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

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.

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)

Techniques Fingerprick (Skin Puncture)

Techniques Fingerprick (Skin Puncture)

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

Techniques - Venipuncture

Techniques - Venipuncture

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.

Techniques - Venipuncture

Techniques - Venipuncture

Techniques - Venipuncture

Techniques - Venipuncture

Techniques - Venipuncture

Bevel near or in vein wall Hematoma formed

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).

Techniques IV Catheter

Basics of Blood Analysis

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.

Basics of Blood Analysis

Enzymatic Assay (Spectrophotometry) (contd)
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.

Basics of Blood Analysis

Basics of Blood Analysis

Basics of Blood Analysis

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.

Basics of Blood Analysis

RIA Tube

Basics of Blood Analysis

Blood RIA Tube

Basics of Blood Analysis

Basics of Blood Analysis

Im the man!

Basics of Blood Analysis

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.

Basics of Blood Analysis

Basics of Blood Analysis

Basics of Blood Analysis

Basics of Blood Analysis

Basics of Blood Analysis

Basics of Blood Analysis

Basics of Blood Analysis

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.

Basics of Blood Analysis