Immunology Solo 2 Lecture 1 Basic Serologic Laboratory Techniques

Serologic Testing

An assay involving serum constituents

Important part of diagnostic testing for viral and bacterial disease

Immunologic Testing

Testing related to antigens and antibodies

Performed in many areas of the lab

Blood specimen preparation

Preanalytical Variables

Specimen types

Delay in testing

Excessive heat

Bacterial contamination

Strong acid or alkali solutions

Lipemia, hemolysis, or icterus

Specimen types

Most immunology assays are done on serum; tube should be allowed to clot before centrifugation occurs; some assays are done on body fluids (cerebrospinal fluid or urine)

Delay in testing

In most cases, immunology testing should be done within 72 hours; if not, serum should be frozen

Excessive heat

Coagulates proteins

Bacterial Contamination

Alters protein molecules

Strong acid or alkali solutions

Must be avoided because these substances have a denaturing effect on serum proteins and make the specimens useless for serologic testing

Lipemia, hemolysis, or icterus

Can make a specimen unacceptable

Inactivation of Complement

Some immunology procedures may require heat inactivation of complement in the serum

Complement in the body fluids can be inactivated by heating at 56 degrees Celsius for 30 minutes

If more than 4 hours has elapsed since inactivation, a specimen can be reheated to 56 degrees Celsius for 10 minutes

Complement may

• Interfere with certain syphilis tests

• Agglutinate latex particles causing a false positive result

• Lyse indicator cells in hemagglutination assays

Manual Pipettes

Pipettes are used to transfer a certain amount of liquid (Sample, reagents, or diluting fluids) while performing certain assays

Graduated Pipettes

Serologic Pipettes

Graduated Pipettes

Delivers the amount of liquid contained between two calibration marks on the pipette

Requires draining between two calibration marks (less accurate)

Serologic Pipettes

Similar to the graduated pipette, but it is calibrated to the tip

Contains a frosted ring at the non-calibrated end

To Contain (TC)

Glassware designed to contain, like graduated cylinders and volumetric flasks, are usually marked with a TC

When liquid is poured from a piece of glassware a small amount remains behind, clinging to the sides of the vessel

A 100ml volumetric flask is designed to hold exactly 100 ml but if the liquid is poured out it will actually deliver a little less than the 100 mL

To Deliver (TD)

Glassware designed to deliver, like pipettes and burets, are marked with a TD

These pieces of glassware account for the small amount of liquid that remains behind

A 100mL pipette contains a little more than 100 mL of liquid, but when the liquid is drained from a pipette exactly 100 mL is delivered

Manual Pipettes: Pipetting Technique

1. Using mechanical suction

2. Wipe off outside of pipette with gauze

3. Adjusting the meniscus

4. Drain into the receiving vessel

Manual Pipettes:

Reading the Meniscus

Manual Pipettes - Step by Step Usage

1. Check the pipette to ascertain its correct size, being careful also to check for broken delivery or suction tips

2. Wearing protective gloves, hold the pipette lightly between the thumb and the last three fingers leaving the index finger free

3. Place the tip of the pipette well below the surface of the liquid to be pipetted

4. Using mechanical suction or an aspirator bulb, carefully draw the liquid up into the pipette until the level of the liquid is well above the calibration mark

5. Quickly cover the suction of the opening at the top of the pipette with the index finger

6. Wipe the outside of the pipette dry with a piece of Kimwipe tissue to remove excess fluid

7. Hold the pipette in a vertical position with the delivery tip of against the inside of the original vessel. Carefully allow the liquid in the pipette to drain by gravity until the bottom of the meniscus is exactly at the calibration mark. To do this, do not entirely remove the index finger from the suction hole end of the pipette; rather, by rolling the finger slightly over the opening, allow slow drainage to take place

8. While still holding the pipette in a vertical position, touch the tip of the pipette to the inside wall of the receiving vessel. Remove the index finger from the top of the pipette to permit free drainage. Remember to keep the pipette in a vertical position for correct drainage. In TD (to deliver) pipettes, a small amount of fluid will remain in the delivery tip

9. To be certain that the drainage is as complete as possible, touch the delivery tip of the pipette to another area on the inside wall of the receiving vessel

Automatic Pipettes

Steps in using piston type automatic micropipette

A: Attaching proper tip size for range of pipette volume and twisting tip as it is pushed onto pipette to give an airtight, continuous seal

B: Holding pipette before use

C: Follow instructions for filling and emptying a pipette tip

If testing cannot be done within ____ hours of collection, a serum specimen should be frozen at -20 degrees Celsius

72 hours

Complement can be inactivated in human serum by heating to ____ degrees Celsius

56

Solution

The mixture of one substance (solute) into another (solvent)

Dilution

Reduces the concentration of the original solute in the solution

Dilutions

To make a dilution, a solvent (also referred to as a diluent) is mixed with a solution to decrease the concentration of the solute

A dilution is the number of parts of the substance in the total number of parts of the final diluted solution

Dilution Examples

If we make a ½ dilution of blood using saline, this would be one part blood and one part saline for a total of two parts:

• 1 mL of blood + 1 mL of saline = 2mL total

A 1/10 dilution would be 1 part blood and 9 parts saline for a total of 10 parts

• 1 mL of blood and 9 mL of saline = 10 mL total

The result obtained from the procedure using a diluted specimen must be multiplied by the dilution factor to get your final result

The dilution factor is the number on the right of the expression; results from a 1/10 dilution would be multiplied by 10 to get the actual result

A patient blood sample is ran on the instrument and reads > 500 mg/dL for glucose. A 1/3 dilution with saline is required in order to get the reading in the dynamic range. How many mL of patient’s blood would be used to prepare a 1/3 dilution in which the total volume is 6 mL

2 mL

A patient blood sample is ran on the instrument and reads > 500 mg/dL for glucose. A 1/3 dilution with saline is required in order to get the reading in the dynamic range. If the analyzer gives a result of 300 mg/dL, how should this patient’s glucose level be reported

900 mg/dL

Serial Dilutions

The stepwise decreasing strength of a substance in solution

Antibody Testing - Testing for Antibody Levels

Acute Phase Specimen

Convalescent Phase Specimen

Acute Phase Specimen

Serum from one with a current infection for which bacterial or viral specific immunoglobulins are measured

Convalescent Phase Specimen

Serum from one who has recovered from an infectious disease and considered to be especially rich in antibodies against the infectious agent

Antibody Testing - Antibody Titer

Refers to the concentration of an antibody

The antibody titer is defined as the reciprocal of the highest dilution of the patient’s serum in which the antibody is still detectable

If serum is being tested for antibody levels with a specific infectious organism:

• One blood sample should be drawn during the acute phase of the illness and

• Another sample drawn during the convalescent phase of the illness

Antibody Titers - Determination of a Titer

Determination of an antibody titer (concentration) for a specific antigen involves two steps

• Preparing a serial dilution of the antibody containing solution (serum)

• Adding an equal volume of antigen suspension to each dilution

High titer indicates that a considerable amount of antibody is present in the serum

For most pathogenic infections, an increase in the patient’s titer of two doubling dilutions, or from a positive result of 1:8 to a positive result of 1:32 over several weeks is an indication of a current infection

This is known as a “four-fold” rise in antibody titer

Streptozyme testing is performed on an initial patient serum dilution of 1:100. Select the statement below that describes the correct dilution procedure

10 uL of patient serum is added to 990 uL of saline diluent

Serum for detection of antibodies should be drawn during the

Acute and convalescent phase of illness