MLT 181 Immunology Chapter 7 Basic Serological Techniques (1)
Chapter 7: Basic Serologic Laboratory Techniques and Clinical Applications
Page 1
Title and Copyright Information
Created by J. Bullard, MPH, MLS (ASCP)
Copyright © 2022, Elsevier Inc.
Page 2: Measurable Student Learning Outcomes
Student Learning Outcomes (SLO):
State basic immunoassay principles in the serology laboratory (Cognitive Level 1)
Perform procedures for testing infectious diseases and immunological disorders (Psychomotor Level 2)
Demonstrate principles of safety and quality assurance in immunology testing (Affective Level 2)
Explain principles and methodologies for serologic diagnosis of infections and immunological disorders (Cognitive Level 1)
Describe standard operating procedures, sample types, glassware, reagents, and test method selection.
Page 3: Building Objectives 1 of 4
At the conclusion of this chapter, readers should be able to:
Identify the parts of a laboratory procedure (Cognitive Level 1)
Describe blood specimen preparation (Cognitive Level 1)
Provide examples of specimen types for immunologic procedures (Cognitive Level 1)
Explain complement inactivation in serum samples (Cognitive Level 2)
Demonstrate pipetting techniques (Cognitive Level 1)
Page 4: Building Objectives 2 of 4
Definitions and Comparisons:
Define dilution (Cognitive Level 1)
Compare acute vs chronic phases of illness (Cognitive Level 3)
Define antibody titer (Cognitive Level 1)
Page 5: Building Objectives 3 of 4
Differentiation and Discussion:
Differentiate testing categories (Cognitive Level 3)
Discuss immunology techniques for rapid testing (HIV, malaria, pregnancy) (Cognitive Level 2)
Describe clinical applications of one POCT assay (Cognitive Level 1)
Analyze case study for assay results interpretation (Cognitive Level 3)
Page 6: Procedures Manual
Description:
Document of current techniques and approved policies available in the laboratory. Must comply with CLSI format.
Page 7: Blood Specimen Preparation
Key Points:
Use an anticoagulated evacuated tube; allow to clot based on protocol
Transfer serum to labeled tube; testing should occur within 72 hours or frozen at -20°C
Heat inactivation may be necessary due to complement interference, which can lead to invalid results.
Page 8: Types of Specimens Tested
Main Points:
Most assays are conducted using serum, but other body fluids may be used.
Lipemia, hemolysis, or bacterial contamination can render specimens unacceptable.
Icteric or turbid serum may be valid for some tests but could interfere with others.
Page 9: Inactivation of Complement
Process:
Inactivation destroys complement activity by heating at 56°C for 30 minutes.
Reactivation possible by reheating if > 4 hours post-inactivation.
Page 10: Specimen Integrity
Description of Gel Use:
Gel density controls separation of blood cells and serum in certain tubes.
Page 11: Pipettes
Types Overview:
Manual Pipettes: Graduated and serologic pipettes.
Semi-Automated Micropipettes for increased efficiency.
Page 12: Manual Pipettes
Features:
TD: Top Capacity mark, to deliver.
Graduated markings for precision in measurement.
Page 13: Automatic Pipettes
Description:
Allow fast, repetitive measurement of solutions with consistent volume,
Include micropipettors, automatic dispensers, and diluter-dispensers.
Page 14: Steps in Using Piston-Type Automatic Micropipette
Method:
Attach proper tip for the pipette
Hold pipette correctly for filling and emptying methods.
Page 15: Diluting Specimens
Definition:
Dilution Factor: Corrects for using diluted samples.
Calculations must multiply result by the reciprocal of the dilution.
Serial Dilutions: Further dilutions in a series (e.g., 1 part sample to 1 part diluent).
Page 16: Serial Dilutions
Process Diagram:
Multiple twofold dilutions illustrated step-by-step.
Page 17: Testing for Antibody Levels
Phases of Specimens:
Acute Phase: Initial disease detection.
Convalescent Phase: Usually assessed about 2 weeks later.
Antibody titer measures the highest dilution at which antibodies are detectable.
Page 18: Interpretation of Antibody Titers
Clinical Significance:
Titer measures evaluate immune status via serial dilutions.
The endpoint dilution expresses the titer as a fraction, with the reciprocal representing the titer value.
Page 19: ASO Antibodies
Characteristics across Varied Timeline:
Peak ASO values and trends over time during infections.
Page 20: Testing Categories
Types Overview:
Waived Tests: Simple and low-risk.
Moderately Complex Tests: Usually automated.
Highly Complex Tests: Require significant judgment and skill.
Provider-Performed Microscopy Tests: Involving slide examinations.
Page 21: Characteristics of Waived Tests
Defined by CLIA 88:
Simple methodologies with negligible error likelihood;
Minimal patient risk if performed incorrectly;
FDA-cleared for home use.
Page 22: Waived Tests Procedures
Requirements:
Identification of tests to be performed,
Procedures for each, confirmatory testing, equipment maintenance, and QC checks.
Page 23: Required Staff Competencies
Competency Assessment must be:
Conducted at orientation and annually.
Include performance on blind specimens and QC monitoring.
Page 24: Malaria Testing
Methods:
Microscopy: Gold standard; Rapid Device Testing implemented in 2007 mandates confirmation by microscopy.
Testing for specific antigens present in infected patients.
Page 28: HIV Testing
Settings for Use:
Employed in clinics and outreach programs; confirmation with 4th-gen immunoassay required due to lower sensitivity of rapid tests.
Page 29: Pregnancy Testing
Development:
Early tests were immunoassays; current tests include solid-phase sandwich immunochromatographic assays for hCG detection.
Page 30: Case Study Summary
Patient Presentation:
9-year-old boy with sore throat - antistreptolysin-O antibody test ordered.
Throat culture confirmed streptococci and positive ASO results; both acute and convalescent serum specimens prepared for titer testing.
Page 31: ASO Test Results Demonstration
Parallel Testing Results:
Acute specimen: Positive at 1:1 dilution; Convalescent: Positive at 1:4 dilution.
Page 32: ASO Questions
Sample Questions:
What did the convalescent specimen demonstrate regarding streptococci infection?
How can comparing acute and chronic specimens aid diagnosis?