October 2021 – Cellular Therapy

A 60-year-old man with a past medical history of AL amyloidosis presented to the therapeutic apheresis treatment unit for collection of an autologous peripheral blood stem cell transplant after five days of mobilization with G-CSF and plerixafor for a peripheral CD34+ of <10 cells/µL. As part of his pre-transplant evaluation, it was noted that he had an asymptomatic cold agglutinin titer of >512. When collected, his day one product “gelled” and did not separate on centrifugation (Figure 1).

Figure 1
Figure 2

What is the most likely problem, and what further steps could be performed to prevent subsequent processing issues?

  • Potential product contamination. Wash product and filter through 170 µm filter prior to cryopreservation. 
  • Cold agglutination of peripheral blood stem cell product. Warm human albumin replacement of plasma in collection and consideration of therapeutic plasma exchange to reduce CAD titer.
  • Elevated neutrophil fraction in product resulting in increased cell free DNA levels resulting in clumping/gelling. Consider treatment with DNase prior to cryopreservation. 
  • Clotting from processing error without heparin addition to product bag. Add anticoagulant and attempt manual massage to break up clots.

The correct answer is ...

Cold agglutination of peripheral blood stem cell product. Warm human albumin replacement of plasma in collection and consideration of therapeutic plasma exchange to reduce CAD titer.

This is a case of cold agglutination of a peripheral blood stem cell product. Cold agglutinins (CA) are common, but rarely present symptomatically due to the low temperatures required for red cell binding. The thermal amplitude is the temperature range at which cold agglutinins will agglutinate and typically falls within 2-4° C. In the setting of cold agglutinin disease (CAD), the thermal amplitude can reach or exceed body temp (28-30° C) leading to clinical symptoms such as acrocyanosis, Raynaud’s and hemolysis. CAs are often seen with a titer of ≥64 during serial dilutions, but symptomatic disease is more frequently seen at a tier of ≥512.

While uncommon, elevated neutrophil fractions with lysis of these cells can result in significant elevations in cell free DNA. This elevated free DNA can cause clumping of the stem cell product, which may cause infusion issues after thawing. In this circumstance, with fresh cells, the collected cells would not be expected to have lysed to such a degree as to cause this phenomenon. Lack of addition of the anticoagulant is a potential processing error that can lead to clotting within a product bag. Normally, red cell agglutinins will be seen, which may break up with addition of anticoagulant after the fact. Lastly, product contamination is always a concern during collection of a product. No information from the clinical vignette points us to a processing error resulting in lack of anticoagulant or introduction of a contaminant during collection. If such concerns were raised, all of these manipulations are reasonable to successfully cryopreserve the collected stem cell product. Given the clinical vignette, the best answer is cold agglutination resulting from the patient’s known CAs.

CAs are predominately pentameric IgM molecules and exist within the intravascular space. Due to the structure of the autoantibody, complement fixation can occur in vivo with frank intravascular hemolysis occurring (Figure 2). Onset of this disease is often insidious; however, unlike the intravascular hemolysis seen with an incompatible red blood cell transfusion. Instead, chronic hemolysis ensues, resulting in hemosiderinuria and complement consumption/deficiency. The specificity (of the antibody) is variable, but most frequently target the “I” antigen; a highly expressed antigen on adult red blood cells starting at 18 to 24 months of age. Bona fide CAD is now recognized as a unique entity associated with a lymphoproliferative disorder with monoclonal lymphocytes.

The presence of CAs during stem cell collection and processing can lead to complications arising from agglutination at cold temperatures below body temperature. In these settings, special manipulations can be done to improve the donation experience and stem cell product processing. The ambient temperature of the room for collection can be raised to improve potential peripheral symptoms in the donor. Additionally, an inline blood warmer can be placed both on the draw and return line of the apheresis machine to prevent agglutination in the machine and post centrifugation prior to reinfusion to the donor. In the most symptomatic of patients/donors, a preemptive therapeutic plasma exchange can be done to reduce circulating cold agglutinins prior to collection. Lastly, special consideration should be made with infusion of these stem cell products. Due to agglutinations at low temperature, these products should be warmed to a higher temperature prior to infusion to prevent complement fixation when entering the intravascular space after thawing.


  • Linz, Walter J. et al. Cold agglutinin disease. Transfusion. Vol. 43,9 (2003):1185. doi:10.1046/j.1537-2995.2003.00502.x
  • Thompson, T.Z., Larsen, R., Savage, N.M., Shikle, J. 2017 A Case of Mixed-Type Autoimmune Hemolytic Anemia with a Complex Autoantibody Specificity including a Ficin Sensitive Cold Agglutinin and a Warm Autoanti-e Treated with Therapeutic Plasma Exchange. College of American Pathologists. Washington, DC.
  • Sigbjørn Berentsen; How I treat cold agglutinin disease. Blood 2021; 137 (10): 1295–1303. doi: https://doi.org/10.1182/blood.2019003809
  • Berentsen S. Cold agglutinin disease. Hematology Am Soc Hematol Educ Program. 2016;2016(1):226-231. doi:10.1182/asheducation-2016.1.226
Thomas Thompson Profile picture square

Thomas (Zach) Thompson, M.D.

Fellow, Cellular Therapy
Mayo Clinic

Margaret DiGuardo profile photo square

Margaret (Maggie) DiGuardo, M.D.

Senior Associate Consultant, Transfusion Medicine
Mayo Clinic

MCL Education (@mmledu)

MCL Education

This post was developed by our Education and Technical Publications Team.