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 see-through 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 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

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 needle- stick injuries

Venipuncture

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 for Venipuncture

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

Common Sites for 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

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)

• 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)