, nursing homes,
rehabilitation centers Regulatory, Ethical, and Legal Issues 
2.Outpatient – physician offices, reference CLIA- stipulates that all laboratories that
laboratories, blood banks (blood donor centers)  perform testing on human specimens for the
purposes of diagnosis, treatment, monitoring,
Personal Appearance – OSHA or screening must be licensed and obtain a
Ethics – refers to good values and actions.  certificate from the CMS. 
HIPAA (Health Insurance portability and CLIA Laboratory Test Classification 
accountability Act) Waived testing
– an act that protects patient information and Provider performed microscopy 
maintains confidentiality of results.  Moderate complexity test
High complexity test
Hospital Patient-Care Areas 
Emergency Department (ED)-Immediate Care JC- is an independent, not-for-profit 
Intensive Care Unit (ICU)- Critically ill patient organization that accredits and certifies more
Cardiac Care Unit-Patient with acute cardiac than 15,000 health-care organizations and
disorder programs in the United States. 
Pediatrics- Children
Nursery -Infants Patient Safety Goal- It is essential that
Short-stay Unit- Outpatient surgery healthcare organizations adhere to these 
Neonatal Intensive Care Unit (NICU)-Newborn goals to maintain their accreditation. 
experiencing difficulty Goals pertaining to the laboratory are: 
Labor and Delivery Room- Childbirth Goal1 Improving the Accuracy of Patient 
Operating Room- Surgical Procedures Identification. 
Psychiatric Unit- Mentally Disturbed Patients Goal 2 Improving the Effectiveness of 
Dialysis Unit- Patient with severe renal disorder Communication among Healthcare Givers.
Medical/Surgical Unit- General patient care Goal 7 Reduce the Risk of Healthcare-
Oncology Center- Cancer Treatment  Associated Infections. 
Goal13 Encourage Patients’ Active Involvement
Clinical Laboratory Personnel  in their Own Case as a Patient Safety Strategy. 
1. Pathologist – is a physician who specializes in
diagnosing disease. CAP is an organization of board-certified 
2. Laboratory administrator\ (Chief Medical
Technologist) - is usually a technologist with an
MEDICAL LABORATORY SCIENCE PRACTICE I

pathologists that advocates high-quality and be free from unwanted exposure to public
cost-effective medical care. The CAP provides view. 
laboratory accreditation and proficiency testing • Release of confidential information is
for laboratories.  considered an invasion of privacy. Entering a
patient’s room without asking permission may
Ethical and Legal Issues  be considered a physical intrusion and an
invasion of privacy. 
CODE OF ETHICS- provide the personal and  Medical Malpractice- is misconduct or lack of
professional rules of performance and moral skill by a health-care professional that results in
behavior as set by members of a profession.  injury to the patient. 
MEDICAL ETHICS OR BIOETHICS- focus on the Negligence- defined as failure to give
patient to ensure that all members of a health- reasonable care by the health-care provider,
care team possess and exhibit the skill, must be proven in a malpractice suit. 
knowledge, training, professionalism, and moral
standards necessary to serve the patient.  Four factors must be proven to claim
negligence: 
Criminal lawsuit- is an action initiated by the Duty: Indicates that there was an established
state for committing an illegal act against the standard of care and proof that it was not
public welfare and can be punishable by  followed. The standard of care can be for the
imprisonment.  procedure and for the training and evaluating
protocols of the phlebotomist performing the
Standard of Care (A duty to protect someone procedure. 
from harm established by standards of the Breach of Duty: The plaintiff (patient) must
profession and expectations of society). show what actually happened and that the
defendant (phlebotomist) failed to perform. It
TORTS- A wrongful act committed by one has to be proven that the defendant knew or
person against another that causes harm to the should have known that this failure could cause
person or his or her property is called a tort.  harm. 
▪ Torts are classified as intentional and Causation: Indicates that the breach of duty
unintentional.  directly caused the injury and that no other
▪ Assault, battery, and defamation are factors could have contributed. 
considered intentional torts. Damages: Actual physical, emotional, or
▪ Negligence and malpractice are considered financial injury had to occur to the plaintiff
unintentional torts.  (patient) because of the negligent act. 

Assault- is the threat to touch another person Respondeat superior (let the master answer) -
without his or her consent and with the establishes that employers are responsible for
intention of causing fear of harm.  their own acts of negligence as well as their
 Battery- is the actual harmful touching of a employees’ acts. 
person without his or her consent. 
Defamation- is spoken or written words that Risk management departments develop
can injure a person’s reputation.  policies to protect patients and employees from
Libel- is false defamatory writing that is preventable injuries and the employer from
published.  financial loss.
Slander- is false and malicious spoken word. 
Invasion of privacy- is the violation of the Sentinel event- is defined as an unexpected
patient’s right to be left alone and the right to occurrence involving death or serious physical
or psychological injury, or the risk there of
MEDICAL LABORATORY SCIENCE PRACTICE I

Sentinel events must be documented for the Tube stoppers are color coded to identify a type
JC.  of additive, absence of additive, or 
special tube property.  
Blood Collection Equipment &Blood Collection
Site Order of draw
PHLEBOTOMY 1 (yellow) = SPS/acid citrate 
CAPILLARY PUNCTURE 2 (light blue) = Sodium citrate
 Drops of blood for testing can be 3 (red) - no Anticoagulant 
obtained by puncturing the capillary 4 (green) = Heparin with Na, Li, ammonium ion
bed of the skin with a lancet or other 5 (lavender) = K2EDTA
sharp device.  6 (white) = K2EDTA with Gel
 Capillary specimen collection (also 7 (gray) = Sodium Fluoride 
called Dermal or Skin Puncture) is 8 (black) = Buffered Sodium Citrate/Oxalate 
especially useful in pediatrics where
removal of larger quantities of blood The ratio of blood to the liquid sodium citrate is
can have serious consequences.  critical and should be 9 to 1 (e.g., 4.5 mL blood
 Collection sites include the fingers of and 0.5 mL sodium citrate). 
adults and children over the age of 2 ▪ When collecting coagulation tests on patients
and the heels of infants.  with polycythemia or hematocrit readings
VENIPUNCTURE greater than 55 percent, the amount of citrate
 The most common way to collect blood anticoagulant should be decreased to prevent
specimens. an increased amount of citrate in the plasma. 
Can be performed by Three Basic Methods: ▪ The increased citrate in the sample will
(a) Evacuated Tube System (ETS)  interfere with the coagulation tests. 
(b) Needle and Syringe
(c) Winged Infusion Set (Butterfly)  Evacuated Tubes:
Evacuated Tube System ▪ Anticoagulants prevent blood from clotting
Multi-sample Needles  and include Ethylene Diamine Tetra 
▪ Allows collection of multiple tubes during Acetic Acid (EDTA), citrates, heparin, and
venipuncture. It has a beveled point on each Oxalates. Each is designed for use in 
end certain types of testing, and it is important to
▪ Parts: bevel (angled point), shaft, lumen, and use the correct one. 
hub.  ▪ Anti-glycolytic Agents prevent glycolysis,
▪ Needles should be visually examined before which can decrease glucose concentration by up
use to determine if any structural defects. to 10 mg/dL per hour. In addition to glucose,
▪ Needles should never be recapped once the sodium fluoride is used to collect ethanol
shield is removed  specimens to prevent an increase in alcohol due
Tube Holder - plastic cylinder with  to fermentation by bacteria. 
a small opening for a needle at one end and a Clot Activators are coagulation factors such as
large opening for tubes at the other.  thrombin and substances such as glass (silica)
▪ Holders are available to accommodate  particles and inert clays like diatomite (Celite)
collection tubes of different sizes.  that enhance clotting by providing more surface
for platelet activation. 
Collection Tubes Thixotropic Gel Separators are inert substances
▪ Evacuated Tubes have a premeasured  contained in or near the bottom of certain
vacuum that automatically draws the  tubes. During centrifugation, the gel lodges
volume of blood indicated on the label.  between the cells and the fluid, forming a
physical barrier that prevents the cells from
 MEDICAL LABORATORY SCIENCE PRACTICE I

metabolizing substances in the serum or Median Cubital Vein

plasma.  ▪ Located near the center of the 
antecubital fossa.
Syringe needles-for venipuncture are 21- to 23-  It is the preferred vein for venipuncture
gauge, sterile, hypodermic needles from 1 to 1 because it is typically large
2/3 inches in length.  Closer to the surface, and the
 Syringe System- An advantage when using most stationary
syringe needles is that blood will appear in the  Easiest and least painful to puncture
hub of the needle when the vein has been and the least likely to bruise. 
successfully entered. 
Cephalic Vein
Butterfly Method  Located in the lateral aspect of the
Winged Infusion Set▪ Is a short needle with a antecubital fossa. 
plastic part resembling butterfly wings and a  It is the second-choice. It is accessible,
length of tubing with a fitting for syringe use or unlikely to roll, less painful, located far
an adapter for ETS use.  enough away from major nerves or
Gauge: 23 gauge most commonly used for arteries, and generally safe to puncture.
phlebotomy  often the only vein that can be felt in
Smaller needles increase the risk of specimen obese patients.
hemolysis.   Basilic Vein
 Located on the medial side of the
THE VASCULAR SYSTEM  antecubital fossa. 
Arteries   It is the last choice in either pattern. 
 Have thick walls to withstand the  Although normally large and easy to
pressure of ventricular contraction, feel, it is not well anchored and rolls
which creates a pulse that can be felt, easily, increasing risk of puncturing a
distinguishing them from veins.  median cutaneous nerve branch or the
 Normal systemic arterial blood is bright brachial artery that is nearby. 
red because it is oxygen rich. SOURCE AND COMPOSITION OF BLOOD
Veins SPECIMENS 
 Have thinner walls than the same-size Arterial Blood 
arteries because blood in them is under ▪ Normally uniform throughout the body.
less pressure.  ▪ Technically difficult and potentially hazardous.
 Consequently, they collapse more ▪ Primarily reserved for BLOOD GAS
easily. Blood is kept moving through EVALUATION.
veins by Venous Blood 
skeletal muscle movement and the opening and closing  Affected by metabolic activity of tissue
of valves that line their inner walls.  it drains.
 Normal systemic venous blood is dark  Lower O2 Content, Cl-, Glucose, pH,
bluish red because it is oxygen poor. CO2, Lactic Acid & NH4
Capillaries  Impaired blood flow can affect other
 Are only one cell thick to allow the analytes. 
exchange of gases and other substances  Capillary Blood 
between the tissues and the blood.   Contains arterial and venous blood plus
 The capillary bed in the skin can easily tissue fluid is highest.
be punctured with a lancet to provide ▪ Warming the site increases it further. 
blood specimens for testing. ▪ Capillary Glucose is normally higher.
Major Antecubital Veins  ▪ Ca+, K+ and Total Protein are normally lower. 
MEDICAL LABORATORY SCIENCE PRACTICE I

▪ Squeezing the site can Falsely Elevate K+ The required information on a requisition
levels.  includes the following: 
TYPES OF BLOOD SPECIMENS SERUM ▪ Patient’s first and last names.
▪ Normally a clear, pale yellow fluid (Non- ▪ Identification Number (In-patient: hospital-
Fasting Serum can be Cloudy) generated; in an outpatient setting it may be a
▪ Separated from clotted blood by laboratory-assigned number.) 
centrifugation.  ▪ Patient’s date of birth.
▪ Routinely used in Chemistry Testing. ▪ Patient’s location.
PLASMA ▪ Ordering health-care provider’s name. 
▪ Normally a clear to slightly hazy, pale yellow ▪ Tests requested. 
fluid. Contains fibrinogen ▪ Requested date and time of sample collection
▪ Separates from cells when blood in an Greeting the Patient
anticoagulant tube is centrifuged.  ▪ A phlebotomist’s professional demeanor
▪ Many Chemistry Tests can be performed on instills confidence and trust in the patient,
either serum or plasma which can effectively ease patient apprehension
▪ Stat and other tests requiring a fast about the procedure. 
turnaround time (TAT) are often collected in ▪ When approaching patients, phlebotomists
tubes containing heparin anticoagulant. should introduce themselves, say that they are
WHOLE BLOOD from the laboratory, and explain that they will
▪ Contains both Cells and Plasma be collecting a blood sample. 
▪ As with plasma, it must be collected in an ▪ The procedure must be explained in
anticoagulant tube to keep it from clotting.  nontechnical terms and in a manner the patient
▪ Whole blood is used for most hematology can understand. 
tests and many point-of-care  ▪ The more relaxed and trusting your patient,
tests (POCTs), especially in acute care and stat the greater chance of a successful atraumatic
situations.  venipuncture. 
*Technical tips Observe any signs on the patient’s door or in
the patient’s room relaying special instructions,
Pre-analytical Consideration and such as Allergic to Latex, Nothing by Mouth
Venipuncture Complications (NPO), Do Not Resuscitate (DNR), Do Not Draw
Requisition Form Blood from (a particular) arm, Infection Control
▪ All phlebotomy procedures begin with the Precautions, or Patient Expired.
receipt of a test requisition form  Special Situation Sleeping 
that is generated by health-care provider.  Patients 
▪ The requisition becomes part of the patient’s ▪ Should be gently awakened and given time to
medical record is to provide the phlebotomist become oriented and have their IDENTITY
with the information needed to correctly VERIFIED before the venipuncture is performed
identify the patient, organize the necessary and secure their INFORMED CONSENT. 
equipment, collect the appropriate samples, ▪ Blood collection from a SLEEPING PATIENT
and provide legal protection.  may result in identification errors or physical
▪ Phlebotomists should NOT COLLECT A SAMPLE injury to the patient and result in a charge of
WITHOUT A REQUISITION  assault and battery (Informed Consent).
FORM. Unconscious Patients 
▪ Phlebotomists should carefully examine all ▪ Unconscious patients should be greeted in the
requisitions for which they are  same manner as conscious patients. 
responsible before leaving the laboratory.  ▪ In this circumstance, nursing personnel are
often present and can assist with the patient, if
necessary. 
MEDICAL LABORATORY SCIENCE PRACTICE I

Psychiatric Units  ▪ Positive patient identification can be made

▪ It is usually preferable to have a nurse assist using barcode technology. 
with patients on the  ▪ Using a wireless hand-held computer, the
psychiatric unit.  phlebotomist positively identifies the 
▪ These patients are often anxious about the patient by scanning the bar code on the 
venipuncture procedure and feel more patient’s hospital ID band.
comfortable when a caregiver with whom they ▪ The system, which is interfaced with the 
are familiar is present.  laboratory information system (LIS). 
▪ Be sure to place blood collection equipment Missing ID Band 
away from the patient. ▪ The phlebotomist must contact the nurse and
Physicians, Clergy, Visitors  request that the patient be banded before the
▪ Physicians, members of the clergy, and visitors drawing of blood. 
may be present when the phlebotomist enters ▪ The nurse’s signature on the requisition form
the room.  verifying identification should be accepted in
▪ When the physician or clergy member is with only emergency situations or according to
the patient, it is preferable to return at another hospital policy. 
time, unless the request is for a stat or timed ▪ Patients in psychiatric units often do not wear
sample.  an identification band. 
Unavailable Patient  Unidentified Emergency Department Patients 
▪ Patients are not always in the room when the ▪ Both the temporary and permanent
phlebotomist arrives.  identification band must be attached to the
▪ The phlebotomist should attempt to locate the patient and confirmed before blood may be 
patient by checking with the nursing station.  collected. 
 ▪ If the sample must be collected at a particular Identification of Young, Cognitively Impaired,
time, it may be possible to draw blood from the or Patients Who Do Not Speak the Language 
patient in the area to which he or she has been ▪ Ask the patient’s nurse, relative, or a friend to
taken.  identify the patient by name, address, and
▪ If this is not possible, the nursing station must identification number or date of birth. 
be notified and the appropriate forms ▪ Document the name of the verifier. 
completed so that the test can be rescheduled.  ▪ This information must be compared with the
▪ The requisition form is usually left at the information on the requisition and the patient’s
nursing station. identification band. 
Patient Identification ▪ Any discrepancies must be resolved before
▪ The most important procedure in phlebotomy collecting the sample. 
is correct identification of the patient. 
▪ Serious diagnostic or treatment errors and Numerous pre-examination variables 
even death can occur when blood is drawn from Variables:
the wrong patient.    Diet 
▪ The Clinical and Laboratory Standards Institute  Posture
(CLSI) recommends two identifiers for patient  Exercise
identification.   Stress
▪ To ensure that blood is drawn from the right  Alcohol
patient, identification is made by comparing  Smoking 
information obtained verbally and from the  Time of Day 
patient’s wrist ID band with the information on  Medications 
the requisition form.  Fever
Bar Code Technology  Physiological Variables: 
 Age
MEDICAL LABORATORY SCIENCE PRACTICE I

 Dehydration ▪ Increased in the proportion of formed

 Pregnancy elements in the blood due to prolonged
 Altitude  application of tourniquet 
 Gender  ▪ Remedy: One minute application of
 Malnutrition  tourniquet 
BASAL STATE  2. Syncope or Fainting 
▪ It refers to an early morning condition before ▪ Due to sudden decrease of blood supply to the
the patient has eaten or become physically brain 
active.  ▪ Remedy: Let the patient lie down, Give spirit
▪ This is a good time to draw blood specimens of ammonia
because the body is at rest and food has not B. Delayed Local Complications 
been ingested during the night. 1. Thrombosis of Veins 
▪ Normal values (reference ranges) for ▪ Formation of blood clots inside the lumen of
laboratory tests are determined from a normal, the vein due to trauma
representative sample of volunteers who are in 2. Thrombophlebitis
a basal state.   ▪ Inflammation of the vein due to thrombus as
Major Tests Affected by Patient Medication  manifested by an inflammatory reaction on the
Major Tests Affected by Technical Variation  outer skin surface 
Site Selection  3. Hematomas
▪ The preferred site for venipuncture is the  Blue or black skin discoloration
antecubital fossa located anterior and below commonly due to repeated trauma or
the bend of the elbow.  puncture of the veins. 
Three major veins  Errors in technique that cause blood to 
the median cubital, the cephalic, and the  leak or be forced into the surrounding 
basilic—are located in this area and, in most tissue and produce hematomas include: 
patients, at least one of these veins can be 1. Failure to remove the tourniquet before 
easily located.  removing the needle.
▪ Vein patterns vary among individuals. The 2. Applying inadequate pressure to the site 
most often seen arrangement of veins in the after removal of the needle. 
antecubital fossa are referred to as the “H- 3. Bending the arm while applying pressure. 
shaped” and “M-shaped” patterns.  4. Excessive probing to obtain blood. 
SPECIAL SITUATIONS IN PHLEBOTOMY 5. Failure to insert the needle far enough into
IV Line on both Arms the vein. 
 Discontinue IV for 2 minutes 6. Inserting the needle through the vein.
 Collect sample below the IV site 7. Selecting a needle too large for the vein. 
 Initial sample (5mL) → discard 8. Using veins that are small and fragile.
IV Fluid Contamination 9. Accidentally puncturing the brachial artery
Increased: C. General Delayed Complications 
Glucose (10% contamination w/ 5% dextrose  Serum Hepatitis/HBV 
→Increased blood glucose by 500 mg/dL)  HIV/AIDS 
Chloride, Potassium, Sodium PREVENTION: 
Decreased: Use of disposable syringe or vacutainer set
 Urea, Creatinine  (Follow the procedures from the Universal
COMPLICATIONS OF BLOOD SAMPLE Precautions in handling infectious
COLLECTION  specimens). 
A. Immediate Local Complications  GUIDELINES FOR SPECIMEN HANDLING 
1. Localized Hemoconcentration or Venous AND PROCESSING 
stasis  SPECIAL TEST REQUIREMENTS 
MEDICAL LABORATORY SCIENCE PRACTICE I

 Transport blood specimens carefully to ▪ It causes interference with large number of

avoid hemolysis. chemical analyses because of turbidity.
 Protect tubes for bilirubin, carotene (Amylase, Bilirubin, Protein). 
from light.
 Transport samples for ACTH, lactic acid, EXAMPLES OF CRITERIA FOR SPECIMEN
ammonia, blood gases in ice slurry. REJECTION
 Maintain tubes in vertical position to 1. Unlabeled or mislabeled samples. 
promote complete clotting.  2. Inadequate volume and collection in the
 Allow serum and gel separator tubes to wrong tube and outdated blood tube. 
clot for 30-60 min before centrifugation 3. Hemolysis, Lipemia, Clotted blood in an
to avoid fibrin strands. anticoagulant tube 
 Centrifuge within 2 hours of collection. 4. Improper handling during transport, such as
 Spin most tubes at 1,000-1,300 RCF for not chilling the sample 
10-15 min. 5. Samples without a requisition form. 
 Spin citrate tubes at 1,500 RCF for 15 6. Contaminated sample containers 
min to produce platelet-poor plasma.  7. Delays in processing the sample. 
 Don’t re-spin primary tubes. Can cause
hemolysis. If re-centrifuging is
necessary, transfer serum/plasma to
Dermal Puncture
 Advances in laboratory instrumentation
another tube.
and the popularity of POINT-OF-CARE
 Keep tubes capped during
TESTING make it possible to perform a
centrifugation to avoid loss of CO2,
majority of laboratory tests on
change of pH, evaporation, or aerosol
microsamples of blood obtained by
formation. 
dermal puncture on both pediatric and
 ▪Don’t re-spin serum separator tubes.
adult patients. 
Serum in contact 
Method of choice for collecting blood from
 with RBC’s under gel can be expressed
infants and children younger than 2 years for
and increased potassium
the following reasons: 
 ▪Separate serum or plasma from cells
1. Locating superficial veins that are large
within 2 hour of collection (exception:
enough to accept even a small-gauge needle is
centrifuged gel tubes)
difficult in these patients, and available veins
 When transferring samples to
may need to be reserved for intravenous
secondary containers, aspirate to avoid
therapy.
cellular contamination. Don’t pour. 
 2. Use of deep veins, such as the femoral vein,
 Separated serum/plasma may be kept
can be dangerous and injury caused by
at RT for 8 hr or at 2-8 deg celsius for 48
restraining the child. 
hours. For longer storage, freeze at -20
3. Drawing excessive amounts of blood from
deg celsius. Avoid repeated freezing and
premature and small infants can rapidly cause
thawing.
anemia, because a 2-pound infant may have a
 Lipemic specimens can be
total blood volume of only 150 mL.
ultracentrifuged at 105 x g to remove
4. Certain tests require capillary blood, such as
chylomicrons (triglycerides).
newborn screening tests and capillary blood
 Don’t freeze whole blood 
gases. 
LIPEMIA OR LACTESCENSE/Milky or Lipemic
Dermal puncture may be required in many
Plasma - occurs when blood samples are
adult patients, including: 
obtained 1-2 hours after eating a fatty meal 
1. Burned or scarred patients
MEDICAL LABORATORY SCIENCE PRACTICE I
2. Patients receiving chemotherapy who require
frequent tests and whose veins must be
reserved for therapy
3. Patients with thrombotic tendencies
4. Geriatric or other patients with very fragile
veins
5. Patients with inaccessible veins
6.  Obese patients
7. Apprehensive patients
8. Patients requiring home glucose monitoring
and point-of-care tests  ▪ Clinical and Laboratory Standards Institute
It may not be possible to obtain a satisfactory
sample by dermal puncture from patients who
are:
1. Severely dehydrated
2. Have poor peripheral circulation or have
3. Swollen fingers
4. Certain tests may not be collected by dermal
puncture because of the larger amount of blood
required such as some coagulation studies that
require plasma, erythrocyte sedimentation
rates, and blood cultures.  ▪ Major sample containers available
Importance of Correct Collection
▪ Correct collection techniques are critical
because of the smaller amount of blood that is
collected and the higher possibility of sample
contamination, microclots, and hemolysis
▪ Hemolysis may occur in dermal puncture for
the following reasons:
1. Excessive squeezing of the puncture site
(“milking”)
2. Newborns have increased numbers of red
blood cells (RBCs) and increased RBC fragility
3. Residual alcohol at the site
4. Vigorous mixing of the microcollection tubes
after collection  ▪ Variety of anticoagulants and additives,
Composition of Capillary Blood
▪ Blood collected by dermal puncture comes
from the capillaries, arterioles, and venules.
▪ Therefore, it is a mixture of arterial and
venous blood and may contain small amounts
of interstitial and intracellular fluids.
▪ Because of arterial pressure, the composition
of this blood more closely resembles arterial
rather than venous blood. 
Comparison of analytes in Capillary blood
1. Glucose- Higher
2. Potassium- Lower 
3. Total protein- Lower
4. Calcium- Lower 
Dermal Puncture Equipment
Dermal Puncture Devices
▪ To prevent contact with bone, the depth of
the puncture is critical
(CLSI) recommends that the incision depth
should not exceed 2.0 mm in a device used to
perform heelsticks.
▪ Distance from skin to vascular area:
a. Newborn: 0.35 to 1.6 mm
b. Adult: 3.0 mm
▪ Punctures should never be performed using an
uncontrolled surgical blade. 
▪ Puncture width: no larger than 2.5 mm
Microsample Container
 
for collection of microsamples. 
▪ Microcollection tubes have largely 
replaced the large-bore glass micropipettes.
Capillary Tubes- frequently referred to as
MICROHEMATOCRIT TUBES
▪ Small tubes used to collect approximately 50
to 75 μL of blood
▪ Red band: Heparinized
▪ Blue band: Plain
▪ Sample is closed with clay sealant or 
a plastic plug.  
Microcollection tubes
▪ MICROTAINER: provide a larger collection
volume and present no danger from broken
glass
including separator gel, are available, and the
tubes are color coded in the same way as
evacuated tubes. 
▪ Some tubes are supplied with a capillary scoop
collector top that is replaced by a color-coded
plastic sealer top after the sample is collected.
▪ Microtainer tubes are designed to hold
approximately 600 μL of blood. 
Dermal Puncture Procedure
MEDICAL LABORATORY SCIENCE PRACTICE I

1. Phlebotomist Preparation 3. Hemolyzes RBCs

▪ must have a requisition form containing the 4. Prevents formation of a rounded blood drop
same information required for the because blood will mix with the alcohol and run
venipuncture. down the finger 
▪ When a sample is collected by dermal Performing the puncture 
puncture, this must be noted on the requisition Sample collection
form ▪ the first drop of blood must be wiped away
▪ Phlebotomists frequently perform dermal with a clean gauze
punctures in the nursery. ▪ should not be obtained by milking of the
▪ Equipment should be kept out of reach of the surrounding tissue, which willrelease tissue
patient at all times.  fluid.
 i. Capillary tubes and Micropipettes: To prevent
Dermal Puncture Procedure the introduction ofair bubbles, capillary tubes
2. Patient Identification and Preparation and micropipettes are held horizontally while
▪ Patients must be identified using: requisition being filled.
form, verbal identification, and ID band.  ii. Microcollection tubes: more than 2 minutes
▪ Very agitated children may need to have their to collect may formmicroclots in an
legs and free hand restrained  anticoagulated microcollection container 
▪ Patient: seated or lying down Order of Collection
▪ Fingerstick: hand supported on a firm surface, 1. Capillary blood gases
palm up, and fingers 2. Blood smear
pointed downward 3. EDTA tubes
▪ Heelstick: infants should be lying on the back 4. Other anticoagulated tubes
with the heel in a downward position  5. Serum tubes 
4. Site selection  Overfilled tube - clot, 
Warming the site Underfilled tube- can cause morphological
▪ Warming dilates the blood vessels and changes in cells. 
increases arterial blood flow. Bandaging the Patient
▪ Moistening a towel with warm water (42°C) or ▪ The finger or heel is elevated and pressure is
activating a commercial heel warmer and applied until the bleeding stops.
covering the site for 3 to 5 minutes effectively  ▪ Confirm that bleeding has stopped before
warms the site. removing the pressure.
▪ The site should not be warmed for longer than 10. Labeling the Sample 
10 minutes or test results may be altered. 
Cleansing the site- cleansed with 70% isopropyl Special Dermal Puncture
alcohol, using a circular motion 1. Collection of Newborn Bilirubin
▪ allowed to dry on the skin for maximum ▪ Bilirubin is a very light-sensitive chemical and
antiseptic action, and the residue may be is rapidly destroyed when exposed to light.
removed with gauze to prevent interference ▪ Hyperbilirubinemia: increased serum bilirubin
with certain tests ▪ Infants who appear jaundiced are frequently
▪ Use of povidone-iodine is not recommended placed under an ultraviolet light (UV) to lower
for dermal punctures because sample the level of circulating bilirubin. 
contamination may elevate some test results,
including bilirubin, phosphorus, uric acid, and Newborn Screening
potassium.  ▪ testing of newborn babies for genetic,
Failure to allow the alcohol to dry: metabolic, hormonal, andfunctional disorders
1.Causes a stinging sensation for the patient that can cause physical disabilities,
2. Contaminates the sample
MEDICAL LABORATORY SCIENCE PRACTICE I

mentalretardation, or even death, if not  The sample is to be collected, analyzed,

detected and treated early. and results reported immediately. 
▪ Screening of newborns for 50 inherited  Stat tests have the highest priority and
metabolic disorders can currently be performed are usually ordered from the
from blood collected by heelstick and placed emergency department or for a
onspecially designed filter paper. critically ill patient whose treatment will
▪ Ideally blood is collected between 24 and 72 be determined by the laboratory result. 
hours after birth, beforethe baby is released STAT (Latin word) statim meaning
from the hospital  immediately.
Capillary Blood Gases Tests that fall into this category include:
▪ Arterial blood is the preferred sample for 1. Crossmatch
blood gases (oxygen andcarbon dioxide 2. Glucose in diabetic ketoacidosis
content) and pH levels in adults 3. Some drug levels such as theophylline
▪ Capillary blood is actually a mixture of venous 4. Amylase in suspected pancreatitis
and arterial blood, with ahigher concentration 5. Creatine kinase in MI
of arterial blood. 6. Hematocrit
▪ Performing deep arterial punctures in 7. Blood gases
newborns and young children isusually not 8. Potassium 
recommended; therefore blood gases are
performed oncapillary blood.  Fasting Samples
Preparation of Blood Smears FASTING PATIENT-Abstinence from food and
▪ Phlebotomists may make smears when one of liquids (except water) for a specified period. 
these tests is ordered anda dermal puncture is NPO (nothing by mouth) PATIENT- is not
performed. allowed to have food or water because of
▪ Blood smears should be collected before other possible complications with anesthesia during
samples to avoid plateletclumping. surgery or certain medical conditions. 
▪ Blood smears should be made within 1 hour of
collection to avoid celldistortion caused by the Timed Samples
EDTA anticoagulant.   Glucose Tolerance Tests
4. Blood Smears for Malaria   Lactose Tolerance Test
5.  Bleeding Time    Diurnal Variation 
Collection Priorities  Therapeutic Drug Monitoring 
Routine Samples 
 Use to diagnose and monitor a patient’s Glucose Tolerance Tests
condition.   Originally these included 2-HOUR
 Routine samples are usually collected POSTPRANDIAL (PP)GLUCOSE TEST 
early in the morning.  Classic GLUCOSE TOLERANCE TEST
 Can be collected throughout the day (GTT)
during scheduled “sweeps” (collection 2-HOUR PP GLUCOSE- compared a patient’s
times) on the floors or from fasting glucose level withthe glucose level 2
outpatients. hours after eating a meal with a high
ASAP Samples“As Soon As Possible.” carbohydratecontent.
 The response time for the collection of Classic GTT required patients to drink a
this test sample is determined by each standard glucose load andreturn for testing on
hospital or clinic and may vary by an hourly basis up to 6 hours in length. 
laboratory tests.    American DiabetesAssociation (ADA)
Stat Samples and the World Health Organization
(WHO) havestandardized and shortened
MEDICAL LABORATORY SCIENCE PRACTICE I
the methods used for the diagnosis of
hyperglycemia.
 2-hour oral glucose tolerance test (OGTT)
-diabetes mellitus
 1-step and 2-step methods- diagnosing
gestational diabetes.
Requirements for GTT:
(1) Ambulatory, Fasting: 8-12 hours.
(2) Unrestricted diet of 150g CHO/day for 3
days.
(3) Smoking, chewing tobacco, alcohol,
sugarless gum, andvigorous exercise should be
avoided before and during the test.
(4) 75 g = adult
 (WHO Standard): 100 g = pregnant  reported to a supervisor and the health-care
GTT Procedure
Step 1. Identify the patient and explain the
procedure.
Step 2. Confirm that the patient has fasted for
12 hours and not morethan 16 hours.
Step 3. Draw a fasting glucose sample. The
fasting blood sample istested before continuing
the procedure to determine whetherthe patient
can safely be given a large amount of glucose.  method for the diagnosis of diabetes
Step 4. Ask the patient to drink the appropriate
flavored glucose solution within 5 minutes.
Small adults and children may haveadjusted
amounts based on 1 g of glucose per kilogram
ofweight.
Step 5. Timing for the remaining collection
times begins when thepatient finishes drinking
the glucose solution . Outpatients aregiven a
copy of the schedule and instructed to
continuefasting, to drink water & to remain in
the drawing station area.  Diabetes
Step 6. Collect remaining samples at the
scheduled times. Timing ofsample collection is
critical, because test results are related tothe
scheduled times; any discrepancies should be
noted onthe requisition.
Step 7. The type of evacuated tubes used for
blood collection mustbe consistent. Blood
samples that will not be tested until the end of
the sequence should be collected in gray
stopper tubes.Consistency of VENIPUNCTURE or
dermal puncture must alsobe maintained. 
Step 8. Corresponding labels containing
routinely required information and sample
order in the test sequence, such as 1-hour, 2-
hour, and 3-hour are placed on the blood
samples.
Step 9. During scheduled sample collections,
phlebotomists shouldalso observe patients for
any changes in their condition, suchas dizziness,
which might indicate a reaction to the
glucose,and should report any changes to a
supervisor. 
Step 10. Some patients may not be able to
tolerate the glucosesolution, and if vomiting
occurs, the time of the vomiting mustbe
providercontacted for a decision concerning
whether to continue thetest.
Step 11. Transport samples to the laboratory
immediately. Samplesnot collected in gray
stopper tubes must be centrifuged ortested
within 2 hours of collection for reliable results.
2-Hour Oral Glucose Tolerance Test
  The OGTT is now the recommended
mellitus.
  The procedure requires the collection
of a fasting glucose sample, havingthe
patient drink a 75-g glucose solution
within 5 minutes and return for an
additional glucose test in 2 hours.
  A result equal to or greater than 200
mg/dL is considered indicative of
diabetes mellitus. 
One- and Two-Step Method for Gestational
 one-step method-utilizes the same procedure
as the diagnostic OGTT used to diagnose
diabetes mellitus.
  A value equal to or less than 140mg/dL
is considered normal. 
The two-step method requires the patient to
receive two tests. First a 50-gglucose challenge
load is administered to the fasting patient and
bloodcollected and tested at 1-hour post
ingestion.
MEDICAL LABORATORY SCIENCE PRACTICE I

   The second test is administered on a after the  medication is given (PEAK

different day and consists of either a75- LEVEL) 
g OGTT or a 100-g 3-hour OGTT based
on institutional protocol andhealth-care TROUGH LEVELS are collected immediately
provider preferences. before the drug is to be given and represent the
   A value of 155 mg/dL in the 2-hour 75- lowest level in the blood and ensure the drug is
g test is considered normal and avalue in the therapeutic (effective) range. 
of 140 mg/dL is considered normal in    The time for collecting PEAK LEVELS
the 3-hour 100-g test. varies with the medication and the
method of administration (30 minutes
 Lactose Tolerance Test after IV, 1 hour after intramuscular, or 1
   A lactose tolerance test evaluates a to 2 hours after oral dosage) ensures
patient’s ability to digest lactose, a milk that the drug is not at a toxic level. 
sugar.   Therapeutic drug monitoring collections are
   Lactose intolerance can be diagnosed often coordinated with the pharmacy,
by a lactose tolerance test. The patient laboratory, and nursing staff. 
is asked to drink a standardized amount  NOTE:
of lactose solution based on body Collection of blood in gel serum separator tubes
weight in place of the glucose.  has caused FALSELY LOW LEVELS for certain
  A blood collection schedule is similar to medications. RED STOPPER TUBES are
a 2-hour GTT.  recommended for Therapeutic Drug Monitoring
  Glucose levels will raise no more than and samples should be transported in an
20 mg/dL from the fasting sample result upright position. 
if the patient is lactose intolerant. 
  Blood Cultures
Diurnal Variation 
  Substances and cell counts primarily  STRICT ASEPTIC TECHNIQUE required and the
affected by diurnal variation are need to collect multiple samples in special
corticosteroids, hormones, serum iron, containers. 
glucose, and white blood cell counts,   A positive blood culture could be from
particularly eosinophils.  skin contamination and not from an
  Phlebotomists are often requested to actual patient infection in the blood. 
draw samples for these tests at specific  Blood cultures are requested on
times, usually corresponding to the patients when symptoms of fever
peak diurnal level.  (Fever of Unknown Origin (FUO)) and
  Certain variations can be substantial.  chills indicate a possible infection of the
  I.E. Plasma cortisol levels drawn blood by pathogenic microorganisms
between 08:00 and 10:00 will be twice (septicemia). 
as high as levels drawn at 16:00.  Factors to consider in Blood Culture:
   Timing of Sample Collection
Therapeutic Drug Monitoring   Collection Equipment 
   To ensure patient safety and  Blood Culture Anticoagulation 
medication effectiveness, the blood  Cleansing the Site 
levels of many therapeutic drugs are  Sample Collection 
monitored. 
   The most beneficial levels are those Timing of Sample Collection
drawn before the next dosage is  Blood cultures are usually ordered stat
given  (TROUGH LEVEL) and shortly or as timed collections. 
MEDICAL LABORATORY SCIENCE PRACTICE I
 Samples are usually collected in sets of
two drawn 30 or 60 minutes apart;
however, timing of sample draws varies
from institution to institution, or just
before the patient’s temperature
reaches its highest point (spike).  the tube or blood culture bottle to
 The concentration of microorganisms
fluctuates and is often highest just
before the patient’s temperature
spikes.
NOTE:
  If antibiotics are to be started
immediately, the sets are drawn at the
same time from different sites.  inhibited.
  Samples collected from different sites
at the same time serve as a control for
possible contamination and must be
labeled as to the collection site, such as
right arm antecubital vein, and their
number in the series (#1 or #2).
Collection Equipment  collection site include 2% iodine
 Blood can be drawn directly into bottles
containing culture media or drawn into
sterile, yellow stopper evacuated tubes
containing the anticoagulant and
sodium polyanethol sulfonate, and
transferred to
culture media in the laboratory.
 Blood can be collected in a syringe and
aseptically transferred to bloodculture
bottles at the bedside using a safety
transfer device.
Occupational Safety and Health Administration
(OSHA)-regulations donot allow direct
inoculation from the syringe to the bottle.  outward 3 to 4 inches in concentric
 A health-care provider may order blood
cultures on a patient who is onantibiotic
therapy
Antimicrobial Removal Devices (ARDs)
containingresin that inactivates antibiotics.
Fastidious Antimicrobial Neutralization (FAN)
Blood Collection System- uses bottles that
contain an activated charcoal that
neutralizesthe antibiotic.
  The recommended procedure is to
collect one blood culture from the IV
line and a second culture by
venipuncture.
Blood Culture Anticoagulation 
   An anticoagulant must be present in
prevent microorganisms from being
trapped within a clot.
  The anticoagulant SODIUM
POLYANETHOL SULFONATE is used for
blood cultures.
  Other anticoagulants should not be
used because bacterial growth may be
 
Cleansing the Site 
  The venipuncture technique for
collecting blood cultures follows
theroutine procedures, except for the
increased aseptic preparation of
thepuncture site.
  Antiseptics for disinfecting the blood
tincture, povidone-iodine, multiple
isopropyl alcohol preps,
andchlorhexidine gluconate, and all are
equally effective in killing bacteria on
the skin. 
   Cleansing of the site typically begins
with vigorous scrubbing of the site for 1
minute using isopropyl alcohol.
   The alcohol is followed by scrubbing
the site with 2% iodine tincture or
povidone-iodine for another minute
starting in the center of the
venipuncture site and progressing
circles.
 Allow the iodine to dry on thesite for at
least 30 seconds.
   The venipuncture site is scrubbed for
30 to 60 seconds in a back-and-forth
motion creating a friction on the skin,
which is effective in skin antisepsis. 
 
Sample Collection 
  Two samples are routinely collected for
each blood culture set, one to be
MEDICAL LABORATORY SCIENCE PRACTICE I

incubated aerobically and the other seconds and allow to air dry for at least 30
anaerobically.  seconds for antisepsis. 
  Syringe: ANAEROBIC BOTTLE should be  Step 10. Assemble equipment while
inoculated first. the  antiseptic is drying. Attach the needle
  Winged Blood Collection Set: AEROBIC BOTTLE to the syringe 
is inoculated first.   Step 11. Remove the plastic cap on
   Filling bottles directly through and the  collection bottle. Confirm the volume
evacuated tube needle and holder of blood required from the label. 
system is not recommended.   Step 12. Clean the top of the bottles with a  70
 NOTE:  percent isopropyl alcohol pad and allow  to dry. 
   Pediatric blood culture volume  Step 13. Reapply the tourniquet and perform
requirements are based on the the venipuncture. Do not repalpate the site
child’s weight and pediatric bottles are without cleansing the palpating finger in the
inoculated. Draw 1 mL of blood for same manner as the puncture site.
every 5 kg (approximately 10 pounds) of  Step 14. Release the tourniquet. Place gauze
patient weight.  over the puncture site, remove the needle, and
   The sample of a child heavier than 45 apply pressure. 
kg is treated as that of an adult.   Step 15. Activate the safety device or remove
   Draw 1 mL of blood on babies the syringe needle with a Point-Lok device.
weighing less than 5 kg, and place all of  Step 16. Attach the transferring device. 
the blood in one pediatric bottle.   Step 17. Inoculate the anaerobic blood  culture
 There should be at least a 1:10 ratio of bottle first when using a syringe or second when
blood to media.  using a winged blood 
  ADULT BLOOD CULTURE BOTTLES usually  collection set. 
require 8 to 10 mL for each.  Step 18. Dispense the correct amount of blood
  PEDIATRIC BOTTLES require 1 to 3 mL for into bottles. Some institutions require
each.  documenting the amount of blood 
  Over filling of bottles-should be avoided  dispensed 
because this may cause false-positive results  Step 19. Mix the blood culture bottles by gentle
with automated systems.  inversion eight times. 
  Under filled blood culture bottles- may cause  Step 20. Fill other collection tubes after
false-negative results.  the blood culture tubes. 
 Blood Culture Sample Collection  Step 21. Clean the iodine off the arm with
 Step 1. Obtain and examine the requisition alcohol if necessary. 
form.  Step 22. Label the samples appropriately and
 Step 2. Greet the patient and explain the include the site of collection.
procedure to be performed. Verify Identification with the patient. 
 Step 3. Use two identifiers to correctly identify  Step 23. Dispose of used equipment and
the patient. supplies in a biohazard container. 
 Step 4. Prepare the patient and verify allergies.  Step 24. Check the venipuncture site for
 Step 5. Select equipment. bleeding and bandage the patient’s arm. 
 Step 6. Wash hands and don gloves.  Step 25. Thank the patient, remove gloves, and
 Step 7. Apply the tourniquet and locate the wash hands. 
venipuncture site.   
 Step 8. Release tourniquet.   Blood Collection From Central Venous
 Step 9. Sterilize the site using chlorhexidine Catheters
gluconate. Creating a friction, rub for 30 to  60
MEDICAL LABORATORY SCIENCE PRACTICE I

   Blood samples may be obtained from laboratory as soon as possible, and

indwelling lines called central venous placed back into the incubator before
catheters (CVCs).  testing.
   This procedure must be performed by    Forensic studies are performed on
specially trained personnel and samples for legal proceedings. The most
physician authorization is required.  common are blood alcohol tests, urine
   CVCs are a special type of catheter drug tests, and DNAanalysis.
that is inserted by a physician or a Documentation of sample handling,
certified health-care professional either the chain of custody, must be strictly
as an internal catheter or external followed. 
catheter into a large blood vessel. 
   It can be used for administration of   Phlebotomists encounter patients of all ages,
fluids, drugs, blood products, and which will require different technical and
nutritional solutions and to obtain communication skills appropriate for each age
blood.  group.
   There are numerous types of CVCs and    Modifications to blood collection
specific procedures must be followed techniques and equipment
for flushing the catheters with saline, arenecessary to successfully
and possibly heparin to prevent accommodate the challenges of
thrombosis, when blood collection is bloodcollection for the pediatric and
completed.  geriatric populations.
  Sterile technique-procedures must be strictly    Phlebotomists must develop and
adhered to when entering IV lines, because they increase their knowledge and skills in
provide a direct path for infectious organisms to working with all age groups of patients
enter the patient’s bloodstream.  while performing blood collection
 Special Sample Handling Procedures procedures. 
   Chilling samples prevents deterioration
of specific analytes such as ammonia, Special Patient Populations: Geriatric
lactic acid, pyruvate, gastrin, Population
adrenocorticotrophic hormone (ACTH),   Normal aging often results in gradual
renins, catecholamines, and hearing loss 
parathyroid hormone.    Failing eyesight is common in the older
   Samples should be immediately placed patient. 
into a crushed ice and water slurry or   Muscle weakness may cause the
uniform ice block after blood patient to drop things or be unable to
collection.  make a fist before venipuncture or to
   Exposure to light will destroy bilirubin; hold the gauze after the venipuncture. 
beta-carotene; vitamins A, B6,    Memory loss  
and  B12, and folate; and porphyrins.   The epithelium and subcutaneous
Wrapping tubes in aluminum foil or tissues become thinner, causing veins
using amber-colored tubes will protect to be less stable and harder to anchor. 
the sample    Epidermal cell replacement in the aging
   Samples for cold agglutinins are patient is delayed, increasing
collected in tubes that have been the chance of infection. 
warmed in a 37°C incubator for 30   Aging veins have decreased collagen
minutes and contain no additive or gel. and elasticity, making them less firm,
   The blood is collected into the more likely to collapse, more difficult to
warmed tube, returned to the
MEDICAL LABORATORY SCIENCE PRACTICE I

anchor and puncture, and more prone   The vacuum pressure in the collection
to hematoma formation.  tube may cause fragile veins to collapse.
  Older patients often feel cold because   A better choice is a winged blood
of the decreased fatty tissue layer, and collection set with a 23-gauge needle
warming of the site may be required.  attachedto a syringe that will allow the
  Arteries and veins often become phlebotomist to control the suction
sclerotic in the older patient, making pressure onthe vein.
them poor sites for venipuncture     A small-gauge needle with a syringe
Disease States  also is an option.
  A patient with Alzheimer’s disease may   If an evacuated tube system is used,
be confused or combative, which can the smallest possible tubes should be
cause problems with identification and filled.
performing the procedure. Assistance  Because of the tendency to develop
from a family member or the patient’s anemia by older patients, the volume
caretaker may be necessary to calm the ofblood collected also should be kept to
patient and hold the arm steady.  the minimum acceptable amount. 
  Stroke patients may have paralysis or Tourniquet Application 
speech impairments requiring   The tourniquet can be placed over the
assistance in positioning and holding patient’s sleeve and must not be
the arm and help with communication.  applied too tight to avoid injury to the
 Patients in a coma should be treated as patient or collapsing the vein. 
if they can hear what is being said.   Gently release the tourniquet after
Assistance will be required when venipuncture without snapping it
holding the arm.  against the patient’s skin to avoid
  Arthritic patients may be in pain or bruising the area. 
unable to straighten the arm and may   Blood pressure cuffs can be used for
require assistance gently positioning the thin patient with small, hard-to-find
and holding the arm. Using a winged veins. 
blood collection set with flexible tubing Site Selection 
will allow the phlebotomist to access   The antecubital fossa may not be the
veins at awkward angles.  best site selection. 
  Older patients may have tremors, as   The veins in the hand or forearm may
evidenced in Parkinson’s disease,  be a better choice.
 and may not be able to hold the arm   Applying heat compresses for 3 to 5
still for the venipuncture procedure.  minutes and stimulating the area with
Emotional Factors  alcohol can make the vein more
Patient Identification  prominent.
  When identifying older patients   To avoid bruising the patient, do not
without identification bands, be sure to tap the vein.
have them state their names.    Other techniques used by
  An elderly patient who is confused or phlebotomists to enhance the
who has difficulty hearing is very likely prominence of veins include massaging
to answer “yes” to any question.  the arm upward from the wrist to the
  When identifying patients, address elbow and briefly hanging the arm
them by their rightful title and not by down. 
their first name. Always be considerate Blood Collection
and thank the patient.    Elderly patients’ veins “roll” easily;
 Equipment Selection  therefore, the skin must be pulled taut,
MEDICAL LABORATORY SCIENCE PRACTICE I

anchored firmly, and the vein   The parents must identify the child if it
punctured in a quick motion.  is an outpatient setting. 
  Loose skin can be pulled taut by   Hospitalized patients will have an
wrapping your hand around the arm identification band. 
from behind.  Techniques for Dealing with Toddlers
  The angle of the needle may need to be   Toddlers have limited language skills
decreased for venipuncture because the and fear of strangers. 
veins are often close to the surface of   It is important to talk to the child
the skin.  calmly and maintain eye contact. 
Bandages   Demonstrate the procedure using toys. 
  Older patients may have increased   Allow children to have their comfort
sensitivities to adhesive bandages and toys or blanket and develop strategies
an increased tendency to bruise.  to distract or entertain them. 
  Therefore, it is preferable to use a self   Reward the child with praise and
adhering pressure dressing bandage stickers. 
(e.g., Coban) because adhesive   Thank the child and parent for their
bandages on the fragile skin of older cooperation. 
patients can actually take off a layer of Techniques for Dealing with Older Children
skin when they are  removed and leave   Older children are more willing to
a raw wound susceptible to infection.  participate. 
Patient/Parent Preparation    Explain the steps of the procedure and
  Pediatric blood collection involves preparing demonstrate the equipment. 
both the child and parent, using certain   Demonstrate and allow the child to
restraining procedures, and special equipment.  touch the tourniquet or other clean
  Pediatric phlebotomy presents equipment. 
emotional as well technical difficulties   Answer their questions honestly. 
and should be performed by only   Never tell a child it will not hurt.
experienced phlebotomists.  Explain that “it will hurt a little bit, but if
  A negative experience can lead to a you hold very still, it will be over
child’s life-long fear of needles. Often, quickly.” 
there is only one chance to attempt a   Give the child permission to cry. 
venipuncture on a child.  Techniques for Dealing with Teenagers
  The phlebotomist must develop   Teenagers are more independent and
interpersonal skills to successfully gain often embarrassed to show their
both the young patients’ and parents’ emotions. 
trust.    Use adult language with teenagers for
Techniques for Dealing with Children  identification and explanation of the
  Techniques for dealing with children procedure. 
vary depending on the child’s age.    Ask them if they have fainted or had
  It is best to establish guidelines and to any reaction to a previous venipuncture
be honest with both the patient and procedure. 
parent.    Encourage them to ask questions about
  Newborns and infants are totally the procedure. 
dependent on their parents.   They may or may not want their
  The phlebotomist should introduce him parents present. 
or herself to the parents and explain the Techniques for Dealing with Children
procedure.  Note:
MEDICAL LABORATORY SCIENCE PRACTICE I

 Utilizing techniques to calm an infant   The minimum amount of blood

and reduce cryingtimes will minimize required for laboratory testing should
the pre-examination effect oftransient be collected from infants and small
elevated white blood cell counts.  children. 
 Methods of Restraint    The amount of blood collected within a
  Older children can usually sit in a 24-hour period must be monitored
drawing chair by themselves.  owing to the small blood volume in
  An infant cradle pad facilitates blood newborns and small children.
collection for infants.    It is recommended that no more than
  Never draw blood from a small child 3% of a child’s blood volume be
without some type of assistance.  collected at one time and no more than
  Physical restraint may be required to 10% in a month. 
immobilize the young child and steady   To quickly estimate a child’s blood
the arm for the venipuncture volume, divide the child’s weight in
procedure.  pounds by 2 to convert to kilograms and
  This can be accomplished by having multiply the kilograms by 100 to get the
someone hold the child or by using a estimated blood volume in milliliters. 
papoose board.    When using an evacuated tube system,
  Either a VERTICAL or HORIZONTAL select the smallest evacuated pediatric
RESTRAINT will work.  tubes available. 
Methods of Restraint: VERTICAL RESTRAINT   Evacuated tubes as small as 1.8 mL are
  In the vertical position, the parent available. 
holds the child in an upright position on   A 23-gauge winged blood collection set
the lap.  needle with a syringe is recommended
  The parent places an arm around the because of the small, fragile veins. 
toddler to hold the arm not being used.   Pain Interventions
  The other arm holds the child’s  A local topical anesthetic, eutectic mixture of
venipuncture arm firmly from behind, local anesthetics (EMLA) 
at the bend of the elbow, in a (Abraxis Pharmaceuticals) is ideal for use on an
downward position.  apprehensive child before venipuncture. 
Methods of Restraint: HORIZONTAL   This emulsion of lidocaine and
RESTRAINT  prilocaine is applied directly to intact
  In the horizontal restraint, the child lies skin and covered with an occlusive
down, with the parent on one side of dressing. 
the bed and the phlebotomist on the   EMLA penetrates to a depth of 5 mm
opposite side.  through the epidermal and dermal layer
  The parent leans over the child holding of the skin. It takes 60 minutes to reach
the near arm and body securely while its optimal effect and lasts for 2 or 3
reaching over the body to hold the hours. 
opposite venipuncture arm for the
phlebotomist.  Research has shown that glucose,  dextrose,
  In some instances, a child may become and sucrose solutions have a  calming effect on
extremely combative. The procedure infants. When administered before or during
should be discontinued to avoid the risk venipuncture  or heelsticks, pain was
of injury to the patient or phlebotomist significantly reduced as opposed to a topical
and the health-care provider notified.  anesthetic. The  duration of crying was
Equipment Selection lessened, which  minimized the temporary
increase in the  white blood cell counts. 
MEDICAL LABORATORY SCIENCE PRACTICE I

 Blood is the transporting fluid of the body

Site Selection   Blood has many important functions and also
CHILDREN OLDER THAN 2 YEARS: Veins Located plays a role in other body functions, including
in the Antecubital Fossa.  the following:
CHILDREN YOUNGER THAN 2 YEARS OF AGE:   Responsible for transporting oxygen
Dorsal Hand Venipuncture. and nutrients to the cellsand tissues of
Small amount of blood: heel stick puncture, the body.
micro sample containers.  Responsible for transporting hormones
Note:  to their target area, sothat the body can
Do not use deep veins. Site selection and function in its proper capacity.
technique is similar to that used for adults can  Responsible for eliminating waste
be used for.  materials (excretion) from the body’s
cells Responsible for maintaining water
Point of Care Testing (POCT) balance for the cells andtissues of the
 Other Names: Near-patient Testing, body
Decentralized Testing, Bedside Testing,  Responsible for transporting antibodies
Alternate Site Testing and protective substances throughout
  Analytical testing performed outside the body so they may attack infecting
the confines of the central laboratory, organisms and pathogens
usually by non-laboratorian personnel  Assists with regulating body
(Nurses, Respiratory Therapists, etc.) temperature
  Use of portable whole blood glucose  Assists with maintaining acid-base
meters for the management of patients. balance 
  the performance of laboratory tests at
the patient’s bedside or nearby rather Blood Composition
than in a central laboratory.   Mixture of fluid and cells
  POCT is particularly beneficial to   About five times thicker than water
patient care in the critical care or   Salty to the taste
intensive care units, operating suites,   Slightly alkaline, with a pH of about 7.4
emergency department, or neonatal The average adult weighing 70 kg
intensive care units.  (approximately 154 pounds) has a blood
  Other POCT locations include satellite volume of about 5 liters (5.3 quarts), of which
laboratories, physician offices, approximately 55% is PLASMA and 45% is
ambulatory clinics, ambulances or FORMED ELEMENTS. 
helicopters, long-term care facilities, A. Plasma
workplace screenings, health fairs,   clear, pale-yellow fluid
dialysis centers, and home settings.    90% water (H2O) and 10% solutes
 Factors that have motivated the (dissolved substances)
practice of POCT include the increased   Gases: oxygen (O2), carbon dioxide
acuteness of inpatient illnesses that (CO2), and nitrogen (N)
require a faster turnaround time (TAT)   Minerals: sodium (Na), potassium (K),
of results and the decreased length of calcium (Ca), and magnesium (Mg)
hospital stays that require the increased   Nutrients: carbohydrates, such as
performance of procedures and care on glucose, and lipids (fats), such as
an outpatient basis.  triglycerides and cholesterol.
  Proteins: Albumin, Antibodies,
Hematology Section  Fibrinogen
MEDICAL LABORATORY SCIENCE PRACTICE I

  Waste products: blood urea nitrogen debris.  Average adult has between
(BUN), creatinine, and uric acid 5,000 and 10,000 WBC per cubic
  Other substances such as vitamins, millimeter of blood
hormones, and drugs   Two main categories:
B. Formed elements: RED BLOOD CELL (RBC) a. GRANULOCYTES: granules are present in the
  MOST NUMEROUS CELLS in the blood, cytoplasm
averaging 4.5 to 5 million per cubic  Neutrophil
millimeter of blood  Eosinophil
  Main function: carry oxygen from the  Basophil
lungs to the cells also carry carbon b. AGRANULOCYTES: no granules are present
dioxide from the cells back to the lungs  Lymphocytes
to be exhaled.  Monocytes
  Resemble the shape of a doughnut
without a hole, appearance is referred PLATELETS / THROMBOCYTES
to as BICONCAVE (7-8 microns) because   SMALLEST IN SIZE of all the cellular
both sides of the red blood cell cave components
inward at the center. (FLEXIBILITY)   Life span: 9-12 days
  Life span: 120 DAYS    Normal adult range is between 150,000
  Ability to transport oxygen and carbon and 450,000 per cubic millimeter of
dioxide occurs as a result of a very blood
important molecule, called   PLATELET PLUG: Bleeding is diminished
HEMOGLOBIN. or halted as a result of platelets sticking
 Hemoglobin is made up of a protein molecule to the site of injury
called globin and an iron compound called   SEROTIN: causes the blood vessels to
heme spasm or narrow and decrease blood
 BRIGHT RED COLOR: when large amounts of loss until the clot forms. 
oxygen attached to the hemoglobin in the RBC
(OXYGENATED) Hemostasis and Blood Coagulation
 DARK BLUISH-RED: when large amounts of   Hemostasis breaks down into HEMO,
carbon dioxide are attached to hemoglobin in meaning “blood” and STASIS,meaning
the RBC (DEOXYGENATED)  “stopping”
HEMOGLOBIN REFERENCE VALUES (ADULT) Following an injury there are four major events
 Male 14–18 grams/100 mL (1 deciliter) involved in stoppingthe flow of blood at the
of blood injured site:
 Female 12–16 grams/100 mL (1   Blood vessel spasm (vasoconstriction)
deciliter) of blood   Platelet plug formation
LEUKOCYTE / WHITE BLOOD CELL (WBC)   Blood clotting (coagulation)
  primarily responsible for destroying  Fibrinolysis or dissolving of the clot and
foreign substances such as pathogens return of the vessel tonormal function
(disease-producing microorganisms)
and removing cellular debris.  * These cells, which are formed in the bone
  not always confined to the vascular marrow, are released into the bloodstream as
spaces to perform their duty. needed to carry oxygen, provide immunity
  DIAPEDESIS: WBC can pass through the against infection, and aid in blood clotting.
thin walls of capillaries to enter the
tissues Sample Collection and Handling
  PHAGOCYTOSIS: WBCs engulf or “eat”   Blood is analyzed in the form of
foreign substances and/or cellular wholeblood, plasma, or serum.
MEDICAL LABORATORY SCIENCE PRACTICE I

  The most common body fluid analyzed ab once it’s formed.

in the hematology section is WHOLE Immunogen-Any substance capable of inducing
 BLOOD (a mixture of cells and plasma) immune 
  A whole blood specimen is obtained response. 
byusing a collection tube with Two Types of Immune System
ananticoagulant (such as EDTA, INNATE / NON ADAPTIVE / NON SPECIFIC
CITRATE)to prevent clotting of the IMMUNITY 
sample.   Ability of an individual to resist
Test Performed  infections by means of normally present
Hematology Section  body functions.
Coagulation  ACQUIRED / ADAPTIVE / SPECIFIC IMMUNITY
 Host response to foreign agents that
Serology (Immunology)  depends on T and B lymphocytes and is
IMMUNOLOGY: The study of the reactions of a characterized by specificity,
host when foreign substances are introduced memory, and recognition of self versus
into the body.  non-self. 
 Can be defined as the study of a host’s Major Mechanisms of the Immune System 
reactions when foreign substances are ACTIVE AND PASSIVE IMMUNITY 
introduced into the body. A foreign Sample Collection and Handling Serology 
substance that induces such an   Sample collection and handling blood
immune response is called an for serological testing is collected in
ANTIGEN.  tubes with red stoppers. 
Immunology as a science has its roots in the   Serum separator tubes are not used
study of immunity, the condition of being when the gel will interfere with the
resistant to infection. antigen-antibody reactions.
SEROLOGY: The study of a non-cellular portion Serology Section 
of the blood known as serum.    The serology (immunology) section
IMMUNITY/ IMMUNE RESPONSE- is a process performs tests to evaluate the body’s
by which a host organism protects itself immune response; that is, the
from attacks by external and internal agents. production of antibodies
  Is necessary to protect the host from (immunoglobulins) and cellular
obvious invaders such as parasites and activation. 
also against external noxious elements   Tests in the serology section detect the
and sun exposure.  presence of antibodies to bacteria,
Terminologies fungi, parasites, viruses, and antibodies
Antigen (Ag)-Foreign substance that stimulates produced against body substances
antibody  (autoimmunity).
production. Large, complex molecules
(MW>10,000), usually protein or Blood Bank Section 
polysaccharide.  Explain the Principles and Procedures in:
Antibody (Ab)- Immunoglobulin produced by  Blood Collection 
plasma cells in   Blood Processing
response to Ag.   Blood Preservation
 Histamine Vasoactive amine released  Blood Storage
from mast cells & basophils during
allergic reaction.  Distribution of Blood Blood Components &
Hapten-Low molecular weight substance that Derivatives
can bind to 
MEDICAL LABORATORY SCIENCE PRACTICE I

Performed the Techniques of BLOOD for a specific number of days, as determined by

GROUPING AND COMPATIBILITY TESTING and PRESERVATIVE SOLUTION(S) used. 
other methods.Includes the concepts in the  APPROVED PRESERVATIVES
operation and organization of the Blood Bank &  ANTICOAGULANTS AND RED CELL ADDITIVES 
Quality Assurance in the performance of   Sample Collection and Handling Blood
Immunohematology tests. Bank   Samples are collected in plain red
(serum), lavender, or pink (plasma)
 BLOOD BANK is a bank of blood and their stopper tubes.
components, gathered as a result of blood  Serum separator tubes containing gel are not
donation, where it is stored with preservative acceptable because the gel will coat the RBCs
for later use in transfusion. Blood banks not and interfere with testing. 
only collect and save the blood for transfusion      Hemolysis also interferes with the
but it also performs the testing to determine interpretation of test results. 
the blood group of patients as well as donors,  Note:
their cross match test and donor screening   Patient identification is critical in the
tests.  blood bank 
  Misidentification or mislabeling of a
HISTORY PERSPECTIVE OF BLOOD BANKING blood bank sample can result in a
1492-First time a blood transfusion was sentinel event. 
recorded in history.  Patient Identification
Blood was taken from three young men &   Must confirm recipient’s ID from
transfused to POPE INNOCENT VII. bracelet ON the patient.
1628- WILLIAM HARVEY: De Motu Cordis   Full patient name and hospital number.
1666- RICHARD LOWER: Transfused blood from   Name of physician 
a dog to another.  The blood bank (BB) is the section of the
1667—Lower and JEAN BAPTISTE DENIS laboratory where blood may be collected,
* Both performed sheep to human transfusion. stored, and prepared for transfusion. 
* Both are unsuccessful and banned.     In the blood bank, blood from patients
BLOOD PRODUCTS/ BLOOD are considered and donors is tested for its blood group
THERAPEUTIC DRUGS because of their use in (ABO) and Rh type, the presence and
treating diseases. The transfusion of blood cells identity of abnormal antibodies and its
is also transplantation, in that the cells must compatibility (crossmatch) for use in a
survive and function after transfusion to a transfusion. 
THERAPEUTIC EFFECT.      Units of blood are collected from donors,
tested for the presence of blood borne
BLOOD PRESERVATION pathogens such as hepatitis viruses and human
GOAL OF BLOOD PRESERVATION: immunodeficiency virus (HIV), and stored for
 Provide a viable and functional blood transfusions. 
components for patients requiring Donor blood may also be separated into
blood transfusion. components including packed cells, platelets,
NOTE:  fresh frozen plasma cryoprecipitate.
 At least 70 % of cells that have   These components are stored
been transfused should remain viable separately and used for patients with
for 24 hours.  specific needs. 
  Patients may come to the blood bank
TO MAINTAIN OPTIMUM VIABILITY: blood is to donate their own blood so that they
STORED in the liquid state between 1 ̊C and 6 C
̊ can receive an AUTOLOGOUS
MEDICAL LABORATORY SCIENCE PRACTICE I

TRANSFUSION if blood is needed during Batch Testing

surgery.   All samples loaded at the same time.
The test between a prospective recipient of a   Single test is conducted on each
blood transfusion and his proposed donor (or sample.
donors) is known as the COMPATIBILITY TEST.  Parallel Testing
Note:    One specimen with more than one test
Universal Donor-Group O is analyzed.
Universal Recipient- Group AB Random Access Testing
Universal Donor for RBC’s- Group O   Able to perform individual tests or
Universal Recipient for RBC’s- Group AB panels.
Universal Donor for Plasma- Group AB   Allows for STAT samples to be added to
Universal Recipient for Plasma-Group 0 run ahead of other specimens. 
Sequential Testing
 Introduction to Clinical Chemistry   Multiple tests analyzed one after
Clinical Chemistry another on a given specimen.
Clinical - Greek word “Kline”, meaning bed Laboratory Information System (LIS)
Also known as    A system of computer software
 Chemical Pathology  designed to handle laboratory data. 
 Clinical Biochemistry  Carry-over
 Medical Biochemistry  The contamination of a sample by a
 Clinical Chemistry previously aspirated sample.
Terminologies:
The discipline originated in the late 19th  Bar Code-A set of vertical bars of varying width
century with the use of simple chemical used to encode information. 
reaction tests for various components of   Used most frequently in the clinical
blood and urine. In the many decades since, laboratory for patient and specimen
other techniques have been applied as science information
and technology have advanced, including the Bar Coding Mechanism- for patient/sample
use and measurement of: identification; used for reagent identification by
  Enzyme Activities an instrument 
  Spectrophotometry Delta Check-An algorithm in which the most
 Electrophoresis recent result of a patient is compared with the
 Immunoassay.  previously determined value.
Sub-specialties: Throughput- Maximum number of tests
General or Routine Chemistry or  Automated generated per hour
Chemistry Turnaround- Amount of time to generate one
Special Chemistry result
 Clinical Endocrinology Dead Volume- Amount of serum that cannot be
 Toxicology  aspirated.
 Therapeutic Drug Monitoring Open Reagent System- System other than
 Tumor Marker manufacturer’s reagents can be utilized for
CLINICAL CHEMISTRY  measurement
 section is the most automated area of Closed Reagent System- The operator can only
the laboratory. Instruments are use the manufacturer’s reagents
computerized and designed to perform Pneumatic Tube System- Transports specimens
single and multiple tests from small quickly from one location to another
amounts of specimen. Dry Chemistry Slide
MEDICAL LABORATORY SCIENCE PRACTICE I

 A multilayered film technology (dry

chemicals) useby automated analyzers.  Discrete Analyzer
 All reagents necessary for a particular  An approach to automated analysis in
test are contained on the “slide.”  which each sample and accompanying
Probe- A mechanized device that automatically reagents is in a separate container.
dips into a sample cup and aspirates a portion   Discrete analyzers have the capability
of the liquid. of running multiple tests one sample at
Rotor- A round device on some automated a time or multiple samples one test at a
analyzers that holds sample cups and is capable time.
of spinning.   Require 2 to 6 uL volume of sample.
Closed Tube Sampling-An instrument’s ability  Random Access Capability (STAT)
to remove the patient’s sample for analysis   Examples: Vitros, Beckman ASTRA
from the primary collection tube by piercing System (4 & 8), Hitachi, Roche Cobas
through the stopper.  Integra 800, Dupont ACA, Abbott ABA-
Integrated Modular System- At least two 100 Bichromatic Analyzer
analytical modules supported by one sample
and reagent processing and delivery system  So, how does the clinical instrument work?and
Multiple Platform- Instrument able to perform how does it measures the analyte?
tests from at least two disciplines.   The four quadrant trays, each holding
Modular Work Cell- At least two instruments ten samples, fit on a tray carrier. 
from a single discipline with one controller.   Sample collection tubes are
 Stand-alone Instrument from a single identified with bar code labels Five-
discipline with automated capability  position Rack Dual sample probes of a
Schematic drawing of MODULAR ANALYTICS chemistry analyzer. Note the liquid
System level sensor to the left of probes. 
  Schematic Drawing of Multiple Platform Wash stations on a chemistry analyzer perform
System the following: 
Three Basic Approaches with Clinical (1) aspirate reaction waste and dispense water; 
Instruments   (2) aspirate and dispense rinse water; 
Continuous Flow Analyzer (3) aspirate rinse water and dispense water for
 Liquids (reagents, diluents, measurement of cell blank; and 
and samples) are pumped through a (4) aspirate cell blank water to dryness. 
system of continuous tubing. 
  Samples are introduced in a SPECTROPHOTOMETRY
sequential manner, following each   Summary of Chemistry Analyzer
other through the same network.  Operations Sample Collection and
  Examples: Simultaneous Handling
Multiple Analyzer (SMA),Technicon Chemistry Tests are performed:
Autoanalyzer II, Three Basic Approaches  Serum (Gel barrier tubes)
with Clinical Instruments  Serum (red, green, gray, royal blue
Centrifugal Analyzer tube)
  Analytic technique that uses the  Plasma 
force generated by centrifugation to  Urine and other body fluids 
transfer and then contain liquids in *Serum and plasma are obtained
separate cuvettes for measurement at by centrifugation, which should be performed
the perimeter of a spinning rotor.  within 1 to 2 hours of collection. 
 Example: Cobas-Bio (RocheDiagnostics,
Centrifichem) Specimens of Concern: 
MEDICAL LABORATORY SCIENCE PRACTICE I

  Hemolyze Specimens; Sodium (Na) Controls blood pressure and blood

  Icteric Specimens;  volume, needed in muscle and
 Lipemic specimens that are cloudy. nerve excitability.
Potassium (K) Regulate fluid balance, muscle
Sample Collection and Handling contraction and nerve signal.
  Fasting samples drawn from patients Iron (Fe) Decreased levels indicate iron
who have not eaten for 8 to 12 hours.  deficiency anemia
  Samples must be allowed to clot fully  Lead (Pb) Elevated levels indicate poisoning
 before centrifugation to Lithium (Li) Monitors antidepressant drug 
ensure complete separation of cells &  Magnesium (Mg) Cation involved in
serum.  neuromuscular excitability of muscle tissue 
  Many chemistry tests require special   Phosphorus (P) Mineral associated with
 collection and handling procedures, skeletal or endocrine disorders 
such as chilling and protection from
light. Sweat contain 50 mmol/L of sodium and 5
mmol/L of potassium
Centrifuge Chemistry Panel Test  ↑ Na: Excess water loss, Conn’s Disease
Comprehensive— Glucose, BUN, Creatinine, (Hyperaldosteronism)
Na+, K+, CO2, Cl-, AST, ALT, Total Protein,  ↓Na: Diuretic use, Renal failure, SIADH,
Albumin, Bilirubin, Ca, and ALP  Aldosterone deficiency
Hepatic- ALP, ALT, AST, Bilirubin (Total and  ↑K: Acidosis, Acute or Chronic Renal
Direct), Total Protein, Albumin/Globulin (TPAG)  failure, Muscular injury, 
Renal- Glucose, BUN, Creatinine, CO2, Cl-, Na+,  ↓K: GI and renal loss, alkalosis
K+, Total Protein, Albumin, Ca, Phosphorous   ↑Cl: RTA, Salicylate intoxication,
Lipid— Cholesterol, Triglycerides, HDL, LDL, Metabolic acidosis, prolonged diarrhea 
VLDL  ↓Cl: Prolong vomit, aldosterone
deficiency, metabolic alkalosis
Tests Performed in the Chemistry Section
 Alanine Aminotransferase (ALT) ↑↑↑ levels Albumin ↓↓ -levels indicate liver or kidney
indicate liver disorders  disorders or malnutrition 
Alkaline Phosphatase (ALP) ↑↑↑ levels Total Protein (TP)- Decreased levels indicate
indicate bone or liver disorders  liver or kidney disorders 
Amylase ↑↑↑ levels indicate pancreatitis  Ammonia- Elevated levels indicate severe liver
Aspartate Aminotransferase (AST) ↑↑↑ levels disorders 
indicate MI or liver disorders  Bilirubin- Elevated levels indicate liver or
Creatine Kinase (CK) ↑↑↑ levels indicate MI hemolytic disorders
or other muscle damage  Blood Urea Nitrogen (BUN)- Elevated levels
Gamma-Glutamyl Transferase (GGT) ↑↑↑ indicate kidney disorders 
levels indicate early liver disorders Creatinine -Elevated levels indicate kidney
Lactic Dehydrogenase (LD [LDH]) ↑↑↑ levels disorders
indicate MI or lung or liver disorders  Creatinine Clearance- Urine and serum test to
Lipase ↑↑↑ levels indicate pancreatitis  measure glomerular filtration rate 
Tests Performed in the Chemistry Section Uric Acid-Elevated levels indicate kidney
Calcium (Ca) Mineral associated with bone, disorders or gout 
musculoskeletal, or endocrine disorders
Chloride (Cl) Maintain proper blood volume, Glucose- Elevated levels indicate diabetes
blood pressure and pH mellitus
MEDICAL LABORATORY SCIENCE PRACTICE I

Glucose Tolerance Test (GTT)- Detects diabetes 1. Urine is a readily available and easily
mellitus or hypoglycemia  collected specimen.
Hemoglobin A1C- Monitors diabetes mellitus 2. Urine contains information, which can be
Cholesterol, TAG, LDL, HDL- Elevated levels obtained by inexpensivelaboratory tests, about
indicate coronary risk  many of the body’s major metabolic functions.
Myoglobin- Early indicator of myocardial   “The testing of urine with procedures
infarction  commonly performed in an expeditious,
Troponin I-Early indicators of myocardial reliable, accurate, safe, and cost-
infarction effective manner.” - the Clinical and
Alcohol- Elevated levels indicate intoxication Laboratory Standards Institute (CLSI)
Arterial Blood Gases (ABGs)-Determine the URINE FORMATION
acidity or alkalinity and oxygen and carbon   The kidney is the only organ with such
dioxide levels of blood.  a noninvasive means by which to
Drug Screening--Detects drug abuse and directly evaluate its status.
monitors therapeutic drugs.  Urine is an ultrafiltrate of plasma
Prostate-specific antigen (PSA)-Screening for  170,00 ml of filtered plasma
prostatic cancer   1,200 ml average urine daily output 

 HISTORY AND IMPORTANCE URINE COMPOSITION

Edwin Smith Surgical Papyrus
 study of urine can be found in the
drawings of  cavemen and in Egyptian
hieroglyphics
Early physicians
  basic observations as color, turbidity,
odor, volume, viscosity, and even
sweetness (by noting that certain
specimens attracted ants).
Hippocrates- wrote a book on “uroscopy.” (5th
century BC)
1140 AD- color charts had been developed that
described, the significance of 20 different colors
17th century- invention of microscope
examination of urinary sediment
Thomas Addis
• developed methods for quantitating the
microscopic sediment.
Richard Bright
• introduced the concept of urinalysis as part of
a doctor’s routine patient examination in 1827.   causes: vomiting, diarrhea, perspiration, severe
1930s- the number and complexity of the tests
performed in a urinalysis had reached a point of
impracticality, and urinalysis began to disappear
from routine examinations.
TWO UNIQUE CHARACTERISTICS OF URINE
SPECIMEN
Normal 
  a. 95% water
  b. 5% solutes
 Solute variations: diet, activity, metabolism,
endocrine, body position
Major organic solute is urea (protein, amino
acid breakdown); also creatinine and uric acid
 Urea and creatinine identify a fluid as
urine
 URINE VOLUME- Determined by body’s state of
hydration
  Influenced by fluid intake, nonrenal
fluid loss, antidiuretic hormone (ADH)
variations, excretion of large amounts
of dissolved solids (e.g., glucose)
Usual daily volume: 1200-1500 mL
Normal range: 600-2000 mL
Anuria- cessation of urine flow
  Severe kidney damage, decreased renal
blood flow
Oliguria- decrease in urine output
burns
  < 1ml/kg/hr → Infants
 < 0.5 ml/kg/hr → Children
  < 400 ml/day → Adults
Nocturia- increased urine excretion at night
 normally 2-3 times more excretion in
the day
MEDICAL LABORATORY SCIENCE PRACTICE I

Polyuria- Increase in daily urine volume   Refrigerate if testing is delayed

 Associated with Diabetes mellitus and   Most problems are caused by bacterial
Diabetes insipidus, artificially induced multiplication
by diuretics, caffeine and alcohol CHANGES IN UNPRESERVED URINE 
a. >2.5 L/ day → Adults Color (Modified/Darkened) - Oxidation or
b. 2.5- 3 mL/kg/day → Children reduction of metabolites
Clarity (Decreased) -Bacterial growth and
Polyuria in Diabetes mellitus vs Diabetes precipitation of amorphous material
insipidus Odor (Increase)- Multiplication of bacteria or
bacterial breakdown of urea to ammonia
SPECIMEN COLLECTION AND HANDLING pH (Increased)- Breakdown of urea to ammonia
Specimen container Recommended: by urease-producing bacteria/loss of CO2
 Disposable Glucose (Decreased) - Glycolysis and bacterial
 Wide mouthed utilization
 Flat bottom Ketones (Decreased)- Volatilization and
 Screw caps bacterial metabolism
 at least 50 ml capacity Bilirubin (Decreased)- Exposure to light/photo
For 24 hour specimen:  oxidation to biliverdin 
 Large plastic container Urobilinogen (Decreased)- Oxidation to urobilin
WHEN WORKING WITH URINESPECIMEN Nitrite (Increased)- Multiplication of nitrate-
LABELLING reducing bacteria
Information on label Specimen should bear the Blood cells & casts (Decreased) -Disintegration
following information: in dilute alkaline urine
✓ Patient’s name Bacteria (Increased)- Multiplication
✓ Age SPECIMEN PRESERVATION
✓ Patient’s ID number Ideal preservative is: 
   Bactericidal
✓ Date and Time of collection
   inhibits urease
✓ Patient’s location/ward
  preserves formed elements
✓ Attending physician
   Does not interfere with chemical
Reminder:  testing
PLACE LABEL ON CONTAINER NOT ON LID
Routine is refrigeration; this is a must for
REQUISITION FORM culture specimens
Must accompany the specimen
 Causes precipitation of amorphous
✓ Information must match label crystals
✓ Time of receipt is stamped on requisition  Must return to room temperature
✓ Other information: type of specimen, for chemical testing
interfering medications   Commercial transport tubes are
available but they must be compatible
SPECIMEN REJECTION with tests
 Unlabeled containers  
  Non-matching labels and requisitions TYPES OF SPECIMEN
 Contaminated specimens: feces, paper   The composition of urine depends on
 Insufficient quantity  the patient’s metabolic state and the
 Delayed or improper transport: ice,  timing and procedure used for
refrigeration collection
SPECIMEN INTEGRITY
 Test within 2 hours of collection 
MEDICAL LABORATORY SCIENCE PRACTICE I
  Patients must be instructed when
special collection techniques are
required
Random Specimen
  Most common type received
 Routine screening for obvious
abnormalities
  May be collected at any time
  Dietary intake and activity may alter
results
 Patients may have to collect a follow-up
specimen  for substances with consistent levels
First Morning Specimen
  Ideal screening specimen
  More concentrated than a random
specimen
  Patient is in a basal state
 Use for orthostatic protein confirmation
and urine pregnancy tests
  Patient collects immediately on arising,
delivers to lab within 2 hours
Fasting Specimen
 Also known as the 8 hour specimen
  actually is second specimen voided –
collected after the first morning
specimen
  Does not contain metabolites from
evening meal
  Recommended for glucose monitoring
 2 hour Postprandial Specimen
 Monitors insulin therapy
 Results can be compared with fasting
urine specimen and blood test results
PROCEDURE:
1. Patient voids before eating routine meal
2. Eats meal
3. Collects next specimen 2 hours after finishing
meal
Glucose Tolerance Specimen
  Institutional option for collection with
blood glucose tolerance test – not
frequently done
  Specimens are collected at the same
intervals as the blood samples
  Used to correlate renal threshold with
patient’s ability to metabolize glucose
Timed Specimen
  Required for quantitative results
  24-hour specimens are needed for
measuring substances with diurnal
variation (results differ in a.m. and p.m.)
and substances that vary with meals,
activity, and body metabolism
  Shorter timed specimens can be used
  Accurate timing is critical for accurate
results
 Calculation for units per 24 hours
includes the volume in milliliters of
urine collected
PRINCIPLE: collection must begin and end with
an empty bladder.
Example (Timed Specimen)
7 AM -patient voids and discards urine
  Patient begins collecting urine 
 7 AM second day – patient voids and
adds this urine to the specimen
container
 Handling of Timed Specimen
  Thoroughly mix specimen and measure
  Save a large enough aliquot to test, and
repeat test if necessary
 Keep specimen on ice or refrigerated
during collection
  Use appropriate and nontoxic
preservatives
  Review instructions with patient
Catheterized Specimens
 Sterile specimen collected from bladder
with a catheter
  Most common test is culture and
sensitivity
  CULTURE FIRST BEFORE PERFORMING
ROUTINE URINALYSIS
 IMPORTANT:
  CULTURE FIRST BEFORE
PERFORMING  ROUTINE URINALYSIS
Midstream Clean-catch Specimen
 Alternative to catheterized specimen
MEDICAL LABORATORY SCIENCE PRACTICE I
  Less contaminated than routine
collection
Provide patient with mild cleansing material
and container and instructions:
1. Wash hands
2. Clean genitalia with supplied cleanser
3. Void into toilet, then into container, and
finish into toilet
4. Do not touch or contaminate inside of    Bags with tubes to a larger container
container
Suprapubic Aspiration
 Completely free of contamination for
culture and cytology
 External needle aspiration from the
bladder
  Possible pediatric specimen  until the time of receipt of laboratory
Prostatitis Specimen
 • Collection similar to midstream clean-
catch
 • 3-glass collection:
Container 1 – first urine passed
Container 2 – midstream urine
Massage prostate to obtain prostatic fluid
Container 3 – remaining urine and fluid
Quantitative cultures on all 3 specimens
  Examine 1 and 3 microscopically for
WBCs
 Stamey-Mears: 4 glass collection
 Initial voided (VB1)
 Midstream (VB2)
 Massaged prostate excretions (EPS)
 Post-massage urine (VB3)
Cultures on all specimens
a. VB1 and VB2 positive: urinary infection
b. Negative cultures on VB1 and VB2
and positive on EPS and VB3 show prostatitis
 EPS examined for WBCs >10-20/ hpf:
abnormal
Pre and Post Massage Test
1. Specimen 1 – midstream clean- catch
specimen
2. Specimen 2 – post-massage specimen
 Prostatitis is indicated by a
quantitative culture result in the second
glass that is 10 times higher than
specimen 1
Pediatric Specimen
 Wee bag: Soft, clear plastic bags, with 
hypoallergenic tape applied to genital area
   Monitor bag frequently
  Clean-catch method with sterile bag
can be used
are available for timed specimens
 Drug Specimen Collection
 Proper collection, labeling, handling
must be documented
  Chain of custody – documentation
from the time of specimen collection
results; standardized form always
accompanies specimen
  Specimen must withstand legal scrutiny
POINTS TO CONSIDER
 Photo ID of urine donor or ID by
employer
  No unauthorized access to specimen
 No adulteration, substitution, or
dilution of specimen
ADULTERATION TEST
  Temperature taken within 4 min must
be 32.5-37.7°C
  Report temperatures outside of range
immediately. Collect another specimen
ASAP
 Inspect urine color for anything unusual
 Follow laboratory instructions for
labeling, packaging, and transport
EXAMINATION PERFORMED PHYSIOLOGY
  Feces are the end product of digestion
  Final digestion occurs in the small
intestine aided by pancreatic enzymes
and bile salts
 Majority of fluids involved in
digestion are reabsorbed, with only
about 150 mL excreted in feces
 Excess water reaching large
intestine:diarrhea 
MEDICAL LABORATORY SCIENCE PRACTICE I
DIARRHEA
 Definition: >200 g stool weight per day with
increased liquid and > 3 movements per day
  Mechanisms of diarrhea:
secretory,osmotic, altered motility
  Laboratory tests: fecal sodium,
potassium, osmolarity, and pH
✓ pH <5.6 indicates sugar malabsorption
✓ Sodium, potassium, osmolarity used to
calculate fecal osmotic gap
MECHANISM OF DIARRHEA
Secretory Diarrhea
 Microbial infections increase secretion
of water and electrolytes
✓ E. coli, Clostridium, Vibrio cholerae,
Salmonella, Shigella,
Staphylococcus, Campylobacter,
Cryptosporidium
 Others include: Drugs,
laxatives, inflammatory bowel
disease/colitis, endocrine disorders,
malignancy, collagen vascular disease
Osmotic Diarrhea
  Incomplete digestion or
reabsorption of food increases water
retention in large intestine
Malnutrition: impaired reabsorption
Maldigestion: impaired digestion of foods
  Others include: Lactose
intolerance, celiac sprue
(malabsorption), amebiasis, antibiotics,
laxatives, antacids
Altered Motility
  Irritable bowel syndrome:
hypermotility and constipation; food,
chemicals, stress, and exercise are
causes Rapid gastric emptying
(dumping syndrome) divided into early
and late dumping syndrome based on
timing of symptoms (30 minutes to 2
hours)
  Others include: Gastrectomy,
gastric bypass, post-vagotomy,
duodenal ulcer, diabetes mellitus
SPECIMEN COLLECTION
 Patients need detailed instructions
 Pea sized specimen for routine
stool examination
 Use clean container and transfer
to laboratory container
  No toilet water contamination
 Ova and parasite containers are used
only for that purpose
  Quantitative collections are 72 hours
✓ 72 hours: time required to pass through
intestine
PHASES OF STOOL EXAMINATION
 Color
  Consistency
 Other notations upon seeing
stool specimen: nematodes, cestodes,
trematodes, etc.
Macroscopic Examination
  Fecal Leukocytes
 >3 Neutrophils/hpf is significant
 Fecal RBC
 Muscle fibers
  Fecal fats
  Parasites, Bacteria
Microscopic Examination
Introduction to Microbiology
MICROBIOLOGY
➢ Study of organisms that are too small to be
seen by the naked eye.
➢ PURPOSE: responsible for the identification
of pathogenic microorganisms and for hospital
infection control. 
HISTORY OF MICROBIOLOGY
LUCRETIUS & GIROLAMO FRACASTORO
  Suggested that diseases were caused
by “INVISIBLE LIVING CREATURES”
ANTON VAN LEEUWENHOEK- “ FIRST TRUE
MICROBIOLOGIST”
 First person to observe and accurately
describe living microorganisms (such as
bacteria and parasite)
 “ANIMALCULES”
LOUIS PASTEUR
MEDICAL LABORATORY SCIENCE PRACTICE I

 Proposed the use of heat in killing  2. Growth requires from several days to several
microorganisms (aseptic technique) weeks. 
IGNAZ SEMMELWEIS  3. Cultures should be maintained in a high-
  “ FATHER OF HANDWASHING humidity environment. 
PROCEDURE” 4. Several techniques are used to obtain culture
JOSEPH LISTER material for slide preparation. 
  Introduced the system of antiseptic  Tease mount method 
surgery.  Cellophane tape method 
 Used PHENOL as anti-microbial agent   The slide culture method uses a block of agar
overlaid with a cover slip. 
GERM THEORY OF DISEASE 
ROBERT KOCH’S POSTULATES BODY SITES AND POSSIBLE FUNGAL
✓The microorganism must be present in every PATHOGENS
case of the disease but absent from a healthy Blood- Candida spp.,Cryptococcus neoformans
host. Cerebrospinal Fluid- Cryptococcus neoformans
✓The suspected microorganism must be Hair, Nail, Skin- Microsporum
isolated from a diseased host and grown in a Epidermophyton,Trichophyton 
pure culture. Lungs-Candida albicans, Aspergillus Histoplasma
✓The same disease must be present when the capsulatum
isolated microorganism is inoculated into a Urine/Genital Tract- Candida albicans 
healthy host.
✓The same organism must be isolated again VIROLOGY- Viruses must be cultured in living
from the diseased host.  cells, and most laboratories send viral
specimens for culturing to specialized reference
MICROBIOLOGY laboratories. 
In large laboratories, the section may be divided Specimen Processing for Diagnosing Viral
into Bacteriology, Mycology,Parasitology, and Diseases 
Virology.  1. Samples should generally come from the
 The microbiology section is responsible infected site. 
for the identification of pathogenic  Skin: Rash site and, depending on the
microorganisms and for hospital virus, serum and urine 
infection control. Sample Collection and   Respiratory: Sputum or throat swabs 
Handling   CNS (e.g., meningitis and encephalitis):
For diagnosis of meningitis,
Phlebotomists are responsible for collecting cerebrospinal fluid (CSF) and serum, as
blood cultures and may be required to obtain well as stool or throat swabs, can be
throat cultures (TCs) and instruct patients in the collected because viruses are
procedure for collecting urine samples for sometimes shed into these sites.
 Urogenital: Needle aspirates and
culture. Specific sterile techniques must be
observed in the collection of culture samples to endocervical and urethral swabs 
  Gastrointestinal tract: Stool samples
prevent bacterial contamination. 
MYCOLOGY- Fungi are identified primarily by and rectal swabs 
  Eye infections: Eye swabs and corneal
culture growth and microscopic morphology. 
scrapings 
Mycology Culture Considerations: 
1. Fungal cultures are incubated at 30°C.  Sample Transport 
 Samples for viral culture must be placed
into a viral transport medium (VTM). 
MEDICAL LABORATORY SCIENCE PRACTICE I

VTM contains:  Slides are made from infected cell

 (a) Buffered saline cultures and examined for cellular
 (b) Protein stabilizers  changes, including clumping, vacuoles,
 (c). Antimicrobials that inhibit bacterial inclusions, granules, cell fusion (i.e.,
and fungal growth  syncytium—multinucleated cell
*Samples for viral cultures can be refrigerated development), and cellular destruction.
in VTM for about 48 hours, but they should These visible changes are referred to
never be frozen at -20°C.  as cytopathic effect (CPE). 
Samples can be stored at -70°C; however, Viral Isolation
infectivity will be  diminished.   Embryonated Eggs (Growth of Virus) 
 Animal Models (used in research
BODY SITES AND POSSIBLE VIRAL AGENTS studies).
Respiratory System-Influenza Virus,   Electron Microscopy (Due to size, most
Parainfluenza Virus, Respiratory Syncytial Virus individual virions can only be seen by
(RSV), Epstein-barrVirus (EBV), Coronavirus, electron microscopy).
Adenovirus  Viral gene probes and nucleic acid
Viral Meningitis- Enterovirus, Echovirus, Herpes amplification (Polymerase Chain
Simplex Virus Type 1 (HSV-1), HSV-2 Reaction [PCR]).
Cutaneous Infections- HSV-1, HSV-2, VZV,  Detection of viral antigen and host
echovirus, measles virus, rubella virus antibody against specific virus. 
Genital Infections- HSV-2 and human BACTERIOLOGY- Identification of bacteria is
papillomaviru Gastroenteritis Rotaviruses, based on morphology, Gram stain reactions,
Norwalk viruses, Adenoviruses oxygen and nutritional requirements, and
Eye Infections- HSV, adenovirus biochemical reactions. 
Neonatal Infections- HSV, CMV, and Rubella General Guidelines for Specimen Collection:
Virus    Specimens should be collected during
the acute or early phases of an illness.
VIRAL IDENTIFICATION:  The correct anatomic site must be
 Histology and Cytology  identified/selected for collection of the
1. Cellular inclusions are diagnostic for many specimen.
viruses.    The site of the infection should be
2. Most DNA viruses replicate in the nucleus, cleansed properly before collection.
they often produce nuclear inclusions.   The quantity of the collected specimen
3. RNA viruses produce cytoplasmic inclusions should be sufficient for diagnostic
(assembled in the cytoplasm). testing.
4. HSV and VZV cause intra-nuclear inclusions.   If possible, specimens should be
5. CMV induces enlarged (cytomegalic) cells collected before antibiotics are
with a basophilic intranuclear inclusion referred administered.
to as "owl eye" inclusion.    Swabs are generally poor specimens
(Except nares and throat
Viral Isolation specimens) compared with tissues or
1. Cell Culture  needle aspirates. 
a. Primary Cell Cultures (Monkey Kidney Cells)     For lesions, wounds and abscesses, the
b. Established Cell Lines (WI-38, MRC-5, IMR- specimen is collected from the
90).  advancing  margin of the lesion
c. Continuous Cell Lines (HeLa, HEp-2, A549, preferably by needle aspiration rather
Vero cells).  than by swabs.
MEDICAL LABORATORY SCIENCE PRACTICE I
   Aspirated specimens must be placed
into a sterile tube or transport vial and
not  squirted onto a swab.
   For patient-collected specimen, clear
instructions should be given by
an authorized healthcare personnel,
and it should be never be assumed that
the  patients knows how to collect a
specimen.
  The specimen must be labelled
accurately with the specific anatomic
site and the patient demographic
profile.
Specimen Container:
1. Specimens for microbiology cultures should
be in sterile containers.
  Stool specimen can be collected IN
CLEAN, NON-STERILE CONTAINER.
2. Swabs are used for specimens from the upper
respiratory tract, external ear, eye and genital
tract.
  Swabs are not recommended for
routine collection.
  If swabs are used, Dacron or calcium
alginate swabs are preferred rather
than cotton-tipped ones.
Specimens Used for Microbiologic Study:
 Most microbiology samples are obtained from
the:  procedure.
 Blood
 Urine
 Throat
 Sputum
 Genitourinary tract  the specimen collection.
 Wounds
 Cerebrospinal fluid
 Feces  the symphysis pubis through the
Blood
  The venipuncture site should be
disinfected with 70% alcohol and
betadine.
  To ensure a complete antisepsis,
betadine should be in contact with the
skin for 30 to 60 seconds.
 Blood should be drawn during the time
of febrile episode.
  For the manner of collection, two sets
should be drawn, from the same drawn.
 No more than three sets should be
drawn in a 24-hour period.
  Aerobic and anaerobic culture bottles
are used. 
Urine (Clean-catch voided Midstream/ Clean-
voided Specimen or CVS)
  The first morning urine is also
preferred because its provides a more
concentrated specimen.
  A pure culture of E. coli that 105
CFU/ml represents a true infection.
 For females, the area should be cleaned
first soap and water, and the labia
should be held apart to begin voiding.
  For males, the glands should be
cleaned with soap and water; the
foreskin should be retracted if
necessary. 
Urine (Straight Catheter)
  After inserting the catheter into the
bladder, the first 15 ml of the urine
should be voided or expelled by the
bladder before collecting the ensuing
urinary stream as specimen.
Urine (Indwelling Catheter)
 The catheter collection port should be
cleaned before starting the collection
 5 ml to 10 ml of urine should be
aspirated with needle and syringe.
Urine (Suprapubic Aspirate)
 The skin should be disinfected prior to
  The authorized healthcare personnel
should aspirate using a needle above
abdominal wall into the full bladder. 
Specimens Used for Bacteriologic Study:
Respiratory Tract (LRT: BAL, Bronchial Brush,
Bronchial Wash)
  An anaerobic culture is appropriate
Respiratory Tract (Sputum)
  Prior to the collection, the patient
should rinse his/her mouth w/ sterile
H2O.
MEDICAL LABORATORY SCIENCE PRACTICE I

  A first-morning specimen is preferred   Exudates should be removed before

for an acid-fast bacilli microscopy. specimen collection.
  For mycobacterial infection, two or   The swab must be moistened with
three consecutive early morning Stuart’s or Amies medium.
sputum specimens should be   The secretion from the mucous
submitted.  membrane of the vagina are swabbed.
  The methods of collecting sputum can Genital Tract (Urethra)
be through deep cough (expectorated   A flexible swab should be inserted two
sputum), or aerosol-induced (induced to four centimeters into the urethra
sputum) for pediatric and and 
uncooperative patients.  rotated for two seconds or
alternatively, some discharge can be
Respiratory Tract (Nasopharyngeal Specimen) collected on a JEMBEC transport
  It is the specimen of choice for the system.
recovery of Bordetella pertussis.   The swab must be moistened with
  For the procedure, a flexible swab is Stuart’s or Amies medium.
inserted through the nose into the Genital Tract (Prostate Glands)
posterior nasopharynx and rotated for   The gland should be cleaned with soap
five seconds. and water.
Respiratory Tract (Pharyngeal/Throat   The swab must be moistened with
Specimen) Stuart’s or Amies medium.
 It is the recommended specimen for the   The secretion on the swab or in the
routine culture of group A streptococci. tube should be collected. 
  For the procedure, the posterior Abscess (Lesions, Wound, Pustule, Ulcer)
pharynx and tonsil should be without   It is preferable to use a needle and
touching the roof and sides of the syringe in collecting specimens to
mouth and tongue.  aspirate the drainage.
Genital Tract (Bartholin Cyst)   Needle aspiration enhances the
 The skin should be disinfected before recovery of anaerobic bacteria.
collecting specimen.  There are two types of abscess:
  The specimen should be aspirated.   (1) Superficial Abscess
Genital Tract(Cervix)  (2) Deep Abscess 
  The mucus should be removed before Body Fluid (Amniotic, Ascites/peritoneal,
collecting the specimen. Joint/Synovial, Pericardial, and Pleural)
  A lubricant should not be used on the   The skin should be disinfected before
speculum and the cervix should be aspirating the sample (needle
thoroughly swabbed.  aspiration).
Genital Tract (Bartholin Cyst)   Concentrating the specimen through
 The skin should be disinfected before centrifugation or filtration may be
collecting specimen. required prior to smearing and
  The specimen should be aspirated.  cultivation
Genital Tract(Cervix)   The sample should be inoculated as
  The mucus should be removed before soon as it is received. 
collecting the specimen. Cerebrospinal Fluid
  A lubricant should not be used on the   The skin should be disinfected before
speculum and the cervix should be aspirating the sample
thoroughly swabbed.   Rapid diagnostic testing like gram
Genital Tract(Vagina) staining and cryptococcal antigen test is
MEDICAL LABORATORY SCIENCE PRACTICE I
the recommended method for this
specimen.
  The required volume is 10 ml
(microbiology and chemical tests).
  The storage should be six hours at 35
degree Celsius.
  Bacteria associated with meningitis
(Neisseria meningitides, Haemophilus
influenza) are fastidious and susceptible
to cold temperature or drying.
  Gram stain can be performed by
cytocentrifugation.  perform identification and susceptibility
Eye (Conjunctiva)
  Samples should be obtained from both
eyes using two separate swabs.  cultured.
Eye (Corneal Scraping)
  An aerobic swab that is moistened with
Stuart’s or Amies medium or pre-
moistened with sterile saline may be
utilized.
  Samples should be obtained from both
eyes using two separate swabs.  culture media.
Gastrointestinal Tract (Gastric Biopsy)
 The specimen is recommended for the
detection and isolation of
Helicobacter pylori.
Gastrointestinal Tract (Rectal Swab)
  For microscopy, methylene blue is
utilized to observe fecal leukocyte.
  For the collection of specimen, a 2.5-
cm swab is inserted through the anal
sphincter.
Gastrointestinal Tract (Stool Culture)
  For out-patient, three specimens are
collected every other day.
  For in-patients, three specimens are
collected every day.
  If a patient has received anti-parasitic
drugs, the specimen collection should
be done after 7 to 10 days.
  For microscopy, methylene blue is
utilized to observe fecal leukocyte.  the specimen and could kill fastidious
Foreign Bodies (IUD)
  It is usually cultured for the detection
of Actinomyces species.
  For the procedure, the skin is
disinfected before the removal of the
IUD.
 Foreign Bodies (Catheters, Pins,
Prosthetic Valves)
  A segment of the catheter (5 cm to 7
cm) is rolled four times across the agar
using a sterile forceps (Maki roll
technique).
  When using catheters and valves, more
than 15 colonies are required to
tests.
  The whole Foley catheter should not be
 
Specimens Preservation:
  If the transport of the specimen to the
laboratory or its processing is 
 delayed the specimen can be
maintained with the use of
preservatives, holding media or even a
 Stool specimen for Clostridium difficile
toxin assay should be collected without
a preservative and can be refrigerated,
but if the specimen cannot be
processed within 48 hours, it should be
stored at - 70 ̊C.
PRESERVATIVES
  Boric acid maintains the appropriate
colony counts for urine specimens at
room temperature for 24 hours.
  Preservatives should not be added on
fecal specimen.
TRANSPORT/ HOLDING MEDIA
 These media maintain the viability of
the microorganism present in a
specimen but do not allow their
multiplication.
  Charcoal is sometimes added to these
media to absorb fatty acids present in
organism like Neisseria gonorrhoeae
and Bordetella pertussis.
ANTICOAGULANTS
MEDICAL LABORATORY SCIENCE PRACTICE I
  These are used to prevent clotting of
specimen including blood, bone  24 hours at room temperature
 marrow and synovial fluid.
  0.025% sodium polyanethol sulfonate
(SPS) is used for a blood culture media
because Neisseria species and some
anaerobic bacteria    URT specimen
 are sensitive to higher concentration.
  Synovial and peritoneal fluids can also
be inoculated into a blood culture broth
media.
 The reasons for using 0.025% SPS as the
preferred coagulant for blood  culture are as
follows:
 to inhibit phagocytosis and complement
activation
 to neutralize antibiotics
 to neutralize the bactericidal effect of
plasma
Specimens Storage:
Refrigerator Temp. (4 ̊C)
 Urine, Stool, Sputa, Swabs, Viral
Specimen
  Anaerobic bacteria should never
be stored in ref. temp.
  Stool isolation of C. difficile toxin can
be stored up to 3 days.
Incubator Temp. (35 ̊C)
  CSF
  Recommended preservation temp.
for samples intended for gram staining,
culture and sensitivity.
Ambient Room Temp(22 to 25 ̊C)  maintained as much as possible to its
  Anaerobic culture, sterile body fluid,
genital specimen, eye and ear swab.
Freezer Temp. (-20 to -70 ̊C)
  Sera for serological studies
  Tissues or specimen that are to be
stored for a long time.
Storage Requirements Prior to Specimen
Process
Plate as soon as possible
 Body fluid
 Bone specimen
 Gastric biopsy
  Suprapubic aspirate of urine
 Prostatic specimen
  Abscess
  Outer ear specimens
  Eye and conjunctiva specimen
  Genital tract specimen
  Tissue
Indefinitely at RT
  Hair, nail,skin scraping
Immediately at 35 ̊C
  Blood
  Bone marrow
  Cornel scraping
Six hours at 35 ̊C 
  CSF (although, immediate processing is 
recommended)
24 hours at 4 ̊C 
  LRT specimen
  Clean-voided midstream urine
  Straight catheter urine
  Indwelling catheter urine
  Foreign devices
72 hours at 4 ̊C
  Rectal swab
  Stool culture
Specimens Transport:
1. Ideally specimen should be transported to
the laboratory immediately and preferably
within 30 minutes of collection and not longer
than two hours.
  Specimens should be transported and
original state when it was collected.
 For anaerobic bacteria, the transport
should not take more than 10 minutes.
 For CSF samples, the transport should
be done immediately and preferably
within 15 minutes after collection.
 Change in oxygen, pH, and temperature
caused by prolonged transport can
prevent the recovery of certain
microorganism and allow the
overgrowth of other.
All specimen container should be leak-proof. All
specimen should be transported to the
laboratory immediately after collection. 
MEDICAL LABORATORY SCIENCE PRACTICE I
  Any delay may decrease the number of
pathogens and hasten the
multiplication of indigenous microbiota.
 All specimen containers should be
placed in sealable, leak proof plastic
bags.
  Patients specimen or culture isolates
must be triple-packed before being
shipped.
  Specimen bags should be marked with
a biohazard label.
Transport Requirements of Specimens
for Microbiological Tests
Immediately at room temperature
 Body fluids Bone
 Inner ear Corneal scrapping
 Foreign bodies Gastric aspirate
 Suprapubic aspirate of urine
 Prostatic sample
W/in 15 minutes at room temperature- CSF
Within 2 hours at room temperature-/Blood or
bone marrow
Immediately at 4 ̊C- Gastric Biopsy
Within 2 hours at 4 ̊C-
Indwelling/straight catheter (Urine sample)
within 24 hours at room temperature
 Abscess
 Outer Ear
 Conjunctiva Specimen  7. The specimen has been reserved for
 Genital tract specimen (JEMBEC)  anaerobic culture from a site known to have
 Hair, Nail, Skin Scrapings
 Respiratory and Tissue specimen  (vagina and mouth).
Within 24 hours at 4 ̊C
 Rectal swab
 Stool culture
 Clean voided midstream urine  10. The specimen being processed has a
Specimens Prioritization:
1 - Critical/Invasive Amniotic fluid, blood, brain,
CSF, heart valves, pericardial fluid
2 - Unpreserved Bones, feces, sputum,
other body fluids not listed under level 1
3-  Quantitation required- Catheter tip, urine,
tissue
4-  Preserved- Urine, feces, and swab in holding
media
NOTE:
 When specimen is received with
multiple requests but the amount of
specimen is insufficient, the clinician
should inform the laboratory staff
which of the tests should be prioritized.
  When multiple specimens arrive at the
same time, priority should be given to
those that are most critical such as CSF,
tissue, blood and sterile fluids.
  Urine, throat, sputa, stool or wound
drainage are specimens that can
be stored and processed later.
 AFB, viral, fungal specimens can be
processed by batch.
Specimens Rejection:
1. The information on the label does not match
the information on the requisition slip.
2. The specimen is transported at an improper
temperature.
3. The transported specimen has not been
placed in the proper medium.
4. The quantity of specimen is not sufficient
(QNS) for testing.
5. The transport of specimen exceeds the two
hours’ post-collection period.
6. The specimen is preserved in a fixative
(formalin).
aerobes as part of the indigenous microbiota
8. The specimen is leaking.
9. The specimen has already dried up upon
transporting to the laboratory.
questionable medical value.
 More than one specimen from the
same source (except for blood) and
from the same patient is submitted on
the same day, A single swab is
submitted with multiple request for
various organism.
 Expectorated sputum in which the gram
stain reveals <25 WBC and > 10
Epithelial Cells. 
MEDICAL LABORATORY SCIENCE PRACTICE I

Critical Panic Results in a Clinical Microbiology Occult blood- Detects nonvisible blood
1. Positive CSF Gram stain and culture (performed on stool samples) 
2. Gram stain suggestive of C. perfringens (gas Ova and Parasites (O & P)- Detects parasitic
gangrene) infection (performed on stool samples) 
3. Positive AFB stain
4. Positive blood culture ANATOMICAL PATHOLOGY 
5. Presence of Streptococcus pyogenes in a  a medical specialty that is concerned
surgical wound with the diagnosis of disease based on
6. Positive antibiotic-resistant bacteria the macroscopic, microscopic,
7. Positive for Legionella, Francisella, and biochemical, immunologic and
Brucella molecular examination of organs and
Gram Stain tissues.
  It is the most commonly used HISTOPATHOLOGY-compound of three Greek
differential stain in the clinical words: 
microbiology laboratory.  histos "tissue"
  Reagent: Crystal Violet, Iodine,  pathos "suffering", 
Acetone-alcohol, Safranin   logia "study of"
Principle:  the microscopic examination of tissue
 Bacteria with thick cell wall containing in order to study the manifestations of
teichoic acid retain the crystal violet- disease.
iodine complex dye after decolorization   refers to the examination of a biopsy
and appear purple, which mean that or surgical specimen by a pathologist,
they are Gram (+). Other bacteria with after the specimen has been processed
thinner cell walls contain and histological sections have been
lipopolysaccharides do not retain the placed onto glass slides. 
dye complex and appear deep pink or SPECIMEN COLLECTION-  commonly performed
red, which means that they are Gram by a surgeon, 
(-).  interventional radiologist, or an interventional
 CULTURE AND SENSITIVITY  cardiologist involving extraction of sample cells
 (C & S) TEST is the primary procedure or tissues.
performed in microbiology.  Physicians gather the specimen from
  It is used to detect and identify the  patient using variety of ways this
microorganisms and to determine the would  include:
most effective antibiotic therapy.   Biopsy
  Results are available within 2 days for  Surgical operation 
most bacteria. 
Tests Performed in the Microbiology Section  BIOPSY 
Acid-fast bacillus (AFB)- Detects acid-fast Types of biopsy:
bacteria, culture including Mycobacterium a. Excisional biopsy: when an entire lump
tuberculosis  or suspicious area is removed.
Blood culture- Detects bacteria and fungi in the b. Incisional biopsy: samples a portion of the
blood  abnormal tissue without attempting to remove
Fungal culture- Detects the presence of and the entire lesion or tumor.
determines the type of fungi  c. Needle aspiration biopsy: when a sample of
Gram stain- Detects the presence of and aids in tissue or fluid is removed with a needle in such
the identification of bacteria  a way that cells are removed without preserving
KOH direct mount technique- For examination the histological architecture of the tissue cells.
of fungal elements (Hypha, Conidia, etc.).  SURGICAL
MEDICAL LABORATORY SCIENCE PRACTICE I

 uses operative manual and instrumental  Patient

techniques on a patient to investigate  Laboratory file
or treat a pathological condition such as RETENTION TIME:
a  disease or injury by removing the  Tissue blocks (paraffin): 3 to 10 years
whole or portion of the target  Pathology slides: indefinite
organ/tissue.   Surgical pathology report: 10 years 
CYTOPATHOLOGY-from Greek words:
SPECIMEN PROCESSING  kytos, "a hollow“
 FIXATION   pathos, "suffering“
 DEHYDRATION  logia, "study of"
  CLEARING / DEALCOHOLIZATION -a branch of pathology that studies
 INFILTRATION / IMPREGNATION and diagnose diseases on the cellular level
 TRIMMING  - discipline was founded by George Nicolas
 EMBEDDING  Papanicolaou in 1928.
 SECTIONING / MICROTOMY -generally used on samples of free cells or tissue
 STAINING fragments aid in the diagnosis of cancer but also
  MOUNTING in the diagnosis of some infectious diseases and
   LABELLING other inflammatory conditions. 
 TISSUE PROCESSING STEPS 
SPECIMEN COLLECTION
SPECIMEN RECEIVING There are two methods of collecting cells  for
  S (surgical), A (autopsy), C (cytology) cytopathologic analysis:
 Specimen should be accompanied with a. Exfoliative cytology: desquamated cells
a requisition form duly filled up by the i. Spontaneous exfoliation- cells are collected
physician after they have been either spontaneously shed
  Check the specimen if fixed/unfixed. by the body.
 Each specimen would be given a unique ii. Mechanical exfoliation- cells are manually
accession code. scraped/brushed off of a surface in the body. 
RETENTION TIME: b. Intervention cytology: intervenes into
  Specimen (tissue): 1 month to 1 year the body for sample collection.
 Requisition form: 2 years  i. Fine needle aspiration cytology- involves use
of a needle (23-27 gauge) attached to a syringe
Requisition form:  is to collect cells from lesions or masses in
  Demographic of the patient various body organs by microcoring, often with
  Date and time of collection the application of negative pressure (suction) to
 Underlying disorder increase yield. 
 Current clinical details ii. Sediment cytology- the sample is collected
 Specimen type and mixed with a fixative taken into a centrifuge
  Manner by which specimen is obtained tube and is centrifuged. 
  Site of origin where the specimen is  The sediment is used for smearing.
obtained 
REPORT/RESULT Methods of smear preparation
 findings made by the pathologist  Streaking
through series of examination  Spreading
performed on the submitted specimen.  Touch preparation / impression
Turnover of results: 24 hours*  Pull apart 
 3 copies SPECIMEN RECEIVING & REPORT
 Doctor RETENTION TIME:
MEDICAL LABORATORY SCIENCE PRACTICE I

Specimen (serum and other body fluids): 48   The term is also used for the complete
hours set of chromosomes in a species or in
 Requisition form: 2 years an individual organism and for a test
Cell blocks (paraffin): 3 to 10 years that detects this complement or
Pathology slides: indefinite measures the number.
Turnover of results: 24 hours*  Cells from bone marrow, blood,
Three copies: amniotic fluid, cord blood, tumor, and
 a. Doctor tissues (including skin, umbilical cord,
 b. Patient chorionic villi, liver and many other
 c. Laboratory file  organ) 
CYTOLOGY PROCESSING STEPS  
 FIXATION CYTOGENETICS PROCESSING STEPS
  STAINING  1. Using cells in culture
  MOUNTING 2. Pre-treating cells in a hypotonic solution,
 LABELLING which swells them and spreads the
chromosomes
CYTOGENETICS-a branch of genetics that is 3. Arresting mitosis in metaphase by a solution
concerned  of colchicine
with how the chromosomes relate to cell 4. Squashing the preparation on the slide
behavior, particularly to their behavior during forcing the chromosomes into a single plane
mitosis and meiosis. 5. Cutting up a photomicrograph and arranging
the result into an indisputable karyogram
HISTORY: RETENTION TIME:
1. Karl Wilhelm von Nägeli: first observed   Cytogenetic slides: 3 years
 chromosomes of plant cells in 1842.  Cytogenetics diagnostic images: 20
2. Walther Flemming: discovered mitosis. years
3. Heinrich Wilhelm Gottfried von Waldeyer:  Cytogenetics reports: 20 years 
coined the term chromosomes   
Levitsky: define the karyotype as the
phenotypic appearance of the
somatic chromosomes.
5. Hans von Winiwarter: reported 47 
chromosomes in spermatogonia and 48 in
oogonia.
6. Theophilus Painter: was not certain whether
the diploid number of man was 46 or 48 and he
correctly insisted on man having an XX/XY
system.
 It took until 1956 until it
became generally accepted that the
karyotype of man included only 46
chromosomes. 

KARYOTYPING
  A karyotype is the number and
appearance of chromosomes in the
nucleus of a eukaryotic cell.