Cut, Cover, or Cultivate: The Art of Designer Blood

By Jay Pennington

Despite current sophisticated crossmatching methods, the risk of clerical errors causing transfusion reactions, coupled with the ever-present challenge of maintaining an adequate supply of compatible blood, keeps researchers seeking alternatives to the ABO “status quo.”

Saturday’s session on “Designer Blood” (5105-S) provided a progress report on the two main avenues to finding answers: 1) cultivation of enucleated human red cells in vitro and 2) chemical modification of the red cell membrane to “silence” its antigenic properties. The modification approach itself can take two paths: 1) conversion of group A and group B red cells to group O cells through treatment with enzymes and 2) masking of antigens by coating them with polyethylene glycol (PEG).

Cultivating Enucleated Red Cells
Mohandas Narla, DSc, from New York Blood Center, began by reviewing nature’s way of growing red cells in marrow before progressing to the application of that knowledge to in-vitro generation of red cells.

Several procedures have attempted to expand CD34+ stem cells ex vivo into fully functioning enucleated red cells. Narla focused on the most significant protocol thus far, in which growth factors and marrow stromal cells were used in combination to successfully produce mature, enucleated cells.
 
The newly cultured cells showed normal hemoglobin content, oxygen uptake and release, and morphology. Narla was optimistic that the problem with in-vitro prototypes in the past—the cells’ short life span—would be resolved with this procedure. 

However, Narla tempered the hope with caution about the challenges still ahead. For example, even with cord blood, the richest source of CD34+ cells available, the question of feasibility remains: Is the relatively modest output that can be produced worth the high expense involved? “To make a practical impact on the blood supply, a huge number of cord blood units will be needed.” Also, 100 percent enucleation is a crucial condition that needs to be met and requires further research to obtain.

Cutting with Enzymes
Next, George Garratty, PhD, FRCPath, of the American Red Cross Blood Services (Southern California Region), outlined the historical and recent developments in the two chemical modification approaches.

One way to handle antibody reactivity is to find a way to “cut off” the offending antigen with specific enzymes. Because both A and B antigens represent extensions of the H antigen, cleaving the immunodominant sugars from the chains—N-acetylgalactosamine for A, and galactose for B—results in red cells that type as O, referred to as “universal” donor or enzymatically converted group O (ECO) cells. 
The greatest success thus far has been with enzyme conversion of type B to type O. The earliest enzyme found to do the job was a galactosidase (B-zyme) from green coffee beans. The results of studies in gibbons, human volunteers and, subsequently, human patients, showed normal cell survival, no transfusion reactions and no antibody evident to the B-zyme (except that an occasional increase in anti-B has been noted). Mass production, however, requires a more efficient enzyme.

Although some success has been found creating ECO cells from type A2 cells, researchers continue to be challenged to find an appropriate A-zyme for A1. Also, crossmatching has shown there are still problems to overcome. Clinical trials of the A ECO red cells are in progress.

Coating with PEG
When PEG is covalently bonded to red cells, it provides a shield so that antibodies do not recognize the antigens, especially with an extra “shell” of water (PEG attracts water molecules) — hence, the term “stealth” red cells. Garratty reported that, in testing, PEG red cells produced weaker reactions with A and B antibodies and much weaker reactions with Rh and other antibodies.
 
During a 1997-1998 study, significant problems were encountered with PEG treatment. PEG red cells stuck to glass or plastic tubes during washing, and they showed nonspecific uptake of proteins. Also, the agglutination of PEG red cells by normal sera was puzzling.
 
Second-generation stealth red cells seemed to rectify most of these issues and demonstrated better shielding of the antigen when a larger crosslinked molecule was used. However, as impressive as the reduction in antigen titers might seem to non-blood-bankers, Garratty noted that significant antibody reaction still occurs. This factor, coupled with recent evidence of antibodies to PEG, has cast doubt about whether the technology will ever be useful in humans. Such complications shouldn’t be surprising, he explained: “If you’re going to fool with Mother Nature, expect problems!”


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