Overview
Blood transfusion services must screen every donation for transfusion-transmissible infections to protect recipients. Mandatory screening covers HIV-1, HIV-2, Hepatitis B (HBsAg), Hepatitis C, Syphilis, and Malaria where epidemiologically indicated.
This lesson covers the serological basis of these screening tests, especially the ELISA methodology (antiglobulin, competitive, and sandwich types), and the interpretation of markers like HBsAg, anti-HBs, HBeAg and CD4:CD8 ratios in HIV progression.
Learning Objectives
After this lesson you will be able to…- Explain the importance of screening blood donations for transfusion-transmitted infections.
- Describe the infectious agents that can be transmitted by blood transfusion.
- Explain the laboratory diagnosis of HIV, Hepatitis B, Syphilis and Malaria in donor blood.
- Describe the three types of ELISA and their principles.
- Perform and interpret laboratory tests for transfusion-transmitted infections.
Clinical Story
Why This MattersA voluntary blood donor's sample tests reactive for HBsAg on the sandwich ELISA screen. The blood bank technologist must correctly interpret this result, quarantine the unit, and understand that this single positive marker — appearing before symptoms or liver enzyme abnormalities — has just prevented a potential post-transfusion hepatitis B infection in a future recipient.
Core Concepts
All blood donations must be screened for HIV-1 and HIV-2 (antigen-antibody combo or antibody), HBsAg for Hepatitis B, HCV antigen-antibody or antibody, and Treponema pallidum antibodies for syphilis. Malaria screening is based on local epidemiological evidence. Only viruses, bacteria and protozoa are known to be transfusion-transmissible; fungal-infected donors are usually too ill to donate.
p24 core antigen appears shortly after exposure; antibodies to gp41 (envelope) and p24 (core) appear within 14–28 days. There is a "window period" before seroconversion when antigen (p24, gp41) may be detectable but antibody is not. The CD4:CD8 ratio is normally about 2:1; in AIDS, HIV destroys CD4 cells and this ratio decreases. Patients with fewer than 200 CD4+ T cells/µL are considered to have AIDS.
Antiglobulin-type ELISA: viral antibody binds immobilized antigen, detected by enzyme-labeled anti-human antibody. Competitive ELISA: test antibody competes with enzyme-linked specific antibody for antigen binding sites — an intense color signifies a NEGATIVE result. Sandwich ELISA: antibody in test sample is bound between immobilized antigen and enzyme-labeled antigen (antigen-antibody-antigen); an intense color signifies a POSITIVE result — this is highly specific.
HBsAg is the first marker to appear, indicating active infection (acute or chronic); persistence beyond 6 months indicates carrier state. Anti-HBs appears after HBsAg clears, indicating recovery and immunity. IgM anti-HBc marks early acute infection; IgG anti-HBc indicates past or chronic infection. HBeAg indicates active/serious chronic infection, while anti-HBe signals resolution.
Non-specific tests (VDRL, RPR) are used for routine syphilis screening due to simplicity and cost; specific confirmatory tests include TPHA, FTA-ABS and TPI. Transfusion-transmitted syphilis is now rare due to cold storage of blood, voluntary donation and antibiotic use. Malaria screening remains challenging — thick and thin blood smear microscopy is the only practical method, though difficult when parasitemia is low.
Laboratory Principle
ELISA relies on the specific binding between antigen and antibody, visualized through an enzyme-labeled conjugate that converts a colorless substrate into a colored product. The intensity of color (optical density), measured by an ELISA reader against a calculated cut-off value, determines whether a sample is reactive or non-reactive for the target infectious marker.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Viral antigen-coated microplate | HIV/HBsAg/HCV specific | Capture antibody or antigen in test sample | 2–8°C |
| Enzyme conjugate (HRP or ALP linked) | Anti-human globulin or specific antibody | Detect bound antigen/antibody | 2–8°C |
| Chromogenic substrate (TMB) | Ready to use | Color development | Protect from light, 2–8°C |
| Stop solution (1N H₂SO₄) | 1 N | Halts enzyme reaction, fixes color | Room temperature |
Step-by-Step Procedure
Add test serum, positive and negative controls to antigen-coated microwells; incubate at defined time/temperature.
Wash wells at least three times with washing fluid to remove unbound serum, and dry wells thoroughly.
Add enzyme-labeled conjugate and incubate; the conjugate binds only to antigen-antibody complexes already formed.
Wash again to remove unbound conjugate, then add chromogenic substrate and incubate in the dark.
Add diluted acid (1N H₂SO₄) to stop the reaction. Read optical density with an ELISA reader after calculating the cut-off value.
Flow Diagram
Quality Control
Every ELISA plate run must include positive and negative controls to validate the cut-off value calculation. Hemolysed, lipemic or contaminated sera must never be used as they interfere with color development.
Blood banks should participate in national external quality assessment schemes (EQAS) for HIV, HBsAg, HCV and syphilis testing to ensure accuracy and comparability across laboratories.
Reference Values
Normal Ranges⚠️ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| HBsAg reactive | Active hepatitis B infection (acute or chronic) | Quarantine unit; confirmatory testing; counsel donor |
| Anti-HBs positive, HBsAg negative | Recovered from infection / immune | Unit may be considered safe per protocol |
| Reactive HIV antibody screen | Possible HIV exposure/infection | Confirmatory Western Blot / repeat testing; counsel donor |
Common Errors & How to Avoid Them
Cause: Antibody screening cannot detect infection before seroconversion (window period).
Prevention: Combine antigen-antibody combo assays and thorough donor history/deferral criteria to reduce residual risk.
Cause: Residual unbound conjugate or serum causes false-positive color development.
Prevention: Wash wells the specified number of times and ensure they are dry before adding the next reagent.
Cause: Confusing color interpretation rules — in competitive ELISA, intense color = negative; in sandwich ELISA, intense color = positive.
Prevention: Always confirm which ELISA format the kit uses before interpreting results.
Laboratory Tips from the Bench
Always follow the manufacturer's kit insert precisely — incubation time, temperature and reagent volumes are validated for that specific kit's cut-off calculation.
Particle agglutination assays are useful in resource-limited settings since they need no expensive equipment and can be read visually.
"Sandwich = Signal; Competitive = Quiet" — in sandwich ELISA more color means positive; in competitive ELISA more color means negative.
Important Notes
Since routine screening of donor blood for malaria parasites is impractical, chemoprophylaxis is recommended for recipients in highly endemic areas.
p24 antigen testing is mainly useful for detecting window-period infection, confirming infection in infants of HIV-positive mothers, and resolving indeterminate Western Blot results.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your KnowledgeAsymptomatic donor presenting for routine voluntary blood donation. No known risk factors reported on donor questionnaire.
A reactive HBsAg result, even in an asymptomatic donor, indicates active hepatitis B infection which could be acute or chronic. The unit must be quarantined and the donor referred for confirmatory testing and clinical evaluation.
- →Donors can be asymptomatic carriers of transfusion-transmissible infections.
- →A reactive screening result always requires quarantine and confirmatory testing.
- →Donor questionnaires alone cannot exclude infection risk — laboratory screening is essential.
Frequently Asked Questions
No practical, sensitive rapid method exists for screening malaria parasites in donor blood at low parasitemia. Microscopy is the only practical method but is time-consuming and insensitive at low parasite density, so chemoprophylaxis of recipients is recommended in endemic areas instead.
Non-specific tests like VDRL can be positive due to viral infections, immunization, lupus erythematosus and dysproteinemias, even without active spirochete infection — hence confirmatory specific tests are needed.
First generation assays use native virus/cell lysates; second generation uses recombinant antigen from cloned viral genes; third generation uses synthetic viral polypeptides — each generation improving specificity and reducing background reactivity.
Quick Revision
10-Minute ReviewKey Takeaways
- Screening blood donations protects recipients from transfusion-transmitted infections.
- HIV, Hepatitis B, Hepatitis C and Syphilis screening is mandatory in blood banks.
- ELISA is the most widely used screening method, available in antiglobulin, competitive and sandwich formats.
- Serological markers like HBsAg, anti-HBs and HBeAg guide interpretation of Hepatitis B infection status.
- Malaria screening relies mainly on microscopy due to lack of a sensitive rapid test.
- The window period is a critical limitation of antibody-based screening tests.
Competency Checklist
Track Your MasteryReferences
- NIOS Hematology and Blood Bank Technique — Lesson 21: Screening for Blood Transfusion Transmitted Diseases.
- WHO. Screening Donated Blood for Transfusion-Transmissible Infections.