University of Arkansas for Medical Sciences

4301 W Markham
Little Rock, AR 72205

Department: Transfusion Services Effective Date: 10-31-2016

Procedure Name: Use of Thiol Reagents (DTT) Replaces Procedure Dated: 04/04/2011
Procedure Number: 237.2 Retired:

Scope: Blood Bank

Purpose/Principle:
Dithiothreitol (DTT) disrupts the disulphide bonds between cysteine amino acid residues. These bonds
maintain the antigen structure necessary for recognition by the corresponding antibody. This treatment
may aid in the identification of certain antibodies. Kell , LW, and Scianna blood groups are known to be
destroyed by DTT treatment.

DTT can also be used to distinguish IgM from IgG antibodies and to disperse autoagglutination.

Definition:
DTT-dithiothreitol
PBS-phosphate buffered saline

Equipment List/Reagents:
1. Normal saline
2. DTT
3. 37ºC incubator
4. Phosphate buffered saline (PBS)
5. Pipettes
6. Test tubes

Specimen Required: NA

Procedure:
I. DTT treatment of RBC’s using 0.2M DTT
A. Wash RBCs to be treated once in Phosphate buffered saline
B. To one volume of washed, packed RBCs, add 4 volumes 0.2 M DTT.
C. Mix well and incubate at 37C for 30-45 minutes.
D. Wash RBCs 4 times with Phosphate buffered saline and resuspend to 3-5%
E. Test treated RBCs with plasma containing antibody.

II. Use of DTT to distinguish IgM from IgG Antibodies using 0.01M DTT
A. Label two tubes, test and dilutional control, respectively.
B. Dispense 1 ml serum or plasma into two test tubes.
C. Add 1 ml PBS to dilutional control.
D. Add 1 ml 0.01 M DTT to test.
E. Mix and incubate at 37°C for 30-60 minutes.
F. Plasma may now be used for desired procedures. IgM antibody should no longer be
reactive. Dilutional control should remain positive.
Document control/Master-Blood Bank procedures Page 1 of 2
237-Use of Thiol Reagents (DTT)
Manual: Vol. IV TS237.2 version: 07/2006, 04/2011, 10/2016
 University of Arkansas for Medical Sciences
4301 W Markham
Little Rock, AR 72205

III. Use of DTT to disperse autoagglutination using 0.01 M DTT.

A. Dilute RBC’s to 50% in PBS.
B. Add equal volume of 0.01 M DTT.
C. Incubate at 37C for 15 minutes.
D. Wash cells 3 times in normal saline, resuspend, and dilute to 2-5%.
E. Test as needed.

Calculations:
Preparation of 0.2 M DTT:
Dissolve 1 gram DTT into 32 ml PBS. Aliquot into 2 ml samples and store at -20ºC or below.

Preparation of 0.01 M DTT:

Dissolve 0.154 gram of DTT in 100 mL PBS. Aliquot into 1 ml samples and store at -20°C.

Results: NA

Limitations/Notes:
Controls should be run with each DTT treatment of RBC.
1. Kell positive cell should be treated and tested with Kell antisera. No reactivity should be observed.
2. D positive cell should be treated and tested with D antisera. Reactivity should be observed.

Record Retention: 10 years

Distribution: Blood Bank

References: 1, 2, 3

Document control/Master-Blood Bank procedures Page 2 of 2

237-Use of Thiol Reagents (DTT)
Manual: Vol. IV TS237.2 version: 07/2006, 04/2011, 10/2016