Procedure number: 212 Page 1 of 4

(Blood Bank Name and address)

Subject: Antibody Detection Test; Saline “Tube Test” Method

ANTIBODY DETECTION TEST; SALINE "TUBE TEST" METHOD

PURPOSE

To detect unexpected RBC antibodies in patient or donor serum or plasma.

PRINCIPLE

The antibody detection test (“antibody screen”) tests the plasma or serum of a patient or
donor for the presence of unexpected RBC antibodies. This procedure describes a standard
“saline tube" indirect antiglobulin test (IAT) method for antibody detection. This form of
IAT is also used for other purposes in antibody identification including panel testing. The
test includes the addition of RBCs (screening cells”) to the test serum, an incubation
period at 37o, and an anti-human globulin (AHG) test phase.

The sensitivity of the antibody screen is limited by the presence or absence of antigens
present on the screening cells. These cells are selected to detect most clinically
significant antibodies. Since screening cells are group O they will not detect anti-A 1 or
other ABO antibodies.

RBCs selected for the tube IAT are typically from a commercial reagent source, but may be
selected from stocks of rare cells or modified in some way such as enzyme treatment, as
described in other procedures. Incubation of the serum and cell mixture may be
performed at other temperatures selected for the purpose of a specific antibody
identification problem (e.g. a room temperature incubation is added when investigating
cold-reactive antibodies). An autocontrol is advisable when the test is performed as part
of an antibody identification protocol. Selection of such procedural variations is described
in procedure #221, "Investigation of a Positive Antibody Detection Test". As with any
antiglobulin test, after incubation, the test RBCs must be thoroughly washed to prevent
neutralization of the AHG serum.

This sensitivity of the procedure also depends on the serum/cell ratio and incubation time
employed. The procedure below calls for 4 drops of serum or plasma and a 30’ incubation
period at 30oC, but many laboratories would use only 2 drops of serum.

MATERIALS AND SPECIMENS

See General Guidelines for serologic testing, procedure # 201

Patient Blood Bank Record, appendix 210.1 or other patient worksheet.

POLICIES

1. An antibody detection test (“antibody screen”) must be performed on all candidates

for transfusion. In an emergency, blood can be issued prior to completion of the
antibody screen with the approval of the patient’s physician.

2. Antibody screening for pretransfusion testing must be performed within 3 days of

specimen collection.

3. For patients/donors with antibody(ies) demonstrated in a previous workup, the

antibody screen test is performed with a cell panel comprised of RBCs selected to
 Procedure number: 212 Page 2 of 4
(Blood Bank Name and address)
Subject: Antibody Detection Test; Saline “Tube Test” Method

rule out clinically significant antibodies other than those the patient has already
formed, along with one RBC which is antigen positive for each antibody the patient
has already formed (refer to procedure #221 for selection of these cells). If the
antibody(ies) become undetectable, the following study may again start with an
antibody screen.

4. An antibody screen must be performed on all whole blood and platelet donors.

PROCEDURE

1. Label an appropriate number of 10x75 or 12x75 mm test tubes (typically 2 or 3

antibody screening cells).

2. Add 4 drops of the serum or plasma to be tested to each of the labeled test tubes.

Note: many variations of this procedure would use 2 or 3 drops of serum or plasma.

3. Mix the 3-5% RBC suspension well and add one drop to the appropriate test tube (see
procedure #202, "Preparation of Red Blood Cells for Analysis" for preparation of
different red cell suspensions).

4. Centrifuge the test tubes at the proper speed and time for the serofuge in use.

5. After centrifugation, examine each tube for agglutination, and record the reaction(s)
(see below). (See procedure #201 for reaction grading criteria).

6. If a room temperature incubation step is to be added, mix the tubes and incubate for
15 to 30 minutes.

Note: this step should not be used in the routine antibody screen, but only in
antibody identification procedures when cold-reactive antibodies such as anti-M or
anti-P1 are suspected.

7. Centrifuge the tubes, examine for agglutination as before, and record your reactions..

8. Mix tubes and incubate at 37oC for 30 minutes.

9. Centrifuge the tubes, examine for agglutination, and record the reactions.

10. Wash each tube with normal saline 4 times. This may be done manually or with an
automatic cell washer.

11. Add 1 or 2 drops of anti-human globulin (typically 1 drop for monoclonal anti-IgG and
1-2 drops for rabbit anti-IgG) serum to each test tube and mix. Centrifuge at the
appropriate speed and time for the serofuge in use.

12. Examine each tube for agglutination. If macroscopic agglutination is not observed, or
if it is equivocal, examine the tube microscopically for agglutination. Record results
in the AHG column.
 Procedure number: 212 Page 3 of 4
(Blood Bank Name and address)
Subject: Antibody Detection Test; Saline “Tube Test” Method

13. To any tube showing a negative AHG result, add one drop of Coombs Control Cells
(“check cells”).

14. Centrifuge, read, and record results in the "CC" column. Any tube resulting in a
negative reaction after the addition of check cells must be repeated.

15. Interpret the results.

INTERPRETATION

No agglutination or hemolysis of the screening cells at any phase of the test is a negative
test result and indicates the absence of a blood group antibody in the serum or plasma
specimen.

The antibody screen is considered positive if there is agglutination of any of the screening
cells at any phase. A positive antibody screen must be investigated as outlined below. If
this form of IAT was performed as part of antibody identification, the significance of the
result is determined by the particular selection of cells and sera, as well as by the test
phase at which reactivity is observed, as described in procedure #221.
Resolution of a Positive Antibody Screen

Initiate antibody identification as outlined in procedure #221, Investigation of a Positive

Antibody Detection Test. For patients urgently needing transfusion, consult the blood bank
physician immediately. Prior to the serologic resolution, the technologist must:
1. Initiate an Immunohematologic Problem Worksheet with patient identifiers, location,
physician, etc.
2. Call the clinical care unit/hospital of the patient to notify them of the positive result
and to request:
a. Additional samples if needed.
b. Patient diagnosis, transfusion history, and medications and enter on the
worksheet.
c. Information on the urgency of the blood request.
 Procedure number: 212 Page 4 of 4
(Blood Bank Name and address)
Subject: Antibody Detection Test; Saline “Tube Test” Method

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