TRANSFUSION-TRANSMISSIBLE INFECTIONS

Transfusion Transmissible Infectious Diseases (TTIDs) are infections that can be transmitted from donor to
recipient through blood or blood components. The primary objective of TTID screening is to ensure that
every unit of collected blood is free from infectious agents, thereby making transfusion safe and risk-free.
Blood collected from donors undergoes mandatory laboratory screening using serological tests, rapid
tests, ELISA, CLIA, and molecular techniques such as NAT before it is released for transfusion.
The microbial agents of importance are those that are transmissible by blood transfusion and can cause
morbidity and mortality in recipients. In order to be transmissible through transfusion, the infectious agent
usually has the following characteristics:
 Presence of the agent in one or more components of blood for long periods and in an infectious form
 Stability at temperatures at which whole blood and blood components are stored
 Generally long incubation period before the appearance of clinical signs and symptoms
 Asymptomatic phase or only mild symptoms in the blood donor, hence not always identifiable during
the blood donor selection process.
Many viruses, bacteria and protozoa can be transmitted by transfusion and new agents that potentially can be
transmitted through transfusion continue to emerge
WHO recommends that, at a minimum, screening of all blood donations should be mandatory for the following
infections and using the following markers:

 HIV-1 and HIV-2: screening for either a combination of HIV antigen-antibody or HIV antibodies
 Hepatitis B: screening for hepatitis B surface antigen (HBsAg)
 Hepatitis C: screening for either a combination of HCV antigen-antibody or HCV antibodies
 Syphilis (Treponema pallidum): screening for specific treponemal antibodies.
 Malaria (Plasmodium Sp.)
The outcomes of the laboratory screening of donations remain the final decision point in the release of blood
components for clinical use; however, even with the high quality assays and systems now available, the
screening process cannot be considered to be totally effective because:
 An infection in donated blood may not be detected due to the collection of the donation during the
window period of infection or failure due to assay sensitivity or error
 There are some emerging infections for which screening is not available or effective
 A donor may be infected with an infectious agent for which donations are not routinely screened; in
such cases, the donor selection process may be able to identify and defer such an individual based on the
symptoms or perceived risk.
Routine screening for generally less clinically significant TTI, such as hepatitis A virus or parvovirus B19 is
generally neither practical nor cost-effective. The screening tests available, if any, may not be appropriate for
blood screening, often being designed primarily to aid the diagnosis of infection in symptomatic individuals. In
these situations, the donor selection process is a significant factor in the identification and deferral of donors
who might harbour these infections in order to prevent them from entering the blood supply.
PURPOSE OF TTID SCREENING
 To prevent transfusion-associated infections.
 To prevent life-threatening complications in immunocompromised, pregnant, or neonatal patients.
 To ensure safe blood supply for the community.
 To reduce window period infection risks by using high-sensitivity molecular testing (NAT).

GENERAL PRINCIPLES OF TTID SCREENING

1. All collected units must be screened before release.
2. Use high-quality, validated kits approved by regulatory authorities.
3. Two-step approach:
o Serological screening (antigen/antibody detection).
o NAT (Nucleic Acid Testing) for HIV, HBV, HCV (reduces window period).
4. Proper sample handling from each blood bag (satellite tube segment).
5. Quality control with positive and negative controls in every run.
6. Documentation of all test results.
7. Donor notification & counseling if the unit tests reactive.
8. Discarding reactive units as per biomedical waste guidelines.

DONOR COUNSELING & MANAGEMENT

(Mandatory Step)
If any TTID test is reactive:

1. Inform donor confidentially

2. Provide pre- and post-test counseling
3. Advise confirmatory tests
4. Refer for medical management
5. Permanently defer from future donations (HIV/HBV/HCV)
6. Maintain privacy & confidentiality

DISPOSAL OF TTID-REACTIVE BLOOD

 All reactive blood components are immediately quarantined.
 Sent for autoclaving and biohazard incineration.
 Proper documentation in TTID register.
COOMBS TEST (Antiglobulin Test)
What is the Coombs test?

The Coombs test — also called the antiglobulin test (AGT) — is a laboratory assay that detects antibodies
and/or complement components that are bound to red blood cell (RBC) membranes but do not, by
themselves, cause visible agglutination.
It uses an antihuman globulin (AHG) reagent that links (“bridges”) Ig molecules or complement fragments on
RBCs to produce visible agglutination. It is the cornerstone test for immune-mediated hemolysis, transfusion
reaction investigation, and pre-/post-transfusion immunohematology.

Why is it needed? — The conceptual problem it solves

 Many clinically important antibodies (especially IgG) coat RBCs in vivo (on the patient’s RBCs) or
sensitize RBCs in vitro but do not cause direct clumping because IgG is small and spaced on the cell
surface — there is insufficient cross-linking to form visible aggregates.
 The Coombs reagent (AHG) contains antibodies against human immunoglobulins and/or
complement. When AHG binds to these Ig/complement molecules already attached to RBCs, it cross-
links the RBCs and produces macroscopically visible agglutination.
 In short: Coombs test reveals “invisible” sensitization of RBCs by using a second antibody to human
Ig/complement.

What is the Coombs test used for?

First, it’s important to note that there are two different types of Coombs tests: direct and indirect.

 Direct Coombs test (sometimes called a direct antiglobulin test) looks at your red blood cells to see if there are
antibodies attached to them. It’s often used to diagnose blood-related conditions such as autoimmune
hemolytic anemia. People with autoimmune hemolytic anemia don’t have enough red blood cells because their
body destroys them too soon.

 Indirect Coombs test tells your provider if there are antibodies present in your bloodstream that could attach to
red blood cells. It’s used as a screening process to see how you’ll react to a blood transfusion. An indirect
Coombs test is also routinely given as part of prenatal testing.

Coombs test during pregnancy and Newborn babies

Your provider will give you an indirect Coombs test if you're pregnant to see if you have antibodies against Rh-positive
blood. For example, if you have an Rh-negative blood type (such as O-, A-, B- or AB-), your red blood cells don’t have the
Rh factor marker on them. But people with Rh-positive blood types do have this specific marker.

If an Rh-negative person is pregnant with an Rh-positive fetus, then Rh sensitization can occur. This means that
your immune system will mistakenly target the Rh factor as a threat and make antibodies to destroy it. It can take a long
time for your body to create antibodies that could harm the fetus. Therefore, during your first pregnancy, the fetus
probably wouldn’t be affected. However, if you get pregnant again with an Rh-positive fetus, the existing antibodies in
your blood could attack its red blood cells. This can result in Rh disease, which is characterized by anemia, jaundice or
other serious health problems. These medical issues usually worsen with every Rh-positive pregnancy you have.

If you’re at risk for Rh sensitization, treatment can almost always prevent this problem. If you’re already
sensitized, then treatment can help protect the fetus. That’s why it’s so important to perform the Coombs test during
your first trimester.
 If your baby is born with jaundice, then your healthcare provider will likely perform a Coombs test. This helps
detect any “foreign” antibodies that have attached to your newborn’s red blood cells.

Immunologic basis / mechanism

1. Sensitization (first step)

o Patient RBCs become coated with human antibodies (commonly IgG) and/or complement fragments
(C3d, C3b) due to immune reactions (alloimmunization or autoantibodies).

o Coating can occur in vivo (e.g., autoimmune hemolysis, HDN, transfusion reaction) or in vitro (patient
serum reacting with reagent RBCs in incubation).

2. Detection (second step)

o The AHG reagent (antihuman IgG ± anti-C3) binds to the Fc portion of human IgG (and/or complement
fragments) attached to RBCs.

o AHG is typically of animal origin (e.g., rabbit IgG) raised against human immunoglobulin/complement
components.

o Binding of multivalent AHG to several IgG molecules on adjacent RBCs creates a lattice visible as
agglutination.

Reagents used

 Antihuman Globulin (AHG) — the essential reagent. There are two common types:
o Polyspecific AHG: contains antibodies to human IgG and complement (usually C3d/C3b).
Used for broad screening.
o Monospecific AHG: contains either anti-IgG or anti-C3d/C3b individually to determine
whether the reaction is due to IgG or complement.
 Coombs control cells (check cells): reagent RBCs coated with a small amount of IgG used to verify
AHG activity when a negative result is obtained (controls for false negatives).
 Wash solution (saline): free of human proteins — used to remove unbound antibodies from RBCs
before adding AHG.
 Saline, incubation media, enhancement media (e.g., LISS or albumin) — used in some testing
formats (note: these relate to modes of performing different Coombs test types).

General workflow concept (high-level, not type-specific)

1. Sample source: RBCs that might be coated (from patient or incubated with serum).
2. Wash RBCs thoroughly with saline to remove unbound antibodies and complement. This is critical —
unbound Ig in suspension will neutralize AHG and cause false negative.
3. Add AHG reagent.
4. Observe for agglutination. Agglutination = positive (RBCs are coated with Ig and/or complement).
5. If negative, add Coombs check cells to the same tube to confirm AHG was active (check cells should
agglutinate; if they do, the negative is true; if not, AHG failure or washing error suspected).

Interpretation (conceptual, without specifying DAT/IAT differences)

 Positive AGT (agglutination observed): RBCs are coated with antibodies and/or complement.
o Clinical implication: immune sensitization of RBCs — potential hemolysis or risk of hemolysis
depending on context.
 o Requires correlation with clinical picture (hemolysis signs, transfusion history, pregnancy
history, drug exposure, etc.) and further testing (identifying antibody specificity, titer,
complement involvement).
 Negative AGT (no agglutination): No detectable human IgG or complement on RBCs, provided AHG
has been validated with check cells.
o If AHG is invalid (check cells fail), result is uninterpretable.

Clinical applications — why Coombs matters

 Investigation of hemolytic anemia: Identifies immune causes (autoimmune hemolytic anemia — warm
or cold types).
 Diagnosis of transfusion reactions: Detects in vivo sensitization of transfused RBCs, confirming
immune hemolysis.
 Hemolytic disease of the fetus and newborn (HDFN): Detects maternal antibodies bound to
fetal/neonatal RBCs.
 Pre-transfusion testing / compatibility workup: Part of antibody investigation and crossmatching
algorithms (indirect antiglobulin concepts).
 Detection of RBC-bound drugs (drug-induced immune hemolysis) — some drugs induce antibodies
that bind RBCs leading to positive Coombs.
 Monitoring therapy and follow-up: A positive or rising DAT may correlate with ongoing immune
hemolysis.

DIRECT ANTIGLOBULIN TEST (DAT/DCT)

Introduction

The Direct Antiglobulin Test (DAT) is a serologic test used in blood banking and immunohematology to detect whether a
patient’s red blood cells (RBCs) are coated in vivo (inside the patient’s body) with immunoglobulins (IgG) and/or
complement components (C3).
It does not detect antibodies in the serum; rather, it identifies antibodies or complement that are already bound to
RBCs circulating inside the body, signifying an ongoing immune-mediated hemolytic process.

Thus, DAT is a direct test of sensitization of RBCs inside the patient, making it essential in diagnosing immune
hemolysis.

Immunological Basis

 In certain immune conditions, the patient’s RBCs become coated with:

o IgG antibodies (warm autoantibodies, alloantibodies)

o Complement (C3) (cold agglutinins, immune complexes)

 These IgG molecules or complement fragments attach to RBC membrane in vivo due to:

o Autoimmune reactions

o Maternal antibodies (in HDN)

o Incompatible transfusion

o Drug-induced immune hemolysis

The amount of coating may be too small to cause visible agglutination, so Anti-human globulin (AHG) reagent is used
to bridge between IgG/complement on neighboring RBCs → visible agglutination occurs.
Principle

1. Patient’s RBCs are washed multiple times to remove unbound antibodies.

2. Anti-human globulin (AHG) reagent is added to the washed RBCs.

3. If IgG or complement is present on RBCs, AHG will bind and cross-link the sensitized RBCs, forming visible
agglutination.

4. Agglutination = Positive DAT (RBCs are coated in vivo).

5. No agglutination = Negative DAT (no significant in vivo coating).

Reagents Used

1. Anti-Human Globulin (AHG) reagent

2. Coombs Control Cells (Check Cells)
3. EDTA anticoagulated whole blood

Procedure (Tube Method) — Step-by-Step

1. Prepare RBC suspension

 Take EDTA blood.

 Wash RBCs 3–4 times with normal saline to remove all free plasma proteins.

2. Add AHG reagent

 Add 2 drops of polyspecific AHG to 1 drop of washed 2–5% RBC suspension.

3. Mix and centrifuge

 Centrifuge at 1000 rpm for 20 seconds (varies by lab protocol).

 Gently resuspend the cell button and examine for agglutination.

4. Read for agglutination

 Grade agglutination from 1+ to 4+.

 Record as positive or negative.

5. Validate negative reactions

 Add Coombs control cells to all negative tubes.

 Centrifuge and check again.

 A valid test must show positive agglutination after adding check cells.

If check cells do not agglutinate, the DAT result is invalid and must be repeated.

Interpretation

Positive DAT

Agglutination seen after AHG addition.

Indicates in vivo coating of RBCs with immunoglobulin and/or complement.

Negative DAT
No agglutination with AHG, but positive check cells ensures test validity.

Indicates no significant antibody or complement attached to RBCs.

Clinical Significance of DAT (Most Important Part)

DAT is positive in conditions where RBCs undergo immune-mediated destruction:

1. Hemolytic Disease of the Newborn (HDN)

 Maternal IgG antibodies cross the placenta and coat fetal RBCs.

 Most common antibodies: Anti-D, Anti-c, Anti-Kell, etc.

 DAT on cord blood confirms immune hemolysis in newborn.

2. Autoimmune Hemolytic Anemia (AIHA)

 Body forms antibodies against its own RBCs.

Warm AIHA

 IgG antibodies

 DAT positive with IgG or IgG+C3.

Cold AIHA

 Caused by IgM cold agglutinins → complement activation

 DAT positive with C3 only.

3. Hemolytic Transfusion Reactions

 When incompatible blood is transfused, patient forms alloantibodies.

 Antibodies bind donor RBCs in vivo → DAT positive.

 Very important test in post-transfusion hemolysis.

4. Drug-induced Hemolytic Anemia

Drugs may cause RBC sensitization by:

 Drug-dependent antibodies (penicillin type)

 Immune complex mechanism (quinidine type)

 Autoantibody induction (methyldopa type)

DAT can be:

 IgG positive

 C3 positive
  Or both

Depending on mechanism.

5. Alloimmune Sensitization in Pregnancy

 Feto-maternal hemorrhage leading to maternal anti-D coating fetal RBCs.

 Confirmed by DAT in fetus/newborn.

6. Hemolysis in Certain Infections

 e.g., Mycoplasma, EBV, malaria → complement coating.

Indirect Antiglobulin Test (IAT)

Introduction

The Indirect Antiglobulin Test (IAT), also known as the Indirect Coombs Test, is a fundamental immunohematological
test used to detect free, unbound IgG antibodies present in a patient’s serum or plasma. These antibodies are capable
of sensitizing red blood cells (RBCs) in vitro, meaning they can bind to RBC antigens when conditions allow.

The IAT works by:

1. Allowing patient serum to react with reagent RBCs possessing specific antigens.

2. If IgG antibodies in the serum bind to these red cell antigens, they remain attached (coated).

3. After washing to remove unbound antibodies, Anti-human globulin (AHG) reagent is added.

4. AHG bridges and agglutinates the IgG-coated RBCs → visible agglutination = positive IAT.

Thus, IAT detects incomplete IgG antibodies that do not cause direct agglutination but can be identified after AHG
addition.

Principle

IAT is based on the fact that IgG antibodies do not directly agglutinate RBCs in saline medium because:

 They are monomeric (small size).

 They do not span the distance between RBCs to cause clumping.

Therefore, sensitized RBCs (coated with IgG or complement) appear as non-agglutinated.

IAT overcomes this by using:

1. Incubation at 37°C → to allow antibody-antigen binding.

2. Washing → removes unbound antibodies that may neutralize AHG.

3. Anti-human globulin (AHG) → bridges the IgG molecules attached to adjacent RBCs and causes visible
agglutination.
Agglutination after AHG addition indicates that the patient’s serum contains IgG antibodies against the antigen
present on test RBCs.

Reagents Used
1. Reagent RBCs
2. Patient Serum/Plasma
3. Enhancement Media (optional)
a. Albumin (22% bovine albumin): Reduces zeta potential.
b. Low Ionic Strength Solution (LISS): Increases the rate of antibody binding.
c. PEG (Polyethylene Glycol): Removes water → concentrates antibodies →
increases sensitivity.
4. AHG (Coombs reagent)
5. Coombs Control Cells (Check Cells)

Detailed Procedure of IAT (Tube Method)

Step 1 – Preparation

 Label tubes appropriately (Screening I, II, III or Panel).

 Add 2 drops of patient serum into each tube.
 Add 1 drop of reagent red cells (screening/panel cells).

Step 2 – Incubation

 Add LISS or other enhancement media (optional but recommended).

 Incubate at 37°C for 15–30 minutes.
This allows IgG antibodies to attach to red cell antigens.

Step 3 – Washing

 Wash the red cells 3–4 times with isotonic saline.

 This is the most critical step.
Unbound IgG antibodies must be completely removed; otherwise, they neutralize AHG.

Step 4 – Addition of AHG

 Add 2 drops of AHG reagent to the washed cell button.

 Mix gently to ensure proper reagent contact.

Step 5 – Centrifugation

 Centrifuge for 15–20 seconds at recommended speed.

 Read immediately for agglutination.

Step 6 – Interpretation
  Agglutination = Positive IAT
Means patient serum contains antibodies that reacted with the test RBCs.
 No agglutination = Negative IAT
Must always add Coombs control cells.

Step 7 – Validating Negative Results

 Add 1 drop Coombs control cells → centrifuge.

 Agglutination must occur.

If not, the test is invalid (faulty AHG reagent, poor washing, or procedural error).

Interpretation of IAT Results

Positive IAT – Agglutination Seen

Indicates IgG antibodies present in the serum against the antigen on test RBCs.

Possible Causes:

1. Clinically significant alloantibodies, such as:

o Anti-D, Anti-C, Anti-E
o Anti-K, Anti-Fya, Anti-Jka
2. Autoantibodies (when using auto-control).
3. Antibodies in pregnancy (maternal IgG crossing placenta).
4. After transfusion (recipient forms antibodies).
5. Delayed hemolytic transfusion reaction.

Negative IAT – No Agglutination

Means no antibody found OR antibody below detectable levels.

Reasons for true negative:

 No unexpected alloantibodies present.

 Antibodies of IgM type (detected by other tests).
 Low-titer/low-affinity antibodies.

Reasons for false negative:

 Inadequate washing.
 AHG neutralized by residual serum.
 Deteriorated AHG reagent.
 Wrong incubation temperature.
Applications of IAT in Blood Banking

1. Antibody Screening

 Detects unexpected antibodies (irregular antibodies).

 Uses 2- or 3-cell screening sets.

2. Antibody Identification

 Using extended antigen-profile panels (11- or 16-cell panels).

 Determines exact antibody specificity.

3. Crossmatching (Compatibility Testing)

 Major crossmatch uses IAT to detect incompatibility between:

o Donor RBCs & recipient serum antibodies
 Prevents hemolytic transfusion reactions.

4. Prenatal Testing

 Detects maternal IgG antibodies capable of crossing placenta → HDN risk.

5. Donor Blood Antibody Screening

 Some donors may have alloantibodies (multiparous women, previously transfused).

6. Red Cell Phenotyping (Indirect AHG Method)

 When testing for weak or low-density antigens.

7. Titers of Alloantibodies

 Anti-D, anti-K etc., in pregnant women.

Crossmatch (Pre-transfusion Compatibility Testing)

Introduction & Purpose

Crossmatching is the final pre-transfusion compatibility test performed to ensure that a specific donor unit is
compatible with a specific recipient. Its purpose is to detect clinically significant antibodies in the recipient’s plasma
that would react with antigens on the donor red cells and to help prevent acute or delayed hemolytic transfusion
reactions.

Key objectives

 Confirm ABO compatibility between donor and recipient.

 Detect alloantibodies in recipient serum that react with donor RBCs.

  Provide a final check (safety net) before issuing blood for transfusion.

Basic Principles

Crossmatch procedures mix recipient serum/plasma (containing antibodies) with donor red cells (bearing
antigens). A reaction (agglutination or hemolysis) indicates incompatibility. There are two conceptual
components:

 Major crossmatch — It involves testing the donor’s red cells with recipient’s serum to determine the
presence of any antibody which may cause hemolysis or agglutination of donor red [Link] is more
important than minor cross match.

recipient serum vs donor RBCs (most important clinically).

 Minor crossmatch — It involves testing of donor’s plasma with recipient’s red cells to determine the
presence of any antibody which may cause hemolysis or agglutination of recipient’s red cells.

donor serum vs recipient RBCs

A valid crossmatch requires prior ABO and Rh typing of donor and recipient, and a recipient antibody screen
(indirect antiglobulin testing / antibody detection). If the recipient has an unexpected antibody, crossmatching is
done using antigen-negative donor units.

Procedure of Cross Matching

There are different methods for cross matching, as shown in table. Among them most commonly used technique is
Anti-human globulin (AHG) cross match.

Major Cross Match

1. Prepare donor and recipient’s blood sample: Donor’s red cells and recipient’s serum/plasma.

2. Prepare 3-5% saline cell suspension of red cells.

3. Label a test tube.

4. Add two drops of recipient’s serum and one drop of donor cell suspension.

5. Mix and incubate the tubes at 37 degree Celsius for about 60 minutes.

6. Decant the serum completely and wash the cells three times in saline.

7. Add two drops of Anti-human Globulin (AHG) and mix. Allow to stand at room temperature for 5 minutes.

8. Centrifuge at 1500 rpm for 1 minute.

9. Observe macroscopically and microscopically for agglutination.

10. If macroscopic agglutination is not observed, transfer a small amount onto a glass slide and examine for
microscopic agglutination. Rouleaux is not an indication of incompatibility.

Minor Cross Match

1. Prepare donor and recipient’s blood sample: Recipient’s red cells and donor’s serum/plasma.
2. Label a test tube.
 3. Add two drops of donor’s serum and one drop of recipient’s cell suspension.
4. Mix and incubate the tubes at 37 degree Celsius for about 60 minutes.
5. Decant the serum completely and wash the cells three times in saline.
6. Add two drops of Anti-human Globulin (AHG) and mix. Allow to stand at room temperature for 5 minutes.
7. Centrifuge at 1500 rpm for 1 minute.
8. Observe macroscopically and microscopically for agglutination.
9. If macroscopic agglutination is not observed, transfer a small amount onto a glass slide and examine for
microscopic agglutination. Rouleaux is not an indication of incompatibility.

Types / Methods of Crossmatch

A. Immediate Spin (IS) Crossmatch — Tube Method (classical)

Purpose: Rapid check for ABO incompatibility before issuing unit (usually used when antibody screen negative).
Materials: Patient serum/plasma, donor packed RBCs (3%–5% suspension), saline, centrifuge, tubes.

Steps

1. Label two tubes: one test tube (patient serum + donor cells) and one control (patient serum + known
compatible cells or saline control per lab protocol).

2. Pipette 2 drops (≈50 µL) of patient serum into test tube.

3. Add 1 drop of donor RBC suspension (3–5%) to the tube.

4. Mix and centrifuge briefly (~1000 rpm for 10–15 seconds) — immediate spin.

5. Gently resuspend the cell button and examine macroscopically for agglutination.

6. Read results: agglutination = incompatible; no agglutination = compatible at IS.

Interpretation

 No agglutination: IS compatible — indicates no ABO or cold-reacting antibodies detectable by IS.

 Agglutination: incompatible — do not transfuse. Perform full IAT crossmatch and antibody identification.

B. Antiglobulin (AHG / IAT) Crossmatch — Tube Method (Full Crossmatch)

Purpose: Detects clinically significant IgG antibodies in patient serum reacting with donor RBC antigens and
complement fixation.

Materials: Patient serum, donor RBCs (3–5% suspension), isotonic saline, incubation (37°C) water bath/incubator,
centrifuge, washing solution, antihuman globulin (AHG) reagent (polyspecific or monospecific), Coombs control cells
(IgG coated cells), test tubes.

Steps (classic tube AHG crossmatch)

1. Label tubes (test and auto/control as required).

2. Add 2 drops (~50 µL) of patient serum to the test tube.

3. Add 1 drop of donor 3–5% RBC suspension.

 4. Mix and incubate at 37°C for 15–60 minutes (common practice: 15–30 minutes depending on lab SOP). This
allows IgG antibodies to bind.

5. After incubation, wash the cells thoroughly (3–4 times) with isotonic saline to remove unbound antibodies.
Washing is critical — residual serum causes false negatives.

6. Add 2 drops of AHG reagent to the washed cell button.

7. Centrifuge (e.g., 1000–1500 rpm for 15–30 seconds depending on SOP).

8. Gently resuspend the cell button and examine for agglutination macroscopically and microscopically.

9. Add Coombs control cells to a separate AHG-negative tube (if AHG tube is negative) to verify AHG reagent
validity. If control cells fail to agglutinate -> invalid AHG result (technical error).

10. Record the result.

Interpretation

 Agglutination at AHG stage: incompatible — clinically significant antibodies present; do not transfuse this
unit.

 No agglutination, valid AHG control positive: compatible — unit can be issued if other requirements satisfied.

C. Gel Column Crossmatch

Principle: Uses microtubes with gel matrix that traps agglutinated RBCs. Standardized, reproducible, and often more
sensitive.

Materials: Gel crossmatch cards (manufacturer reagents), patient serum, donor RBC suspension (0.8–1% depending
on product), centrifuge for gel cards.

Steps

1. Add specified volume of patient serum (or plasma) into the gel microtube.

2. Add donor RBC suspension to the gel microtube.

3. Incubate at 37°C if required by protocol (some are immediate).

4. Centrifuge the gel card per manufacturer instructions (e.g., 10 minutes at specific rpm).

5. Read: RBCs will migrate through gel; agglutinated RBCs remain at top or within gel, while non-agglutinated
cells form a pellet at the bottom.

Interpretation

 Cells at bottom = compatible (negative).

 Cells trapped in gel or at top = incompatible (positive).

STANDARD OPERATING PROCEDURE (SOP): CROSSMATCHING

1. PURPOSE

To detect serologic incompatibility between donor red blood cells and recipient serum/plasma before transfusion.
Crossmatching ensures that no clinically significant antibodies in the recipient will react with donor RBC antigens and
cause hemolysis or shortened RBC survival.

2. SCOPE

This SOP applies to all crossmatch procedures performed in the blood bank, including:

 Immediate Spin Crossmatch (IS)

 Antiglobulin Crossmatch (AHG / IAT Crossmatch)

(Only procedural steps included; theory & types were already explained)

3. RESPONSIBILITIES

 Technical staff / MLTs: Perform crossmatching according to SOP.

 Senior technologist / Officer: Review, validate results, troubleshoot discrepancies.

 Pathologist / Transfusion Medicine Specialist: Authorize release of blood units.

4. MATERIALS & REAGENTS

 Donor red cell segment from blood bag

 Recipient serum or plasma

 LISS (Low Ionic Strength Solution)

 Anti-human globulin (Coombs) reagent

 Normal saline (0.9% NaCl)

 Test tubes (12 × 75 mm)

 Wash bottle

 Centrifuge capable of 1000 rpm

 Pipettes

 Marker pens

 37°C incubator / water bath

 Microscope

 Agglutination grading chart

5. PRE-ANALYTICAL REQUIREMENTS

5.1 Recipient Sample Requirements

 Collect clotted blood or EDTA plasma sample.

 Sample must be properly labeled (name, ID, date, time, collector).

 Check sample age:

o If patient has been transfused or pregnant in last 3 months → valid for 24 hours.

o Otherwise → valid for 72 hours.

 Perform ABO & Rh typing on the recipient.

 Perform antibody screen (IAT) on the recipient.

5.2 Donor Unit Requirements

 Verify integrity of the blood bag.

 Check expiry date.

 Perform ABO & Rh confirmatory typing on the donor.

 Take a segment from donor tubing (never open the main bag).

 Label test tubes with donor unit number.

6. PRINCIPLE

Crossmatching involves combining recipient serum with donor RBCs to detect agglutination or hemolysis.

 IS crossmatch: Detects ABO incompatibility using immediate centrifugation at room temperature.

 IAT crossmatch: Detects IgG antibodies reactive at 37°C and AHG phase.
This SOP covers both.

7. PROCEDURE

A. IMMEDIATE SPIN (IS) CROSSMATCH

(Used when antibody screen is negative & no history of antibodies)

7.1 Preparation of Donor Red Cells

1. Remove ~1–2 drops of donor blood from the tubing segment.

2. Prepare a 2–5% RBC suspension in normal saline.

7.2 Test Setup

Label two tubes:

  Tube 1 – IS Crossmatch

 Tube 2 – Auto Control (optional in IS, mandatory in AHG)

Add in Tube 1:

 2 drops recipient serum/plasma

 1 drop donor 2–5% RBC suspension

7.3 Mixing & Centrifugation

 Mix gently.

 Centrifuge at 1000 rpm for 1 minute.

7.4 Reading

 Gently resuspend the cell button.

 Observe for agglutination or hemolysis.

Interpretation

 No agglutination / no hemolysis → IS Compatible.

 Any agglutination or hemolysis → Incompatible (do not issue).

B. ANTIGLOBULIN CROSSMATCH (IAT / AHG CROSSMATCH)

(Mandatory if antibody screen is positive, or patient has antibody history, or for infants, or when clinically indicated)

7.5 Preparation

Use three tubes:

1. IAT Crossmatch

2. Auto Control (patient serum + patient RBCs)

3. Reagent Control (if using polyspecific AHG)

7.6 Steps

Step 1 — Room Temperature Phase

In the IAT Crossmatch tube:

 Add 2 drops recipient serum

 Add 1 drop donor 2–5% RBC suspension

Mix.
No reading required at this stage (unless performing full three-phase crossmatch).

Step 2 — 37°C Incubation Phase

1. Add 2 drops of LISS (or follow reagent manufacturer’s instructions).

2. Mix well.

3. Incubate at 37°C for 15–30 minutes.

Purpose:

 Encourages IgG antibodies to sensitize RBCs where present.

Step 3 — Washing Phase

After incubation:

1. Centrifuge for 1 minute.

2. Decant supernatant completely.

3. Wash the cell button with normal saline three times.

Washing is critical — even trace serum will neutralize AHG reagent.

After final wash:

 Shake the tube to remove excess saline.

Step 4 — AHG Phase

1. Add 2 drops of Anti-Human Globulin reagent.

2. Mix gently.

3. Centrifuge at 1000 rpm for 1 minute.

4. Resuspend the cell button.

Step 5 — Reading

 Examine for macroscopic agglutination.

 If negative, check microscopically under low power.

Step 6 — Coombs Control Check (CCC)

If the AHG result is negative, add:

 1 drop Coombs control cells (IgG-coated RBCs)

 Centrifuge and read.

Result:

 Must show 1+ or more agglutination → validates negative result.

 If no agglutination → invalid test, repeat entire AHG phase.

7.7 Interpretation (IAT Crossmatch)

 No agglutination → Compatible

 Any agglutination (1+ to 4+) or hemolysis → Incompatible

o Do NOT issue blood

o Investigate antibody, resolve incompatibility

8. GRADING OF AGGLUTINATION

Grade Description

4+ One solid clump

3+ Several large clumps

2+ Many medium clumps; background clear

1+ Small clumps; background cloudy

± Doubtful, very small aggregates

0 No agglutination

Compatibility Testing (Pre-transfusion Testing)

1. Introduction & Purpose
Compatibility testing (crossmatch) is the process performed before blood transfusion to ensure that donor red
cells are compatible with the recipient’s plasma/serum, minimizing the risk of hemolytic transfusion reactions.
It confirms ABO/Rh compatibility, detects clinically significant alloantibodies not revealed by initial screening,
and verifies that the selected unit is safe for transfusion.

Primary goals

 Prevent acute hemolytic transfusion reactions (AHTR).

 Identify incompatible combinations due to unexpected antibodies.
 Provide evidence of compatibility for legal/clinical documentation.
2. Basic Principles
Compatibility depends on:

1. ABO and Rh typing of donor and recipient — absolute requirement.

2. Antibody screen (recipient serum vs screening cells) — detects unexpected alloantibodies.
3. Crossmatch (donor RBCs vs recipient serum) — detects serologic incompatibility from antibodies in
recipient serum reacting with donor RBC antigens.

If antibody screen is negative and no recent transfusion/pregnancy/antibody history, many centres perform an
electronic crossmatch or immediate spin (IS) crossmatch. If antibody screen is positive or history indicates
clinically significant antibodies, perform full antiglobulin crossmatch (AHG, 37°C IAT).

3. Components / Tests Included in Compatibility Testing

 Review of patient ID & clinical details (transfusion history, pregnancy, recent transfusions).
 Verification of two independent blood samples (sample validity).
 Confirmation of ABO/Rh typing (forward and reverse).
 Antibody screen (IAT) using at least a three-cell panel.
 Crossmatch (type chosen according to antibody status):
o Immediate spin (IS) — detects ABO incompatibility
o Full crossmatch (AHG/37°C IAT) — detects IgG antibodies and complement-binding
antibodies
o Electronic crossmatch (computer) — when allowed by policy and prerequisites met
o Gel matrix crossmatch — same principles as tube IAT but using gel cards

4. Pre-requisites & Safety Checks

 Two unique patient identifiers on the sample and request (name + hospital ID + DOB).
 Specimen age: usually collected within 72 hours for transfusion testing (may vary by policy). If >72 h
or patient recently transfused/pregnant/antibody history, draw new sample.
 Sample type: EDTA plasma or serum; some tests prefer serum for AHG testing.
 Blood bank records: previous antibody records must be reviewed.
 Positive patient ID at bedside: for specimen collection.
 Donor unit selection: ensure unit has full identification labels (unit number, ABO/Rh, expiry,
preservative).
 Informed consent per institutional policy.

5. Interpretation & Reporting

 Compatible (No agglutination + Valid Controls): unit may be issued for transfusion. Document:
patient ID, unit number, method, operator, date/time.
 Incompatible: Do NOT transfuse. Actions:
o Repeat ABO/Rh testing on both donor and recipient.
o Repeat antibody screen and identification; perform panel to identify specificity.
o Select antigen-negative or crossmatch compatible units.
o Notify clinician; document adverse findings and investigation steps.
  Invalid test (failed control): repeat testing; do not issue unit until valid.

ANTIBODY SCREENING & IDENTIFICATION

(Detailed, Comprehensive, and Fully Elaborated Explanation)

INTRODUCTION
Red cell antibodies other than ABO antibodies are known as irregular antibodies or unexpected antibodies.
These may be:

 Clinically significant (cause transfusion reactions or HDN)

 Clinically insignificant (cold-reactive, naturally occurring, non-reactive at 37°C)

Antibody Screening & Identification is performed to:

 Detect unexpected antibodies in patient serum/plasma

 Identify the specific antibody (or antibodies)
 Ensure safe transfusion, prevent HDN, and avoid incompatible transfusions

This testing relies on the antigen–antibody reaction using reagent RBC panels with known antigen profiles.

COMPONENTS OF ANTIBODY SCREENING &

IDENTIFICATION
Before going to procedures, understand the components used:

1. Screening Cells
 These are reagent red cells from group O donors to avoid ABO interactions.
 Available as:
o 2-cell panel (minimum required)
o 3-cell panel (preferred)
 Each cell has a pre-printed antigen profile, including Rh, Kell, Kidd, Duffy, MNS, Lewis, P, etc.

Purpose

To detect presence or absence of unexpected antibodies in patient plasma.

2. Antibody Identification Panel
 A panel of 10–16 different group O red cells, each with known antigen composition.
 More detailed than screening cells.
 Each cell’s phenotype sheet (antigen profile) includes:
o Antigens present
o Dosage expression
o Phase of reactivity (RT, 37°C, AHG)

Purpose

To identify which antibody/antibodies are present.

3. Enhancement Media
Used to increase sensitivity of tests:

 LISS (Low Ionic Strength Solution)

 PEG (Polyethylene Glycol)
 Albumin (22% bovine)
 Enzymes (Papain, Ficin)

Each medium enhances or destroys different antigen–antibody reactions.

4. Testing Phases
Antibody detection is performed in three phases, as in IAT:

1. Immediate Spin (IS) — Room Temperature

o Detects cold IgM antibodies
o Example: Anti-M, anti-N, anti-Lea, anti-Leb
2. 37°C Incubation Phase
o Detects warm reactive antibodies
o Example: Anti-Rh, anti-Kell, anti-Kidd
3. Antiglobulin (AHG) Phase
o Detects IgG antibodies
o Most clinically significant antibodies are detected here.

ANTIBODY SCREENING
Purpose
To detect unexpected antibodies in patient serum BEFORE transfusion or pregnancy.

METHOD — STEPWISE PROCEDURE (Tube

Method)
Materials

 Patient serum/plasma
 Screening reagent cells (2 or 3 cell panel)
 LISS or PEG
 AHG reagent (Anti-IgG or polyspecific)
 Centrifuge, test tubes, pipettes
 Coombs control cells

Procedure (Tube Method)

1. Immediate Spin Phase (Room Temperature)

1. Add 1–2 drops of patient serum to a labeled test tube.

2. Add 1 drop of screening cells.
3. Mix and centrifuge immediately.
4. Observe for agglutination or hemolysis.

2. 37°C Incubation Phase

1. Add 2 drops of LISS to enhance reaction.

2. Incubate at 37°C for 10–15 minutes.
3. Centrifuge and examine for agglutination.

3. AHG Phase

1. Wash the tube 3–4 times with normal saline.

2. Add 2 drops of AHG reagent.
3. Centrifuge and observe for agglutination.
4. Add Coombs control cells to negative tubes to validate test.

Interpretation of Screening
Negative Screen
  No agglutination in any phase
 Indicates absence of unexpected clinically significant antibodies

Positive Screen

 Agglutination in one or more phases

 Indicates unexpected antibody present
 Proceed to Antibody Identification Panel

ANTIBODY IDENTIFICATION
When screening is positive, next step is to identify the exact antibody using an antibody identification panel
(10–16 cells).

This is the most critical interpretation skill in Immunohematology.

STEPWISE PROCEDURE (Tube Method)

1. Setup

 Prepare one tube for EACH panel cell (10–16 tubes).

 Add patient serum + reagent cell exactly like screening.

2. Perform all three phases

 IS (cold antibodies)
 37°C (warm antibodies)
 AHG (IgG antibodies)

3. Record Results

Prepare a reaction grid, marking:

 0 (negative)
 1+, 2+, 3+, 4+
for each panel cell at each phase.

INTERPRETATION OF RESULTS (THE

CRITICAL PART)
1. Look for a Pattern
Antibodies show characteristic patterns of reactivity.

Example:

Panel Cell C c E e K Fya S M Result

1 + - + - - - + - 3+

2 - + - + - + - + 0

3 + - - - + - + - 3+

If all cells positive for antigen X show agglutination → antibody against X.

2. Use Rule of Three (3+3 Rule)

For antibody identification to be confirmed:

 Must have 3 antigen-positive cells reacting

 Must have 3 antigen-negative cells showing no reaction

This ensures statistical validity.

3. Check Dosage Effect

Some antibodies react more strongly with homozygous cells:

 Kidd (Jka, Jkb)

 Duffy (Fya, Fyb)
 MNS (M, N, S)

If stronger reactions with homozygous cells → supports identification.

4. Consider Phase of Reactivity

 Cold IgM: react at IS or RT (Anti-M, N, I, P1)

 Warm IgG: react at 37°C and AHG (Anti-D, C, E, K, Jka, Fya)
 Enzyme sensitive/destroyed antibodies help rule out or confirm:
o Destroyed by enzymes: M, N, S, Fya, Fyb
o Enhanced by enzymes: Rh, Kidd, Lewis, P

5. Exclusion
Mark off antigens present on non-reactive panel cells
→ eliminates antibodies that SHOULD HAVE reacted.

6. Confirmatory Testing

 Additional selected cells may be used to confirm multi-antibody combinations

 Enzyme panel may be used
 Phenotype the patient (if not transfused recently)

COMMON CLINICALLY SIGNIFICANT

ANTIBODIES IDENTIFIED
Warm IgG Antibodies (AHG Phase)
 Rh system: Anti-D, C, c, E, e
 Kell system: Anti-K
 Duffy system: Anti-Fya, Fyb
 Kidd system: Anti-Jka, Jkb
 MNS system: Anti-S, s

Cold IgM Antibodies (RT Phase)

 Anti-M, N
 Anti-Lea, Leb
 Anti-I, i
 Anti-P1

CLINICAL IMPORTANCE
1. Prevents Transfusion Reaction

Identifies antibodies that may hemolyze donor RBCs.

2. Prevents HDN

Especially Anti-D, C, E, Kell, Duffy.

3. Prevents Autoantibody Interference

By differentiating autoantibodies from alloantibodies.

4. Safe Component Selection

Allows selection of antigen-negative units for transfusion.