Cellular and Molecular Pathology – Essay Example
Cellular and Molecular Pathology – Essay Example
The presence of lumps in the liver can be felt as a patient lies flat with the liver being swollen. Other cases present an enlarged spleen and ability to hear a unique sound/noise when a stethoscope is used to listen to the blood vessels. The noise is usually caused by tumor pressure on the vessels.
Liver biopsy is the appropriate method that can be used in obtaining materials for the study. In this case, a definite diagnosis is provided. It is appropriate since it deals with the actual tissues and fluids from the liver thus giving appropriate results rather than the suspect results as provided by the physical method of testing.
Liver biopsy is done through obtaining a sample of the liver or rather a tissue fluid using a fine needle. The obtained tissue or fluid is prepared and checked under the observation microscope for the presence of cancer cells. In many incidences, about 70% biopsy shows a positive result for cancer (Brown, 2010). Cellular and Molecular Pathology – Essay Example.
However, in fewer situations, there are risks involved whereby about 0.4% of the cases, some patients develop severe blood loss since a number of tumors are supplied with major and numerous blood vessels thus the heavy bleeding.
Fixation is the process by which obtained tissue samples are preserved in a life-like state preventing damage and distortions and is always carried out sooner after tissue removal through surgeries and immediately after death in the case of autopsies. There are several fixatives such as alcohol, mercurials, oxidizing agents, picrates, and aldehydes. In this case, I would prefer the use of formaldehyde which is also regarded as a combination of formalin and glutaraldehyde.
The choice is coherently based on the neutral nature of the formaldehyde solution and the ability to penetrate the cells of the tissue thus encouraging visibility during the observation time. Formaldehyde has a standard solution hydrogen potential at 10% buffered formalin. The buffer is significant in the prevention of acidity that would in turn cause precipitation of the formol-heme tint in the tissues. Formalin has an osmotic pressure that is equal to that of the mammalian cells thus preventing the tissue structure changes due to its reaction.
Cellular and Molecular Pathology – Essay ExampleIn obtaining the right tissue for the examination, there are two distinct methods that can be employed. That is the physical examination technique and the liver biopsy technique. During physical examination, the medical history of the suspected patient is checked and more attention is paid to the patient’s abdomen. Presence of lumps in the liver can be felt as a patient lies flat with the liver being swollen. Other cases present an enlarged spleen and ability to hear a unique sound/noise when a stethoscope is used to listen to the blood vessels. The noise is usually caused by tumor pressure on the vessels. Cellular and Molecular Pathology – Essay Example. Liver biopsy is the appropriate method that can be used in obtaining materials for the study. In this case, a definite diagnosis is provided. It is appropriate since it deals with the actual tissues and fluids from the liver thus giving appropriate results rather than the suspect results as provided by the physical method of testing.
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Molecular testing is becoming an important part of the diagnosis of any patient with cancer. The challenge to laboratories is to meet this need, using reliable methods and processes to ensure that patients receive a timely and accurate report on which their treatment will be based. The aim of this paper is to provide minimum requirements for the management of molecular pathology laboratories. This general guidance should be augmented by the specific guidance available for different tumour types and tests. Preanalytical considerations are important, and careful consideration of the way in which specimens are obtained and reach the laboratory is necessary. Sample receipt and handling follow standard operating procedures, but some alterations may be necessary if molecular testing is to be performed, for instance to control tissue fixation. DNA and RNA extraction can be standardised and should be checked for quality and quantity of output on a regular basis. The choice of analytical method(s) depends on clinical requirements, desired turnaround time, and expertise available. Internal quality control, regular internal audit of the whole testing process, laboratory accreditation, and continual participation in external quality assessment schemes are prerequisites for delivery of a reliable service. A molecular pathology report should accurately convey the information the clinician needs to treat the patient with sufficient information to allow for correct interpretation of the result. Molecular pathology is developing rapidly, and further detailed evidence-based recommendations are required for many of the topics covered here. Cellular and Molecular Pathology – Essay Example.
Introduction
The management of individual cancer patients is increasingly influenced by specific features of the tumour, often due to specific genetic alterations.1 The concept of personalised medicine for cancer is not new, being variously known as individualised, stratified, or precision medicine. Examples are now commonplace, from oestrogen receptor and ERBB2 (HER2) amplification in breast cancer to EGFR mutations or ALKrearrangements in lung cancer, BRAF mutations in malignant melanoma, RAS mutations in colorectal cancer, and BCR/ABL1 in Philadelphia positive chronic myeloid leukaemia. Methods to individualise the status of a tumour include immunohistochemistry, and molecular pathology, which is generally taken to mean the analysis of nucleic acids from tissue, cells or fluids.2
Molecular testing is therefore becoming an essential part of the work-up for many if not most patients with solid and haematological tumours. As a consequence, most hospitals with an active oncology practice require access to laboratories that provide the necessary information on the genetic make-up of a tumour from biopsy material. This trend is likely to strengthen as new drugs become available and those on the market start to be used in combination or sequentially to overcome resistance mechanisms.
The challenge to the pathologist is to go beyond diagnosis and classification to produce the information required to guide treatment accurately and to do so in as short a time as possible. Cellular and Molecular Pathology – Essay Example.The simplest way to think about the requirements for laboratories offering these services is to consider the pathway from patient to result, and the requirements at each stage (figure 1). Inevitably, there will be differences between healthcare systems and laboratories in how they organise their work. The purpose of this framework is to assist molecular pathological laboratories in providing the best service to patients. Clear responsibilities for requesting molecular analysis, preanalytical sample handling, nucleic acid extraction and analysis, and reporting of results are prerequisites for the operation of a safe and efficient service.
Workflow for laboratories undertaking molecular pathology for cancer patients from formalin-fixed paraffin-embedded (FFPE) tissue samples. Cellular and Molecular Pathology – Essay Example.
One frequently asked question revolves around the number of samples any laboratory should be handling to be considered reliable. There are, at present, no data to answer this question, but we know from other settings (eg, ERBB2 (HER2) testing3 4) that it is not wise to consider setting up services for small numbers of patients, and indeed it is difficult to achieve a cost-effective solution for small numbers of samples. Equally, some genes are rarely mutated, and as laboratories adopt panel testing as the norm, even the largest laboratories are likely to find that they are reporting some mutations very rarely. Our belief is that the key to this question lies in the number of samples submitted for testing, and the clinical and logistic requirements of the service. The practicalities of testing and clinical needs may, therefore, make the decision straightforward for most tests.
Given that the number of patients requiring testing is likely to increase, most accredited laboratories are likely to offer a reliable service if they adhere to the recommendations covered in the subsequent sections, though monitoring of this by national bodies is recommended. It should be noted that there is separate guidance available for the various steps in this pathway, and for individual testing needs. An excellent example, based on a systematic review, is the guidance recently issued by the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology (AMP), for ALK and EGFR testing in lung cancer.5This paper is intended to provide an overview to guide laboratories in operating a reliable service: we have incorporated many of their recommendations and those from other relevant publications2 6––10 and the ISO15189 guidance.
Requesting molecular analysis
Multiple medical disciplines (eg, surgery, oncology or pathology) may request molecular analysis to define a treatment strategy for individual patients, though in practice this will usually be the oncologist. In many cases, a multidisciplinary team (tumour board) will make the decision to request a test. The requesting process should ensure that the request is made appropriately, and that every patient who needs a test is offered one in a timely manner. This is one of the most difficult aspects of operating a molecular pathology laboratory: it is important to ensure that tests are requested on all patients who need one, but equally that unnecessary tests are not performed. Cellular and Molecular Pathology – Essay Example. Molecular testing is expensive, although costs are currently decreasing, and oncologists or multidisciplinary teams may feel that they need to manage demand, particularly if they manage the budget. However, automatic (reflex) testing by pathologists based on diagnosis and tissue availability within the pathology department can be more efficient, particularly if >10% of patients with a particular diagnosis require testing (Cree, unpublished). The decision to implement reflex testing should be based on a business plan, taking into account the costs in time and money of specimen retrieval from pathology archives when the result becomes clinically necessary.6 Additionally, the amount of tissue that can be obtained versus that required for molecular pathology should be included in considerations of the sampling strategy: this is a particular problem in lung cancer patients. There is also a need for flexibility: technology and requirements are changing rapidly and frequent reassessment of clinical need is essential. Good communication is required between the laboratory, the oncologists, and surgeons to ensure that necessary tests are requested timely.
Previous treatment (eg, chemotherapy) can change gene expression11 and mutation status,12––14 and should be documented on the request form. It is important to discuss any changes to current protocols with surgeons and pathologists, who require clear identification of resection margins, but also need to accept the requirement for molecular analysis. Equally, interpretation of molecular analysis requires knowledge of the diagnosis and available treatment strategies.
Preanalytical standards
The pathology laboratory has to be able to handle multiple sample types. This raises a number of challenges when the needs of molecular analysis are included within a diagnostic pathway, though many of the issues are common to any diagnostic test. At every stage, there is potential for samples to be misidentified, and careful attention should be given to this danger. Cellular and Molecular Pathology – Essay Example.
It is the responsibility of the person taking the specimen to identify the patient, the sample required, ensure that the sample is correctly labelled, fixed in formalin (where necessary), and/or dispatched to the laboratory. However, when samples are being acquired in particular situations, such as within an operating theatre, the primary responsibility of the operating theatre staff is to the patient, not the sample, and thought should be given to the need to implement and operate fail-safe procedures to ensure that samples are treated correctly in order to ensure that reliable and accurate diagnosis and test results can be obtained.
The preanalytical process has recently been examined in some detail by the SPIDIA project (http://www.spidia.eu), the results of which we understand are due to be incorporated into ISO guidance (ISO 15189).
Tissue: The majority of diagnostic biopsies are small and are fixed rapidly when placed in neutral buffered formalin (4% formaldehyde), but larger surgical specimens require controlled fixation.15 16 It is probably optimal for the fixation process to be controlled by the pathology laboratory. This may involve transport of fresh specimens to the pathology laboratory, preferably under vacuum if this takes more than an hour (see below). Fresh tumour samples may be taken for molecular analysis (or tissue banking) in the operating theatre, but preferably or after receipt in the pathology laboratory according to defined protocols. It is best to open bowel specimens and (where possible) to incise larger masses to allow the penetration of fixative. Formalin only penetrates tissue at around 1 mm/h, and fixation will only start when penetration occurs. Formalin should only be used 24 h after dilution to 4% w/v, in order to reduce the effect of polymerisation, and ensure a stable 4% concentration. Cold ischaemia occurs between removal of tissue and its fixation, and is a particular problem for large surgical specimens.Cellular and Molecular Pathology – Essay Example. It alters levels of gene expression (at the RNA and protein level), and is a major consideration for molecular pathology.17 Control of fixation time and temperature is recommended for molecular analysis of proteins such as ERBB2 (HER2) or for the analysis of gene expression. Vacuum packing of tissue and transfer to the laboratory at 4°C (Tissue Safe) is one option attracting attention, and can be very useful for sites distant to the hospital laboratory.18 Cooling should be as rapid as possible and cold fixation preserves RNA well, though cold shock-induced changes can be a consideration for some biomarkers.19
Standard operating procedures (SOP) should be designed for tissue handling which incorporate aspects listed in the box 1.
Cytology samples should also be handled according to defined SOPs. In some centres, pathologists are involved in performing fine needle aspirations, which aids handling of the sample. Fixation is usually in an alcoholic fixative, which preserves nucleic acids relatively intact. This renders such samples suitable for most molecular analysis, though some problems have been noted in human papillomavirus (HPV) and non-small cell lung cancer (NSCLC) testing.20 21 22 23
Liquid biopsy: EDTA blood samples can be used to extract nucleic acids from cells (buffy coat) and plasma. The leukocyte fraction provides a ready source of germline DNA and has long been used to generate molecular data for haematologic neoplasia. Viruses and bacteria can be isolated and identified by molecular methods from buffy coat preparations. Cellular and Molecular Pathology – Essay Example. Cell-free DNA can be used for mutation analysis and, although such methods have yet to enter routine practice, they show considerable promise.13 24 25 The preference is for plasma from EDTA blood, sent directly to the molecular pathology laboratory or to the blood sciences laboratory. Serum contains DNA from leukocytes and is less suitable. It is feasible to use samples taken for routine haematology measurements, but lithium heparin tubes should be avoided, as lithium is a PCR inhibitor.
Caution should be exercised with RNA as measurement of levels can be affected by continuing production in cells after the specimen has been taken, and ongoing degradation by RNAse activity. Preventative measures should be taken to minimise such degradation of RNA, but rapid transport of samples on ice to laboratories is not a standard procedure in most hospitals, and establishing a cold chain or adding inhibitors to the samples as they are taken may be difficult. Specialised blood collection tubes are available to preserve RNA (eg, PAXgene, PreAnalytix/Qiagen; Valencia, California, USA), but their clinical use requires validation with the tests concerned.
In clinical laboratories, all processes must have SOPs. It is important that these are used and adhered to using work instructions and checklists where necessary.
Sample receipt and handling
There should be a dedicated process for the receipt of samples, which should be suitably staffed and allow accession numbers to be allocated to samples as they enter the laboratory (as per ISO 15189, and ISO 9001). Most Laboratory Management Information Systems (LIMS) allow this, but few have as yet acknowledged the need for integration of molecular pathology needs. Barcoding of samples is good practice and is encouraged. All samples should be tracked from patient to report. Cellular and Molecular Pathology – Essay Example.
Tissue, blood and fluid processing within the laboratory should be performed to defined SOPs (see ISO 15189). While most tissue biopsy samples are received fixed, fresh tissue can also be sent to the laboratory for intraoperative frozen sectioning or molecular analysis. This requires separate SOPs and dedicated portering, nursing and laboratory staff.
Tissue samples go through a macroscopic examination and cut-up stage, when blocks are taken and placed in bar-coded and/or numbered plastic cassettes for processing. Spare tissue in formalin can be retained at this stage. Vacuum or microwave-based processors are temperature controlled devices which ensure excellent penetration of graded alcohols and xylene substitutes, allowing paraffin embedding. The temperature for different machines and protocols can vary substantially, and this can affect nucleic acid recovery (Cree, unpublished). The use of fixed tissue that has been previously frozen is not advised, and decalcification reduces DNA and particularly RNA recovery. Both have been found to produce no or suboptimal results.26 27 28 Close collaboration between clinical teams and histopathology laboratories is advised, and it is critical that changes in processing are communicated to the molecular pathology laboratory and adapted where necessary.
Histopathological diagnosis is made from stained sections cut from paraffin blocks at 4–6 μm according to minimum dataset requirements (see http://www.rcpath.org/publications-media/publications/datasets/datasets-TP.htm). For molecular testing, it is essential to take strict precautions to prevent cross-contamination between samples. It is best practice to replace the knife (blades) regularly (ideally before each new formalin-fixed paraffin-embedded (FFPE) tissue block is cut). Additionally, disposable plasticware should be used to transfer sections to glass slides (if sections are used). Cellular and Molecular Pathology – Essay Example.The use of waterbaths to stretch sections can be avoided by using a droplet of PCR quality water on glass slides. Care should be taken that decontamination procedures do not reduce DNA or RNA levels required for assay when sections are cut. For example, the use of DNAzap wipes on microtome blades immediately before cutting sections can reduce DNA levels substantially, and should be avoided. The use of substances likely to inhibit PCR should be avoided.29
It is recommended that the pathologist marks the area of the section containing neoplasia on the H&E slide at the time of diagnosis for macrodissection or microdissection, if used. It may be necessary to mark multiple areas of the neoplasia for manual microdissection to account for heterogeneity of neoplastic cell content within large tumours. As an alternative to microdissection, 1 mm punches of marked areas may be used.30 Laser capture microdissection is largely a research tool and not necessary for routine molecular pathology.
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The histopathologist should estimate the percentage of neoplastic cells present in the sample (or part of the sample) selected for DNA/RNA extraction. This estimation can be important in determining success or failure of subsequent testing, as it may define the lower limit of detection and a minimum percentage of neoplastic cells present can be applied to many tests.31 If necrosis is present, this should be noted, but is best avoided in samples for molecular analysis. Widely dispersed tumour cells within a sample, or lymphangitis carcinomatosa, are both notorious examples in which a low percentage of neoplastic cells can produce false negative results. Where the pathologist is directly responsible for requesting the molecular analysis, such ‘reflex’ testing permits rapid turnaround times and prevents the need for retrieval of blocks at a later date, reducing test turnaround time, reducing staff time and therefore cost.Cellular and Molecular Pathology – Essay Example. This needs to be balanced against unnecessary testing in patients who do not need further treatment, or who need treatment at a later date when testing for mutations in alternative genes might be needed. Clear responsibilities for the assessment of neoplastic cell content and macrodissection or microdissection are required: this is an essential role of the histopathologist. The estimated percentage of neoplastic cells present in the tissue used for DNA/RNA extraction should be mentioned in the pathology report.
Blood samples (EDTA) are usually spun down at low speed to pellet red cells and retrieve the buffy coat, and plasma fractions. Processing should begin as soon as possible (ideally within 30 min) after sampling. Collection time should be indicated on the tube, and the elapsed period between sampling and processing should be recorded. Cells are usually removed from plasma by centrifugation at 1000–2000 g for 10 min using a refrigerated centrifuge. Centrifugation at 2000 g for 15 min also depletes platelets in the plasma sample. Again, a protocol should be strictly observed. It is feasible to use density centrifugation to obtain white cells, exosomes, and plasma from the same sample.32 Specimens can be flagged on many LIMS for molecular analysis and reflex testing may be feasible. Aliquoted plasma samples can be stored at −80°C.
SOPs should be established for secure and, where possible, controlled collection, handling, and storage of tissue and blood samples, and their fractions (including extracted nucleic acids) (see ISO 15189 and any national guidelines). Few laboratories maintain their stores of tissue blocks and slides in temperature controlled environments. Equally, blood samples are usually discarded within days of receipt. Blood and blood-derived products are considered biohazards and should be handled accordingly. Fresh tissue poses similar risk, but FFPE tissue is regarded as safe for handling, though chemicals involved in tissue processing need careful storage and handling.Cellular and Molecular Pathology – Essay Example.
