Immunology Serological Testing

Define plasma

Liquid portion of blood that contains water, plasma proteins, nutrients, electrolytes, hormones and coagulation factors

Define Serum

Liquid portion of blood MINUS clotting factors

Describe the difference between plasma isolation and serum isolation

1. Plasma isolation

-Collect blood in antigcoagulated tubes (EDTA, heparin)

-Centrifuge

2. Serum Isolation

-Collect blood in tube with NO anitcoagulant

-Incubate sample until blood fractionation

How do we store serum if testing delay?

1. Between 2-8degrees C up to 72 hours

2. Frozen at 20 degrees C or below

How do we inactivate complement for testing?

Heat sample to 56 degrees C for 30 mins

What are the two acceptable serum tubes?

Red and yellow

(Plain tube and Gel tube)

=No anticoagulants

TD (to deliver) pipette:

Dispense an exact volume

TC (to contain) pipette:

Hold a specific volume but do not deliver that exact volume

Volumetric pipette:

Delivers only one volume

Graduated pipette:

Marked at regular increments to allow varying amounts

Micropippetes:

Deliver small volumes

Define dilution:

The process of decreasing the concentration of solutes in solution by adding more diluent

Solute:

SUbstance dissolved in a solution

Diluent:

The liquid being added to dilute the solute

How to calculate concentration:

C= Amount of solute/volume of solution

What is the dilution formula:

C1V1 = C2V2

You have a 100% stock hemolysin reagent. You need 10 mL of a

5% solution for a complement fixation test.

• How much stock reagent do you use?

• How much diluent (DI-water) do you add?

1. 0.5 ml

2. 9.5 ml

Describe Serial dilutions:

Dilution factor is the same between tubes in a series

-Each tube contains 1/n the concentration of the solute as previous tube

What can serial dilutions be often used for?

Antibody titer

-Reciprocal of the last dilution in the series that shows antigen-antibody reaction

Define Diagnostic Sensitivity:

How good a test is detecting disease when present

Define Diagnostic Specificity:

How good a test is at ruling out diseases when its absent

Which combination would describe the ideal diagnostic test?

High sensitivity and High specificity

What does Negative predictive value tell us?

The probability that a negative test result truly means no disease

What does Positive predictive value tell us?

The probability that a positive test result truly means disease present

What is the formula for sensitivity?

Use the Has disease (+) column

%= True positive/ True positive + False Negative

*Detects disease when present

What is the formula for specificity?

Use Does not have disease column (-)

%= True negative/ True negative + false positive

*Detects when disease is truly absent

A test with high sensitivity will cause:

Few false negative

What is the memory hook SnOUT?

Highly sensitive test helps rule out disease

A test with high specificity means?

Very few false positive

What is the memory hook SpIN?

A highly specific test helps rule disease in

Sensitivity and specificity have what kind of relationship?

Trade off

High sensitivity =

Few false negative but more false positives

High specificity =

Few false positive but more false negatives

What is the formula for PPV?

%= TP/TP + FP x100

What is the formula for NPV?

%= TN/TN + FN

Serial dilutions are used to determine?

Titer or strength of antibody