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Topic 2:

Phlebotomy and Basic Blood Analysis

HPHE 6720Dr. Cheatham

Laboratory Manual Section 09

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Outline

What is phlebotomy Review of Universal Precautions Phlebotomy Techniques

Fingerprick Venipuncture IV catheter

Basics of blood analysis Enzymatic reactions (spectrophotometer) ELISA RIA

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Universal Precautions

Refers to the practice of avoiding contact with patients'bodily fluids, by means of the wearing of nonporousarticles such as medical gloves, goggles, and face shields.

Medical instruments, especially scalpels and hypodermicneedles should be handled carefully and disposed of properly in a sharps container.

Under Universal Precautions all patients are consideredto be possible carriers of blood-borne pathogens. Theguideline recommends wearing gloves when collectingor handling blood and body fluids contaminated withblood and wearing face shields when there is danger of blood splashing on mucous membranes and whendisposing of all needles and sharp objects in puncture-

resistant containers.

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Universal Precautions

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Techniques Fingerprick (Skin Puncture)

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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

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Supplies: Gauze, alcohol pads, tourniquet,

Needle holder Needles

The higher the gauge, the smaller theneedle.

Typically 22 to 25 G is used. Vacutainers

Different colored vacutainers havedifferent anti-coagulants (or none at all).

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

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The difference between plasma and serum Plasma

Clear watery component of blood Is collected in a tube that has some form of anti-

coagulant 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.

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Bevel near or in

vein wallHematoma formed

Needle passedthrough vein Vein collapsed

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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).

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Techniques IV Catheter

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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 tomeasure.

This chemical reaction typically results in a colorchange in the chemical reagent.

The amount of this color change is measured by thespectrophotometer.

The more color change, the more of the compound in theblood that you are measuring.

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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 bloodcompound.

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Radioimmunoassay (RIA) To perform a radioimmunoassay, a known quantity of an antigen is made

radioactive, frequently by labeling it with gamma-radioactive isotopes of iodineattached 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 theserum to compete with the radiolabeled antigen for antibody binding sites.

As the concentration of "cold" antigen is increased, more of it binds to theantibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen.

The bound antigens are then separated from the unbound ones, and theradioactivity of the free antigen remaining in the supernatant is measured. Abinding curve can then be plotted, and the exact amount of antigen in thepatient's serum can be determined.

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RIA Tube

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RIA Tube

Blood

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Im theman!

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Enzyme-Linked ImmunoSorbent Assay (ELISA) In simple terms, in ELISA an unknown amount of

antigen is affixed to a surface, and then a specificantibody 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 convertto some detectable signal.

Thus in the case of fluorescence ELISA, when light of theappropriate wavelength is shone upon the sample, anyantigen/antibody complexes will fluoresce so that theamount of antigen in the sample can be inferredthrough the magnitude of the fluorescence.

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A sandwich ELISA . (1) Plate is coated with a capture antibody; (2) sample is added, andany antigen present binds to capture antibody; (3) detecting antibody is added, and bindsto 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.

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