ARTIFICIAL BLOOD, HOPE FOR THE FUTURE?

A sigh of relief, the tension is relieved as the gynaecologist took the baby out with great  difficulty through the incision. The  indication for caesarean section was severe fetal distress and the OT atmosphere has now turned pleasant as the paediatrician reported that the APGAR score is 9. The anaesthesiologist is busy preparing  oxytocin infusion and sedation.The patient who was given regional anaesthesia is now anxious to see her baby.But the enjoyment did not last long as the surgical team noted profuse bleeding with a flabby uterus not responding to ergometrine or oxytocin.A request for issuing blood was  sent to blood bank but the request was returned ,stating that the husband refused any kind of blood transfusion as he is a strict Jehovah's witness which he didn't mention at the time of taking the consent!

 Jehovah's witnesses, a fellowship of more than 1 million americans, object to the administration of blood in any form for any indication.This objection is based on "THE HOLY BIBLE"  Acts 15:28,29 which states "For it has seemed good to the Holy Spirit and to us to lay on you no greater burden than these requirements: that you abstain from what has been sacrificed to idols, and from blood, and from what has been strangled, and from sexual immorality. If you keep yourselves from these, you will do well. Farewell.” According to them blood removed from the body should be discarded "You should pour it upon the ground as water"  (Deuteronomy 12:24.)This makes pre operative blood conservation technique like preoperative phlebotomy and storage impractical

The search for an oxygen carrying blood substitute started with world war II as the military realized the difficulties of whole blood transport and storage.Subsequently several trials were made to bring out an ideal blood substitute using chemicals which can carry oxygen.
1)Perfluorocarbon compounds:Of the various substances that carry or facilitate the transport of oxygen the perfluorocarbons gave the most promising results.They were created by replacing H2 atoms of hydrocarbons with flurine. This was following the first real success in  "fluid breathing system" proposed by Leland Clark in 1966. He found that oxygen and carbondioxide are very much soluble in fluorocarbon liquids and can be used for artificial ventilation of the lungs which are immersed in these compounds where satisfactory oxygen uptake  and giving out of CO2 takes place by the alveoli through the liquid media, in the absence of external oxygen supply.He performed his experiment in anaesthetised rat where the animal is paralysed intubated and immersed in PF liquid.After bubbling oxygen through the liquid this is pumped into the animal's lungs and recirculated. Most of the animals kept in the fluid for upto an hour survived for several weaks. Subsequently trials were made in humans during war time.               Figure: Demonstrating a living mouse immersed  in  perfluorocarbon compound along with a goldfish.

• Fluosol-DA was the most notable compound among PFCs, regarded as a first generation PFC.It contained perfluorodecalin and perfluorotripropylamine emulsified with Pluronic F-68  It was approved by the FDA for use in percutaneous transluminal coronary angioplasty initially but was subsequently withdrawn(see below)
• They have high affinity for oxygen approximately 10-20 times greater than plasma.The  oxygen content of PFCs  is directly proportional to oxygen partial pressure and are most efficient as oxygen carriers at a partial pressure of more than 300mmHg, which limited their use, as patients needed high inspired oxygen concentration. A short intravascular half life, unstable at extreme temperatures, Low oxygen carrying capacity ,poor shelf life and adverse effects such as acute complement activation and disruption of pulmonary surfactant, all lead to their withdrawal  from the market. But still these compounds have found some place in "Liquid ventilation" of lungs in ARDS.
• Perfluorocarbons demonstrate a linear oxygen dissociation curve in contrast to the sigmoid dissociation curve of blood and oxygen transport is enhanced by elevated PO2. Because of this linear relationship the oxygen delivery to the capillaries remained unsatisfactory as most of the oxygen get dissociated before the blood reaches the capillaries.
• Second generation PFCs: A mixture of perfluorooctyl bromide and perfluorodecyl bromide was introduced later on,  which used egg yolk lecithin as emulsifier.This compound was termed Oxygent. The oxygen carrying capacity was 2-3 times that of Fluosol-DA and was more stable at room temperature. This product is now under phase 3 clinical trial.Febrile reactions, cerebrovascular insufficiency  and transient thrombocytopenia are the adverse reactions observed with oxygent.
• Other PFC emulsions that have been developed include Perftoran; Oxycyte ( entering phase II trials), and Oxyfluor

2)Hemoglobin based oxygen carriers;(HBOC) or Stroma free Hb.

• The finding that native Hb is not antigenic, led to the development of hemoglobin solutions or stroma free hemoglobin.The red cells are removed and hemoglobin isolated and maintained as solution.They withstood sterilisation and had a shelf life of 2 years.Rapid administration (as they are not antigenic) and easy availability were other advantages. But they are not as effective at oxygenation as packed RBCs  because of their high affinity to oxygen.They have a reduced circulatory half life as the Hb tetramers are split to dimers and are cleared from blood stream by glomeruar filtration and uptake by RE system.They are hyperosmolar solutions and significantly increase plasma oncotic pressure. Renal dysfunction,coagulopathy  and liver dysfunction are the other side effects observed.They bind to nitric oxide and can cause hypertension. Several approaches were made to prevent the toxicity and to decrease the avidity of Hb to oxygen ,and as a result modified hemoglobins were introduced.
• Crosslinked hemoglobins: Diaspirin cross-linked hemoglobin (DCLHb) is the prototype molecule of this category of blood substitutes. Stability of the molecule is achieved by cross-linking between the two alpha chains and thus dissociation is prevented and the circulating half life is extended. Both intermolecular and intramolecular crosslinkage was experimented. It was found that a bifunctional agent, 2-Nor-2-formylpyridoxal 5-phosphate which is also a 2,3-DPG analogue can intramolecularly crosslink the 2 beta subunits of the hemoglobin molecules and the resulted compound has less oxygen affinity and high oxygen carrying capacity.Diaspirin cross linked Hb(DCLHb) made from outdated human blood has a shelf life of approximately 9 months when frozen and 24 hours when refrigerated. The intravascular half-life is 2-12 hours and is dose dependant but the raise in colloid oncotic pressure was a concern. Eventhough it increased blood oxygen content, intense vasoconstriction causing high vascular resistance, reduced cardiac output, and high heart rate were undesirable for the critically ill patients and the manufacturers halted further development of DCLHb in 1998.
• Polymerised Hemoglobins: The next step in the development was to polymerise the crosslinked tetramers to reduce oncotic pressure and to avoid filtration by kidneys.The crosslinking agent was glutaraldehyde. Since it was difficult to control the Hb-glutaraldehyde polymerization reaction which yielded products of various molecular sizes and the viscosity of the solution was high, the results were not promising.The polymerisation reaction is as below

                    H-CO-(CH2)3-CO-H + HB-NH2 = HB-NH-CO-(CH2)3-CO-NH-HB

                  glutaraldehyde                hemoglobin         Crosslinked Hemoglobin

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  PolyHeme: is a first-generation pyridoxylated polymerized hemoglobin made from outdated human  blood   and is currently being evaluated in a phase III clinical trial that is enrolling patients.The purified Hemoglobin is chemically modified into a polymerised form and incorporated into solution. 1 gram of Polyheme contains 50 grams of modified hemoglobin.which is almost same as that of blood and the use of a 2,3-DPG analogue, pyridoxal phosphate, to crosslink hemoglobin improved the P50. It has a half-life of 24 hours, a shelf life longer than 12 months when refrigerated, and a p50 of 28-30 mm Hg.Polymerised polynitroxyl Hb, (Hemozyme) is a vasodilator, an antioxidant and an antiinflammatory agent and can be used in ischemia reperfusion and inflammation.Also beneficial in post reperfusion multi organ dysfunction syndrome.Other first-generation polymerized hemoglobin products include HbOC-201, Hemopure and HemoLink. Hemopure is a polymerized form of bovine hemoglobin with a p-50 of 30 mm Hg that is closer to human hemoglobin compared to stroma-free hemoglobin. It has an intravascular half-life of 8-23 hours and a shelf life of 36 months at room temperature. Hemopure is approved in South Africa for the treatment of anemic patients for surgery to avoid or reduce transfusion needs but following phase II trials the US withhold its use due to adverse reactions including strokes and myocardial infarction.
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  Conjugated Hemoglobins:Conjugation of Hb is done by covalent binding of Hb to a biocompatible polymer, such as polysaccharide, in order to increase its overall size.This process achieved same results as that of polymerisation with regard to stability and improved oxygenation. Polyethylene glycol modified Human Hb (Hemospan) has the advantage of low affinity and increased oxygen transport capacity.Pegylation(using poly ethylene glycol) of hemoglobin prevents immunological reactions and these compounds claim the longest circulating half life of upto 12-20 hrs.

3) Encapsulated hemoglobin: Artificial red blood cells. Efforts have been made to encapsulate hemoglobin within a lipid-membrane to create a compound capable of carrying oxygen while not being associated with significant vasoconstriction. These liposomes appear to be retained in plasma for a significant period. These have P50 and oxygen dissociation curve similar to red blood cells, since 2-3-DPG is reatained inside Hemoglobin also stays inside as tetramers. These artificial red blood cells do not have blood group antigens on the membrane and therefore do not participate in antigen antibody reactions.The major problem with these artificial cells is that they are rapidly cleared off the circulation by the reticuloendothelial system resulting in reduced circulatory half life.Preparation of smaller submicron lipid membrane artificial red blood cells resulted in improvements in circulation time.Other than activation of the reticuloendothelial system these artificial cells are difficult to produce and they stimulate the complement pathway. They also activate platelets.

4) Recombinant human hemoglobin:

The next generation HBOCS.The first recombinant hemoglobin product, rHb 1.1 (Optro, Somatogen and Eli Lilly) was a genetically engineered variant of human hemoglobin, Hemoglobin Presbyterian, with modifications to decrease its oxygen affinity. The human gene responsible for producing Hb is copied and modified with the intent to enhance the stability and and functionality of the Hb including reduced side effects.Copies of this gene are then inserted into the DNA of a highly controlled population of E-Coli and the bacteria produce Hb which is subsequently purified.The product had an intravascular half-life of 2-19 hours and a shelf life of 18 months when refrigerated. The adverse effect profile was similar to DCLHb and consisted of vasoconstriction, GI distress, fever, chills, and backache. Currently, the production of this compound has been discontinued due to safety concerns.

Ideal blood substitute: Should satisfy the following criteria

• High oxygen carrying capacity
• lack of antigenecity
• Stable shelf life
• Stable and prolonged intravascular half life of weeks to months.
• Free of infectious complications
• Free of adverse effects or systemic toxicity
• Easy to produce in large scale
• Cost effective

Advantages of blood substitutes:

• Universal compatibility; helpful in emergency
• Purity and non toxicity; free of infectious agents and allergens
• Storage and shelf life; need not be refrigerated and ideal in remote areas, fields, and disaster situations.
• Availability; readily available in large quantities
• predictability; has ingredients which are known and understood and the effects can be predicted

Adverse Effects Related to Blood Substitutes(In general)

• Adverse effects associated with hemoglobin-based oxygen carriers include hypertension, abdominal pain, skin rash, diarrhea, jaundice, hemoglobinuria, oliguria, fever, stroke, and laboratory anomalies such as an elevation in lipase levels. Although most of these side effects were transient and clinically asymptomatic, many clinical trials involving these agents have been discontinued or held due to the associated adverse effects

The general benefits of HBOCs over transfused red bloodcells:

• No prior planning needed eg emergency use
• Faster & better O2 distribution
• Ready to use
• No equipment needed
• Long shelf life
• No refrigeration required
• Universally compatible
• Immediately offloads oxygen
• No 2,3-DPG
• Can be used by Jehovah's Witnesses

Other applications of PFCs:Other than oxygen carrying agents in hemorrhage or anaemia, they could be incorporated into solutions used in open heart surgery, and in supplying devascularized organs with oxygen prior to transplantation.They may be used to perfuse the myocardium or brain tissue in heart attacks and strokes, oxygenating obstructed regions due to capillary blockage and for mechanical ventilation in ARDS.In cancer therapy they are helpful to increase oxygenation of tumours which may help radiation therapy. Chemotherapeutic drugs could be added to the PFC and delivered to the target organ affected by cancer.It was also observed that toxic doses of PFCs resulted in necrosis of tumour cells.They had promising results in the treatment of fungal or bacterial infections of skin and GI tract, carbon monoxide poisoning, Alcheimer's disease and medical imaging.Clinical conditions like a/c MI, cardiac failure,thromboembolism, air embolism,hepatic failure, a/c renal failure and preservation of donor organs etc also make use of the oxygen carrying capacity of these compounds.


In spite of many years of research, the ideal blood substitute continues to elude researchers.Continued research helped them to reduce the complications and limitations associated with HBOCs. Initial problems in the development which were related to potential toxicity, poor half life, poor oxygen carrying capacity and limitations with animal studies.( which may not reflect human response). Most of the recent attempts are focussed to bridge this gap to prove the safety of these agents in humans and many of these problems have now been solved by extensive basic studies on Hemoglobin. Crosslinked hemoglobin would be the first modified hemoglobin available for routine clinical use.Hopefully, as better blood substitutes are developed and enter routine clinical use, the need for blood transfusions in the operative and trauma settings will decrease.

Recent advances: Researchers are hunting around stem cells for the possible production of hemoglobin. Major research is underway in the UK and US to utilize stem cells to generate blood without a donor. That is, stem cells can be used to generate synthetic blood in a lab which is very much identical to human blood in all aspects.The blood taken from newly cut umbilical cords or adult stem cells, such as induced pluripotent stem cells (iPSCs) can  be used to generate Hb. .According to telegraph, The British scientists are planning a ground-breaking research project to create synthetic human blood from embryonic stem cells, The three-year project will be led by the Scottish National Blood Transfusion Service and includes NHS Blood and Transplant and the Wellcome Trust, the world's biggest medical research charity. The artificial blood will be made from the stem cells of human embryos left over from IVF treatment.Such blood produced from stem cells would have the benefit of not being at risk of infected with viruses such as HIV and hepatitis.The project lead Professor Marc Turner stated "We could provide an unlimited supply of blood in this way" The idea is to produce O -ve red cells in the laboratory from stem cells.  Fig. by courtesy of BBC UK

1) Embryo created from IVF is tested for O-negative blood group, then allowed to develop for several days until stem cells can be extracted
2) Stem cells are cultured in laboratory with nutrients to stimulate red blood cell creation
3) Nuclei are removed in final stage to produce oxygen-carrying mature blood cells. Trillions of these will be needed to build up a blood bank.
References:
1) Sara J Grethlein, MD, Arun Rajan, MD, Blood substitutes:
http://emedicine.medscape.com/article/207801-overview
2)  http://www.telegraph.co.uk/.../British-scientists-to-create-synthetic-blood-from-embryonic-stem-cells.html
3)  www.onenewspage.co.uk/news/Health/.../Synthetic-blood-from-embryos-bid.htm
4)  community.advanceweb.com/.../artificial-blood-why-and-when.aspx
5)   Benesch R, Benesch RE, Yung S, Edalji R. Hemoglobin covalently bridged across the polyphosphate binding site. Biochem Biophys Res Commun 63:1123, 1975.
6)   Blood substitutes excerpt:eMedicine.com,inc.
7)   Scott MG, KucikDF,GoodnoughLT: Blood substitutes evolution and future applications, ClinChem 1997; sept43(9);1724-31
8)   Cohn SM: Blood substitutes in surgery.Surgery2000;127(6):599-602
9)   Anaesthesia UK web site.