Chapter 15 Specimen Collection And Laboratory Procedures

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Chapter 15 Specimen Collection And Laboratory
Procedures
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Microscope

• One or more lense that makes an enlarged image of
  an object.

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Compound Microscope

• Lets light pass through an object and then
  through two or more lenses.

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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
  see-through double convex lens curved to cause
  images to appear larger. 
• Monocular microscope has one eyepiece
• Binocular microscopes have two

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Objectives

• The objectives are screwed into a circular
  revolving nosepiece that is revolved by hand

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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

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Objectives

• The 4X objective is used for scanning a slide
• This is lung tissue(pneumonia) 4X

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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

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Objectives

• The 40X objective is used for red and white blood
  cell counts
• This is a WBC

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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.

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10X, 40X, 100X
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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

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Light

• In order for the microscope to work, light must
  pass upward through the material being viewed and
  into the objective lens

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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

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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

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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. 

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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

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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.

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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 low-power-(10X) objective into position
• Still looking for the side, use the coarse
  adjustment knob, lower the objective until it
  nears the stage or stops

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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)

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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

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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

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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

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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

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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.

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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 needle- stick
  injuries

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Venipuncture

• Venous  venipuncture surgical puncture of a
  vein
• 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

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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 patients 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)

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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

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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
• hand and wrist

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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

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Butterfly Winged Infusion Collection Set

• The butterfly winged infusion device consists of
  needle with plastic wings,
• plastic tubing, and an adapter
• Its 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

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Butterfly Winged Infusion Collection Set

• The butterfly winged infusion device consists of
  needle with plastic wings, plastic tubing, and an
  adapter
• Its 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

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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)

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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

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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

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Methods Of Performing A Venipuncture

• There are two methods of performing this
  procedure
• Syringe method
• Needle and vacuum tube method

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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
  patients 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)

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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

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Vacuum Method

• This method is probably the most popular because
  it is so convenient

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Gauge for Venipuncture

• Gauge average size for venipuncture needles is 21
  or 22 gauge in size

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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

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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

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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(contains the anticoagulant EDTA, which
  binds to calcium, preventing the formation of a
  clot)
• Gray(contains two additives sodium fluoride and
  potassium oxalate)
• Young red-headed boys greet little girls

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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

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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.

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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

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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

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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

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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

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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)

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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 RBCs 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 RBCs meet the plasma.

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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

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Erythrocyte Sedimentation Rate(ESR)

• ERS is a test to indicate
• Acute infections
• Chronic infections
• Rheumatoid arthritis
• Monitoring inflammation
• Page 791(normal values and how to perform an ESR)

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Introduction

• Measuring the blood level of glucose, a simple
  sugar, is probably the most commonly performed
  blood chemistry test in the lab

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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)

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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

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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

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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

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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

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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 lt 100)
• HDL (high-density lipoprotein cholesterol, also
  called "good" cholesterol gt60)
• 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 lt150)

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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)

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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

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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

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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)

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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

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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 childs diaper

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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

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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

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• 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

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Turbidity/Clarity

• TurbidityNormally 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.

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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.

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Specific Gravity
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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

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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)

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Microscopic Properties of Urinalysis
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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

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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

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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

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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

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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)