1. Cold Agglutinin Screen vs Cold Agglutinin Titer
What is the financial impact of implementing a cold agglutinin screen for the diagnosis of cold agglutinin disease?
Penny Szklarski, MLT
Vanderbilt University Medical Center
I wanted to undertake this research project for a few reasons. The first being my own personal career growth. When empty nest syndrome set in, I realized that I had a little extra time on my hands, so I started looking for a project and I came up with this.

2. Healthcare Costs
In the ever changing financial environment of healthcare, we are all looking for ways to cut costs. In April 2012, Vanderbilt transfusion medicine implemented a cold agglutinin screen for the diagnosis of cold agglutinin disease. The screen includes an IgG and C3d DAT and saline antibody screens with autocontrol at room temperature and 30c. After the screen was implemented there did not seem to be much change in the ordering practices of clinicians and titers were still being ordered for diagnosis; therefore we decided to take a closer look at what was being ordered.

3. Anemia
The World Health Organization defines anemia as a hemoglobin level less than 13 g/dl in men and less than 12 g/dl in premenopausal, nonpregnant women.
AABB Technical Manual – 18th Edition
There are basically only 3 causes of anemia; blood loss, increased RBC destruction (hemolysis) and decreased production of RBCs. Each of these causes include a number of etiologies that require specific and appropriate therapy.

4. Autoimmune Hemolytic Anemia (AIHA)
Warm AIHA
Cold agglutinin disease
Mixed-type AIHA
Paroxysmal cold hemoglobinuria
AABB Technical Manual – 18th Edition
Autoimmune hemolytic anemia is the shortening of red cell survival as a result of an immune-mediated response. There are 4 subtypes of autoimmune hemolytic anemias. Cold agglutinin disease is one of those.

5. What is Cold Agglutinin Disease?
Cold Agglutinin Disease is rare and accounts for 15%
of patients with autoimmune
hemolytic anemia (AIHA)
Blood, 15 August Volume 122, Number 7
Cold agglutinin disease is rare and accounts for 15% of patients with autoimmune hemolytic anemia. It can be primary (idiopathic) or secondary (related to an underlying condition).

6. The IgM molecule is large enough to span the distance between the RBCs and overcome the natural repulsive forces between the cells. When blood shifts toward the peripheral circulation and cools, IgM transiently binds the RBC membrane.

7. Bound IgM Molecule
Once bound, the IgM molecule activates the complement cascade, binding C3b to the cell surface. As the coated cells return toward the core body, IgM dissociates and the cells lose the surface membrane by receptor specific macrophages present in the liver and the spleen, resulting in extravascular hemolysis and perhaps some degree of intravascular hemolysis. The severity depends on the thermal amplitude rather than the serum concentration of IgM.

8. Polyclonal Antibodies Monoclonal Antibodies
Typically seen in post infectious setting
Most commonly seen in pediatrics
Mycoplasma
Long term disease
Often resists treatment
May be associated with an underlying lymphoproliferative disorder
Seen in geriatrics
Blood, 15 August 2013 – Volume 122, Number 7
The cold agglutinin IgM molecules can be polyclonal or monoclonal. Polyclonal antibodies are typically seen in the post infectious setting (mycoplasma, EBV, influenza, it can be viral or bacterial) and are self resolving. Polyclonal IgM antibodies are most commonly seen in children.
Monoclonal IgM antibodies are seen in older adults. It is long term and often resists treatment. It may be associated with an underlying lymphoproliferative disorder. The monoclonal IgM antibodies are directed against I/i carbohydrate antigens on the RBC surface.

9. Wright Stain Blood Smears
Normal
Cold Agglutinin Disease

10. Wright Stain Blood Smears
Normal
Cold Agglutinin Disease

11. Specimen tubes from a patient with cold agglutinin disease
Notice the macroscopic agglutination of the red cells in the tube on the left. The plasma is quite icteric indicating that hemolysis has occurred.
Photo complements of
Dr. Garrett Booth, Vanderbilt
University Medical Center

12. Primary Study Hypothesis
Was the assumption correct that providers were ordering cold agglutinin titers instead of cold agglutinin screens?
It seemed that even with the implementation of the cold agglutinin screen for diagnosing cold agglutinin disease, providers were still ordering titers independent of the screen for diagnostic purposes. Was this truly the case?

13. Research Methodology
Institutional Review Board (IRB) approval was obtained.
Retrospective chart review was performed
Titers from 1/1/2010 to 10/31/2013
Screens from 4/2012 to 10/31/2013
Cold agglutinin titers of >1:64 were considered clinically significant
I performed a IRB approved retrospective chart review identifying all cold agglutinin titers from 1/1/2010 to 10/31/2013 and all cold agglutinin screens since implementation through 10/31/2013. The electronic medical record of each patient was reviewed for diagnosis, age and preliminary testing. Titers of >1:64 were considered clinically significant.

14. Limitations Ordering provider’s field of expertise
Small sample size due to the rarity of the disease
There were some limitations with this study. Due to the rarity of the disease, the sample size was quite small even though the time period was almost 3 years. Also, the ordering provider’s field of expertise seem to be a factor in the study.

15. What is the most cost effective blood bank test for the diagnosis of cold agglutinin disease?

16. Cold agglutinin titer Titers are performed at 4°C
Low titers of cold agglutinins can be found in normal individuals
Titers are performed at 4C only. Since low titers of cold agglutinins can be found normally, a titer alone would not give a definitive diagnosis.

17. How common is cold agglutinins in blood donors?
A recent study revealed that 5% of donors tested for cold agglutinins had titers of >1:4
Retrospective review of 276 healthy donors
(2014 Transfusion, 54:
A recent study published in Transfusion in May 2014 demonstrated that 5% of the 276 healthy donors tested had titers of >1:4 up to 1:32 in a retrospective review.

18. Cold agglutinin screen
IgG and C3d Direct Antiglobulin Test (DAT)
Room temperature and 30°C saline antibody screens with auto controls
Thermal amplitude if positive
*Immune Hemolytic Anemias – Second Edition Lawrence D. Petz, MD George Garratty, PhD
The cold agglutinin screen we implemented at Vanderbilt was developed using Immune Hemolytic Anemias – Second Edition authored by Dr. Lawrence Petz and Dr. George Garratty. The screen includes DAT (IgG and C3d) as well as room temperature and 30C saline antibody screens with auto controls. Thermal amplitude testing is performed on all positive screens. If you remember, the severity is determined by the thermal amplitude and not the serum concentration of the IgM antibody.

19. Diagnostic for Cold Agglutinin Disease
70% of all clinically significant cold agglutinins react at 37°C
100% of all clinically significant cold agglutinins react at 30°C
Positive C3D and Negative IgG DAT
Positive RT and 30°C saline antibody screen with positive auto controls
Immune Hemolytic Anemias-Second Edition , Dr .Lawrence Petz and Dr. George Garratty
Cold agglutinin diagnosis is strongly supported when there is positive C3d and negative IgG DAT, positive room temperature and positive 30C saline antibody screen with positive auto controls. Testing at 30C has been found to correlate well with cold agglutinin disease as 70% of all clinically significant cold agglutinins react at 37C but 100% of all clinically significant cold agglutinins react at 30C.

20. Results: Cold Agglutinin Titer
80 Titers were ordered prior to inception of the screen
5 (6.3%) were clinically significant
2 were diagnosed with Stevens-Johnson Syndrome
1 was diagnosed with B-Cell Lymphoma
2 were newly diagnosed with Cold Agglutinin Disease
68 (85%) had a negative C3d
My findings showed 80 cold agglutinin titers ordered prior to the inception of the screen, of which, 5 (6.3%) were clinically significant. 2 were diagnosed with Stevens-Johnson Syndrome, 1 with B-cell lymphoma and 2 newly diagnosed cold agglutinin disease. 68 (85%) of the 80 ordered titers had a negative C3d.

21. Why is a negative C3d significant?
Cold agglutinin disease is strongly supported with a positive C3d and a negative IgG DAT
Why is a negative C3d significant? Remember a diagnosis of cold agglutinin disease is strongly supported when there is a positive C3d and a negative IgG DAT. That is not to say that a patient can not have cold agglutinin disease with a negative C3d because there is always someone who does not want to follow the established rules, but it is highly unlikely. That means that 85% of those titers were most likely ordered unnecessarily.

22. Results: Cold Agglutinin Screen
17 Cold agglutinin screens were ordered.
7 (41%) were positive requiring thermal amplitude
3 (17.6%) diagnosed with cold agglutinin disease
1 diagnosed with Systemic Lupus Erythematosus
1 diagnosed with Myelofibrosis
2 were outpatients and lost to follow-up
The cold agglutinin screen includes a C3d and IgG DAT, thus eliminating the need for the added cost of a titer if the screen is negative. Since the implementation of the screen, there were 17 screens ordered. Reflexively, thermal amplitude testing is performed on all positive screens. Of the 17 screens, 7 (41%) needed the extended workup with thermal amplitude. Ultimately, 3 (17.6%) were diagnosed with cold agglutinin disease, one with lupus, one with myleofibrosis and 2 were outpatients and were lostnto follow-up.

23. Since implementation of the cold agglutinin screen.
38 Titers were ordered independent of the screen
3 (7.8%) were true positive with titers of >1:64
Since the implementation of the screen, 38 titers were ordered independent of the screen, of which, 3 (7.8%) were true positives with titers of >1:64 and all were previously diagnosed with cold agglutinin disease.

24. What are the financial implications of ordering titers instead of screens for diagnosis?

25. Cost Analysis Cold Agglutinin Titer Cold Agglutinin Screen
Consumables
Cost per Unit
Units
Total
10X75 Tubes $ 11 $
Screen Cell 1 $ 10 $
Total Consumables $
Salary and Benefits
Hrly Rate
Hrs
Tech Time $
0.35 $
Total Cost per Test $
Consumables
Cost per Unit
Units
Total
10X75 Tubes $ 1 $
12x75 Tubes $ $
MTS Gel Card $ $
C3d Reagent $ $
Total Consumables $
Salary and Benefits
Hrly Rate
Hrs
Tech Time $
0.48 $
Total Cost per Test $
The cost at our facility for a cold agglutinin titer is $16.06/titer and a cold agglutinin screen is $17.32/ screen. This includes consumables, reagents and labor. It seems a titer would be more cost effective as it cost $1.26 less than the screen, but a titer alone will not result in a definitive diagnosis as low titers of cold agglutinins may exist in normal, healthy individuals.

26. Positive Pretest Probability
6.3% using the titer
17.6% using the screen
The study demonstrated the pretest probability went from 6.3% using the cold agglutinin titer to 17.6% using the cold agglutinin screen, meaning there is a much higher success rate of diagnosis with the screen as opposed to using the shot in the dark titer approach.

27. Cost of inappropriately ordered tests
38 Independently ordered titers
35 (92.1%) rendered no new cold agglutinin disease diagnosis at a cost of $562.10
Of the 38 independently ordered titers since the implementation of the screen, 35 (92.1%) revealed no new diagnosis of cold agglutinin disease at a cost of $ The 3 true positive titers were appropriately ordered for monitoring of previously diagnosed cold agglutinin disease. The only true need for ordering a titer alone would be to monitor patients previously diagnosed with cold agglutinin disease.

28. Conclusion Cold agglutinin disease is rare
Knowledge of correct test for diagnosis is essential
Inappropriate testing cost money
Inappropriate testing takes time away from more important projects
Cold agglutinin disease is rare and can be difficult to diagnose. Educating clinicians as to the best diagnostic test to order could save money and time, improving patient care. Inappropriately ordered tests take time away that would be of more use on other projects.

29. Acknowledgements
Garrett Booth, M.D., Associate Medical Director Allison Paroskie, M.D, Associate Medical Director Emily Coberly, M.D., Transfusion Medicine Fellow Stephanie Sephel, MLS, SBB, Transfusion Medicine Manager Mary Johnson, MLS, SBB, Transfusion Medicine Supervisor The entire Vanderbilt Transfusion Medicine Staff

30. Thank You!