Blood plasma fractionation
Encyclopedia
Blood plasma fractionation
refers to the general processes of separating the various components of blood plasma
, which in turn is a component of blood obtained through blood fractionation
.
is the liquid component of whole blood
, and makes up approximately 55% of the total blood volume. It is composed primarily of water with small amounts of minerals, salts, ions, nutrients, and proteins in solution. In whole blood, red blood cells, leukocytes, and platelets are suspended within the plasma.
, immunoglobulins, and clotting proteins such as fibrinogen
. Albumin constitutes about 60% of the total protein in plasma and is present at concentrations between 35 and 55 mg/mL. It is the main contributor to osmotic pressure
of the blood and it functions as a carrier molecule for molecules with low water solubility
such as lipid soluble hormone
s, enzyme
s, fatty acids, metal ions, and pharmaceutical compounds. Albumin is structurally stable due to its seventeen disulfide bonds and unique in that it has the highest water solubility and the lowest isoelectric point (pI)
of the plasma proteins. Due to the structural integrity of albumin it remains stable under conditions where most other proteins denature
.
, and during plasma exchange. Since albumin is the most abundant protein in the plasma its use may be the most well known, but many other proteins, although present in low concentrations, can have important clinical uses. See table below.
Examples of Plasma Components for Clinical Use>
Plasma Component
Reasons for Use
factor VIII
hemophilia A
factor IX complex
hemophilia B
anticoagulant
overdose
factor II and factor X
deficiencies
liver disease
immunoglobulin
passive prophylaxis
some types of immune thrombocytopenic purpura
antithrombin III
congenital deficiency
disseminated intravascular coagulation
alpha-I-antitrypsin
hereditary deficiencies
emphysema
and COPD
cirrhosis
or transfusion
, the plasma component must be highly pure. The first practical large-scale method of blood plasma fractionation was developed by Edwin J. Cohn during World War II
. It is known as the Cohn process
(or Cohn method). This process is also known as cold ethanol fractionation as it involves gradually increasing the concentration
of ethanol
in the solution
at 5oC and 3oC. The Cohn Process exploits differences in properties of the various plasma proteins, specifically, the high solubility
and low pI
of albumin. As the ethanol concentration is increased in stages from 0% to 40% the [pH] is lowered from neutral (pH ~ 7) to about 4.8, which is near the pI of albumin. At each stage certain proteins are precipitated out
of the solution and removed. The final precipitate is purified albumin. Several variations to this process exist, including an adapted method by Nitschmann and Kistler that uses less steps, and replaces centrifugation
and bulk freezing with filtration
and diafiltration.
Some newer methods of albumin purification add additional purification steps to the Cohn Process and its variations, while others incorporate chromatography
, with some methods being purely chromatographic. Chromatographic albumin processing as an alternative to the Cohn Process emerged in the early 1980s, however, it was not widely adopted until later due to the inadequate availability of large scale chromatography equipment. Methods incorporating chromatography generally begin with cryodepleted plasma undergoing buffer exchange via either diafiltration or buffer exchange chromatography, to prepare the plasma for following ion exchange chromatography
steps. After ion exchange there are generally further chromatographic purification steps and buffer exchange.
For further information see chromatography in blood processing
.
of diseases, and separation of plasma is a necessary step in the expansion of the human plasma proteome
.
is the primary method for discovery and detection of biomarkers in plasma. This involves the separation of plasma proteins on a gel by exploiting differences in their size and pI
. Potential disease biomarkers may be present in plasma at very low concentrations, so, plasma samples must undergo preparation procedures for accurate results to be obtained using 2D Electrophoresis. These preparation procedures aim to remove contaminants that may interfere with detection of biomarkers, solubilize the proteins so they are able to undergo 2D Electrophoresis
analysis, and prepare plasma with minimal loss of low concentration proteins, but optimal removal of high abundance proteins.
The future of laboratory diagnostics are headed toward lab-on-a-chip
technology, which will bring the laboratory to the point-of-care
. This involves integration of all of the steps in the analytical process, from the initial removal of plasma from whole blood to the final analytical result, on a small microfluidic device
. This is advantageous because it reduces turn around time, allows for the control of variables by automation
, and removes the labor intensive and sample wasting steps in current diagnostic processes.
or reverse phase liquid chromatography (RPLC) with high efficiency cation exchange chromatography
and subsequent tandem mass spectrometry
for protein identification.
Fractionation
See also: Fractionated spacecraftFractionation is a separation process in which a certain quantity of a mixture is divided up in a number of smaller quantities in which the composition changes according to a gradient. Fractions are collected based on differences in a specific property of the...
refers to the general processes of separating the various components of blood plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
, which in turn is a component of blood obtained through blood fractionation
Blood fractionation
Blood fractionation is the process of fractionating whole blood, or separating it into its component parts. This is typically done by centrifuging the blood.The resulting components are:...
.
Blood plasma
Blood plasmaBlood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
is the liquid component of whole blood
Whole blood
Whole blood is a term used in transfusion medicine for human blood from a standard blood donation. The blood is typically combined with an anticoagulant during the collection process, but is generally otherwise unprocessed...
, and makes up approximately 55% of the total blood volume. It is composed primarily of water with small amounts of minerals, salts, ions, nutrients, and proteins in solution. In whole blood, red blood cells, leukocytes, and platelets are suspended within the plasma.
Plasma proteins
Plasma contains a large variety of proteins including albuminSerum albumin
Serum albumin, often referred to simply as albumin is a protein that in humans is encoded by the ALB gene.Serum albumin is the most abundant plasma protein in mammals. Albumin is essential for maintaining the osmotic pressure needed for proper distribution of body fluids between intravascular...
, immunoglobulins, and clotting proteins such as fibrinogen
Fibrinogen
Fibrinogen is a soluble plasma glycoprotein, synthesised by the liver, that is converted by thrombin into fibrin during blood coagulation. This is achieved through processes in the coagulation cascade that activate the zymogen prothrombin to the serine protease thrombin, which is responsible for...
. Albumin constitutes about 60% of the total protein in plasma and is present at concentrations between 35 and 55 mg/mL. It is the main contributor to osmotic pressure
Osmotic pressure
Osmotic pressure is the pressure which needs to be applied to a solution to prevent the inward flow of water across a semipermeable membrane....
of the blood and it functions as a carrier molecule for molecules with low water solubility
Solubility
Solubility is the property of a solid, liquid, or gaseous chemical substance called solute to dissolve in a solid, liquid, or gaseous solvent to form a homogeneous solution of the solute in the solvent. The solubility of a substance fundamentally depends on the used solvent as well as on...
such as lipid soluble hormone
Hormone
A hormone is a chemical released by a cell or a gland in one part of the body that sends out messages that affect cells in other parts of the organism. Only a small amount of hormone is required to alter cell metabolism. In essence, it is a chemical messenger that transports a signal from one...
s, enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s, fatty acids, metal ions, and pharmaceutical compounds. Albumin is structurally stable due to its seventeen disulfide bonds and unique in that it has the highest water solubility and the lowest isoelectric point (pI)
Isoelectric point
The isoelectric point , sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge....
of the plasma proteins. Due to the structural integrity of albumin it remains stable under conditions where most other proteins denature
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
.
Plasma proteins for clinical use
Many of the proteins in plasma have important therapeutic uses. Albumin is commonly used to replenish and maintain blood volume after traumatic injury, during surgerySurgery
Surgery is an ancient medical specialty that uses operative manual and instrumental techniques on a patient to investigate and/or treat a pathological condition such as disease or injury, or to help improve bodily function or appearance.An act of performing surgery may be called a surgical...
, and during plasma exchange. Since albumin is the most abundant protein in the plasma its use may be the most well known, but many other proteins, although present in low concentrations, can have important clinical uses. See table below.
Factor VIII
Factor VIII is an essential blood clotting factor also known as anti-hemophilic factor . In humans, Factor VIII is encoded by the F8 gene...
Factor IX
Factor IX is one of the serine proteases of the coagulation system; it belongs to peptidase family S1. Deficiency of this protein causes hemophilia B. It was discovered in 1952 after a young boy named Stephen Christmas was found to be lacking this exact factor, leading to...
anticoagulant
Anticoagulant
An anticoagulant is a substance that prevents coagulation of blood. A group of pharmaceuticals called anticoagulants can be used in vivo as a medication for thrombotic disorders. Some anticoagulants are used in medical equipment, such as test tubes, blood transfusion bags, and renal dialysis...
overdose
factor II and factor X
Factor X
Factor X, also known by the eponym Stuart-Prower factor or as prothrombinase, is an enzyme of the coagulation cascade. It is a serine endopeptidase .-Physiology:...
deficiencies
liver disease
Liver disease
Liver disease is a broad term describing any single number of diseases affecting the liver.-Diseases:* Hepatitis, inflammation of the liver, caused mainly by various viruses but also by some poisons , autoimmunity or hereditary conditions...
some types of immune thrombocytopenic purpura
Antithrombin III deficiency
Antithrombin III deficiency is a rare hereditary disorder that generally comes to light when a patient suffers recurrent venous thrombosis and pulmonary embolism...
disseminated intravascular coagulation
Disseminated intravascular coagulation
Disseminated intravascular coagulation , also known as disseminated intravascular coagulopathy or consumptive coagulopathy, is a pathological activation of coagulation mechanisms that happens in response to a variety of diseases. DIC leads to the formation of small blood clots inside the blood...
emphysema
Emphysema
Emphysema is a long-term, progressive disease of the lungs that primarily causes shortness of breath. In people with emphysema, the tissues necessary to support the physical shape and function of the lungs are destroyed. It is included in a group of diseases called chronic obstructive pulmonary...
and COPD
Chronic obstructive pulmonary disease
Chronic obstructive pulmonary disease , also known as chronic obstructive lung disease , chronic obstructive airway disease , chronic airflow limitation and chronic obstructive respiratory disease , is the co-occurrence of chronic bronchitis and emphysema, a pair of commonly co-existing diseases...
cirrhosis
Cirrhosis
Cirrhosis is a consequence of chronic liver disease characterized by replacement of liver tissue by fibrosis, scar tissue and regenerative nodules , leading to loss of liver function...
Plasma processing
When the ultimate goal of plasma processing is a purified plasma component for injectionInjection (medicine)
An injection is an infusion method of putting fluid into the body, usually with a hollow needle and a syringe which is pierced through the skin to a sufficient depth for the material to be forced into the body...
or transfusion
Blood transfusion
Blood transfusion is the process of receiving blood products into one's circulation intravenously. Transfusions are used in a variety of medical conditions to replace lost components of the blood...
, the plasma component must be highly pure. The first practical large-scale method of blood plasma fractionation was developed by Edwin J. Cohn during World War II
World War II
World War II, or the Second World War , was a global conflict lasting from 1939 to 1945, involving most of the world's nations—including all of the great powers—eventually forming two opposing military alliances: the Allies and the Axis...
. It is known as the Cohn process
Cohn process
The Cohn process is a series of purification steps with the purpose of extracting albumin from blood plasma. The process is based on the differential solubility of albumin and other plasma proteins based on pH, ethanol concentration, temperature, ionic strength, and protein concentration. Albumin...
(or Cohn method). This process is also known as cold ethanol fractionation as it involves gradually increasing the concentration
Concentration
In chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration...
of ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
in the solution
Solution
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...
at 5oC and 3oC. The Cohn Process exploits differences in properties of the various plasma proteins, specifically, the high solubility
Solubility
Solubility is the property of a solid, liquid, or gaseous chemical substance called solute to dissolve in a solid, liquid, or gaseous solvent to form a homogeneous solution of the solute in the solvent. The solubility of a substance fundamentally depends on the used solvent as well as on...
and low pI
Isoelectric point
The isoelectric point , sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge....
of albumin. As the ethanol concentration is increased in stages from 0% to 40% the [pH] is lowered from neutral (pH ~ 7) to about 4.8, which is near the pI of albumin. At each stage certain proteins are precipitated out
Precipitation (chemistry)
Precipitation is the formation of a solid in a solution or inside anothersolid during a chemical reaction or by diffusion in a solid. When the reaction occurs in a liquid, the solid formed is called the precipitate, or when compacted by a centrifuge, a pellet. The liquid remaining above the solid...
of the solution and removed. The final precipitate is purified albumin. Several variations to this process exist, including an adapted method by Nitschmann and Kistler that uses less steps, and replaces centrifugation
Centrifugation
Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture...
and bulk freezing with filtration
Filtration
Filtration is commonly the mechanical or physical operation which is used for the separation of solids from fluids by interposing a medium through which only the fluid can pass...
and diafiltration.
Some newer methods of albumin purification add additional purification steps to the Cohn Process and its variations, while others incorporate chromatography
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....
, with some methods being purely chromatographic. Chromatographic albumin processing as an alternative to the Cohn Process emerged in the early 1980s, however, it was not widely adopted until later due to the inadequate availability of large scale chromatography equipment. Methods incorporating chromatography generally begin with cryodepleted plasma undergoing buffer exchange via either diafiltration or buffer exchange chromatography, to prepare the plasma for following ion exchange chromatography
Ion exchange chromatography
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their charge. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be injected is usually called a sample, and the...
steps. After ion exchange there are generally further chromatographic purification steps and buffer exchange.
For further information see chromatography in blood processing
Chromatography in blood processing
Chromatographic techniques have been used in blood processing and purification since the 1980s. It has emerged as an effective method of purifying blood components for therapeutic use.-Human blood plasma:...
.
Plasma for analytical uses
In addition to the clinical uses of a variety of plasma proteins, plasma has many analytical uses. Plasma contains many biomarkers that can play a role in clinical diagnosisMedical diagnosis
Medical diagnosis refers both to the process of attempting to determine or identify a possible disease or disorder , and to the opinion reached by this process...
of diseases, and separation of plasma is a necessary step in the expansion of the human plasma proteome
Proteome
The proteome is the entire set of proteins expressed by a genome, cell, tissue or organism. More specifically, it is the set of expressed proteins in a given type of cells or an organism at a given time under defined conditions. The term is a portmanteau of proteins and genome.The term has been...
.
Plasma in clinical diagnosis
Plasma contains an abundance of proteins many of which can be used as biomarkers, indicating the presence of certain diseases in an individual. Currently, 2D ElectrophoresisTwo-dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins...
is the primary method for discovery and detection of biomarkers in plasma. This involves the separation of plasma proteins on a gel by exploiting differences in their size and pI
Isoelectric point
The isoelectric point , sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge....
. Potential disease biomarkers may be present in plasma at very low concentrations, so, plasma samples must undergo preparation procedures for accurate results to be obtained using 2D Electrophoresis. These preparation procedures aim to remove contaminants that may interfere with detection of biomarkers, solubilize the proteins so they are able to undergo 2D Electrophoresis
Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins...
analysis, and prepare plasma with minimal loss of low concentration proteins, but optimal removal of high abundance proteins.
The future of laboratory diagnostics are headed toward lab-on-a-chip
Lab-on-a-chip
A lab-on-a-chip is a device that integrates one or several laboratory functions on a single chip of only millimeters to a few square centimeters in size. LOCs deal with the handling of extremely small fluid volumes down to less than pico liters. Lab-on-a-chip devices are a subset of MEMS devices...
technology, which will bring the laboratory to the point-of-care
Point-of-care testing
Point-of-care testing is defined as medical testing at or near the site of patient care. The driving notion behind POCT is to bring the test conveniently and immediately to the patient. This increases the likelihood that the patient, physician, and care team will receive the results quicker,...
. This involves integration of all of the steps in the analytical process, from the initial removal of plasma from whole blood to the final analytical result, on a small microfluidic device
Microfluidics
Microfluidics deals with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter, scale.Typically, micro means one of the following features:* small volumes...
. This is advantageous because it reduces turn around time, allows for the control of variables by automation
Automation
Automation is the use of control systems and information technologies to reduce the need for human work in the production of goods and services. In the scope of industrialization, automation is a step beyond mechanization...
, and removes the labor intensive and sample wasting steps in current diagnostic processes.
Expansion of the human plasma proteome
The human plasma proteome may contain thousands of proteins, however, identifying them presents challenges due to the wide range of concentrations present. Some low abundance proteins may be present in picogram (pg/mL) quantities, while high abundance proteins can be present in milligram (mg/mL) quantities. Many efforts to expand the human plasma proteome overcome this difficulty by coupling some type of high performance liquid chromatography (HPLC)High performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
or reverse phase liquid chromatography (RPLC) with high efficiency cation exchange chromatography
Ion exchange chromatography
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their charge. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be injected is usually called a sample, and the...
and subsequent tandem mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
for protein identification.