TaqMan?
Related Terms
Alloimmune thrombocytopenia, amniocytes, fluorescence reporter probe, genotyping, HPA, human platelet antigen, human platelet-specific antigen, hybridization, LightCycler?, melting curve analysis, NAIT, neonatal alloimmune thrombocytopenia, PCR, platelet alloantigens, platelet glycoproteins, platelet-specific antigens, platelet transfusion refractoriness, polymerase chain reaction, post-transfusion purpura, real-time PCR, PTA, single nucleotide polymorphism, single strand conformation polymorphism, SNP, SSCP, TaqMan?, white blood cells.
Background
Platelet antigen genotyping is a method used to determine the genetic makeup of an individual with respect to human platelet antigen (HPA). The genetic makeup, or genotype of an organism, comprises the entire complex of genes. In a platelet antigen genotyping assay (analysis technique), the alleles responsible for HPA are determined and characterized, which aids in the detection, diagnosis, and treatment of certain medical conditions.
Genes: Genes (deoxyribonucleic acid, or DNA) are considered the building blocks of life because they provide instructions for all cells in the body. Genes, which are located inside cells, control an organism's development and functions by instructing cells to make new molecules (usually proteins). Proteins are organic (carbon-containing) compounds made of amino acids; the sequence of the amino acids in a protein is defined by a gene. Proteins are required for the growth and maintenance of the body. Alleles are two or more alternative forms of a gene that may occur alternatively at a given site on a chromosome. Chromosomes carry hereditary information in the form of genes.
DNA is a long, thread-like (double-helix) molecule made up of large numbers of nucleotides. Nucleotides are the building blocks of DNA and are made of nitrogen bases, sugars, and phosphate. Nitrogen bases are of two types: purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and thymine (T). Long strands of nucleotides form nucleic acids. The sequence of bases in DNA serves as the carrier of genetic (hereditary) information.
Platelets: Platelets (thrombocytes) are small, colorless, disc-shaped cell fragments in the blood, derived from large cells in the bone marrow known as megakaryocytes. Platelets promote blood clotting, or coagulation, when blood vessels are damaged.
Antigens: An antigen is any substance, such as a virus, bacterium, toxin, or foreign protein, which triggers an immune response in the body. The immune response is in the form of proteins that are specific to an antigen; they are known as antibodies or immune bodies.
Human platelet antigens: The external layer of a platelet membrane contains a series of complex molecules known as glycoproteins (a protein and carbohydrate compound). These glycoproteins (GP) express several types of (polymorphic) antigenic determinants on their surface, which are called human platelet-specific antigens (HPA). Antigenic determinants are located at specific regions on the surface of an antigen capable of evoking an immune response and may combine with a specific antibody to counter that response (e.g., HPA-1, HPA-2, etc.). HPA are chiefly found on the platelets and megakaryocytes. A mutation is a permanent variation in a DNA sequence of a gene, and mutations that occur in more than one percent of the general population are called polymorphisms.
Platelet-specific alloantigens are passed on from both the parents to children in an autosomal dominant manner. Alloantigens are like other antigens, but are present only in some individuals of a species. A human has 22 pairs of chromosomes, which are also called as autosomes, and one pair of sex chromosomes (X and Y chromosome). Normally, a child inherits only one chromosome of each pair from a parent, i.e., one from the mother and the other from the father. Autosomal dominant inheritance refers to a dominant allele passed on from parent to child on any autosomal chromosome. A dominant allele is one that is expressed even if only one copy is present or that masks the expression of the other allele derived from the second parent.
About 24 types of HPA have been defined and are grouped under six biallelic (allele-pairing) systems as HPA-1, -2, -3, -4, -5, and -15. The frequency of each type of HPA allele varies in different population groups. For example, HPA-2 and HPA-5 are more common among African-Americans than whites. The allelic forms of HPA are as a result of single nucleotide polymorphisms (SNPs) in the genes encoding the relevant platelet proteins. SNPs are DNA sequence variations that occur when a single nucleotide in the genome sequence is altered.
Platelet alloimmune disorders: Alloantibodies against platelet alloantigens play an important role in immune-mediated platelet disorders such as neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP), and platelet transfusion refractoriness (PTA). NAIT in fetuses and newborns is associated with thrombocytopenia, a condition characterized by very low levels of platelets in the blood. This condition is caused by destruction of fetal platelets by maternal antibodies elicited during pregnancy, as these antibodies are directed against fetal-specific platelet antigens that are inherited from the father. The platelet-specific antigens inherited from father are different from those present in the mother.
PTP is another form of alloimmune thrombocytopenia that is characterized by sudden onset of severe destruction of platelets by platelet alloantibodies and a decrease in platelet counts in the blood within 5-10 days of transfusion of blood products. Decreased platelets in the blood may result in purpura, i.e., bleeding underneath the skin, giving an appearance of red or purple colored spots on the skin. Platelet transfusion refractoriness is due to immune- or non-immune-related destruction of platelets that have been transfused, which results in the failure of satisfactory therapeutic responses than expected from the platelet transfusions. One of the immune-related causes for the destruction of platelets in PTA is due to HPA antibodies. The non-immune-related cause for the destruction of platelets may be due to sepsis (bacterial infection of the bloodstream or body tissues), drugs (e.g., amphotericin B), etc.
Indications: Platelet antigen genotyping is indicated in patients with post-transfusion purpura or in patients with unsatisfactory therapeutic response to platelet transfusion as a result of refractoriness. This test is also done as a prenatal test (i.e., during pregnancy) to determine maternal and paternal platelet alloantigen incompatibilities in suspected NAIT; when parents have had a prior affected pregnancy; and in female patients planning for a pregnancy who have a sister with a previously affected pregnancy. It is also indicated to confirm specific antibodies in patients with new platelet alloantigens and in persons with very low platelet counts in the blood not sufficient for serologic typing procedures, which detect platelet alloantibodies in a blood sample.
Methods
Deoxyribonucleic acid (DNA) is a long, thread-like, double-helix molecule made up of large numbers of nucleotides. Nucleotides are the building blocks of DNA and are made of nitrogen bases, sugars, and phosphate. Nitrogen bases are of two types: purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and thymine (T). Long strands of nucleotides form nucleic acids. The sequence of bases in DNA serves as the carrier of genetic (hereditary) information.
Platelets are small fragments of cellular material in the blood. The external layer of a platelet membrane contains a series of complex molecules known as glycoproteins (a protein and carbohydrate compound). These glycoproteins (GP) express several types of (polymorphic) antigenic determinants on their surface, which are called human platelet-specific antigens (HPA). An antigen is any substance, such as a virus, bacterium, toxin, or foreign protein, which triggers an immune system response in the body. The immune response is in the form of proteins that are specific to an antigen; they are known as antibodies or immune bodies. Antigenic determinants are located at specific regions on the surface of an antigen capable of evoking an immune response and may combine with a specific antibody to counter that response (e.g., HPA-1, HPA-2, etc.). The allelic forms of HPA are as a result of single nucleotide polymorphisms (SNPs) in the genes encoding the relevant platelet proteins. SNPs are DNA sequence variations that occur when a single nucleotide in the genome sequence is altered. A mutation is a permanent variation in a DNA sequence of a gene, and mutations that occur in more than one percent of the general population are called polymorphisms.
Platelet antigen genotyping is a method to determine the genetic makeup of an individual with respect to human platelet antigen. The genetic makeup, or genotype of an organism, is comprised of the entire complex of genes. In a platelet antigen genotyping assay (an analysis technique), the alleles responsible for human platelet antigen are determined and characterized, which aids in the detection, diagnosis, and treatment of certain medical conditions or disorders.
The initial step in the platelet antigen genotyping method involves the isolation or extraction of DNA from the sample of interest, such as blood or amniotic fluid. Amniotic fluid is the fluid surrounding the developing fetus (the child in the womb) that is found within the amniotic sac in the mother's womb. The purified DNA is used as a template (i.e., serves as a pattern for synthesis) for further analysis.
PCR-SSCP method: Conventional methods to detect single nucleotide polymorphisms (SNPs) associated with the development of platelet-specific antigens (alloantigens) use polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique, which is labor-intensive and time-consuming.
PCR is an efficient and sensitive laboratory technique to amplify (produce multiple copies of) a specific sequence of DNA into billions of copies. The amplification process is done in the presence of sequence-specific oligonucleotide primers and DNA polymerase enzymes in controlled conditions (e.g., temperature, time required, etc.). Oligonucleotide primer is a sequence of nucleotides, usually of 20-50 bases, that is complementary to a target DNA sequence and serves as a starting point for DNA replication. DNA polymerase is an enzyme that makes new DNA strands using preexisting DNA strands as a template, thereby assisting in DNA replication. An enzyme refers to a protein substance that helps in a biochemical reaction. Radioactive molecules are also introduced into the PCR product during the amplification process. This assists in seeing the PCR products at a later stage.
The PCR product, which is a double-stranded DNA, is then separated into single strands of DNA by a process called denaturation.
SSCP is a process of electrophoretic separation of single-stranded nucleic acids (denatured PCR products) based on the difference in a single base pair (e.g., a single nucleotide polymorphism, or SNP). Electrophoresis is a technique in which charged particles, such as nucleic acids, move under the influence of an applied electric field in a liquid or jelly-like medium made of tiny particles dispersed uniformly. In SSCP, a neutral (nondenaturing) polyacrylamide gel is used for electrophoresis. Polyacrylamide is a separation medium that is highly water-absorbent and made of polymers (repeated subunits) of acrylamide (a chemical compound).
The mobility of a single-stranded DNA is dependent on the length of the fragment and its structural arrangement (called conformation). Any change in the DNA sequence will alter the conformation; thus molecules of different conformations will move at different rates and get separated on a gel electrophoresis. SSCP takes advantage of this feature to show sequence variations due to SNPs.
The electrophoresis step is followed by visualization of the separated DNA fragments, which may be done with the help of autoradiography. Autoradiography is a technique where the separated DNA fragments that are labeled (attached) with radioactive molecules are visualized by developing a film exposed to radiation (similar to an X-ray).
DNA Sequencing: DNA sequencing is a process in which the exact sequence of nucleotides in a sample of DNA is determined. Usually Sanger's method, which is also called dideoxy or the chain termination method, is used to detect the platelet alloantigens.
In Sanger's method of DNA sequencing the initial step involves extraction of high-quality DNA from the sample of interest (e.g., blood), followed by PCR in the presence of fluorescent-labeled (attached) dideoxynucleotide triphosphate (ddNTP). High-quality DNA refers to the sample DNA that is not contaminated from other DNA sources and/or the sample DNA is not broken into very minute fragments, as they may give false results. ddNTPs are synthetic nucleotides that are structurally somewhat different from regular nucleotides and function as DNA chain terminators (stoppers) during the synthesis of a DNA sequence. The end reaction product is a set of DNA sequences differing in length by one nucleotide; the last nucleotide base in each sequence is the unique fluorescent-labeled ddNTP.
The reaction products are run on the electrophoresis, which separates the DNA fragments based on their size. During this, fluorescent detection systems identify the nucleotide base. These fluorescent signals are fed into and analyzed by a computer, identifying the exact sequence of DNA. The whole process is automated, and the resultant information of the DNA sequence is compared with other sequences by various computer programs, thereby spotting the single base pair changes (SNPs) in the sample DNA sequence.
Conventional DNA sequencing is difficult, time-consuming, and expensive, but with the development of automated DNA sequencers and newer detection methods (described above) this technique has become very competitive in detecting gene mutations. A permanent variation in a DNA sequence of a gene is called a mutation. Other advantages of direct (automated) DNA sequencing is the complete information it provides in a single experiment, such as the type of the SNP, the exact location of SNP on the DNA sequence, and the sequence context of each polymorphism associated with the platelet alloantigens.
Newer typing techniques: Newer methods to detect SNPs associated with platelet alloantigens have developed considerably in terms of automation and modifications in PCR techniques, electrophoresis, and detection methods, making them fast and efficient.
Platelet antigen genotyping is performed on a real-time PCR platform (e.g., LightCycler?, TaqMan?, etc.) with a fluorescence detection system using hybridization probes and a melting curve analysis technique. This is explained as follows:
Real-time PCR (RT-PCR), also called real-time quantitative PCR (RTq-PCR), is a polymerase chain reaction (PCR)-based laboratory technique that enables both detection and quantification of a specific sequence in a DNA sample simultaneously. The amplified DNA can be measured and also viewed in real time after each amplification cycle.The advantages of RT-PCR over conventional PCR are due to its fast, efficient, accurate analysis and measurement of the quantity of DNA in real time. RT-PCR is fully automated with a fluorescent detection system, eliminating the use of radioactive molecules and gel electrophoresis.
The amplified reaction products of PCR (or amplicons) can be detected and measured by two commonly used methods. One method follows the use of fluorescent dyes (e.g., SYBR green) during the reaction process, which bind with the amplicon. Another method uses fluorescent reporter probes or fluorescent hybridization probes, which are modified single-stranded oligonucleotide probes that fluoresce (release light) when paired with complementary DNA templates (using hybridization). A probe is a nucleic acid sequence used to detect complementary nucleic acid sequences on the target DNA.
Real-time PCR along with the monitoring and quantification of DNA synthesis also determines the melting point of the amplicon at the end of the amplification reactions. Each double-stranded DNA has its own specific melting temperature, which is the temperature at which 50% of the DNA becomes single-stranded. After PCR amplification a ''melt curve'' is generated by raising the temperature by a fraction of a degree; the change in the fluorescence (light emitted) is measured. All PCR products for a particular primer should have the same melting temperature and a constant emission of fluorescence. Any single mismatch between the labeled probe and the amplicon will significantly reduce the melting temperature as well as the fluorescence intensity, which is detected and analyzed with a suitable computer program. Hence, the melting curve technique may be used to detect and analyze any mismatched base pairs, which are representative of gene variations or SNPs associated with platelet alloantigens. The whole process is automated and analyzed within 45 minutes.
The advantage of platelet antigen genotyping in comparison to standard serological platelet typing tests is that the genotyping may be performed even if the samples have low platelet counts, as it uses DNA isolated from white blood cells (cell components of blood), whereas serological methods use platelets.
Research
Platelets are small fragments of cellular material in the blood. The external layer of a platelet membrane contains a series of complex molecules known as glycoproteins (a protein and carbohydrate compound). These glycoproteins (GP) express several types of (polymorphic) antigenic determinants on their surface, which are called human platelet-specific antigens (HPA). An antigen is any substance, such as a virus, bacterium, toxin, or foreign protein, which triggers an immune system response in the body. The immune response is in the form of proteins that are specific to an antigen; they are known as antibodies or immune bodies. Antigenic determinants are located at specific regions on the surface of an antigen capable of evoking an immune response and may combine with a specific antibody to counter that response (e.g., HPA-1, HPA-2, etc.). A mutation is a permanent variation in a DNA sequence of a gene, and mutations that occur in more than 1% of the general population are called polymorphisms.
Newer polymorphisms associated with human platelet-specific antigen (HPA) are currently being researched, which will aid in the early detection of HPA-related transfusion incompatibilities and transfusion refractoriness, thereby helping to monitor and treat such conditions. Transfusion is a process of transferring whole blood or its components from one individual (a donor) to another (a recipient). Transfusion incompatibility refers to the reaction of the recipient's body to a transfusion of blood or its components that is not compatible with its own blood. Platelet transfusion refractoriness is due to immune- or nonimmune-related destruction of platelets that have been transfused, which results in the failure of satisfactory therapeutic (treatment) responses to the platelet transfusions. One of the immune-related causes for the destruction of platelets in PTA is due to HPA antibodies. The nonimmune-related cause for the destruction of platelets may be due to sepsis (bacterial infection of the bloodstream or body tissues), drugs (e.g., amphotericin B), etc.
Ongoing research of HPA as a risk factor for myocardial infarction (heart attack) and cardiovascular (heart) diseases is being conducted to develop alternative therapies to manage these heart conditions.
Studies are underway to understand the role of HPA in relation to various platelet disorders, and transplantation of solid organs and bone marrow for better therapeutic management of these conditions.
Implications
Platelet antigen genotyping identifies the types of human platelet-specific antigens (HPA), which initiate the development of antibodies in the body and play an important role in immune-mediated platelet disorders such as platelet transfusion refractoriness (PTA), neonatal alloimmune thrombocytopenia (NAIT), and post-transfusion purpura (PTP). An antigen is any substance, such as a virus, bacterium, toxin, or foreign protein, which triggers an immune system response in the body. The immune response is in the form of proteins that are specific to an antigen; they are known as antibodies or immune bodies. Platelets or thrombocytes are small, disc-shaped cell fragments in the blood that promote blood clotting.
Neonatal alloimmune thrombocytopenia (NAIT): NAIT is caused by the destruction of platelets in the fetus (the child in the womb) by maternal antibodies triggered during pregnancy, as these antibodies are directed against fetal-specific platelet antigens that are inherited from the father. The platelet-specific antigens inherited from father are different from those present in the mother. There is a 50% chance that NAIT may occur during the first pregnancy and about an 80% chance of it occurring in subsequent pregnancies.
NAIT is seen in fetuses and newborns as thrombocytopenia (a condition that causes very low levels of platelets in the blood). Usually it has no symptoms or mild symptoms, which may be seen as bruises, peticheae (small purple spots on the body surface), spontaneous or prolonged bleeding, and hematoma at injection sites. A hematoma is collection of blood, usually clotted, which is caused by bleeding. In some newborns, it may be severe and accompanied by bleeding complications such as intracranial hemorrhage (bleeding within the skull), which may lead to death.
If NAIT is suspected early during the first pregnancy, it should be confirmed by maternal and fetal antibody tests as well as by HPA typing (for the mother, father, and fetus), followed by suitable treatment management for both mother and fetus. As the reoccurrence of NAIT is both high and increasingly severe in subsequent pregnancies, HPA typing may aid in preventing or monitoring this condition in the next pregnancy. HPA typing is also indicated for the sisters of affected women, as their fetuses are at high risk of developing NAIT.
Post-transfusion purpura (PTP): PTP is another form of alloimmune thrombocytopenia, characterized by sudden onset of the severe destruction of platelets and a decrease in platelet counts in the blood within 5-10 days of the transfusion of blood products. Transfusion is a process of transferring whole blood or components from one individual (a donor) to another (a recipient). Decreased platelets in the blood may result in purpura, i.e., bleeding underneath the skin giving an appearance of red or purple colored spots on the skin.
Typically, PTP is seen in persons who have been exposed to HPA by an earlier blood transfusion containing platelets (a process known as sensitization). The fresh blood transfusion triggers an immune response in an already-sensitized person by stimulating the production of HPA antibodies. These antibodies destroy the platelets in the person's blood, thereby reducing the platelet count. Due to severe decrease in platelet counts, hemorrhages (bleeding) are common, which may be fatal. The most commonly associated HPA responsible for PTP is HPA-1a/b. PTP due to HPA-3, -4, -5, and -15 have also been reported in the literature. Thus, HPA genotyping would be helpful in sensitized individuals and in persons receiving repeated blood transfusions so as to prevent the development of PTP
Platelet transfusion refractoriness (PTA): PTA is due to immune- or nonimmune-related destruction of platelets that have been transfused, resulting in failure of satisfactory treatment responses to platelet transfusions, hence these patients are known as refractory to platelet transfusions. Platelet transfusions are usually given to patients with very low platelet counts (due to conditions such as certain cancers of blood), patients on chemotherapy (cancer treatment), etc. One of the immune-related causes for the destruction of platelets in PTA is HPA antibodies. The nonimmune-related cause for the destruction of platelets may be sepsis (bacterial infection of the bloodstream or body tissues), drugs (e.g., amphotericin B), etc.
Identification of HPA genotyping is important in PTA, as the platelet transfusion dose may have to be increased in patients with unsatisfactory treatment response. In some patients transfusions may have to be discontinued so as to improve response
Coronary thrombosis: Several recently conducted studies have indicated that HPA may represent a genetic risk factor for coronary thrombosis and coronary heart disease. Coronary thrombosis is a blockage of the coronary artery (the blood vessel supplying blood to the heart) by a blood clot, leading to myocardial infarction (heart attack), a major cause of illness and death. HPA-1 and HPA-3 polymorphisms have been associated with the development of coronary thrombosis. Thus, platelet antigen genotyping may aid in taking suitable precautions to prevent or control the development of these heart conditions.
Limitations
The single strand conformation polymorphism (SSCP) technique can detect single nucleotide polymorphisms (SNPs) from an optimal fragment size in the range of 150-200 bp (base pairs) but not in larger fragments. Hence, SSCP is not suitable to detect SNPs in a single gene or multiple SNPs in a single experiment. SSCP is also very time-consuming and may become very expensive if used to detect multiple SNPs or to scan the whole gene to detect new SNPs. DNA is a long, thread-like (double-helix) molecule made up of large numbers of nucleotides, and it serves as the carrier of genetic (hereditary) information. A base pair refers to the pair of nitrogen bases that connects the complementary strands of DNA by hydrogen bonds such as adenine-thymine and cytosine-guanine. SNPs are DNA sequence variations that occur when a single nucleotide in the genome sequence is altered. SSCP is a technique to detect the SNPs associated with human platelet antigens (HPA).
Another disadvantage of SSCP is that the mobility difference between the fragment sizes does not match the amount of sequence difference (changes in the sequence), thus it only indicates whether PCR products are either identical or different. This information may not be sufficient to detect SNPs. PCR is an efficient and sensitive laboratory technique to amplify (produce multiple copies of) a specific sequence of DNA into billions of copies.
The disadvantage of the use of DNA sequencing to detect SNPs is that it requires very high-quality DNA and is expensive, which is being overcome by advances in technology. Another major drawback with this method is that only about 400 base pairs of sequencing data can be generated by DNA sequencing in a series of experiments, restricting it to smaller fragment sizes, thereby increasing cost and time.
Future research
Accumulated knowledge of the past and present research on human platelet-specific antigen and its alloantibodies will be useful for the development of cell-based therapies and regulations, and for the adaptation of immune-based therapies of both acquired and hereditary diseases. Cell-based therapies use stem cells, which have the ability to self-replicate and give rise to specialized cells, in the treatment of certain blood disorders and blood cancers. Immune-based therapies are treatment methods where a person's immune system is regulated by stimulating or inhibiting the growth and activity of various immune system cells.
Author information
This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).
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