The need for blood substitutes

Risks associated with blood transfusions have been minimised with the development of highly sensitive tests for blood-borne pathogens such as hepatitis and the immunodeficiency virus. So why do we need an alternative?

Social problems associated with transfusions

  • The populations of developed nations are aging, and since most donor blood goes to people over the age of 65 for surgery, the ratio of donor blood requirements to population is increasing. If the demand for blood remains at the current per capita level in elderly patients and rates of donation do not change, a significant shortage of approximately 4 million units of blood will occur by the year 2030 (US).
  • Donor blood is very expensive - $NZ200 per unit. This covers: obtaining the donor blood, transportation, extensive testing, processing and storage. Ideally an alternative would drastically lower this cost.
  • In countries like the US, donor incentives, which are needed to reach donor targets, attract a high proportion of donors whose blood is at high risk of infection. These are donors such as drug addicts with HIV or the homeless with Hepatitis C.
  • One final but significant social reason for the search for alternatives is the existence of certain patients who cannot receive transfusions because of religious beliefs.

Technical reasons for an alternative to transfusions

  • Blood needs to undergo extensive testing and processing before it can be used in human patients. The blood needs to be grouped according to ABO surface antigens and Rhesus factor, and must also be screened for infectious agents such as HIV and Hepatitis.
  • Whole blood presents difficulties with sterilisation, as it cannot be treated with traditional sterilisation methods.
  • Donor blood also presents logistical difficulties, when considering refrigerated transport - it is not practical to have supplies of donor blood at the front line of a war for example.
  • Donor blood only has a shelf life of 3 weeks, due to diminishing clinical effectiveness and bacterial contamination.



Qualities of an effective blood substitute

  • The ideal blood substitute would have the correct affinity for O2, strong enough to bind it in the lungs, and weak enough to release it to the tissues.
  • As the substitute would lack red blood cells (RBC), there would be no surface antigens to match, thereby eliminating another step in the processing.
  • Ideally, it would be guaranteed disease free, by either advanced filtration of the artificial blood or through animal-free production methods such as recombinant protein production in E. coli or yeast.
  • A blood substitute should perhaps not be of animal origin so patients of all religions could be treated. It could be stored indefinitely at room temperature, perhaps in a lyophilized state. This way it could be stockpiled for war and disasters.
  • One final requirement is an extended intravascular dwell time. Ideally it needs to last long enough for the body to replace the artificial blood with new blood cells.



Approaches

Current approaches fall under two main categories:

Perfluorochemical emulsions

Perfluorocarbons are organic compounds where the hydrogen atoms have been replaced with fluorine (CF2)n. These compounds have the ability to transport oxygen and carbon dioxide without reacting with them, making them good candidates for haemoglobin replacements. Perfluorocarbons are simple to manufacture in large quantities and can easily be sterilised to prevent disease transmission. An important potential use of PFCs is for patients whose religion does not allow the use of animal blood products and their derivatives.

Modified haemoglobin products

The three approaches in this category include:

In the first two approaches, the RBC membrane is filtered out, which means the products are compatible with all blood types.

Pure haemoglobin itself cannot be used as a blood substitute as it breaks down into a toxic dimer, and is quickly filtered out by the kidneys. Furthermore, the absence of the 2,3-DPG co-factor, which is concentrated inside the RBCs, shifts the O2 affinity as to render it incapable of releasing O2 to the tissues.

Biopure

Biopure, a large corporation in the US started work on ″hemopure″. They discovered that ″freeform″ bovine Hb has a similar O2 affinity as human Hb encapsulated in {red blood cells|RBC]s. Now the upside of using cow blood is that it is available in abundance as a waste product of slaughterhouses. The problem was that purified bovine Hb still dissociated into the toxic dimers.

The obvious solution to this was to cross-link the tetramer to prevent dissociation. This was done using gluteraldehyde, which cross-links the proteins covalently into a stable quaternary structure. This product works very well and was licensed in 2001 for veterinary use. Hemopure is also approved for use in humans in Africa, as blood transfusions are extremely risky due to high levels of HIV contamination in donor bloodstocks.

There are several problems with Biopure's approach, like the need for managed herds of beef with documentation assuring the origin, medical history, feed (no mammalian protein) and young age of the cattle. Public perception of the product is another problem because of the perceived risk of transmissible spongiform encephalopathy agents. Another problem with hemopure is that Hindu patients cannot accept the bovine products due to their religious beliefs.

Baxter

Baxter is an US/Swiss corporation who also decided to research blood substitutes. They took the approach of using human Hb as a starting point. Human blood is also available in abundance as it has a limited shelf life for transfusions and old stock is normally discarded.

The Hb was purified and cross-linked using gluteraldehyde as was the bovine Hb. Clinical trials went ahead but when stage III was reached and analysed, it was discovered that more people were dying when using their product than when using the placebo!!!. By then Baxter had invested too much money and did not want to research any further by taking the trials to 1000 people, so product development ceased. Not to be outdone, Baxter instead bought Somatagen, who had been working on a novel approach to Hb therapeutics.

Somatagen

When Somatagen lost the confidence of investors, they were working on a recombinant Hb protein in E. coli. When this protein was cross-linked to prevent dissociation, it was discovered that the O2 binding increased, which decreased the efficiency of O2 transport to the tissues.

The researchers reacted with a mutation to Aspβ108 → Lys, which lowered the O2 binding. When combined with the cross-linking effect, it produced an overall O2 affinity close to the ideal 28mmHg. This process was named ″Optro″.

Clinical studies revealed that the new, mutated Hb interfered with blood pressure regulation. This was as far as they had come when investors lost confidence.

Enter Baxter - when Baxter bought Somatagen they immediately began work on discovering how blood pressure was being affected. They discovered that the recombinant Hb was scavenging Nitric Oxide (NO), an important molecule produced by the vascular endothelium, which regulates a host of biochemical reactions, including vascular smooth muscle relaxation.

The researchers have since mutated the NO binding site on the synthetic Hb and the new product is currently undergoing clinical trials.




Conclusions

Despite the short intravascular dwell time, blood substitutes have many superior traits such as long-term storage at room temperature, no blood type mismatching, no infectious virus, etc. These traits, which far outweigh the limitations of blood transfusions, are a big step in the advancement of clinical medicine.



References

VAMVAKAS, E.C., TASWELL, H.F.; Epidemiology of Blood Transfusions; Transfusion; 31:355;(1994).

CHANG, T.M.S.; Blood Substitutes: Principles, Methods, Products and Clinical Trials; Karker Landes Systems; 1997.

SQUIRES, J.E.; Artificial Blood; Science; 295; Feb (2002); p1002-1005

STOKSTAD, E.; Not Blood Simple; Science; 295; Feb (2002); p1003


* Shamelessly noded homework from a post graduate paper Microbial Biotechnology, entitled: "Modern Approaches to the Replacement of Blood Transfusion" The University of Auckland


Special Thanks to Muse for helpful commments.

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