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Chapter 15 Specimen Collection
And Laboratory Procedures
Microscope
One or more lense that makes an enlarged image of an object.
Compound Microscope
• Lets light pass through an object and then
through two or more lenses.
Oculars
• The ocular lens, also known as
the eyepiece lens, is the part of
a compound microscope that a
user looks into to see a
magnified image. It is a seethrough double convex lens
curved to cause images to
appear larger.
• Monocular microscope has one
eyepiece
• Binocular microscopes have
two
Objectives
• The objectives are
screwed into a circular
revolving nosepiece
that is revolved by
hand
Objectives
• Objectives are the lens
farthest from the eye and
closest to the specimen
• A microscope has 3 to 4
objectives
• 4x, 10x, 40x, 100x
• Always begin focusing a
microscope on the lowest
power and then move to
the next higher power and
refocus
Objectives
• The 4X objective is
used for scanning a
slide
• This is lung
tissue(pneumonia) 4X
Objectives
• The 10X objective is used
to count cells and to scan
urine sediment
• This is the most common
objective. ALWAYS START
WITH THE LOW POWER
OBJECTIVE! Low power
lens gives the widest field
of view and makes it
easier to find
the specimen when you
look through the
microscope
Objectives
• The 40X objective is
used for red and white
blood cell counts
• This is a WBC
Objective
• The 100X has the
greatest power and is
use to identify
characteristics for
bacteria and blood
smears
• Finding
the specimen at high
power, without first
centering it in the field
of view at low power,
is nearly impossible.
10X, 40X, 100X
Stage
• The stage is the
platform that holds the
slide to be viewed
• The hole on the stage
allows light to enter
from below, passing
through the specimen
to the lens system
Light
• In order for the
microscope to work,
light must pass
upward through the
material being viewed
and into the objective
lens
How to use a microscope
• Focusing the microscope
is done by moving the
objective up or down
relative to the stage.
Movement of the stage is
controlled by the round
knobs, on both sides of the
microscope
• This permits resting your
arms on the table while
focusing
•
Course and Fine Adjustments
Knobs
The coarse adjustment
focus knob moves the
objective
quickly(usually the
larger knob)
• It moves the tube and
lenses closer and
farther away from the
specimen
• This knob is used first
to bring the specimen
into focus
Course and Fine Adjustments
Knobs
• The fine adjustment knob
is used to fine-tune the
focus of your specimen
after using the coarse
adjustment knob.
• It moves the objective
much more slower with
only a short distance.
Focus slowly. It is easy
to focus right past
the correct focus point if
you are going too fast.
Eyepiece Adjustment
• When using a binocular
microscope, it is necessary
to adjust the eyepieces to
your own eye span and
visual acuity. A gentle
push inward or pull
outward with adjust the
distance between the
eyepieces to accommodate
your eye span
Light Adjustment
• The right level of light
is essential for a clear
image.
• Too little light will
obscure details in
darkness, while too
much light will
produce a blinding
glare without the
contrast necessary to
distinguish features.
•
Putting It All Together
• To focus the microscope for lab work using the 10X
objective:
• Maximum distance between the stage and objective
• Clamp the slide on the stage
• Turn on the light
• Raise the condenser to its highest position
• Look at the side not through the oculars, rotate the lowpower-(10X) objective into position
• Still looking for the side, use the coarse adjustment knob,
lower the objective until it nears the stage or stops
Putting It All Together
• Look through the eyepiece and reverse the
direction of the course focus adjustment knob until
the slide comes into focus
• Adjust the fine focus until you attain the clearest
possible image
• Adjust the condenser(light source) until the image
is clear
• Slide the specimen with the mechanical knobs(left
and right, toward or away from you)
Key Points
• Start with the lowest power first
• Always clip your objective(slide) in the center of the light source
• Use the course adjustment knob(larger knob) to focus and move the
stage(now you are done with this knob)
• You can use the fine adjustment knob if needed(depending on your
eyesight)
• Then you can turn the nosepiece and use a higher power objective
• Never use the course adjustment again(in the higher power) This will
raise the stage and could crush the slide and break the microscope
• Again only then will you use the fine adjustment knob to focus
• Always put the objective back on the lowest power before you remove
the slide and before you turn off the microscope
• Cover the microscope
Microscope Care
• The microscope is a
delicate, expensive
instrument that is easily
damaged by dust, oil,
light, and falls
• Arm-part of the
microscope is the part that
you carry the microscope
with on hand
• Base-always support the
microscope with the other
hand underneath the base
Cleaning the microscope
• Only clean your
microscope lenses when
they are dirty and only use
lens paper
• Blow dust form the glass
with a syringe
• Clean only the outside
surface of the lens
• Never touch the lens
• Never substitute the lens
paper(this may scratch the
lens
Skin Puncture
• Dermal puncture or capillary puncture:
venipuncture is the most frequent performed
phlebotomy procedure.
• However, because current laboratory
instruments and procedures enable us to use
smaller and smaller amounts of blood,
obtaining micro samples by capillary or
dermal(skin) puncture is also popular
• Always use the ring/middle finger
Fingerstick Sites
• When performing a finger-stick, select a site on the central,
fleshy portion of the ring or middle finger
• The incision should be slightly off the middle of the finger and
never to close to the nail bed
• Avoid the index finger due to a large number of nerves(more
painful)
• The little(fifth) finger(decrease mass)
• The thumb is too course
(harder to obtain blood)
Finger-sticks should not be performed on
infants younger than 6 months of age
due to the short distance between the
finger bone and the skin surface.
Skin Puncture
• Lancets are designed to control the depth of
the dermal puncture
• Safety lancets must be used, so that the
blade retracts after use to prevent needlestick injuries
Venipuncture
• Phlebotomy Chair
• For all blood collection procedures, the patient should be
lying down or seated in a phlebotomy chair
• In an outpatient setting, lock the arm mechanism, this
prevents the patient from falling if the patient faints
• It is critical to make the patient safe and comfortable
during the procedure
Tourniquets
• Tourniquets are used to help pool the blood in the veins, causing
the veins to enlarge
• In addition, the pooling of veins make it easier for the vein to
tolerate the vacuum suction of the tubes withdrawing blood
• Place the tourniquet on the patient’s upper arm, about 3 inches
above the elbow
• Ask about latex allergies
• Disinfect the tourniquets with 70% alcohol after ever use
• Never leave a tourniquet on longer than 1 minute(this can also
alter blood test results)
• If it is necessary to wait longer than a minute, remove the
tourniquet and reapply it when ready
• ALWAYS remove the tourniquet before removing
the needle from the vein(this will cause a
hematoma)
Venipuncture
• Venipuncture method as called phlebotomy,
take blood directly from a vein, most
commonly
in the antecubital area
of the arm
The inner arm at the
bend of the elbow
Common Sites
Venipuncture
•
•
•
•
•
The vein used most often are the
Median cubital vein
Cephalic vein
Basilic vein
Other sites
sometimes used
are the lower
forearm, back of
Common Sites for
hand and wrist
Venipuncture
Antiseptics
• 70 percent alcohol wipes(most common)
• Alcohol prevents the introduction of
microorganisms into the body during
venipunture
• It is critical when using alcohol to allow the
alcohol to completely air dry
• Betadine is the other antiseptic commonly
used for blood alcohol levels, blood
cultures, and blood donations
Butterfly Winged Infusion
Collection Set
• The butterfly winged infusion device consists of needle
with plastic wings,
plastic tubing, and an adapter
• It’s easy-to-grasp and flexible
• It can be attached to either
a needle holder or a syringe
• The needle gauge is usually 19 to 23 gauge and ¾ of an
inch long
• This can be helpful when collecting a specimen from a
neonate, child, elderly, cancer patient, or severely
traumatized patient
Butterfly Winged Infusion
Collection Set
• The butterfly winged infusion device consists of needle
with plastic wings, plastic tubing, and an adapter
• It’s easy-to-grasp and flexible
• It can be attached to either
a needle holder or a syringe
• The needle gauge is usually 19 to 23 gauge and ¾ of an
inch long
• This can be helpful when collecting a specimen from a
neonate, child, elderly, cancer patient, or severely
traumatized patient
Butterfly Utilizing Tube System
• When using a butterfly needle with an
evacuated tube, use a 10 to 15 degree angle,
this is slightly lower than the standard needle
• With a butterfly set you can grasp the butterfly
by the wings(easy to grasp)
Butterfly Winged Infusion
Collection Set
• When using a butterfly needle, you
will know when the needle has
entered the vein because you will
observe a “flash” of blood on the
shaft of the tubing
• Patients may request the butterfly needle, because the length of the
needle is shorter and looks smaller
• Inform the patient that the gauge is about the same, therefore, there
should no difference in pain with the two needles
Hematoma
• Venipuncture must be always be done carefully to avoid
causing an hematoma(collection of blood just under the
skin)
• To prevent a hematoma:
• Puncture only the uppermost wall of the vein
• Remove the tourniquet before removing the needle
• Use the major superficial veins
• Make sure the needle fully penetrates the upper most wall
of the vein. (Partial penetration may allow blood to leak
into the soft tissue surrounding the vein by way of the
needle bevel)
• Apply pressure to the venipuncture site
Methods Of Performing A
Venipuncture
• There are two methods of performing this
procedure:
• Syringe method
• Needle and vacuum tube method
Syringe Draw
• A syringe is a plastic barrel and plunger set that is manually pulled to
control the flow of blood
• Syringes come variety of sizes; 10 mL an 20 mL are the most
commonly used in the venipuncture procedure(do not use any larger or
smaller syringe)
• Prior to attaching the syringe it is important to “prime” the syringe.
Pull the plunger in and out of the syringe to ensure it moves smoothly
• It is critical that the plunger be pushed back into place before
beginning the phlebotomy procedure to avoid injecting air into the
patient’s vein
• You can control the rate at which the blood is being withdrawn
• You should observe the vein during the procedure and make any
adjustments to the rate of collection as needed (collapsed vein)
• Once the blood has been collected in the syringe, you must engage the
needle safety mechanism, remove the needle, and replace it with a
transfer device before transferring the blood to the tubes. Allow the
tubes to fill without applying pressure(this will hemolysis the blood)
Hemolysis
• Hemolysis is the breakdown of red blood cells.
• This can cause alterations to blood test results and give an inaccurate
reading
• Hemolyzes blood samples are generally discarded
• Causes:
• An improper choice in the venipuncture site
• Prolonged tourniquet time
• Cleansing the venipuncture site with alcohol and not allowing the site
to dry
• Pulling the plunger of a syringe back too fast
• Transferring into a tube by pushing down on the syringe plunger in
order to force blood into a tube
• Vigorous mixing or shaking of a specimen
• Having the patient pump their hand
Vacuum Method
• This method is probably the most popular
because it is so convenient
Gauge for Venipuncture
• Gauge average size for venipuncture
needles is 21 or 22 gauge in size
Blood Collect Tubes
• A variety of different tubes are required
when performing venipuncture. Tubes
vary in size, volume, and additives
• The color of the rubber stopper located at
the top of the tube indicates what additive
is found in the tube
• Many tubes contain an anticoagulant, a
chemical that interferes with the clotting
process and prevents a clot from being
formed in the tube
Blood Collect Tubes
• These tubes have a “vacuum” a specified
amount of air has been taken out by the
manufacturer so that when the tube is
pierced by the needle, the tube will fill with
a predetermined amount of blood
• This ensures that only the proper amount of
blood enters the tube
Order Of Draw
• When several blood specimens are ordered, they should be
drawn into the color-coded stopper tubes in the order to
prevent contamination from the additives in the other tubes
• Yellow(for blood cultures)
• Red/red-gray(no additive or anticoagulant is used)
• Blue(contains the anticoagulant sodium citrate)
• Green (contains one of three different anticoagulants:
sodium heparin, lithium heparin, or ammonium heparin)
• Lavender
• Gray(contains two additives: sodium fluoride and
potassium oxalate)
•
Young red-headed boys greet little girls
Explain What You Are Going To
Do
• It is important to explain what you are
going to do
• Ask the patient if they have any questions
• Remember that you what the patient to feel
comfortable and relaxed
Laboratory Classification And
Regulation
•
•
•
•
POC: physician's office laboratory
Waived test: moderately complex tests(rapid strep's, UA)
Non-waived tests: complex tests with multiple steps(HIV)
CLIA: Clinical Laboratory Improvement Amendments:
Law passed in 1988, establishing quality standards for all
non-research laboratory testing performed on specimens
derived from humans for the purpose of providing
information for the diagnosis, prevention, treatment of
disease, or impairment of, or assessment of health.
Hemoglobin And Hematocrit
• Hemoglobin: is the protein
molecule in red blood
cells that
carries oxygen from the
lungs to the body's tissues
and returns carbon dioxide
from the tissues to the
lungs
• The HemoCue is one
example of the instrument
commonly used to
measure hemoglobin with
capillary blood
Hemoglobin
• Hemoglobin is measured if anemia is
suspected in the patient(usually along with
hematocrit)
• The normal range for females: 12 to 16 g/dl
• The normal range for males: 14 to 18 g/dl
The Hematocrit
• The hematocrit is a simple yet reliable test to
measure the percent volume of red blood cells per
volume of whole blood
• Given as a percent, it is often used as an indirect
measure of hemoglobin
Cellular Layers
• To measure the volume of red
blood cells, you first must
separate them from other blood
components by high-speed
centrifugation
• During centrifugation, the red
blood cells are packed at the
bottom of the tube
• Packed cell volume, or PCV is
another
name for hematocrit
• On top of the red blood cells are
white blood cells and
platelets(whitish-tan
layer)called the buffy coat
• Plasma is at the top of the tube
•
The Microhematocrit Method
• Capillary puncture or venipuncture can be used to
obtain microhematocrit blood sample
• Heparinized(red tip) capillary tubes should be used
when collecting capillary blood samples
• They are filled three-fourths full
(horizontally, avoid air bubbles)
• The clean end is sealed with clay
• A gloved index finger should be
placed over the open end to
prevent blood from flowing into
the sealing material(must be done)
How To Use A Hematocrit reader
1.Place the bottom of the tube along the red
line below the scale.
2. Be sure that the place where the white
hematocrit sealer meets the RBC’s is on the
line at the bottom of the scale itself.
• 3. Move the tube along those lines until the
top of the plasma intersects the top line of
the scale. (If the tube is very full, you may
need to use the scale on the right side.)
4. The value of the hematocrit is read on
the line where the RBC’s meet the plasma.
The Erythrocyte Sedimentation
Rate(ESR)
• The ERS(ESR and sed rate) is the rate at
which red blood cells settle out of plasma
when placed in a vertical tube
• The ESR is calculated by measuring the
distance the red blood cells travel through the
plasma during a given interval of time
Erythrocyte Sedimentation
Rate(ESR)
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•
•
•
•
ERS is a test to indicate:
Acute infections
Chronic infections
Rheumatoid arthritis
Monitoring inflammation
Page 791(normal values and how to
perform an ESR)
Introduction
• Measuring the blood level of glucose, a
simple sugar, is probably the most
commonly performed blood chemistry test
in the lab
Blood Glucose And Disease
• The normal glucose level in a random blood
sample is 70 to 110 mg/dl
• If the patient has fasted, before the test, the
normal level is 70 to 120 mg/dl
• Blood glucose levels outside the normal
range may indicate pathology(diabetes)
DM Type 1
• The more severe form of diabetes, type 1(insulin-dependent or
juvenile-onset) diabetes
• Type 1 is comprises about 5 to 10% of all cases an usually
strikes before age 25
• Type 1 diabetes requires administration of insulin to manage
the disease
• This occurs when the pancreas does not produce enough
insulin to properly control blood sugar levels.
• Type 1 diabetics lack the insulin hormone
DM Type 2
• Type 2 diabetes or adult-onset can usually be
controlled by diet and oral hypoglycemic
medications
• It is the less severe form of the disease and the
more common, comprising 90 to 95% of all
cases if DM
• It usually has a gradual onset and generally
affects adults over age 40
• Patients with this form of the disease often are
obese
Glucose Tolerance Tests
• When fasting blood glucose levels are not
definitive for a diagnosis of diabetes or when
there is unexplained glycosuria, a glucose
tolerance test(GTT) may be ordered
• Fast for 8 to 12 hours then you drink a liquid
containing a certain amount of glucose. Your
blood will be taken before you do this, and
again every 30 to 60 minutes after you drink
the solution
• A GTT is done on pregnant women to
determine gestational diabetes
Hemoglobin A1C
• The hemoglobin A1C test, detects
hyperglycemia that may be missed in type 1
patients who have wide swings in there
blood glucose levels
• This is a test over a three month period
Cholesterol Testing
• Cholesterol is a steroid normally found in the body
• Normal cholesterol should be under 200
• LDL (low-density lipoprotein cholesterol, also called "bad"
cholesterol < 100)
• HDL (high-density lipoprotein cholesterol, also called
"good" cholesterol >60)
• Triglycerides (fats carried in the blood from the food we
eat. Excess calories, alcohol, or sugar in the body are
converted into triglycerides and stored in fat cells
throughout the body <150)
Infectious Mononucleosis
• Also called mono
• This serious disease most often affects teenagers and
young adults(kissing disease)
• It is caused by the Epstein-Barr virus, and it has flu-like
symptoms, including fever, fatigue, weakness, swollen
glands, sore throat, and headache(also effect liver, spleen)
Newborn Screening
• This is a screening done with capillary
blood from the heel of the foot
• Done at birth and then at 14 day old
• Tests for metabolic disorders inherited
disorders can hinder an infant's normal
physical and mental development in a
variety of ways. And parents can pass along
the gene for a certain disorder without even
knowing that they're carriers
• Some states test for 50 different disorders
Introduction
• Urine specimens can provide a great deal of
information about both the urinary tract system and
other systems(DM,HBP,liver,drugs)
• This is very useful test and can disclose evidence of
disease, even some that have not caused signs or
symptoms
• The urine samples must be correctly collected and
maintained
Urinalysis
• Urine may be analyzed for its physical, chemical, or
microscopic properties
• When all three types of analysis are made, this is
referred to as a urinalysis(UA)
• Urine can also be tested for a just few tests(urine
glucose level)
• A urinalysis is performed for three general reasons:
• 1. Screening for urine glucose
• 2. Diagnosis of suspected disease(bacteria UTI)
• 3. Monitoring the course of treatment (UTI antibiotics)
First Morning/24 hour Urine
Specimens
• First Morning Specimen This is the specimen of
choice for urinalysis and microscopic analysis, since
the urine is generally more concentrated (due to the
length of time the urine is allowed to remain in the
bladder)
• Urine for 24 hours
• Purpose: This procedure checks the function of your
kidneys or measures certain products in your urine
• A special container is used to collect the urine.
• A preservative is in the container.
• Refrigerate the container during
collection, the urine must be kept cold
Pediatric Collection System
• These are available for infants to young
children
• Consists of a plastic bag with an adhesive
backing around the opening
• This system can under the child’s diaper
Collecting The Sample
• Some patient who are not able to follow the
urine collection procedures may require
urinary cauterization, which involves placing a
tube called a catheter through the urethra into
the bladder to obtain a urine specimen
Physical Examination
• All routine urinalysis should begin with a
physical examination of the urine sample.
This examination includes assessment
of volume, odor, and appearance (color
and turbidity), and specific gravity
• While odor is not generally recorded, it may
be noted
• The appearance of urine depends on its color and its
turbidity or clarity(cloudiness)
• Color- Urine color usually is determined by concentration,
color can be yellow(light dark), amber, red, brown, orange,
green, or blue
• The amount of fluids an individual drinks is an important
factor
• The more fluids a person drinks, the more dilute the urine
Turbidity/Clarity
• Turbidity—Normally freshly voided urine
is clear to hazy in appearance. A cloudiness,
may be due to a number nonpathological
causes or to a disease(blood, proteins,
mucus, bacterial infection). The turbidity of
urine should always be recorded and
microscopically explained.
Specific Gravity
• Shows how well the kidneys balance the
amount of water in urine. The higher the
specific gravity, the more solid material is
in the urine. When you drink a lot of fluid,
your kidneys make urine with a high
amount of water in it which has a low
specific gravity. When you do not drink
fluids, your kidneys make urine with a
small amount of water in it which has a high
specific gravity.
Specific Gravity
Chemical Urinalysis
• The chemical portion of the this test
provides information regarding the presence
or absence of several chemicals including
glucose, ketones, protein, and blood
Chemical Urinalysis
• Urine pH: should be slightly acidic to fight off bacteria
• Protein: is normally not found in the urine(hard exercise, pregnancy,
kidney disease)
• Ketones: when fat is broken down for energy, negative ketones is
normal(DM, low carbohydrate diet)
• Bilirubin and Urobilinogen: negative is normal(liver problems or
disease)
• Hematuria(blood in the urine): a normal urine sample should not have
any detectable blood(kidney or prostate problems)
• Nitrite: is normally not found in urine(positive means bacteria possibly
UTI)
• Glucose: Negative is normal (when the blood sugar level is very high,
as in uncontrolled diabetes, the sugar spills over into the urine)
Microscopic Properties of
Urinalysis
Urine Sediment
• Urine sediment is a centrifuged deposit suitable for
microscopic examination for the presence of
erythrocytes(RBC), leukocytes(WBC), casts,
crystals, bacteria, fungi, parasites, mucous, and
sperm(males)
• Next to actual biopsies of kidney tissue, microscopic
findings are the best indicators of renal disease
• This is referred to “liquid biopsy” for this reason
• Page 809
Pregnancy Testing
• This is a test to determine the presence(or
absence) of human chorionic gonadotropin
hormone or HCG
• This is a waived test with 3 drops of urine
and timed for 3 minutes
Culture Media
• A tentative diagnosis may be followed up
with a lab test to confirm the diagnosis with
a culture
• A blood test may be done, or a specimen
from the infected area may be examined
directly
Throat Cultures
• Strep throat is cause by Group A strep
(GAS), a strain of streptococcus bacteria
• The Group A Strep Test is easy to perform,
but needs to be precisely followed(steps)
• Without treatment(antibiotics) of the
original strep infection, rheumatic fever
may result
Strep Throat
• First the throat is swabbed with a
sterile, nonabsorbent rayon-fiber swab
• The swab is inserted into with the reagents
to extract the Strep A antigen(If there is a
strep A antigen the test will be positive)