Procedure number: 212
Page 1 of 3
(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-A1 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 30 oC, 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 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 number: 212
Page 2 of 3
(Blood Bank Name and address)
Subject: Antibody Detection Test; Saline “Tube Test” Method
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.
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.
Procedure number: 212
Page 3 of 3
(Blood Bank Name and address)
Subject: Antibody Detection Test; Saline “Tube Test” Method
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.
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