Antiglobulin Test & Antibody Screening

Antiglobulin Test
Antiglobulin test is one of the most important

serological tests done in a routine blood transfusion laboratory. It utilizes the anti-human globulin (AHG) reagent to bring about agglutination of red cells coated with immunoglobulin or complement component, which do not show any agglutination in saline.

Antiglobulin Test
Principle
Mostly incomplete antibodies are IgG which attach to the red cell

membrane by he Fab portion.

The two arm of IgG molecule are unable to bridge the gap between

red cells separated from each other negative charge on their surface. do not form lattice.

Sensitization of the cells occurs but no agglutination seen RBCs

Addition of AHG reagent results in the Fab portion of the AHG

molecule combining with the Fc portion of two adjacent IgG molecules bridging the gap between the red cells and causing agglutination.

Antiglobulin Test

The light-colored antibody molecule represents the anti-globulin reagent that binds with the Fc portion of the IgG antibody attached to the red blood cells

Antiglobulin Test

Complement-coated red blood cells

Antiglobulin Test

The light-colored antibody molecule represents the antiglobulin reagent that binds with the complement attached to the red blood cells.

REAGENT PREPARATION
Polyclonal or monoclonal sources
Polyclonal - Animals hyperimmunized with human globulins; bled

for antisera to obtain high titered, high avidity specificity to human Immunoglobolins

Monoclonal - Hybridoma cells from mice; collection of culture or ascites fluid yields antisera

mice hyperimmunized with human globulins prepare cell suspension from spleens; fuse with immortalized myeloma cells screen hybridoma clones for desirable specificities and affinities maintain cultures of clones in vivo or in vitro Product is highly regulated for potency

Specificity types
Polyspecific Anti-human Globulin blend of

Anti-IgG & Anti-C3b, -C3d Monospecific reagents Anti-IgG alone or AntiC3b,-C3d alone Reagent does not contain antibodies to IgM. Information about IgM coating of cells comes from the presence of C3 coating the cells since IgM is a strong complement activator.

Interpretation of Antiglobulin Tests

Whether the cells have been coated, or sensitized, in vivo or in vitro the

final interpretation is based on the following

Positive Antiglobulin Test

Wash cells three times to remove unbound antibody

Only antibody attached to the cells remain

Add Anti-Human Globulin

Visible Agglutination in the test tube

Grade the reaction strength

Summary of the reaction:

Antigen-antibody reaction, which can take place either

in vivo or in vitro Cells coated with IgG antibody and/or complement Cells washed 3-4X to remove unbound or free antibody or complement The only antibody or complement left is attached to red cells AHG (Coombs serum) added Antibodies in Coombs serum react with antibodies or complement on red cells, causing agglutination If no agglutination add Coombs control reagent cells* (CCC).

Negative Antiglobulin Test

Antibodies are not attached to the antigens during incubation

Wash the cells 3 times to remove any unattached antibodies.

Add Anti-human globulin

Negative Antiglobulin Test

No visible agglutination and therefore a negative test

Add Coombs Control - Check Cells

Check cells agglutinated & original test cells remain unagglutinated.

Check Cells
Coombs Control Agglutinated by Anti-Human Globulin
Coombs Control - Check Cells tell you if you did the test

properly when test is negative

No antigen-antibody reaction occurred. No attachment of antibody or complement to red cells Cells washed three to four times = all plasma or serum antibodies were washed away. Anti-human globulin - Coombs serum added, which would react with antibody-coated cells if present. But no agglutination because no antibodies or complement on red cells for the anti-human globulin to react with

Check Cells
Must add Coombs Control Check Cells to negative

reactions CCC are cells coated with IgG antibody Will react with antibodies in Coombs serum still "floating around" in the tube. Agglutination will now result Agglutination following addition of CCC verifies negative result

False-Negative Reactions
False-negative reactions can occur when antigen-

antibody reactions have occurred but WASHING IS INADEQUATE and free antibody remains when the antihuman globulin is added. Anti-human globulin (Coombs) antibody prefers to react first with free antibody and then with antibody-coated cells If the free antibody has already reacted with the antihuman globulin, no free Coombs serum to react with Coombs Control Check Cells (CCC)

False-Negative Reactions
Inadequate cell washing will lead to unbound antibody

remaining in the red cell suspension that are available to neutralize the AHG (Coombs serum) so it will not react with red cells bound with antibody.
Delay in adding Coombs serum after washing step will

lead to antibody eluting off detaching from cell while cells are sitting in saline. Now free antibody present in the saline neutralizes the AHG, Coombs, serum so it will not be able to react with the cells bound with antibody.

False-Negative Reactions
Small fibrin clot among the cells that were not washed

away will have immunoglobulins & complement present. The antibodies and complement in the fibrin clot neutralizes AHG - Coombs serum leading to a negative test.
Inactive AHG (Coombs serum) or the failure to add

AHG (Coombs serum) will also be detected by a negative reaction when adding Coombs Control Check Cells.

False-Negative Reactions
There are also false negatives NOT detected by negative

Coombs Control Cells that include:

Too heavy cell suspension Delay during cell washing procedure leading to antibody eluting off cells and then the antibody is washed away during the remaining washes Improper centrifugation can either lead to loss of cells during the washing or the need to shake too hard during re suspension.

False positives
False positive reactions can also occur when performing this

test not detected by the use of Coombs Control Check Cells. Reasons for a false positive reaction could be the following: Using improper sample (clotted cells instead of EDTA for Direct Antiglobulin Test - DAT) Spontaneous agglutination (cells heavily coated with IgM) Non-specific agglutination ("sticky cells") All of these reactions would be the result of cells appearing to agglutinate not actually agglutinating.

Types of Antiglobulin Tests

There are two types of antiglobulin tests

- Direct - Indirect

TEST SENSITIVITY
DAT detects about 150 to 500 IgG or C3d molecules/cell IAT detects about 100 to 200 IgG or C3d molecules/cell

FACTORS AFFECTING SENSITIVITY

Ratio of serum to cells (increasing ratio by adding

more serum may increase sensitivity) Temperature (37oC is optimal) Incubation time in saline (30 to 60 min) in LISS (10 to 15 min) Washing must be thorough (else, neutralization of AHG) and rapid (else, elution of bound Abs) Centrifugation (3500 RPM for 20 seconds)

Agents that Enhance Agglutination

22% albumin decreases zeta potential by buffering allows Ab-coated cells to come closer together Low Ionic Strength Solution (LISS) decreases zeta potential Polyethylene glycol (PEG) removes water, concentrating Ab use monospecific AHG with anti-IgG (else, false positives)

Direct Antiglobulin Test (DAT)

Direct antiglobulin test is used to detect in-vivo

sensitization (coating) of red cells with immune antibody or the complement component (C3d or C3c) in:

Diagnosis of haemolytic disease of the newborn (HIDN) Diagnosis of autoimmune haemolytic anemia (AlHA) Investigation of haemolytic transfusion reaction Investigation of drug induced red cell sensitization

Direct Antiglobulin Test (DAT)

Principle
When the blood is drawn the antibodies and/or

complement have already attached to the red cells. Those red cells from the EDTA tube will be washed 3 or more times and a 3% cell suspension is made. A drop of cell suspension and the anti-human globulin are mixed in a tube and then centrifuged. If agglutination occurs it indicates the patient has a positive D A T due to antibody coating the cells in vivo. If IgM antibodies involved DAT will be identified by complement binding since the polyspecific antisera has both anti-IgG & anti-C3.

Direct Antiglobulin Test (DAT)

Technique
Add patient cells from EDTA tube Wash these cells with saline 3-4 X & make a 3% cell suspension. Add a drop of 3% cell suspension to a clean, labeled tube. Add drop of Polyspecific AHG (Coombs serum) to the tube.

If test positive with polyspecific reagent set up again using

monospecific reagents antibody or complement or both coating the cells. To make it more sensitive allow all negatives to incubate 5 minutes to enhance complement coating. Read all negatives microscopically to detect weak coating.

Direct Antiglobulin Test (DAT)

In-vitro complement coating frequently happens when sample

clots or cools down due to weak cold-acting auto-antibodies like anti-I Prevent by using EDTA tube to tie up Ca+ and Mg+ ions and prevent complement activation in vitro. Positive DAT obtain the following information on the patient: Diagnosis AIHA, HDN Medications Recent transfusion history of both red cell and plasma components Other lab values that may indicate red cell destruction (hematocrit, bilirubin, LDH)

Clinical Causes of Positive DAT

Normal person with unexplainable reasons for a

positive DAT
Transfusion reaction work-ups DAT is performed

On post-transfusion specimen patient's antibodies and/or complement may coat the transfused donor cells. These reactions are usually a weak positive or mixed field agglutination testing is done on mixed population of patient & donor cells.

Clinical Causes of Positive DAT

Warm-acting Autoimmune disease

lead to patient antibodies coating their own cells. This results in a strong positive result. Cold-acting autoimmune hemolytic anemia It is due to IgM antibodies that in turn activate complement. The complement-coated cells are detected by the antiglobulin reagent.

Clinical Causes of Positive DAT

Hemolytic disease of the newborn

is due to the mother's IgG antibodies crossing the placenta and coating the antigens on the fetal red blood cells. Cord blood collected at the time of birth would be tested Need to followed up by a heel stick of EDTA blood. The reaction is usually a strong positive.

Clinical Causes of Positive DAT

Complement on the red cells

May be the result of antigen-antibody reactions which may not involve red cells. Complement can also be activated if immune complexes are present in the plasma & the activated complement attaches to the red cells.

Clinical Causes of Positive DAT

Passive transfer of antibody from donor units of plasma or platelets

Attach to the patient's red cells Recipients are given ABO compatible blood other unexpected red cell antibodies may not be detected. These antibodies in donor plasma can coat antigens on patient cells Particularly when group AB, A, or B receive group O plasma products (and possibly platelets)

Clinical Causes of Positive DAT

ABO mismatched transplants

Particularly of bone marrow can occur if an universal "O" donor bone marrow is given to an A, B, or AB recipient. "Passenger lymphocytes" from group O donor organ make antibody to group AB, A, or B recipient cells & can activate complement. It is also more common for "O" individuals to make an IgG anti-A,B, which would also contribute to a positive DAT.

Clinical Causes of Positive DAT

Sensitization of red cells due to medications
Drugs like penicillin and cephalosporins usually

involves non-specific coating of red cells. Other drugs like tetracyclines, antihistamines and sulphonamides cause the development of immune complexes that are capable of activating complement. Some drugs, like ibuproten, levodopa and methyldopa, are also known to cause autoimmunity. If a patient has a positive DAT, drug-induced problems should be considered.

Indirect Antiglobulin Testing

The indirect antiglobulin test is one of the most important

and commonly used techniques in immunohematology. It is used commonly for the detection of: Weak D's in donor units & pregnant females who type D Negative during ABO & Rh typing. The presence or absence of antigens on red cells from particularly the Kell, Kidd, and Duffy Blood Group systems. Unexpected, clinically significant antibodies in the patient's serum during the antibody screening procedure & antibody identification procedure

Indirect Antiglobulin Testing

Principle The purpose of the indirect antiglobulin test is to detect In vitro sensitization of red cells This is done when sensitization does not lead to direct agglutination. This occurs when there are : Too few antigens on the red cell Too few antibodies in the serum Antibodies are in the IgG class

Summary of the Indirect Antiglobulin Technique

Incubate cells with serum at 37oC for the

recommended time. (Usually 15 to 30 minutes.) After incubation wash the cells three to four times. Add AHG Coombs reagent, centrifuge and read for agglutination. If the test is negative add Coombs Control Check Cells to check for false negatives

Indirect Antiglobulin Testing

Uses: Screening Serum for Unexpected Antibodies

Involves patient serum plus reagent red cells (Screening Cells) The patient's serum potentially has unknown antibody. Screening Cells have known antigens for the common clinically significant antibodies. If there is agglutination after Coombs step with either (or both) Screening Cells patient has an unexpected antibody.

Indirect Antiglobulin Testing

Uses:
If antibody screen positive, must do additional tests to

specifically identify antibody The uses for antibody screen are: Testing donor plasma to make sure no unexpected antibodies will be transfused to the recipient. Testing recipient serum before transfusion to make sure patient has no unexpected antibodies to react with donor cells. Testing maternal serum to make sure pregnant mother has no antibodies to react with fetal cells to cause HDN.

Antibody Screening
PRINCIPLE AND APPLICATIONS
The patient's serum is tested for the presence of clinically

significant antibodies using IAT method. The serum is tested against unpooled Group O cells selected to possess the relevant blood group antigens. The antibody screen is: Routinely performed as part of compatibility testing; It is also performed upon request on prenatal patients, As part of an organ transplant workup

Antibody Screening
SAMPLE Patient's serum is tested:

10 ml clotted sample is preferred (4 or 5 ml is acceptable) 2 ml clotted sample is acceptable from a newborn. The sample should be tested when fresh Old or improperly stored samples may lose complement activity & lead to false negatives

Antibody Screening
REAGENTS, EQUIPMENT, AND SUPPLIES

12 x 75 mm test tubes Lighted agglutination viewer Plastic test tube holder Reagent Screening Cells I & II, and III Indelible marking pen PEG Large bore disposable pipettes Anti-Human Globulin (Coombs serum) 37oC waterbath or heat block Coombs Control Cells Serofuge Wash bottle with physiologic saline

Antibody Screening
Screening Cells Characteristics
Screening cells are cells from two or three individual donors. These cells are Group O and must contain the following

antigens: D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb, Jka, Jkb. The most common clinically significant antigens must be present in order to detect the clinically significant antibodies. It is preferred that many antigens as possible be homozygous on the red cells because a double dose of the antigen results in stronger reactions and therefore can detect weaker antibodies.

Antibody Screening
PROCEDURE Verify that patient information on the sample matches information on the worksheet. Centrifuge the sample and separate the serum to a labeled tube. Prepare a washed 3% cell suspension from the patient's cells. Label 3 tubes Patient last name/or #, I Patient last name/or #, II Patient last name/or #, III

Antibody Screening
PROCEDURE (conte.)
Using a large bore pipette, add 2 drops of patient serum to all

tubes. Add one drop of Screening Cell I to tube I; add one drop Screening Cell II to the II tube; add one drop Screening Cell III to the III tube. Shake all tubes to mix and centrifuge at high speed the time appropriate for the saline spin calibration in the serofuge. Gently resuspend and examine macroscopically for agglutination using the lighted agglutination viewer. Record all negative reactions as ; grade positive reactions on a scale of 1+ to 4+.

Antibody Screening
PROCEDURE (cont.)
Again holding the dropper at a consistent angle add 2

drops of BSA (Bovine Serum Albumin) to all tubes. Shake to mix and incubate at 37oC for 30 - 45 minutes. Wash all tubes 4 times decanting well after each wash, mixing the cell button well between washes, blotting the last drop of saline after the final wash.

Antibody Screening
PROCEDURE (cont.)
Immediately add one drop AHG to each tube, shake to mix,

and centrifuge 15 20 seconds Immediately resuspend gently and check macroscopically for agglutination Read and record results as described above on the worksheet. Add one drop Coombs Control Cells to all negative reactions and centrifuge 15-30 seconds in the serofuge. Resuspend and macroscopically examine for agglutination. There must be agglutination in this step or the test is invalid. Record results.


SexRh MNSs P Lewis Kell Duffy Kidd li nk ed X
b

37o

AG C

CC T

P1

Le

Le
a b

K K k p Jsa
a

Fy

Fy
a

Jk
b

Jk
a

Se g
a

x / /

1 2 3

+ + 0

+ 0 0

0 + 0

0 + +

+ 0 +

+ + +

0 0 +

+ + 0

+ + +

+ + +

+ 0 0

0 + +

0 0 +

+ + +

0 0 0

0 0 0

+ + 0

0 + +

+ + 0

+ 0 +

+ + +

Antibody Screening
INTERPRETATION
The lack of agglutination indicates the absence of

antibodies to antigens on the reagent test cells test is reported negative. Agglutination in either Screening Cell tube prior to the addition of Coombs Control Cells indicates the presence of unexpected alloantibodies Test is reported positive and Further testing is necessary to identify the antibody(ies).

Antibody Screening
INTERPRETATION
Agglutination in the autocontrol (AUTO) indicates coating

of cells circulating in the patient. This may be due to an antibody to medications, autoantibody, passively transfused alloantibody, alloantibody coating transfused cells in the patient. Perform a DAT and obtain the patient's medication list, diagnosis and transfusion history

Antibody Screening
NOTES AND PRECAUTIONS

AHG must be added to the cells immediately following

washing. Antibodies may elute from the cells if they are allowed to sit in saline without the addition of AHG. The Coombs Control Cells must be positive at the end of the procedure. Reasons for negative results here include: Inadequate washing of the cells in the Coombs phase. Residual serum neutralizes AHG. A small fibrin clot has formed in the tube, neutralizing AHG.

Antibody Screening

NOTES AND PRECAUTIONS

Weak or questionable reactions may be enhanced by: Increasing the amount of serum used to 3-4 drops. Extend the incubation time to at least 20 minutes. Using enzyme treated cells. Repeat testing using PEG to the test mixture. See manufacturer's directions.

Antibody Screening
NOTES AND PRECAUTIONS

Occasionally, a moderately strong reaction at room

temperature persists weakly in Coombs. This may not be clinically significant if it is due to complement binding at room temperature from a coldreactive antibody. To determine if the reaction is due to complement binding, do a pre-warmed screen

ANTIBODY IDENTIFICATION

Uses a panel of RBCs (type O) of known Ag content to

determine unknown Ab specificity Applications Providing information for donor unit selection for recipients with unexpected Abs Working up a case of HDN Working up a case of AIHA Samples, most reagents (except cells) and protocols usually same as those for Ab screen

SELECTING COMPATIBLE UNITS FOR PATIENT WITH ANTIBODY

If patient has an alloAb, he/she needs units that are

negative for that Ag

Select random ABO/Rh compatible units, perform

compatibility test and, if negative, check units for Ag of interest using known antiserum
How many random donor units will it take to find needed

units? Use known Ag frequencies to determine

Red Cell Antigen Typing

Red cell antigen typing involves patient cells & reagent antiserum.

The patient's cells are the unknown antigen and the reagent antiserum

is the known antibody. The antiglobulin technique is used for antigen typing for weak D clinically significant antibodies like the Kell, Kidd & Duffy antibodies. If there is agglutination after the addition of anti-human globulin patient cells had that specific antigen. Specific procedure varies depending on the antigen being tested brand of antiserum being used Always read and follow directions in product insert carefully

Red Cell Antigen Typing

Uses for red cell antigen typing are:
Typing donors for antigen if patient has antibody units

that are negative for that antigen are required. Verifying that patient is negative for antigen if he/she has made the antibody. Typing patient to see what antigens he/she lacks so can predict what antibodies he/she is capable of making

Red Cell Antigen Typing

Controls
When performing red cell antigen testing always run known

positive and negative controls. This will verify that antiserum is acting properly and helps you interpret your test results. The positive control should be heterozygous for the antigen to ensure antiserum is capable of detecting weaker antigens.