1. Chapter 15 Specimen Collection And Laboratory Procedures

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

3. Compound Microscope
Lets light pass through an object and then through two or more lenses.

4. 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

5. Objectives
The objectives are screwed into a circular revolving nosepiece that is revolved by hand

6. 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

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

8. 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

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

10. 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.

11. 10X, 40X, 100X

12. 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

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

14. 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

15. 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

16. 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. 

17. 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

18. 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.

19. 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

20. 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)

21. 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

22. 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

23. 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

24. 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

25. 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.

26. 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

28. 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

29. 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)

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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)

36. 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

37. 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

38. Methods Of Performing A Venipuncture
There are two methods of performing this procedure:
Syringe method
Needle and vacuum tube method

39. 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)

40. 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

41. Vacuum Method
This method is probably the most popular because it is so convenient

42. Gauge for Venipuncture
Gauge average size for venipuncture needles is 21 or 22 gauge in size

43. 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

44. 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

45. 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

47. 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

48. 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.

49. 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

50. 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

51. 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

52. 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

53. 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)

54. 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.

56. 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

57. 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)

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

59. 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)

60. 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

61. 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

62. 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

63. 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

64. 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)

65. 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)

66. 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

67. 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

68. 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)

69. 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

70. 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

71. 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

72. 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

73. 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

74. 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.

75. 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.

76. Specific Gravity

77. 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

78. 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)

79. Microscopic Properties of Urinalysis

80. 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

81. 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

82. 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

83. 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

84. 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)