SYNTHETIC BLOOD

BLOODThis is the true story of the amazing defenders inside your body, a brave band of cells that keep you healthy by constantly battling against all kinds of invading germs. Every minute, every hour, everyday of your life, they are fighting……..

HaemetologyThe study of blood

Haemopoiesis

The production of blood cells, which in normal healthy adults occurs in the bone marrow and the lymphatic system. The specific blood cells that are produced and eventually released into the blood stream are the red cells or erythrocytes, the various types of white cells or leukocytes and the platelets or thrombocytes.

Erythropoiesis

The production of red blood cells. Red cells contain haemoglobin, which is needed for the transportation of oxygen in the blood stream to all parts of the body.

The blood is really the transport system of the body, as it carries materials from one organ to another.

It takes oxygen from the lungs to the heart and then to the working muscles.

It takes carbon dioxide from the working muscles to the heart and then to the lungs.

It also takes soluble food from the intestine and waste products from the kidneys.

The average human has 5 litres of blood.

What are the 4 main components of blood ?

1.RED BLOOD CELLS- Red blood cells are made from red bone marrow. The pigment that gives the cells their colour is called HAEMOGLOBIN and it attracts OXYGEN. It picks up oxygen in the lungs and delivers it to the working muscles.

One cubic millimetre of healthy blood contains about 5 million red blood cells ( this is known as the “BLOOD COUNT” ).

Haemoglobin (Hb)

•Human Hb-64 kDa tetrametric protein

•Consists of two α subunits and β-globin sub units.

•Each α and β subunits contain an iron- heme group that binds to oxygen molecule allowing for transport.

2. PLASMA- A pale straw coloured liquid consisting of 90% water.

3. WHITE BLOOD CELLS

4. BLOOD PLATELETS- Formed in the red bone marrow and helps in the clotting of blood.

Artificial BloodThe blood of the future

• Blood substitutes or synthetic blood are currently labeled as “oxygen carriers”.

This is because they are unable to mimic many of the other functions of blood.

• Main function- replace lost blood volume and oxygen carrying capacity.

The History of Artificial Blood• Milk was one of the first substances used as a blood

substitute in order to treat patients with Asiatic cholera.

• After several patients died by receiving milk transfusions, other substances were discovered as potentials:– Salt or saline solutions: used primarily as a plasma volume

expander, rather than as artificial blood– Hemoglobin isolated from red blood cells– Animal plasma could be used as a substitute for human

blood• However, since many of the materials in animal plasma

are toxic to humans, this posed a problem to using it as a substitute

• The problem of not having a workable substitute led to Ringer’s Solution…

Ringer’s Solution• Created by the physiologist Sydney Ringer in 1882• Ringer’s solution is a saline solution which is able

to prolong the life of cells in the body• Solution contains sodium chloride, potassium

chloride, calcium chloride, and sodium bicarbonate: designed in the concentrations found in the human body to keep the heart pumping even outside of the body

• This solution resembles blood serum• Often used to culture animal cells

Blood Grouping: Karl Landsteiner

• Karl Landsteiner was a world-renowned immunologist and pathologist in the 1900s.

•contributed to the world of medicine in the fields of anatomy, histology, and immunology

• 1909: Landsteiner classified human blood into four different groups: A, B, AB, and O

•Recognized the agglutinins in the blood

•This has become the basis for modern blood typing today

• received the Nobel Prize for Physiology or Medicine in 1930

Artificial Blood vs. Blood Substitutes

Artificial Blood Blood Substitutes• the main purpose of artificial blood is to act as normal blood in the body, providing a long-term solution to blood loss or distortion• no working artificial blood has been created in the status quo

• the main purpose of a blood substitute is to provide temporary support to the circulatory system when necessary.• blood substitutes generally are focused on the role of transporting oxygen for short-term cases such as blood transfusions or surgeries• blood substitutes are generally simpler since they are only focused on one of the several functions of real blood• several types of blood substitutes have been found including:

–Hemoglobin-based oxygen carriers–Perfluorocarbon emulsions

Ideal Characteristics of Artificial Blood

• Safe to use

• Increased availability that would rival that of donated blood

• Volume expansion

• Compatible in the human body

• Able to transport and release oxygen where needed

• Storable and durable for longer time periods

• Pathogen free

• Long shelf life

Problems Currently Associated with Artificial Blood and Blood Substitutes

• Bodily immune systems may sometimes react negatively to the foreign blood that is inserted into the body.

• Trauma/Shock Patients– Since these patients are often frequent recipients of blood

substitutes or plasma during surgery, it becomes challenging to understand which types of blood substitutes have affected which problem in the patient’s body.

• Currently no real working source of artificial blood exists that can perform the multifarious tasks of real human blood cells.

Types of Blood Substitutes1.Perfluorocarbon (PFC) emulsions

• Perfluorocarbons are derived from a group of hydrocarbons in which the hydrogen atoms are replaced by fluorine atoms.– PFCs are chemically inert due to the strength of the carbon-

fluorine bonds– PFCs can dissolve 40 to 70 % oxygen per unit volume, almost 3

times more than blood. • Used to create artificial blood during surgeries

Process of production:– Water, salts, and phospholipids surfactant are added and

emulsified through high-pressure homogenization– Purified through high temperatures of steam.

• Common PFCs:– Perfluorodecalin, Perflubron

Perfluorocarbons (PFC) emulsions

• Structure: – Perfluorocarbon core– Surrounded by a

phospholipid surfactant.• A surfactant is “a

substance that reduces the surface tension of the liquid in which it is dissolved”

• In this application, the perfluorocarbon is used as a part of an emulsion, typically using Pluronic F-68 or egg yolk phospholipids (lethicin) as surfactants, in water.

• For example, Fluosol-DC:

Ingredient w/v% Perfluorodecalin 25.0

Yolk phospholids 3.6 Fatty acid (emulsion stabilizer) trace

D-Sorbitol (emulsion stabilizer) 3.5 NaCl 0.204 KCl 0.010

MgCl2 0.007 Sodium lactate 0.105

Perfluorocarbons (PFC) emulsions

Advantages• PFCs do not react with oxygen.•PFCs allow easy transportation of the oxygen to the body.• they allow increased solubility of oxygen in plasma.•PFCs minimize the effects of factors like pH and temperature in blood circulation.

Disadvantages• often causes flu-like symptoms.

– this is often caused by phagocytosis of the perfluorocarbon emulsion by the recipient organism’s immune system

• unable to remain mixed as aqueous solutions – thus, they must be prepared as emulsions for use in patients.• a decrease in blood platelet count.• PFC products cannot be used by the human body, and must be discarded this takes approximately 18-24 months.• because PFCs absorb oxygen passively, patients must breathe at a linear rate to ensure oxygenation of tissues.

Types of Blood SubstitutesHemoglobin-based Oxygen Carriers (HBOCs)

• Hemoglobin-based Oxygen Carriers were created as a mechanism to mimic the oxygen-carrying role of hemoglobin in the body, while still reducing the need for real human hemoglobin.

• Unmodified and modified haemoglobin solutions Stroma free Hb and cross-linked Hb-

It is prepared from outdated RBC. Hb dissociate into dimers and monomers in solution. This solution has higher affinity for oxygen and cleared from plasma easily. This disadvantage overcome by modifying this Hb by pyridoxilation which reduces its oxygen affinty

Advantages•Available in much larger quantities•Can be stored for long durations•Can be administered rapidly without typing or cross-matching blood types•Can be sterilized via pasteurization

Disadvantages• reduced circulation half-life• disrupts certain physiological structures, especially the gastrointestinal tract and normal red blood cell hemoglobin.• the release of free radicals into the body

Microencapsulated Hb-

Stroma free Hb is enclosed in synthetic membranes (phospholipids)

Hb is not exposed to extracellular environment.

Synthetic membrane more stable than biological RBC.

No blood group antigens and and artificial cells do not react with blood group antibodies.

Experimental Design – MethodsHemoglobin Modification Methods

• Ensuring the stability of hemoglobin– When hemoglobin is left outside a cell, it has the tendency

to break into its individual parts, instead of remaining as an entire hemoglobin protein.

– Methods have been discovered to retain the stability of hemoglobin including:

Hemoglobin-based Oxygen Carriers (HBOCs)

• Currently, experimentation is occurring to develop vesicles which carry the Hemoglobin before inserting it into the body as an HBOC

• There are several advantages to HBOC carrier vesicles• Prevents the denaturation of the Hemoglobin• The vesicles themselves are made of purified Hb and lipids, making

them compatible with the human immune system

• Current Setbacks with Hb Vesicle Technology• Materials such as nylon, gelatin, and gum arabic have been tried but

the human immune system, specifically the reticuloendothelial system, removed the vesicle promptly as a natural bodily response.

• Later, the theory of phospholipid vessels was used to create a vesicle using phospholipids

• Researchers are trying to develop a way to create liposome sacks to carry the HBOCs.

Types of Blood Substitutes

This figure displays the different forms of blood substitutes that can be used to simulate the actions of a red blood cell

Can we make a blood substitute using Biotechnological techniques?

• In order to create a blood substitute that would effectively transport oxygen throughout the body, the hemoglobin gene would have to be inserted in a plasmid vector and multiplied in a specific medium.

• The hemoglobin gene would be inserted into the plasmid to carry the cloned gene into E. Coli cells– This would allow for expression of large amounts of the hemoglobin

protein

Escherichia Coli P678-54• The particular strain of the bacteria E. Coli P678-54 has the unique

property of dividing abnormally – it forms a mother cell and a daughter cell. – The mother cell is similar to a regular cell in that it contains

chromosomal DNA. However, it contains ALL of the cell’s DNA– The daughter cell is achromosomal. It is smaller, and contains no

chromosomal DNA. • The daughter cell is often referred to as a “mini cell”

• Because E. Coli P678-54 has the property of dividing abnormally, it would be ideal if used as a Gene Delivery Vehicle, or GDV.

• Since E. Coli P678-54 has a tendency to divide into mother cells as well as mini cells, these mini cells could be ideal for the creation of hemoglobin-based oxygen carriers.

Applications

• PFCs are now licensed for use in Percutaneous Transluminal Coronary Angioplasty

• In military, during war situations

• For anaemic patients

• For surgery and blood transfusion

End notes

• Only three products of blood substitutes are available for human use in only two countries South Africa and Russia

• Polyheme - HBOC developed by the American company named

Northfield.

• Hemospan - HBOC developed by the American company named

Sangart.

• No blood substitutes launched in India yet

Reference:• “Ringer's solution." Encyclopedia Britannica. 2007. Encyclopedia Britannica Online.

23 July 2007  <http://www.britannica.com/eb/article-9063724>.

• Practice of safe blood transfusion, Dr. R.N Makroo, 1999, pg 189-192.

• Transfusion,( 2008)48 :574-575

• Vox Sanguinis, ISBT Science Series, (2008)3, 1-9

• Artificial blood: What is it? Will I use it? Bruce J. Leone M.D.

http://www.demsonline.org/jax-medicine/1998 journals/december 98/artificial blood.htm

• wikipedia.com

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