Phlebotomy and Basic Blood Analysis

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1 Topic 2: Phlebotomy and Basic Blood Analysis Laboratory Manual Section 09 HPHE 6720 Dr. Cheatham Outline What is phlebotomy Review of Universal Precautions Phlebotomy Techniques Fingerprick Venipuncture IV catheter Basics of blood analysis Enzymatic reactions (spectrophotometer) ELISA RIA 1

2 What is Phlebotomy? Derived from the Greek words: Phlebo (relates to veins) Tomy (relates to cutting) The incision of a vein for blood letting (i.e. blood collection) Has sometimes been expanded to include other types of samples (capillary, arterial) Universal Precautions Refers to the practice of avoiding contact with patients' bodily fluids, by means of the wearing of nonporous articles such as medical gloves, goggles, and face shields. Medical instruments, especially scalpels and hypodermic needles should be handled carefully and disposed of properly in a sharps container. Under Universal Precautions all patients are considered to be possible carriers of blood borne pathogens. The guideline recommends wearing gloves when collecting or handling blood and body fluids contaminated with blood and wearing face shields when there is danger of blood splashing on mucous membranes and when disposing of all needles and sharp objects in punctureresistant containers. 2

Universal Precautions Techniques Fingerprick (Skin Puncture) Indications: Useful when a small amount of blood can be obtained and adequately tested.

3 Universal Precautions Techniques Fingerprick (Skin Puncture) Indications: Useful when a small amount of blood can be obtained and adequately tested. Hematocrit, blood glucose, hemoglobin Composition of skin puncture blood: Composed of blood from arterioles, venules, capillaries, and some interstitial fluid Sites: Typically the second, third, or fourth finger is used. Avoid the fifth finger (pinky) 3

4 Techniques Fingerprick (Skin Puncture) Techniques Fingerprick (Skin Puncture) 4

Techniques Fingerprick (Skin Puncture) Techniques Venipuncture Indications: Used when more blood is needed than can be provided for by a skin puncture.

5 Techniques Fingerprick (Skin Puncture) Techniques Venipuncture Indications: Used when more blood is needed than can be provided for by a skin puncture. Used mostly due to superficiality of veins and low pressure. Composition of Blood: Venous blood only Sites: Use antecubital space if possible Basilic, cephalic, and median cubital veins Never draw from legs, ankles or feet unless physician gives approval 5

6 Techniques Venipuncture Techniques Venipuncture 6

Techniques Venipuncture Supplies: Gauze, alcohol pads, tourniquet, Needle holder Needles The higher the gauge, the smaller the

What you are measuring in the blood determines what vacutainer(s) to use.

has some form of anticoagulant Therefore, plasma still has clotting factors.

7 Techniques Venipuncture Supplies: Gauze, alcohol pads, tourniquet, Needle holder Needles The higher the gauge, the smaller the needle. Typically 22 to 25 G is used. Vacutainers Different colored vacutainers have different anti coagulants (or none at all). What you are measuring in the blood determines what vacutainer(s) to use. Techniques Venipuncture The difference between plasma and serum Plasma Clear watery component of blood Is collected in a tube that has some form of anticoagulant Therefore, plasma still has clotting factors. Serum Clear watery component of blood Is collected in a tube with an accelerated clotting agent or a tube with nothing in it. Tube is allowed to sit (typically around 15 min) to allow for clotting to occur Therefore, serum has no clotting factors. 7

8 Techniques Venipuncture Techniques Venipuncture 8

9 Techniques Venipuncture Techniques Venipuncture 9

Techniques Venipuncture Hematoma formed Bevel near or in vein wall Needle

Often used when multiple blood samples are needed over time.

Procedures very similar to venipuncture Needle is inserted, then removed.

10 Techniques Venipuncture Hematoma formed Bevel near or in vein wall Needle passed through vein Vein collapsed Techniques IV Catheter Indications: Often used when multiple blood samples are needed over time. Reduces number of injections. Procedures very similar to venipuncture Needle is inserted, then removed. What remains is a flexible tube. Catheter must be flushed with saline or heparin/saline in order to keep catheter free flowing (no clots). 10

This chemical reaction typically results in a color change in the chemical reagent.

11 Techniques IV Catheter Enzymatic Assay (Spectrophotometry) A biological sample (often blood) is mixed with a chemical reagent. This chemical reagent causes a chemical reaction with the compound in the blood you want to measure. This chemical reaction typically results in a color change in the chemical reagent. The amount of this color change is measured by the spectrophotometer. The more color change, the more of the compound in the blood that you are measuring. 11

12 Enzymatic Assay (Spectrophotometry) (cont d) The color change in the reagent is specific to a certain wavelength of light. The spectrophotometer sends that wavelength of light through the chemical reagent. The more light that is absorbed, the greater the color change and the greater quantity of the blood compound. 12

Radioimmunoassay (RIA) To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma radioactive isotopes of iodine attached to tyrosine.

Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added.

13 Radioimmunoassay (RIA) To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma radioactive isotopes of iodine attached to tyrosine. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen for antibody binding sites. As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibodybound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured. A binding curve can then be plotted, and the exact amount of antigen in the patient's serum can be determined. 13

14 RIA Tube Blood RIA Tube 14

15 I m the man! 15

Enzyme Linked ImmunoSorbent Assay (ELISA) In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind

16 Enzyme Linked ImmunoSorbent Assay (ELISA) In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence. 16

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19 A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form. 19

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Patient Preparation Unique patient preparation requirements are listed under each test in the Test Directory.

Patient Preparation Unique patient preparation requirements are listed under each test in the Test Directory. Specimen Collection Lab results are only as good as the specimen provided. Patient preparation, venipuncture technique, specimen handling, and transportation can all affect the quantity of results. Health