Basic Serologic Laboratory: Techniques and Clinical Applications ( LECT )

Serologic testing

is crucial for diagnosing viral and bacterial infections in clinical laboratories. It encompasses various immunologic tests across fields such as microbiology, cytopathology, and immunohematology.

Procedures Manual

• A complete document of current techniques & approved policies

• Must be accessible at all times in the lab bench area

• Serves as a guide and reference for lab personnel.

• Ensures standardized procedures

• Promotes accuracy and safety

• Must be reviewed periodically by all personnel

What is the importance of the. Manual?

• Collect blood in a plain evacuated tube (no anticoagulant)

• Allow blood to clot naturally

• Remove serum promptly after clotting

What are the Blood Specimen Preparations?

a plain evacuated tube

Blood should be collected in?,

~ 1 hour

complete retraction occurs at what time?

Applicator stick

Loosen clot from sides using?

10 minutes

Tube should be centrifuge for?

Pasteur pipette

Used to transfer serum

Recentrifuge

What to do if serum is contaminated with RBCs

coagulates proteins

Excessive heat can cause?

Alters proteins

Bacterial contamination can cause?

freeze at -20°C

It not tested in 72 hours specimen should be?

SERUM

MAIN SPECIMEN:

• Lipemia (fat in blood)

• Hemolysis (RBC rupture)

• Bacterial contamination

Problems that make serum unacceptable:

URINE

Used for Pregnancy tests (hCG detection) and Urinary tract infection testing

destroying complement activity

Inactivation can cause

Complement inactivation

Required for certain immunology tests

• Syphilis tests

• Latex agglutination assays

• Hemagglutination assays

Complement can interfere with:

Syphilis

Can cause false results

Latex agglutination assays

Cause False positive due to particle clumping

Hemagglutination assays

Lysis of indicator cells

56 °C for 30 minutes

Serum should be heated at.

10minutes

>4 hours have passed since inactivation -> complement activity may return and should be heated again for ?

Traditional glass pipettes

• Less common, but sometimes still needed

Automatic pipettes (semiautomated)

Widely used in labs today

Allow fast, repetitive measurements, Deliver equal volumes consistently

Sampling-diluting pipette

Micropipettors

• Two main types of automatic pipettes

Sampling-diluting pipette

measures sample + adds diluent

Micropipettors

- very small, deliver precise volumes

Automatic or semiautomatic pipetting devices

• Used for:

• Rapid, repetitive measurements

Methods for Dilution

Different dilution methods may be employed, such as single dilutions or serial dilutions.

Single dilutions

involve a simple one-step dilution

Multiply the original concentration × dilution 1 x dilution 2 x

General Rule To calculate the final dilution

Antibody Titers

measures amount of a specific type of antibody present in the patients plasma

is important in diagnosing infectious diseases.

Acute serum

early stage

Convalescent serum

later stage

Serial dilution

each dilution made from the previous one

Used to determine antibody concentration.

endpoint dilution

= where antibody is last detected.

High titer

• high antibody concentration.

fourfold rise in titer

A sign of active infection.

Titers

Checking antibody levels in the blood to see if they are adequate against a specific disease

Pregnancy

Testing

First rapid POCT test

• Detects hCG (human chorionic gonadotropin)

Precipitation

Soluble antigen + soluble antibody • insoluble visible complex.

Agglutination

Particulate antigen (cells, beads) + specific antibody • clumping or aggregation.

Affinity

= strength of one antibody binding site to one antigen site.

• binding site (Fab) and one antigen epitope.

• Held by weak bonds at very short distances.

• Depends on specific fit

Avidity

overall binding strength when multiple sites interact.

• Sum of all affinities between multiple binding sites.

• Measures stability of the antigen-antibody complex.

• High avidity can compensate for low affinity.

Precipitation Curve Overview

The amount of precipitate depends on the ratio of antigen to antibody.

• Zone of Antibody Excess

• Zone of Equivalence

• Zone of Antigen Excess

Three main zones:

Prozone Phenomenon

• Too much antibody

• Antigen binds only to one/few antibody sites • no cross-linking

• Many free antibodies left in solution

• Result: False-negative possible

Zone of Equivalence

A reaction in which the optimum concentration of antigen and antibody is present

Postzone Phenomenon

• Too much antigen

• All antibody sites saturated with single antigen molecules

• No lattice network formed

• Result: False-negative possible

Prozone fix

dilute antibody and retest

Postzone fix

. : repeat test later (allow more antibody production)

Turbidimetry

The measurement of light transmitted through a suspension of particles

Nephelometry

Measures light scattered at an angle (not straight through).

• The more complexes, the more light is scattered.

Rate Nephelometry

• Measures how fast scattering increases after antibody is added.

• Antibody kept constant - rate depends on antigen concentration.

• Immunoglobulins (IgG, IgA, IgM, IgE)

• Kappa & Lambda light chains

• Complement proteins

• C-reactive protein (CRP), haptoglobin, ceruloplasmin

Rate Nephelometry Used to measure

Radial Immunodiffusion

• Single-diffusion technique.

• Antibody is mixed uniformly in agarose gel.

Precipitation ring forms at zone of equivalence.

• Immunoglobulins (IgG, IgA subclasses, . IgM, IgE)

• Complement components

Radial Immunodiffusion Used to measure?

Ouchterlony Double Diffusion

• A classic immunodiffusion technique.

• Both antigen and antibody diffuse in two dimensions (horizontally & vertically).

• Uses agarose gel with wells cut into it.

Immunoelectrophoresis

separation of molecules by electric charge.

Immunoelectrophoresis (Classic Method)

A double diffusion technique + electrophoresis.

Immunofixation Electrophoresis (Modern Method)

• Patient serum applied to 6 lanes.

Compare reactions in 5 lanes to reference.

IgM

• Large, pentameric -> strong lattice formation

• Best reaction at 4-27°C

ABO antibodies (IgM) = agglutinate at room temp

IgG

• Smaller, less flexible -> weaker|

• Best reaction at 30-37°C

• Responsible for clinically significant blood group reactions

Direct Agglutination

detecting antibodies by visible clumping

Passive Agglutination

• Uses carrier particles (latex, RBCs, gelatin, synthetic beads)|

• Antigen is coated onto the particle

Reverse Passive Agglutination

Antibody rather than antigen is attached to a carrier particle