Exeley Inc.

Kisiel, Anna

Jęckowska, Katarzyna

Polish

CHITINASES AS THE KEY TO THE INTERACTION BETWEEN PLANTS AND MICROORGANISMS

Xiao, Feng

Sun, Hao

English

Analysis and Design of Image Segmentation Algorithm Based on Super-pixel and Graph Cut

Bub, C.B.

170 IMMUNOHEMATOLOGY, Volume 33, Number 4, 2017
Dithiothreitol treatment of red blood cells
C.B. Bub
Dithiothreitol (DTT), a reducing reagent, has multiple 
applications in blood bank testing. DTT disrupts the bridging of 
the disulfide bonds between amino acid residues necessary for 
structural conformation of some proteins and the bonds holding 
an IgM molecule in the pentameric formation. DTT treatment 
of red blood cells (RBCs) can denature or modify certain blood 
group antigens—in particular, those in the Kell, Lutheran, YT, 
JMH, LW, Cromer, Indian, Dombrock, and Knops systems—and 
prevent recognition by the corresponding antibodies. It also 
destroys RBC CD38, allowing DTT-treated RBCs to be used to 
avoid testing interference by therapeutic anti-CD38 preparations. 
DTT treatment can be used to disperse spontaneous agglutination 
of RBCs caused by heavy IgM autoantibody coating that 
invalidates ABO/Rh cell grouping and direct antiglobulin tests. 
Immunohematology 2017;33:170–172.
Key Words: dithiothreitol (DTT), indirect antiglobulin test, 
Kell system, daratumumab
Principle
Dithiothreitol (DTT) is a reducing agent capable of 
irreversibly cleaving accessible disulfide bonds when the 
solution pH is >7. DTT treatment of red blood cells (RBCs) 
will modify the tertiary structure of protein-based erythrocyte 
membrane antigens if their confirmation depends on disulfide 
bonds.1 Antigens of the following blood group systems are 
destroyed or weakened by 0.2 M DTT treatment: KEL, IN, 
JMH, YT, LU, MER2, KN, DO, CROM, and LW.2,3 Antibodies 
directed at antigens in these systems will not react or will be 
significantly weaker with the treated RBCs.
DTT will also cleave the disulfide bonds that connect 
the monomeric subunits and the J chain of the IgM antibody 
pentameric form. When heavy coating of RBCs with IgM 
autoantibody causes spontaneous agglutination, DTT 
treatment will disrupt the IgM structure and disperse the 
agglutination. 
Indications
DTT-treated reagent RBCs can be used in antibody 
identification to suggest the possible blood group specificity 
of an unidentified antibody based on whether reactivity is 
affected by the treatment. When a known antibody is directed 
at a DTT-sensitive antigen, treated RBCs may be used to detect 
or exclude underlying antibodies to DTT-resistant antigens. 
Treatment of autologous RBCs with 0.01 M DTT can resolve 
spontaneous agglutination due to potent IgM autoantibodies 
that interfere with ABO/Rh and direct antiglobulin testing.4
Monoclonal anti-CD38 (daratumumab), approved by the 
U.S. Food and Drug Administration for treatment of multiple 
myeloma, targets the CD38 antigen on malignant plasma cells. 
CD38 is also weakly expressed on all normal RBCs, including 
those in RBC reagents used in pretransfusion testing. 
This expression complicates the identification of clinically 
significant RBC antibodies because the plasma/serum of such 
patients will react with most or all RBCs in antibody detection 
Treatment to Destroy RBC Antigens
Reagents/Supplies
Reagents Supplies
• PBS, pH 7.3
• 0.2 M DTT
• RBC test cell
• K+ RBCs (control cell)
• Anti-K (reagent or serum/plasma 
specimen)
• Test tubes
• Pipettes
• Centrifuge
• pH meter
• Water bath or 37°C 
incubator
RBC = red blood cell; PBS = phosphate-buffered saline;  
DTT = dithiothreitol.
Procedural Steps
0.2 M DTT Preparation
• Dissolve 1 g DTT in 32 mL PBS, pH 8.0. Adjust final pH of DTT to 
8.0 with 0.1 M HCl/0.1 M NaOH as needed. 
• Aliquot and store at –20°C or colder for up to 12 months.
Procedure
• Wash 1 volume of the test RBCs and control RBCs with PBS, 
pH 7.3. Decant.
• Add 4 volumes of 0.2 M DTT, pH 8.0.
• Incubate at 37°C for 30 minutes.
• Wash three to four times with PBS.
• Resuspend the cells to a 2–5 percent suspension in PBS.
• Test DTT-treated cells with serum containing the antibody in 
question. Test K+ treated RBCs with anti-K.
DTT = dithiothreitol; PBS = phosphate-buffered saline; RBCs = red 
blood cells.
S
E
R
O
L
O
G
IC
 M
E
T
H
O
D
 R
E
V
IE
W
IMMUNOHEMATOLOGY, Volume 33, Number 4, 2017 171
and identification tests performed by the indirect antiglobulin 
test.5 Because CD38 configuration depends on disulfide bonds, 
DTT treatment of reagent RBCs is used to denature the red 
cell CD38 and avoid this interference.6 A large volume of RBCs 
can be treated with DTT and stored in Alsever’s solution, since 
the stability of common RBC antigens has been demonstrated 
for up to 14 days.7 Serologic investigations within this time 
period are more efficiently performed when pretreated cells 
are available. 
Procedure to Destroy RBC Antigens 
In a properly labeled tube, place 1 volume of the patient's 
packed RBCs (approximately 50 μL). Wash the RBCs three 
times with phosphate-buffered saline (PBS), pH 7.3. Decant 
the supernatant completely after the last wash. Add 4 volumes 
of 0.2 M DTT (approximately 200 μL) to 1 volume of RBCs 
to be treated. Mix well, and incubate at 37°C for 30 minutes. 
Wash the RBCs four times with PBS. If marked hemolysis 
occurs, repeat the procedure with fresh RBCs and a smaller 
volume of DTT. Resuspend the RBCs to a 2–5 percent 
suspension if the testing is performed in the tube or to the 
concentration required for non-tube test methods. Assess the 
batch for completeness of treatment using anti-K as described 
in the quality control section. If the reactivity of the test serum 
is eliminated, appropriate RBC samples can be treated and 
tested to exclude other clinically significant alloantibodies.
Procedure to Disperse Spontaneous Agglutination
In a properly labeled tube, wash autologous RBCs three 
times in saline. Washing with warm saline can also aid in 
removing cold-reacting IgM autoantibodies. Dilute washed 
RBCs to a 50 percent suspension in PBS. Add an equal volume 
of 0.01 M DTT. Mix and incubate at 37°C for 15 minutes. 
Wash treated RBCs at least three times in PBS. Resuspend 
an aliquot of the RBCs to a 2–5 percent suspension or to the 
concentration required by the test method being performed. 
Test the RBCs for removal of the spontaneous agglutination as 
described in the quality control section.
Limitations
As in all serologic procedures, factors such as contaminated 
materials or inadequate incubation time, temperature, or 
centrifugation may produce false results. Preparation of the 
0.2 M DTT at pH 8.0 is required for irreversible denaturation 
of antigens.2 Reagent preparations at a lower pH may cause 
inadequate and reversible reduction of disulfide bonds. Treated 
RBCs cannot be typed for any antigens destroyed by DTT.
Antibodies to antigens destroyed or weakened by DTT 
cannot be excluded on nonreactive treated RBCs. Because 
anti-K is the most frequently encountered clinically important 
antibody in this group, K– RBCs should be selected for 
transfusion if the patient’s RBCs are K– or the K antigen status 
is unknown, unless anti-K has been excluded in other tests. 
Quality Control
When performing treatment to destroy RBC antigens, 
K+ RBCs should be DTT-treated with each test batch. The 
treated and untreated K+ RBCs are then tested with anti-K. 
The treated RBCs should be nonreactive; otherwise, the DTT 
treatment was not adequate. Other antigens of the Kell system 
can also serve as controls.1 
If dispersal of spontaneous agglutination is being 
performed, test the treated RBCs using 6 percent albumin. No 
agglutination should be present if the treatment was successful.
DTT-modified RBCs
Treatment to Disperse Spontaneous 
Agglutination
Reagents/Supplies
Reagents Supplies
• PBS, pH 7.3
• 0.2 M DTT
• 6% albumin
• Test tubes
• Pipettes
• Centrifuge
• Water bath or 37°C 
incubator
PBS = phosphate-buffered saline; DTT = dithiothreitol.
Procedural Steps
0.01 M DTT Preparation
• Dilute 1 volume 0.2 M DTT with 19 volumes PBS, pH 7.3.
Procedure
• Wash autologous RBCs at least three times with PBS and dilute to 
a 50% concentration in PBS.
• Add an equal volume of 0.01 M DTT to the RBC suspension.
• Incubate at 37°C for 15 minutes.
• Wash RBCs three to four times in PBS.
• Resuspend the RBCs to a 2–5 percent suspension.
• Test the treated RBCs with 6% albumin (immediate-spin test) to 
assess for dispersal of spontaneous agglutination.
DTT = dithiothreitol; PBS = phosphate-buffered saline; RBCs = red 
blood cells.
172 IMMUNOHEMATOLOGY, Volume 33, Number 4, 2017
C.B. Bub
Summary
DTT treatment of RBCs is an informative method to aid 
in antibody identification and to determine whether a serum 
contains additional alloantibodies when an antibody to a 
DTT-sensitive antigen is present. It also destroys CD38 on the 
RBC surface, thereby avoiding interference from anti-CD38 
in pretransfusion testing. Donor units typed as K– must be 
transfused when antibody detection/identification has been 
performed using DTT-treated RBCs, unless anti-K is excluded 
by another method.
Spontaneous agglutination of autologous RBCs causing 
false-positive results in ABO/Rh and direct antiglobulin 
testing can be resolved by using DTT to degrade the IgM 
antibody coating the RBC.
References
 1. Fung MK, Grossman BJ, Hillyer CD, Westhoff CM. Technical 
manual. 19th ed. Bethesda, MD: American Association of 
Blood Banks, 2017: Method 3–18.
 2. Branch DR, Muensch HA, Sy Siok Hian S, Petz LD. Disulfide 
bonds are a requirement for Kell and Cartwright (Yta) blood 
group antigen integrity. Br J Haematol 1983;54:573–8.
 3. Reid ME, Lomas-Francis C, Olsson M. The blood group antigen 
factsbook. 3rd ed. London:Elsevier Academic Press, 2012.
 4. Fung MK, Grossman BJ, Hillyer CD, Westhoff CM. Eds. 
Technical manual. 19th ed. Bethesda, MD: American 
Association of Blood Banks, 2017: Method 2–18.
 5. Murphy MF, Dumont LJ, Greinacher A. Interference of new 
drugs with compatibility testing for blood transfusion. N Engl 
J Med 2016;375:295–6.
 6. Chapuy CI, Aguad MD, Nicholson RT, Aubuchon JP, Cohn CS, 
Delaney M, et al. International validation of a dithiothreitol 
(DTT)-based method to resolve the daratumumab 
interference with blood compatibility testing. Transfusion 
2016;56:2964–72.
 7. Disbro WL. Stability guidelines for dithiothreitol-treated red 
blood cell reagents used for antibody detection methods in 
patients treated with daratumumab. Immunohematology 
2017;33:105–9.
Carolina Bonet Bub, MD, PhD, Hematology and Hemotherapy 
Physician, Hematology and Cell Therapy Department, Hospital 
Israelita Albert Einstein, 627 – 3º andar, Bloco E, CEP: 05651-901 
Sao Paulo, SP, Brazil.
The editorial staff of Immunohematology welcomes manuscripts 
pertaining to blood group serology and molecular genetics for 
consideration for publication. We are especially interested in 
review articles, case reports, papers on platelet and white cell 
serology, scientific articles covering original investigations or new 
blood group alleles, papers on molecular testing, and papers on 
new methods for use in the blood bank. To obtain instructions for 
submitting scientific articles, case reports, and review articles, 
see Instructions for Authors in every issue of Immunohematology 
or e-mail a request to immuno@redcross.org. Include fax and 
phone numbers and e-mail address with all manuscripts 
and correspondence. E-mail all manuscripts to immuno@
redcross.org.
Manuscripts
Notice to Readers
Immunohematology is printed on acid-free paper.
For information concerning Immunohematology or the 
Immunohematology Methods and Procedures manual, 
contact us by e-mail at immuno@redcross.org.


Dithiothreitol treatment of red blood cells

BARTNICKA, Małgorzata

ADAPTATION OF FLATS IN CONCRETE SLAB BUILDINGS FOR PEOPLE WITH DISABILITIES. ISSUES, COSTS, REIMBURSEMENT OPTIONS, COMPARISONS

HASANZADEH, Ali

NUMERICAL ANALYSIS OF THE RESPONSE OF PILE-RAFT SYSTEMS CONSIDERING THE APPLICATION OF CEMENT AND POLYPROPYLENE FIBER TREATMENT

Jędrzejczak, Joanna

Majkowska-Zwolińska, Beata

Ryglewicz, Danuta

Nagańska, Ewa

Mazurkiewicz-Bełdzińska, Maria

Recommendations of the Polish Society of Epileptology for the treatment of epileptic seizure in adult patients in Poland: an update

Kopka, Marcin

Cannabinoids in the treatment of epilepsy – an updated review

Shuping, Xu

Shuang, Wang

yu, Guo

Xiaohui, Su

Research of Network Closed-loop Control System Based on the Model Predictive Control

Benson, Stuart

Cable, Gordon

Workman, Lorna

CHALLENGES IN ANAESTHESIA DURING SPACE EXPLORATION MISSIONS

Pędraszewski, Piotr

Wlaźlak, Edyta

Wlaźlak, Wiktor

Krzycka, Magda

Pająk, Paulina

Surkont, Grzegorz

The role of TVT position in relation to the pubic symphysis in eliminating the symptoms of stress urinary incontinence and urethral funneling

Not a member yet