LABORATORY FUNDAMENTALS

COLLEGE OF MEDICAL LABORATORY SCIENCE

OUR LADY OF FATIMA UNIVERSITY
FUNDAMENTALS OF LABORATORY
Laboratory Safety Rules
Specimen Collection and Considerations
Quality Management
LABORATORY SAFETY RULES
GENERAL LABORATORY SAFETY
RULES
 LABORATORY SAFETY

• Recognition of hazards
• Application of common sense
• Safety-focused attitude
• Good housekeeping in all laboratory work and storage areas
EMPLOYER’S RESPONSIBILITIES
• Establish laboratory work methods and safety
policies
• Provide supervision and guidance to employees
• Provide safety information, training, personal
protective equipment, and medical surveillance to
employees
• Provide and maintain equipment and laboratory
facilities that are adequate for the tasks required
 EMPLOYEE’S RESPONSIBILITIES
• Know and comply with the established laboratory work safety
methods.
• Have a positive attitude toward supervisors, co-workers, facilities, and
safety training.
• Give prompt notification of unsafe conditions or practices to the
immediate supervisor and ensure that unsafe conditions and practices
are corrected.
• Engage in the conduct of safe work practices and use of personal
protective equipment.
SAFETY EQUIPMENT
• Developed specifically for use in the laboratory
• Safety showers (deliver 30 to 50 gallons of water
per minute at 20 to 50 pounds per square inch
(psi) and be located in areas where corrosive
liquids are stored or used)
• Eyewash stations(within 100 feet or 10 s travel),
fire extinguishers  periodically tested and
inspected
• Others: fire blankets, spill kits, and first aid
supplies.
• Mechanical pipetting device must be used to
manipulate liquids
 BIOLOGICAL HAZARD

 ROT in breaking the chain of infection

 Susceptible – infected host in continuing infection cycle
 Health care setting as a source of potential pathogen
 CDC  universal precautions (1987)
 Blood and body fluid precautions should be consistently used for all patients
 Potentially infectious materials:
 Body fluids
 Semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid,
amniotic fluid, saliva in dental procedures, any body fluid that is visibly contaminated with blood, and all
body fluids
 Unfixed tissues, organs or blood slides
 Hiv-containing cell or tissue cultures, organ cultures, and HIV- or hbv-containing culture medium or other
solutions; and blood, organs, or other tissues from experimental animals infected with HIV or HBV.
 BIOLOGICAL HAZARD
1. All samples and other body fluids should be transported, handled, and processed using
strict precautions
2. Gloves, gowns and face protection must be used if splash or splattering is likely to occur
3. Specimens should be ―capped‖ during centrifugation
4. Eating, drinking, smoking, applying cosmetics, wearing of contact lenses
5. Appropriate signs to identify hazards
6. Any blood, body fluid, or other potentially infectious material spill must be cleaned up
 IN CLEANING POTENTIALLY
INFECTIOUS MATERIAL SPILL, YOU
MUST....
• Wear appropriate protective equipment
• Use mechanical devices to pick-up broken glass or other sharp objects
• Absorb spills with paper towels, gauze pad, or tissue
• Clean the spill site using a common aqueous detergent
• Disinfect the spill site using approved disinfectant or 10% bleach using
appropriate contact time
• Dispose all materials in appropriate biohazard containers
PREPARATION OF DILUTED
HOUSEHOLD BLEACH
 BIOLOGICAL HAZARD
5. OSHA blood-borne pathogens standard requires written ―exposure control plan‖- this plan
must cover specific preventative measures including exposure evaluation, engineering
controls, work practice controls, and administrative oversight of the program.
6. Categories of exposure:
A. Category I - employees are all exposed to a specific hazard as a regular part of their job.
B. Category II - employees who are occasionally exposed to a hazard.
C. Category III - employees have no occupational exposure to a hazard.

7. Biological safety cabinets should be installed to facilitate manipulations of infectious

materials
CHEMICAL SAFETY
• 1. Plan and implement a written hazard communication program.
• 2. Obtain sdss for each hazardous compound present in the workplace and have the
sdss readily accessible to employees.
• 3. Educate all employees annually on how to interpret chemical labels, sdss, and
health hazards of the chemicals and how to work safely with the chemicals.
• 4. Maintain hazard-warning labels on containers received or filled on-site.
CHEMICAL HAZARDS
• CHP must include:
• 1. Criteria for and methods of monitoring chemical exposure
• 2. SOP for handling hazardous chemicals
• 3. Criteria for implementing engineering controls (fume hoods)
• 4. Use of PPE and other hygiene practices
• 5. Special precautions for extremely hazardous chemicals
• 6. Specific measures to ensure that fume hoods and other protective equipment are working
properly
• 7. Provision for employee information and training
• 8. Provision for medical consultation and examination
• 9. Designation of a chemical hygiene officer responsible for implementation of the CHP.
 CHEMICAL SAFETY
MSDS
• Product name and identification.
• Hazardous ingredients.
• Permissible exposure limit (pel).
• Physical and chemical data
• Health hazard data and carcinogenic potential.
• Primary routes of entry
• Fire and explosion hazards.
• Reactivity data
• Spill and disposal procedures.
• Ppe recommendations.
• Handling.
• Emergency and first aid procedures.
• Storage and transportation precautions.
• Chemical manufacturer’s name, address, and telephone number
• Special information section
 CHEMICAL WASTE DISPOSAL

• Flush water-soluble substances down the drain with large quantities of

water
• Strong acids and bases should be neutralized before disposal
• Foul smelling chemicals should never be disposed down the drain
• Flammable solvents  collected in approved containers
• Flammable material  specially designed incinerators
• Solid chemicals  landfill
 RADIATION HAZARDS
• Radioactivity is encountered in the clinical laboratory when procedures using
radioisotopes are performed.
• Radioactivity present in the clinical laboratory is very small
• Time
• Distance
• Shielding
• Protect both the environment and the personnel
• All areas where radioactive materials are used or stored must be posted with
caution signs, and traffic in these areas should be restricted to essential personnel
only.
• Records must be maintained for the length of employment plus 30 years.
 FIRE HAZARD
• Fire is a chemical reaction that involves the rapid oxidation of
a combustible material or fuel, with the subsequent liberation
of heat and light.
• In the clinical chemistry laboratory, all the elements essential
for fire to begin are present—fuel, heat or ignition source, and
oxygen (air).
• The fire triangle has been modified into a three dimensional
pyramid known as the fire tetrahedron.
• This provide additional means by which fires may be
prevented or extinguished. A fire will extinguish if any of the
three basic elements are removed.
FIRE TETRAHEDRON
FIRE OR EXPLOSIVE HAZARDS
ELECTRICAL SAFETY
• Lock-out or tag malfunctioning electrical or
mechanical equipment until serviced
• Know how to knock a shocked person loose
using a non-conductive material
ELECTRICAL PRECAUTIONARY
PROCEDURES:
• Be particularly careful when operating high-voltage equipment, such as
electrophoresis apparatus.
• Check for frayed electrical cords.
• Promptly report any malfunctions or equipment
• Do not work on “live” electrical equipment.
• Never operate electrical equipment with wet hands.
• Know the exact location of the electrical control panel for the electricity
to your work area.
• Use only approved extension cords and do not overload circuits. (Some
local regulations prohibit the use of any extension cord.)
 SHARP AND
PHYSICAL HAZARDS
Sharp hazards
• Includes needles, lancets, and broken glassware
• Must be disposed in puncture – resistant containers
• Use mechanical device to pick-up sharps

Physical hazards
• Avoid running in rooms and hallways
• Watch for wet floors
• Bend the knees when lifting heavy objects
• Keep long hair pulled back
• Avoid dangling jewelry
• Maintain a clean, organized work area
TYPES OF SAFETY HAZARDS
PHLEBOTOMY AND CONSIDERATIONS

College of Medical Laboratory Science

Our Lady Fatima University
GENERAL PRECAUTIONS
IN COLLECTING BLOOD SAMPLE
GENERAL PRECAUTIONS IN COLLETING BLOOD SAMPLE

SITES TO BE AVOIDED
VASCULAR ACCESS DEVICE & EDEMATOUS
AREA
 INTRAVENOUS LINE
 ARTERIAL LINE
 ARTERIOVENOUS SHUNT OR FISTULA
 HEPARIN OR SALINE LOCK
 CENTRAL VASCULAR ACCESS DEVICE
GENERAL PRECAUTIONS IN COLLETING BLOOD SAMPLE

SITES TO BE AVOIDED
BURNED, SCARRED & TATTOOED
AREAS
GENERAL PRECAUTIONS IN COLLETING BLOOD SAMPLE

SITES TO BE AVOIDED
HEMATOMA
GENERAL PRECAUTIONS IN COLLETING BLOOD SAMPLE

SITES TO BE AVOIDED
POST-MASTECTOMY SITE
GENERAL METHODS OF BLOOD SAMPLE COLLECTION

PHLEBOTOMY
 SKIN PUNCTURE
 ARTERIAL PUNCTURE
 VENIPUNCTURE
SKIN PUNCTURE
• Dermal puncture, capillary puncture, prick method
• For micromethod, ultramicromethod and nanoliter
method
• The method of choice for
• Pediatrics
• Geriatrics
• Obese patients

• Sites of puncture
• Palmar surfaces of fingertips
• Lateral plantar heel surface
• Plantar surface of the big toe
• Earlobes
MATERIALS & EQUIPMENT

• 70% ISOPROPYL ALCOHOL

• ABSORBENT COTTON SPONGES/BALLS OR SWABS
• GAUZE PAD
• STERILE PUNCTURE INSTRUMENT (LANCET)
• MICRO COLLECTION TUBES

ORDER OF DRAW
 EDTA SPECIMENS
 OTHER ADDITIVE SPECIMENS
 NON-ADDITIVE SPECIMEN
PROCEDURE
• Position patient and select an appropriate puncture site
• Warm the site using warm towel if applicable
• Cleanse the puncture site
• Prepare equipment
• Make a puncture perpendicular to the skin surface
• Wipe away the first drop with clean gauze pad
• Collect blood specimen in a suitable container
• After collection, apply pressure
• Label specimen and dispose used materials
VENIPUNCTURE

• For macro-method
• Sites of collection:
• Veins in the antecubital fossa
• Median cubital vein
• Cephalic vein
• Basilic vein
• Vein of the longitudinal sinus, sagittal
sinus
• Femoral vein, wrist vein
• Great saphenous vein
• Veins on the dorsal portion of the hand
MATERIALS & EQUIPMENT
Tourniquet
 A tourniquet is applied to a patient’s
arm during venipuncture
 It should be tight enough to restrict
venous flow but not arterial flow.
 One (1) minute application rule
MATERIALS & EQUIPMENT
Syringe sytem
Includes a plastic syringe, a needle, and a transfer
device
 Syringe needles are available in a wide range of
gauges and lengths for many different uses.
 Syringes have a barrel with graduated markings and a
plunger that fits snuggly into it.
 Transfer device is like a tube holder with a needle
inside.
MATERIALS & EQUIPMENT
Evacuated tubes

 Tubes with pre-measured vacuum that

automatically draws the volume of blood
indicated on the label.
 Tube stoppers are color coded to
identify a type of additive, absence of
additive
ORDER OF DRAW
• BLOOD CULTURE
• CITRATE
• NON-ADDITIVE
• HEPARIN
• EDTA
• NA FLUORIDE
• NA OXALATE
VENIPUNCTURE PROCEDURE
VENIPUNCTURE
• Select the puncture site
• Apply the tourniquet
• Select the vein
• Remove the tourniquet
• Cleanse the area
• Re-apply the tourniquet
• Inspect the needle
• Perform venipuncture
• Release the tourniquet
• Position the gauze over the puncture site
• Remove the needle and apply pressure
 COMPLICATIONS OF VENIPUNCTURE

Immediate local complications

• Hemoconcentration or venous stasis. False increase in the number formed
elements in blood due to decrease plasma volume
• Remedy: one minute tourniquet application

• Syncope or fainting
• Transient loss of consciousness due to lack of oxygen in the brain & results in an
inability to stay in an upright position
• Remedy: patient’s head is lowered between legs & instructed to breath
deeply; give spirit of ammonia
 COMPLICATIONS OF VENIPUNCTURE
Delayed local complications
• Thrombosis of veins
• Formation of blood clots inside the lumen of the vein due to trauma
• Thrombophlebitis
• Inflammation of the vein caused by thrombus
• Hematomas
• Blue or black skin discoloration commonly due to repeated trauma or puncture of the veins
 COMPLICATIONS OF VENIPUNCTURE

GENERAL DELAYED COMPLICATIONS

 SERUM HEPATITIS
 AIDS
 REASONS FOR SPECIMEN REJECTION
• Hemolyzed, lipemic
• Clots in anticoagulated tube
• Non-fasting specimen
• Wrong blood collection tube
• Improper transportation
• Discrepancies between requisition and specimen label
• Unlabeled or mislabeled specimen
• Contaminated specimen/ leaking specimen
HEMATOLOGY
HEMATOLOGY
Smear Preparation
Hemoglobin and Hematocrit
Erythrocyte Sedimentation Rate
Osmotic Fragility Test
Reticulocyte count, LE Preparation
RBC Indices Computation
Automation
SMEAR PREPARATION
BLOOD SMEAR

• Hematological smear
• Drop of blood spread thin on a microscope slide
• EDTA blood specimen
 TYPES OF BLOOD SMEAR

1. The cover glass smear.

2. The wedge smear .
3. The spun smear.
• The are two additional types of blood smear used for
specific purposes
1. Buffy coat smear for WBCs < 1.0×109/L
2. Thick blood smears for blood parasites .
PROCEDURE
BLOOD SMEAR
BLOOD SMEAR
BLOOD SMEAR
BLOOD SMEAR
 CHARACTERISTICS OF IDEAL BLOOD SMEAR

• Gradual transition from thick to thin

• 2/3 to ¾ length
• Visible lateral edges
• Without irregularities, holes or streak
• Feathery edge
LIMITATIONS

• The angle between the slides is dependent upon the size of the
blood drop and viscosity of the blood. The optimal angle is 45
degrees.
• The larger the drop of blood and lower the haematocrit, the higher
the angle needs to be so the blood smear is not too long.
• Blood with a higher haematocrit needs to have a lower angle so the
smear is not too short and thick
• Glass slides must be clean; otherwise, this results in imperfect
distribution of cells and improper staining.
• Once the drop of blood has contact on the slide, the smear needs to
be made immediately. Otherwise, the blood will clump and dry,
again resulting in uneven distribution of WBC and platelets.
PROCEDURE

• Smear preparation
• Fixation of smear using methanol
• Stain using Wright or Wright-Giemsa stain
• Place 1 drop of cedar-wood oil on the edge of the
smear
• Use OIO (100x) and perform zigzag method
• Count and classify 100 WBC using Schilling’s counter
too acidic suitable too basic
MICROSCOPY FOR DIFF COUNT
HEMOGLOBIN AND HEMATOCRIT
OXYGEN DISSOCIATION CURVE
REFERENCE VALUE OF HEMOGLOBIN

MALE : 13.5 TO 17.5 g/dL

FEMALE : 12.0 to 16.0 g/dL
Methods of Hemoglobin Determination

• ACID HEMATIN METHOD

• CYANMETHEMOGLOBIN METHOD
ACID HEMATIN METHOD

• PRINCIPLE: Haemoglobin is converted to acid

hematin with dilute 0.1 N HCl resulting to
brownish yellow colour produced upon
addition of distilled water is matched with the
colour standard in the comparator block
CYANMETHEMOGLOBIN METHOD

• PRINCIPLE: Haemoglobin is converted from

ferrous to ferric state to form methemoglobin
by the action of ferricyanide. Methemoglobin
then combines with potassium cyanide to
produce the stable cyanmethemoglobin which
is measured spectrophotometrically,
CYANMETHEMOGLOBIN AS THE METHOD OF CHOICE

• Cyanmethemoglobin is stable in dilutions

• Cyanmethemoglobin standard are readily available
• All hemoglobin derivatives except sulfhemoglobin are measured
with their conversion to cyanmethemoglobin
• The spectrum of cyanmethemoglobin is such that other types of
spectrophotometers can be used for reading colour intensity
produced
ACID HEMATIN METHOD: PROCEDURE

• Introduced 0.1 N HCl solution up to 2 mark of the Sahli graduated

tube
• Using the Sahli pipette, draw blood up to 0.02cc mark
• Dispense the measured volume of the blood into the graduated
tube. Gently draw up and down to rinse the pipette and also to mix
the resulting colloidal suspension
• Thoroughly mix with the use of the stirrer and allow to stand for 5
minutes
• Add distilled water drop by drop, stirring after each addition and
compare with the comparator block
• When the resulting solution compares with the colour standard in
the comparator block, get the reading in % scale and in grams
• Write your result
CYANMETHEMOGLOBIN METHOD: PROCEDURE

• Place 5cc of Drabkin’s reagent into a test tube

• Using Sahli pipette, draw blood to 0.02cc mark.
• Dispense the blood to the test tube
• Gently draw up and down to rinse the pipette and also to mix the resulting
colloidal suspension
• Cover and mix well by inversion. Let it stand for 5 minutes
• Transfer the mixture to a cuvette
• Set the spectrophotometers to 100% transmittance at 540nm wavelength
• Use Cyanmethemoglobin reagent as a blank
• Continue reading the patient’s sampe, and record the percentage
transmittance
• The hemoglobin concentration is obtained from a calibration curve
prepared with the use of standards
HEMATOCRIT

• The packed cell volume (PCV) is a measurement of the ratio of the

volume occupied by the RBCs to the volume of whole blood in a
sample of capillary or venous blood.
• A whole blood sample in an anticoagulated tube is centrifuged at
10,000 to 13,000 rpm for 5 minutes.
• Following centrifugation, this ratio is measured and expressed as a
percentage or decimal fraction
• The hematocrit percentage is read below the buffy coat layer
• Clinically, the PCV is used to detect anemia, polycythemia,
hemodilution, or hemoconcentration. In conjunction with an
erythrocyte count, the PCV is used to calculate the mean
corpuscular volume (MCV).
• The PCV is also used in conjunction with the hemoglobin
concentration to calculate the mean corpuscular hemoglobin
concentration (MCHC).
REFERENCE VALUE OF HEMATOCRIT

MALE : 42% TO 52 %
FEMALE : 37 % TO 47%
NEWBORN : 53 % TO 65%
CHILD : 30 % TO 43 %
Methods of Hgb Determination

• MACROMETHOD
• MICROMETHOD
MACROMETHOD: PROCEDURE

• Mix the blood

• With the use of long stem pipette, fill the wintrobe tube to the 10
mark
• NOTE: NO AIR BUBBLES
• Centrifuge blood for 30 minutes
• Read the height of the PCV on the scale at the right side of the
tube.
MICROMETHOD: PROCEDURE

• After necessary preparations, make capillary puncture and produce

a rounded drop of blood
• In a horizontal position, put one end of capillary tube on the drop
of blood and fill the tube about 2/3 full
• Seal one end of the capillary tube with the clay at a 90 degree angle
• Assemble the tube in microhematocit centrifuge in such a way that
the unsealed end is nearest the center of the centrifuge
• Spin at 10, 000 to 15, 000 rpm for 5 minutes
• Using the microhematocrit reader, determine the HCT
• The reading on the window of the hematocrit reader corresponds
to the hematocrit value
VARIOUS CONSIDERATIONS

• Hematocrits are run in duplicate and must agree within1%

• Incomplete sealing will give falsely low results
• If the buffy coat is included in the reading, hamatocrit will be
falsely elevated
• The microhematocrit centrifuge should never be forced to
stop by applying pressure to the metal coverplate. This will
cause RBC layer o sling forward and will result in a falsely
elevated value
• Hematocrit is usually 3x the hemoglobin value