The impact of cytogenetics and molecular testing in diagnostic hematology

Abstract

Aim: This is to review the impact of cytogenetics and molecular testing in the diagnosis of haematological disorders, Its efficacy and rapid turnaround time in the diagnosis of malignant haematology.

Methodology: Relevant literature was retrieved to highlight the principles of Fluorescence in situ hybridization (FISH) and polymerase chain reaction(PCR), indications and applications, derivable clinical relevance, sources of error and necessary steps towards effective diagnosis in haematology.

Conclusion: When conducted properly and with appropriate controls, molecular testing in the clinical laboratory provides a rapid and accurate means of diagnosing and generating prognoses for many hematologic diseases and infections. Patient care will improve as a result of rapid turnaround time for diagnostic tests and more individualized patient treatment resulting from prognostic indicators identified by molecular testing.

Keywords: Impact, cytogenetic, molecular, diagnostic haematology

Corresponding Author: Peter Ubah Okeke

School of Sciences & Engineering

Atlantic International University Hawaii- USA

Term Paper

Introduction

Cytogenetics is the study of chromosomes, their structure and their inheritance. There are more than 30,000 genes in the human genome most of which reside on the 46 chromosomes normally found in each somatic cell.

Chromosome disorders are classified as structural or numerical and involve the loss or gain or the rearrangement of a piece of a chromosome or the entire chromosome. Buckton K E et al (1980) observed chromosomal abnormalities in about 0.6% of all live births. The gain or loss of an entire chromosome other than a sex chromosome is usually incompatible with life and accounts for about 505 of first trimester spontaneous abortions Boué J et al (1975).

In leukemia, cytogenetic abnormalities are observed in more than 50% of bone marrow specimens. These recurring abnormalities often define the leukemia and indicate clinical prognosis. However, cytogenetic analysis is so crucial to the diagnosis and management of hematological neoplasms that it is necessary for hematologists to understand the reports that are received and hematologists are often involved in the collection of appropriate samples.

Why chromosome testing?

This is because chromosome anomalies are major causes of reproductive loss and birth defects and also non random chromosome abnormalities are recognized in many forms of cancer. Medical doctors could order a test of karyotyping for analysis of mental retardation, infertility, Down syndrome, short stature, fetal loss and cancer. This testing will help in early detection of such disorders and solution could be sought timely. Analysis of cytogenetics could be carried out on the following;

• 1. Skin fibroblasts

• 2. Bone marrow cells

• 3. Blood cells

• 4. Serous effusions cells

• 5. Lymph node aspirates etc.

Chromosome banding

Testing of each individual chromosome is made possible by staining the chromosome with dye. Hence chromosome means colored body deriving its name from a Greek word. Caspersson et al (1969) were the first investigators to stain chromosomes successfully with a fluorochrome dye. Using quinicrine mustard which binds to adenine- thymine( AT) – rich areas of the chromosomes, they were able to distinguish a banding pattern, called Q-banding. Other stains are used to identify chromosomes but in contrast to Q-banding, these methods normally necessitate some pretreatment of the slide to be tested. Giemsa (G) bands can be obtained by pretreating the chromosomes with the proteolytic enzymes- trypsin. R- banding requires pretreatment of the chromosomes with hot 80oc to 90oc alkali and subsequent staining with Giemsa. R- banding is often useful for the study of structural changes of the ends of the chromosomes or telomeres.

Laboratory testing of chromosomes— Metaphase analysis

After banding slides are scanned under the light microscope with a low power objective lens (10x). When a metaphase has been selected for analysis, a 63x or 100x oil immersion objectives is used. Each metaphase is analyzed first for chromosome number. Then followed by its banding pattern. We know that normal somatic cell contains 46 chromosomes, which includes 2 sex chromosomes. Any variation in number and banding pattern is recorded by the laboratory technologist incharge of testing. At least 20 metaphase cells are analyzed from leukocyte cultures. Computer imaging or photography is used to confirm and record the microscopic analysis. Metaphase cells are selected for imaging on the basis of;

• 1. Containing the modal number of chromosomes

• 2 Having sharply banded chromosomes

• 3 Containing no artifacts

• 4 Having little or no chromosome overlap

Non-Radioactive In Situ Hybridization( FISH)

Fluorescence In Situ hybridization (FISH) is a molecular technique commonly used in cytogenetic laboratories. FISH studies are a valuable adjunct to the diagnostic workup and can be used for prognostic stratification response to treatment and also minimal residual disease. In FISH, the DNA or RNA probe is labeled directly with a fluorophor or indirectly labeled with a hapten such a biotin. Target DNA is treated with heat and formamide to denature the double stranded DNA rendering it single stranded.

After hybridization, the unbound and non-specifically bound probe is removed by a series of stringent washes and the cells are counterstained for visualization. If using a hapten label, specific antibodies to the hapten are applied to the cells. These antibodies carry a fluorescent tag. After the antibodies bind to the DNA or RNA the cells are washed and an antifade is applied and using a fluorescence microscope, In situ hybridization can be used to identify individual chromosomes.

Specific loci probes can be used to detect structural and numerical abnormalities in cancer -chronic myelogenous leukemia. Using FISH, dividing metaphase and nondividing interphase cells can be analyzed with FISH. The advantages of FISH include, that many cells can be examined -useful for detecting residual disease states. FISH can be performed in a shorter period of time- may be critical in confirming a diagnosis of acute promyelocytic leukemia. The main disadvantage is that only those abnormalities that are specifically sought will be found. Whereas conventional cytogenetic analysis permits all chromosomes to be evaluated.

Cancer cytogenetics

Cytogenetic analysis of malignant cells can help determine the diagnosis and probable prognosis of a hematologic malignancy, assist the oncologist in the selection of appropriate therapy and aid in monitoring the effects of therapy. Cytogenetic analysis of cancer involving other organs can be performed from solid tissue obtained during surgery or by needle biopsy. Chromosomal defects in cancer include a wide range of numeric abnormalities and structural rearrangements.

Concepts of molecular testing in diagnostic hematology

The application of molecular biology methods to the clinical laboratory enhances the diagnostic system in identifying an increasing number of diseases. Molecular testing also enables clinicians to monitor disease progression during treatment and to make more accurate prognoses.

There are 3 main areas of hematopathology molecular testing which includes:

• 1. Detecting chromosomal translocation in hematologic malignancies, and inherited hematologic disorders.

• 2. Identifying hematologically important infectious agents.

• 3. Monitoring minimal residual disease (MRD) after cancer treatment.

DNA – STRUCTURE AND FUNCTION

The central dogma in molecular biology testing is that information stored in the DNA is converted to a message (messenger RNA [mRNA]) that is translated into a functional unit (protein). This DNA to mRNA to protein relationship is very important to carry out cellular functions while preserving a record of the stored information. The copying of the stored sequences of DNA to form messages occurs by a process called transcription; DNA is transcribed to mRNA. The mature message is converted to a peptide (Protein) sequence by translation. The ability to store information depends on DNA structure. Structural units that carry the message for a single protein are called genes. Calladin C R et al (2004) observed that human somatic cells contain about 20,000 to 25,000 genes along 2m of DNA.

Application of molecular testing in hematology

This is grouped into 3:

• 1. Detection of inherited hematologic disorders;

• Hemoglobinopathies (sickle cell anaemia, Hbcc, Hbsc, ß and a – thalassemias).

• Coagulopathies (Haemophilia A and B, factor V Leiden mutation).

• Erythrocytic disorders, lipid storage diseases and Neutrophil disorders.

• 2. Hematologic malignancies possessing p53 alterations;

• Myelodysplastic syndrome

• Acute and chronic Leukaemia

• Lymphomas.

• 3. Detection of Hematologically important pathogens;

• Parasitic pathogens (malaria, leishmania, filariasis and trypanosomiasis).

• Fungal and Bacterial Pathogens of importance.

• Viral pathogens (cytomegalovirus, (CMV), Epstein – Barr, HIV – 1 and 2 etc).

TRANSCRIPTION AND TRANSLATION

Primary mRNA segments are composed of introns and exons. Introns are intervening sequences in the coding portions of genes whose functions remain unclear, although recent evidence suggests they play a role in regulation of gene expression Le Hir et al (2003). The exons provide the nucleotide sequences that encode the protein product. Before mRNA can serve as a template for translation, introns are spliced out of the primary transcript and the exons are joined. The nature mRNA is formed after other modifications Such as addition of a 5´cap and a tail of many adenine nucleotides Lewin B (2004). The mRNA leaves the nucleus and enters the cytoplasm to be translated into protein.

DNA replication

After cells carry out their functions, they divide via mitosis or die via apoptosis, also called programmed cell death. The cell cycle progresses through a sequence of phases. Interphase comprises the G1, S and G2 phases. During the G1 phase, the cell grows rapidly and performs its cellular functions. The S phase is the synthesis phase, the point in the cell cycle where DNA is replicated. The G2 phase is the period when the cell produces materials necessary for cell division. The M phase refers to mitosis, producing two identical daughter cells, each progeny having received one entire set of the DNA that was replicated during S phase. Some cells exit the cell cycle during the G1 phase and enter a phase called Go. Cells in Go normally do not re-enter the cell cycle and remain alive until apoptosis or necrosis occurs. For molecular purposes, the substrate for DNA polymerase is the free hydroxyl group located on the 3 carbon of a deoxyribonucleotide. DNA polymerase recognizes this hydroxyl group and catalyzes the joining of the complementary deoxyribonucleotide. DNA is read 3" to 5" by DNA polymerase, and the complementary strand is synthesized 5" to 3`.A primer provides the free 3" hydroxyl group required for DNA polymerase activity.

Primers are short polymers of nucleotides that are complementary to the template strand. However, it is worthy to state that DNA polymerase uses the primers to initiate the formation of the complementary strand, continuing until it meets a previously hybridized primer. The cell cycle is a highly regulated process. At certain critical points within the cell cycle, decisions are made to continue with cell division or begin cell death via apoptosis. This decision may depend on the state of the DNA replicated. Normally, the cell detect errors made during replication and corrects them or begins the process of apoptosis to prevent the production of cells containing errors within genes. If the sensing molecules within a cell fail, cell division may continue. If debilitating mutations in the genes that mediate cell cycle control are replicated, this may result in the beginning stages of tumour formation. One protein responsible for sensing damaged DNA is P53, a tumour suppressor protein. Damaged cells with increased P53 protein arrest cell division at G1, allowing time for DNA repair. Cells with mutant P53 protein are unable to arrest cells in G1 and continue the process of cell division with damaged DNA Kastan M B et al (1992). If the cell can repair the DNA damage, the cell cycle continues. If the cell damage is too severe, the cell undergoes apoptosis. Hematological malignancies such as 21%. CML kelma Z et al (1989), 23% of CLL Amiel A et al (1997) and 17% of ALL Zhou M et al (2000) are associated with P53 mutations or deletions.

DNA synthesis and accurate cell cycle control demands that the integrity of the nucleotide sequence is maintained during DNA replication.

Specimens for DNA isolation and testing

Most molecular testing begins with the isolation of DNA or RNA from the patient sample. To test for a mutation, the patient"s DNA is isolated. The patient sample for human DNA testing includes:

• 1. Peripheral blood samples

• 2. Bone marrow

• 3. Tissue biopsy

• 4. Needle Aspirates

• 5. Cheek swabs

• 6. Cerebrospinal fluid (CSF)

3 ml whole blood collected into routine EDTA is sufficient. The white blood cells are separated from the rest of the blood components and a lyses solution (detergent and proteinase enzyme) ruptures the WBC. A concentrated salt solution removes the cellular debris and proteins leaving the DNA with the aid of a precipitating agent – isopropanol and taken out of solution. If a delay in testing is inevitable, it could be stored frozen indefinitely at – 80ºc. DNA extraction of dispersed cells from bone marrow and needle aspirate specimens follow similar procedures Bartlett J M S et al (2003).

Polymerase chain reaction

The principal technique used in the clinical molecular laboratory is the polymerase chain reaction (PCR). It is an enzyme based method for reproducing large numbers of a target sequence of DNA Mullis K B & Faloona FA (1987), enabling detection of the sequence of interest from a small amount of starting material. As with DNA replication amplification of a specific nucleotide sequence by PCR requires the use of primers the other reactants in the PCR reaction are a DNA polymerase called Taq polymerase isolated from the thermophilic bacterium thermus aquaticus and deoxyribonucleotides. The steps of a PCR reaction include denaturing the DNA, allowing hydrogen bonding of the primers to their complementary sequences on the template and adding nucleotides to the primers through DNA polymerase activity. High temperature (95ºc) destroys the hydrogen bonding between the strands of DNA (denaturation).

A lower temperature 40ºc to 60ºc allows hydrogen bonding of the primer to the target (annealing) and must be optimized for each primer set. Finally, 72ºc is the optimum temperature for addition of nucleotides by the DNA polymerase (extension). And to facilitate these changes in temperature during the PCR reaction, an instrument called a thermocycler is used to modulate and monitor the temperature of the reaction tubes accurately. When the double stranded DNA template is denatured, one primer anneal to the 5´ – to – 3´ strand and the other primer anneals to the 3´ – to – 5´ strand. The Taq polymerase recognizes the hydroxyl group of the primers reads the template strand and catalyzes the formation of the phosphodiester bond joining the first complementary deoxyribonucleotide to the primer.

In the second PCR run, the temperatures changes are repeated, and the extended product becomes the template for the production of a daughter strand. After the second cycle, the daughter strand is bounded by the primer sequences at the 5´ and 3´ends, producing a fragment of DNA of the desired length. In subsequent cycles (30 – 40) i.e. total cycles, this DNA of specific length and sequence is reproduced millions of times. Proper controls are an essential feature in conducting molecular test using PCR. The three controls required for PCR are:

• A) Positive

• B) Negative

• C) "No – DNA" control

All these three controls should be included in each PCR run and assessment. The no – DNA control contains all the reagents used in the PCR, except that water is substituted for the DNA sample. Comparing the sample DNA lanes with that of controls on gel electrophoresis determines whether the target DNA sequence is present in the patient"s DNA.

REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION

Some molecular hematologic testing procedures require mRNA for analysis. Because the translation of mRNA produces proteins, altered ribonucleotide sequences within mRNA may result in an altered protein. Examples of this are leukaemia involving the Philadelphia chromosome, seen in 95% of CML cases, 20% of Adult ALL cases, 5% of pediatric ALL cases Dewald & Ketterling (2005) and rare cases of Acute myelogenous leukaemia McClure & Litz (1994). The classic Ph results from a reciprocal translocation of the ABL gene on chromosome 9 to that of chromosome 22, producing a BCR-ABL hybrid gene Keung Y K et al (2004).

Transcription of this target produces chimeric mRNA comprising portions of the BCR and the ABL genes. The translation of this chimeric mRNA results in a fusion protein that may alter the normal control of a cell´s cycle, eventually resulting in hematologic malignancy Clark S S et al (1987). A procedure known as reverse transcription PCR (RT-PCR) can be performed to detect the presence of the chimeric mRNA and is the preferred template compared with genomic DNA. In RT-PCR, the reverse transcriptase enzyme produces complementary DNA from mRNA present in a sample of total RNA that has been extracted from a patient´s cells. PCR amplifies the number of these complementary DNA molecules. RT-PCR requires the following:

Oligo(dT), Random or specific primer, Enzyme reverse transcriptase, Deoxyribonucleotides, primers, mRNA template and Taq polymerase. The first step in the procedure involves producing an RNA-cDNA hybrid using the enzyme reverse transcriptase and a primer. The primer called oligo (dT) consists of many thymine nucleotides. Most mRNAs possess a string of adenine nucleotides on the 3´end called a poly A tail. This oligo (dT) primer anneals to the poly A tail of any mRNA present. Reverse transcriptase recognizes the hydroxyl group on the last nucleotide of the primer and reads the mRNA template strand, then adds the correct complementary deoxyribonucleotide. Reverse transcriptase continues up the mRNA template strand, joining the complementary deoxyribonucleotides to the growing cDNA strand to form the mRNA-cDNA hybrid. Heat denaturation breaks the hydrogen bonds between the mRNA-cDNA hybrids, separating the two strands. The cDNA strand can act as a template for replication. The next step involves amplifying the single-stranded cDNA using primers specific for a target sequence in the BCR gene and ABL gene. DNA polymerase extends the primers, forming a double stranded cDNA of the target chimeric gene. The cycling continues, resulting in millions of copies of cDNA representing the sequence of interest Preudhomme C et al (1999).

DETECTING AMPLIFIED DNA

Several methods are available for detecting the amplified target DNA. These methods include:

1. Gel electrophoresis using ethidium bromide, fluorescence or autoradiography for visualization.

2. The use of restriction endonuclease enzymes followed by gel electrophoresis.

3. Nucleic acid hybridization of a known sequence to the amplified DNA.

4. DNA sequencing and Fluorescent probes and dyes for real-time PCR.

Gel electrophoresis

Nucleic acids possess an overall negative charge owing to the phosphate groups of the backbone. In addition, the size of the amplified target DNA sequence consists of a specific number of deoxyribonucleotides. DNA lengths are measured in BP or kilobase pairs (kb) which are equal to 1000 BP. Gel electrophoresis provides the means of separating the amplified DNA fragments based on these attributes. To electrophoreses, DNA or RNA, the nucleic acids are sieved through a polyacrylamide or agarose matrix by passing an electric current through the gel bathed in a conducting salt solution. During preparation, polyacrylamide or agarose matrices gel by cross linking produces pores. The pores of an agarose gel are larger than that of a polyacrylamide gel. This difference in pore size determines the gel required for electrophoresis. When separating larger DNA fragments (500bp to >50bp), an agarose gel is most effective. For smaller DNA fragments (5-1000bp), a polyacrylamide gel is used Sambrook & Russel(2001). Whether using an agarose or a polyacrylamide gel, the process of gel electrophoresis is the same. The amplified DNA, the appropriate controls and a size marker are loaded into the sample wells. An electrical current supplied by the power source moves the negatively charged DNA toward the positive terminal. The fragments migrate through the gel based on their size, three- dimensional shape and mass- to- charge ratio. Smaller DNA fragments move faster through the gel than the larger fragments. A size marker of DNA fragments of known sizes runs along with the amplified DNA samples with which the sample DNA fragments are compared.

Detecting the controls, size marker and the patient DNA fragments typically involves the use of ethidium bromide, sybr-green or autoradiography. Ethidium bromide a hydrophobic molecule about the same size as a DNA or RNA base, intercalates between the bases of a DNA double helix or between bases of a segment of secondary structure of a single DNA or RNA strand, labelling the nucleic acid such that it fluoresces when exposed to UV light. Ethidium bromide may be added during the preparation of the gel or after electrophoresis by soaking the gel in an ethidium bromide- containing buffer. Placing the gel under UV light causes the ethidium bromide to fluoresces orange illuminating the nucleic acid bands. Ethidium bromide is a mutagen; so many laboratories instead use another fluorescing molecule called sybr-green. Sybr-green binds to the minor groove of nucleic acid helices and lacks the health risks associated with ethidium bromide.

Another method of visualizing the DNA fragments is autoradiography. During PCR, one of the deoxyribonucleotides, usually the adenine nucleotide contains a radioactive alpha phosphate group that is incorporated during the elongation step. After gel electrophoresis, X-ray film is placed over the dried gel. The radioactivity present in the amplified DNA fragments exposes the film, producing a banding pattern that is interpreted by the technologist Lodish et al (2000). The use of gel electrophoresis alone, detecting the bands by any of the described method, is most appropriate when the presence or absence of the target is the goal of the test. This is often the case in assessing infectious diseases, such as cytomegalovirus infection. Plasma or leucocytes or both are the sample for DNA isolation and commercially available primers are available the PCR reaction.

Autoradiography also can be performed after incorporation of an enzyme- conjugated nucleotide into the growing chain or hybridization of a probe containing an enzyme- conjugated nucleotide to the nucleic acid. The enzyme most commonly used are horseradish peroxidase or alkaline phosphatise, which can cleave luminal or other synthetic chemiluminescent substrate with the release of visible light. This light is confined to the bands that contain horseradish peroxidise- conjugated DNAs. This methodology avoids the health hazards associated with radioactivity and is now commonly used in clinical laboratory practice.

In the clinical laboratory practice, automation is described for increasing reproducibility and efficiency, issues that often drive test design of any type. In terms of commercial kits, detection of the amplicon is often not left to human interpretation of the gel, with the storage and reproducibility issues that that accompany it. Rather fluorescently labelled nucleotides often are incorporated into the growing PCR products and relative fluorescence intensity is monitored to assess the presence or absence of the target band. Likewise, many other molecular based testing kits use the incorporation of fluorescent molecules for tracking and detecting amplicons, for quantification, for sequence analysis, and for presence or absence of target determinations.

Restriction endonucleases

One method to determine whether an amplified target DNA contains a mutation of interest uses enzymes called restriction endonucleases (also called restriction enzymes). These enzymes are present in bacteria and serve to cut (restrict) foreign DNA that may enter the bacterium. Today, they are commonly used in molecular biology procedures. Type-11 restriction endonucleases recognize specific nucleotides sequences and cut both strands of the target DNA at these specific points that is, restriction fragments. Hundreds of restriction endonucleases are commercially available allowing restriction of many different DNA sequences. The number of restriction fragments produced depends on the number of restriction sites present in the amplified target DNA Ross D W(1990). For example, one restriction site produces two restriction fragments, two restriction sites produces three restriction fragments, and so on. If a mutation creates or destroys a sequence pattern recognized by a specific restriction enzyme, that enzyme can be used to detect the mutation in an amplicons population. The term restriction fragment length polymorphism refers to the use of restriction endonucleases to identify mutations within genes by detecting changes in the length of the resulting bands after restriction. An example of this is the factor V Leiden mutation. Individuals possessing this mutation have an increased risk of venous thrombosis. The factor V mutation results from the replacement of guanine with adenine within the factor V gene. The mutation destroys a restriction site detected by the restriction endonucleases, Mnl1. The factor V amplicon is 223bp, housing two Mnl1 sites. One site is destroyed by the mutation. After PCR and incubation with Mnl1, the amplicon is reduced to restriction fragments of specific sizes.

Polyacrylamide gel electrophoresis separates the fragments followed by detection using ethidium bromide. Restriction of wide-type factor V amplicon generates fragments that are 37bp 82bp, and 104bp long, whereas the mutant gene has restriction fragments with length of 82bp and 141bp. A normal individual possesses two copies of the wild-type factor V gene. Three bands with the sizes of 37bp, 82bp, and 104bp are present after gel electrophoresis. An individual homozygous for the factor V mutation possess two copies of the mutated factor gene, resulting in the presence of two bands with sizes of 82bp and 141bp. An individual heterozygous for the factor V mutation possesses one normal and one mutated factor V gene. These individuals have four bands present with the sizes of 37bp, 82bp, 104bp and 141bp.

Nucleic acid hybridization

Another method to detect the DNA targets is by hybridization of a nucleic acid probe to the sample or PCR product. This method makes use of gel electrophoresis and may or not uses restriction endonucleases in addition to the hybridization Southern E M(1975). A southern blot can use isolated DNA from a patient sample without amplification by PCR but also can be performed on amplified DNA. After the isolation or amplification of the target DNA, a restriction endonuclease called EcorR1 cuts the DNA gel electrophoresis separates the fragments and the DNA is depurinated with acid to nick the DNA fragments. Sodium hydroxide denatures the DNA within the gel; producing single- stranded DNA without changing the DNA´s nucleotide sequence or two- dimensional position. The single- stranded DNA is transferred into a nitrocellulose filter by electrical current or capillary action so that the nitrocellulose filter exactly represents the gel. Methods used to affix the single-stranded DNA permanently to the nitrocellulose filter include baking and UV cross linking.

In the classic southern blot, detection of the band containing the DNA sequence of interest involves using a radioactive or enzyme – conjugated (horseradish peroxidase or alkaline phosphatise), single stranded probe complementary to the target DNA sequence Thomas PS (1980). Hybridization of this single stranded probe to the target DNA is followed by washing off the unhybridized probe. Detecting any bands labelled with the radioactive probe requires apposing the blot to film, called autoradiography. Southern blotting may be performed using hybridization of a fluorescently labelled probe instead of a radioactive one, which eliminates the hazards of working with radioactive labels Dowton & Slaugh (1995). An excellent application of the southern blot technique for hematologic testing is the B-cell IgH immunoglobulin gene or T-cell receptor gene rearrangement assays. Each B-cell or T-cell possesses a unique sequence for its IgH or T-cell receptor gene as a result of a complex process of rearrangement, joining together distinct segments of the gene from clusters of segments with unique sequences. Each B-cell and its progeny produce a specific antibody to an antigen, and each T-cell and its progeny possess a specific cell receptor to an antigen. Lymphoproliferative disorders arise from a malignant transformation of a B-cell or T-cell. In other words, a cell containing damaged DNA continues its cell cycle when it should have gone through apoptosis. These damaged cells no longer are under the control of the normal regulators for cell division and continue to divide uncontrollably, forming a malignant population of cells. Because the cells within the malignant population contain the same gene rearrangement, the cells are monoclonal. Gene rearrangement analysis can determine whether a monoclonal population of cells exists and whether the cell lineage for lymphoproliferative disorders is B or T-cell. The appropriate specimen for gene rearrangement analysis is bone marrow, blood or tissue from the suspect site. After DNA extraction from the specimen, the restriction enzymes EcoR1, BamH1 and Hind111 cut the DNA into fragments, agarose gel electrophoresis separates the DNA restriction fragments. The DNA is depurinated, denatured and transferred to a nitrocellulose filter. The hybridization step uses probes specific for nucleotide sequences in either B-cell or T-cell genes and autoradiography visualizes the banding pattern. The presence of a distinct band represents a monoclonal population of cells. A polyclonal population consisting of varied cells with unique gene rearrangements appears as a smear with no distinct banding pattern Noorali et al ( 2003). Gene rearrangement analysis provides physicians with an important tool to diagnose and monitor lymphoproliferative disorders in their patients. Single – stranded DNA probes also can be used to detect sequences of DNA in amplified samples by specific hybridization. The procedures are similar to southern blotting but use PCR products instead of genomic DNA. These DNAs are blotted into nitrocellulose by placing the denatured sample directly on the filter or transferred to the filter after gel electrophoresis. Under the proper conditions, the single stranded probe hybridizes to the single- stranded target DNA (if present in the sample), and the labelled probe is detected according to the label used.

In the clinical setting, radioactivity is not the method of choice for labelling nucleic acid probes for health and safety reasons, but there are many alternative labelling and detection kits and reagents commercially available. Radioactivity is incorporated into the nucleic acid during the synthesis of the probe by using a nucleotide building block that is conjugated to the radioactive molecule. The radioactivity can be detected by apposing to the film; thus it is an example of a direct label. Labels refer to molecules conjugated to the nucleic acid that can be visualized in some manner and can be direct or indirect. Directly labelling the DNA means that the molecule incorporated into the nucleic acid can be visualized on its own, whereas indirect labels require additional molecules to be visualized. Fluorogenic molecules can be conjugated to nucleotides for incorporation into DNA probes as direct labels. Acridinium ester is a chemiluminescence label for nucleic acids. After it is conjugated to the probe and the probe is hybridized to the target, sodium tetra borate solution containing 1% Triton X-100 degrades the acridinium ester on unhybridized probes, whereas base pairing of the probe to the target protects the ester. The bound ester is detected by a brief emission of light after addition of hydrogen peroxide.

Two commonly used indirect labels for nucleic acids are biotin and digoxygenin. Biotin is conjugated to nucleotides for inclusion in single-stranded probes. The biotin can bind a protein called avidin and the avidin is linked to a fluorescent molecule or to an enzyme for detection.

In a similar fashion, digoxygenin is incorporated into probe sequences and bound by an antidigoxygenin antibody conjugated to a fluorescent molecule or enzyme. Alkaline phosphatise and horseradish peroxidise are the enzymes most commonly used in indirect labelling systems. Their enzymatic activities are used to cleave substrates into visible compounds as pigments or as chemiluminescence.

DNA sequencing

The ability to read the sequence of nucleic acid has been just as important to the development of molecular biology as PCR. A combination of these two important methods (cycle sequencing) has made DNA sequencing more efficient and its analysis less subjective. DNA sequencing is a procedure whereby the sequence of the nucleotide bases in a strand of DNA is determined and it is applied in molecular testing to assess amplified sequences for insertions, deletions or mutations such as factor V Leiden mutation or the beta-globin mutation.

Cycle sequencing is based on dideoxynucleotide terminator sequencing. As discussed earlier, the addition of nucleotides to growing polymer requires a 3´hydroxyl group on the last added nucleotide and a triphosphate group on the 5´end of the next nucleotide to be polymerised. If a nucleotide also lacks the 3´hydroxyl group (a dideoxynucleotide), it can be incorporated into a strand of DNA, it cannot be added to, so the fragment is terminated at that base. If small amounts of dideoxyadenosine triphosphate, dideoxycytosine triphosphate, dideoxyguanine triphosphates and dideoxythymine triphosphate are include in the PCR reaction over numerous strands and numerous cycles eventually a nested series of fragments is produced with strands that terminate at each successive base. The dideoxynucleotides are fluorescently labelled, a different colour for each base. Capillary electrophoresis of the nested series of fragments moves the labels through a detecting laser one at a time based on their length. The sequence of the detected colours would reveal the nucleotide sequence complementary to the template strand. DNA is double stranded, so using two primers for the PCR run would produce two series of nested fragments and the detector would read two bases at each position. The PCR reaction is done with one primer in a reaction called single-sided PCR. Using the sickle cell mutation as an example, DNA sequencing of either strand, but not both strands shows whether the mutation (adenine to thymine) is present in the DNA. Each cell has two copies (alleles) of somatic gene; sequencing produces a nested series of fragments from each allele. If the patient is homozygote for this gene, the two nested series of fragments are identical, whether wide-type or mutant. If the patient is heterozygote for this gene, wild -type and mutant fragments are produced in the single sided PCR reaction and two nested series of fragments are produced. In analysis of this sequence, adenine and thymine signals are present at the position of the mutation, but because only half the templates contain each sequence, the signals of the adenine and thymine are half as strong. Sequencing is a reliable method for detection of mutations or single-nucleotide polymorphisms in DNA, but it is expensive and requires significant instrumentation, so it is not often used in clinical laboratory practice

Assessment of minimal residual disease

Advances in molecular testing in the diagnosis of hematologic malignancies have led to methods for assessing the effectiveness of treatments and as prognostic indicators for disease remission

Molecular techniques such as PCR, real – time quantitative PCR (RQ – PCR), cytogenetic and flow cytometry are more sensitive and can identify cells in hematologic specimens.

The low level of disease in patients who are in a state of clinical remission is called minimal residual disease (MRD). With lower levels of MRD detection by molecular methods, even to the point of molecular remission, the incidence of relapse is recognized earlier.

Detection of low levels of disease in patients in clinical remission also can help doctors with treatment decisions and provide indications of possible drug resistance.

PCR technologies have the ability to detect a single malignant cell in a population of 1 million cells, making it a sensitive method for MRD monitoring. Gene rearrangements and chromosomal deletions, insertions and translocations can be detected by PCR, whether they are generalized to a cancer type or specific to the individual.

Conclusion

When conducted properly and with appropriate controls, molecular testing in the clinical laboratory provides a rapid, sensitive and accurate means of diagnosing and generating prognoses for many hematologic diseases and infections. Patient care will improve as a result of rapid turnaround time for diagnostic tests and more individualized patient treatment resulting from prognostic indicators identified by molecular testing.

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Autor:

Okeke Peter Ubah