The Liquid Biopsies and Traditional Histopathological Approaches - Report Example

Are liquid biopsies going to replace traditional histopathological testing? Name Department Institution 1. Introduction Histopathology has always been an important component in patient care. The use of histopathology specimens not only improved diagnosis of patients but helped shaped important decisions in the care and treatment of patients. For instance, the information gained from the specimens helps to decide on the therapy to use on a patient or whether to change they therapy and start a different approach to therapy. Furthermore, the specimens are a critical component in the collection and storage of patients’ data1. As a result the data can establish treatment follow up procedures such as the follow up routines or surgical procedures. The specimens provide information about the patient’s response to therapy and which therapy areas effective among others. Traditional histopathological approaches have be used and relied upon for over a century for diagnosis and management of malignant and benign cancers. However, at the moment the traditional histpathological processes seem to be giving way to liquid biopsies. Both biopsies serve the same diagnosis and testing functions but they use different approaches. Liquid biopsies are liquid biomarkers in the blood of patients which can be used by physicians to test for benign and malignant cancers, monitor patients response to therapy. They can further use it to detect any acquired resistance to target drugs. The biopsies include microRNA, circulation tumor proteins and DNA, and circulating tumor cells. On the other hand, the traditional biopsies involve tissues biopsies and can also be used in diagnosis of cancer and other diseases, monitoring response to diseases, as well as, monitoring for residual infections2. 2. An overview of tradition and liquid biopsies and emerging issues Precision therapies that target specific genes are set to transform the way cancer patients receive care and treatment. There is even development towards personalized medicine which is likely to facilitate treatment of many cancers and chronic illnesses. However such processes are limited by challenges in acquisition of biopsy materials. The conventional methods of acquiring biopsy materials limit the ability of clinicians to establish the molecular status of their patients. This also limits their ability to diagnose and monitor cancer. The developments in liquid biopsy are posed to change this dilemma. The challenge with traditional biopsies is that limited tissue availability. Biopsies used different biomarkers and therefore, acquisition of biopsies materials depend on the target biomarkers. Unfortunately it is also difficult to identify candidates that qualify for different biopsies tests. Thus tissue availability becomes an enormous challenge for the dependability of traditional biopsies. Literature is full of examples of limitation of the traditional biopsies. For instance, according to the estimations as reported by Rosen on 57% of biopsies on lung andenocarcinoma had adequate genes for pathology staining and diagnosis3. Similarly Kris et al. reporting the findings of their study noted that only 66 percent of specimens tested positive for gene mutations, yet all the specimens had oncogenic indicators of mutations4. Also Amir et al reporting findings of a study noted that molecular testing for bone biopsy sample from sites commonly affected by breast cancer metastasis was only 36 percent accurate5. The traditional histological procedures relay of tissue samples that provide limited information about a patient’s response to threapy6. When collected tissue do not contain adequate tumor cells to facilitate genomic testing they fail to represent the fail reveal the prevailing genomic status of the cancer or tumor. However, liquid biopsies provide a solution to these challenges. The greatest problem that liquid biopsies solve is the problem of tissue availability. Liquid biopsy can detect tumor material for small samples of body fluids such as raw blood. The process involves an evaluation of circulation tumor cells (CTC) or circulating tumor DNA5,6. The approaches improve the efficiency with which the doctors can interrogate the tumor materials. Both method can be complimentary to each other and as result can facilitate determine whether a patient is eligible for target therapy. The greatest challenge with tumor material in the blood is that they are in small quantities. Separating and evaluating such small components has always been a challenge, however new developments have proved that it is possible to generate massive information about the patients’ status of from the little components. The components also provide an effective non invasive way of checking the patient’s health status7. At the moment clinical oncologists have adopted the use of liquid biopsy to gain quick real time data of cancer patients. Unlike the traditional biopsies, liquid biopsies have consistence in generating personalized data and can facilitate personalized treatment8. Liquid biopsy is not limited to blood alone but can use other body fluids9. Current studies indicate the cerebral spinal fluid may be effective at testing proteins from CNS tumors9. Liquid biopsies may also use miRNA profiles in plasma10,11,12. 3. Technical aspects, their advantages and disadvantages 3.1. Methods and techniques in traditional Biopsies For safe and effective functioning, all aspects of histopathology laboratory activities and procedures require accurate evaluation and monitoring. Quality control must cover pre-analytical, analytical and post-analytical activities13. The safety measures should cover the personal and the environment as well. This is because there are potential biohazards, chemical hazards, chemical hazards, equipment related accidents, as well as risk of fire and electrical hazards13. 3.1.1. Histological procedures There are key standard procedures used in performing histopathology. Histological procedures are an example of basic histopathology techniques. The procedure can provide quality samples that allow effective use of a light microscope to interrogated changes in animal or human tissues. Tissues collected from an individual are routinely fixed using 10% natural formalin and embedded in paraffin. The tissues are manually sectioned using microtome to yield 4–5 µm-thick paraffin sections14. The sections are dewaxed and subsequently stained using hematoxylin and eosin (H&E). Conversely, the dewaxed section can be put to task for other process such including special stain, in situ hybridization or immunohistochemistry among others. The process must yield standard, interpretable sections. Histopatholgy procedures have evolved over centuries along with the microscope and by the end of the nineteenth century most of the histology techniques had been documented. Such methods mostly relied on physicochemical reactions to facilitate tissues preservation, sectioning and staining. Some of these processes are partial automate but most of them are done manually and involve delicate resource intensive steps. 3.1.2. Immunocytochemistry Immunohistochemistry (ICH) is a technique that uses antibodies to detect antigens in tissue samples using antibody binding principles and radioactive substance or dyes that illuminate the antigen under a microscope principles 15. The procedure is important for detecting cancer using cancer specific antigens. ICH require use of appropriates biopsies, thus the availability of such biopsies is very influential. The biopsies have to be processed into sections using a microtome. Then, the Sections of the processed biopsies have to be incubated with a relevant antibody15. This is followed by visualizing the antibody binding site under a florescent or ordinary microscope using a marker such as enzyme, colloidal gold, fluorescent dye, or radioactive element that can be directly associated with the primary antibody or an appropriate related secondary antibody. The use of IHC was hinted at since 1930s but active studies commence in 1940s and shaped its evolution and development15. ICH is now an important tool in detecting biomarkers in biopsies applicable for detecting and monitoring cancers16. There are various advantage and disadvantages associated with ICH processes. In relation to the antibodies used, the advantages and disadvantages are as follows17. Antibody Advantage Disadvantage Monoclonal Permanent and reproducible results The antibody preparation is against non-purified antigen High specificity; the apposite combination of antibodies recognizes more epitops There is cross-reactivity when the same epitome is preset on more proteins Lacks validity since it fails to recognize more that a single antigen or epitope Polyclonal It is easy to prepare Has high validity There is non-specific reaction for natural antibodies Antigen specific purification is needed to enable detection Antibody purification is necessary Variable reproducibility Background due to nonspecific reactions Limited production 3.1.3. In situ hybridization In situ hybridization (ISH) is a hybridization technique that uses labeling of complimentary RNA, DNA or modified nucleic strands to interrogate a histological section with precision in locating specific mutations or abnormalities within such sections18. ISH works based on the underlying principle that adequate preservation of nucleic acids within a histologic specimen makes the discernable when a complimentary nucleic acid strand to which the report molecule can attach is applied. Through reporter molecule visualization, the RNA and DNA sequences in heterogeneous cell population such as those found in tissue samples become localized. In addition, riboprobes facilitate the location and assessment of the extent of gene expression. In situ hybridization facilitates interrogation for RNA (riboprobes) single-strand DNA (ssDNA) double-strand DNA (dsDNA) and synthetic oligonucleotides (PNA, LNA)18. For labeling, the approach uses radioactive isotopes and non-radioactive labels. Various ISH approaches and probes have various advantages and disadvantages19,20 Methods and probes Advantages Disadvantage Fluorescence in situ hybridization Works with all tissue types in research and clinical settings Works with archival specimen up to 7 years old Does not interfere with specimen structure Fast (48-72hrs) Supports variety of fixatives with variable results Facilitate automated and amenable analyses Reliable unbiased results Facilitate manual review of specimen images Acetic acid zinc formalin and formalin result in over 95% success rate Facilitate decalcified bone marrow core and B5 biopsy with over 50% success rate. Ineffective evaluation of chromosomal complement Manual scoring Difficulty in signal enumeration Oligonunleotied Does not require expertise in molecular biology or cloning It is stable There is good penetration of tissue There is no self-hybridizations Developed based on recipe for amino acid data Depends on published sequences Has unstable hybrids, Need for DNA synthesizer Low specific activities or sensitivity Limited labeling approaches RNA High specific activities Stable hybrids Unhybridized probe No reanealing No need for probe denaturation Limited tissue penetration Need for sub cloning DNA (double strand) Ease of use Needs no subcloning Range of labeling methods High specific activities Can achieve signal amplification During hybridization reanealing is necessary (due to probe availability reduction ) Need for probe denaturaiton to extend length increase tissue penetration which is usually decreased Hybrids less stable that RNA probes DNA (single strand) No reaneal No denaturation Technical complexities Hybrids unstable compared to RNA Sub cloning is needed 3.1.4. H&E Stains and Special Stains H&E stain and special stains serve an important role in tissue-based diagnosis. Pathologists have been using Hamatoxylin-Eosin (H&E) staining for over a century now21. There are various established methods for using this stain that available literature on this staining indicates that it will still be preferred by pathologists for over fifty years to come22. Hematoxlin and Eosin stain differently. They stain cell nuclei in blue and cytoplasm and connective tissue pink respectively. Special stains come in handy where H&E staining cannot reveal all information needed in biopsies. The term special stain refers to a range of stains that can be used in place of H&E stain. The stains use can be applied using a number of techniques to make various components easily visible under the light microscope. The stains functionality takes advantage of the intra- and extracellular chemical reactions involving the dyes and the tissue components. Ideally, these methods employ chemicals or dyes that have an enormous affinity for the components under investigation. The stains can thus make various structures, tissues and microorganisms to get stained and thus become visible. Although immunohistochemistry staining is an advance form of staining compared to traditional staining, ICH stains are sometime considered to be part of the special stains. The stains are monoclonal antibodies that target specific proteins and can be amplified through cloning. Antibodies get marked or labeled by brown or red counter stains. As a result the target cells become visible via a light microscope. 3.2. Liquid biopsies methods and techniques There are also various method use for conducting liquid biopsies. The primary techniques used for the testing for tumors under liquid biopsies are circulating tumor cells (CTCs), cell free DNA (cfDNA) and exosomes. Circulating tumor cells (CTCs) CTCs are cells that are released by a tumor into the patient’s body vascular. Such cell may serve as propagation materials for new tumors to develop in other body organs, which is referred to as metastasis23. Studies have shown presence of CTC in a number of metastatic carcinoma in lung cancer, colorectal, prostrate and breast cancer. One disadvantage with CTC is a low level of sensitivity24. However development such as enrichment-free assays, negative selection, positive selection and physical properties can effectively isolate rare CTCs. On the other had enrichment to increase sensitivity also present a challenge since it can decrease specificity. CTS cell have cytokeratin positive, epithelial cell adhesion molecule (EpCAM) positive, which is a viable nucleus, but do not have CD45. However, EpCAM and other indicators may not express on CTCs23. They may also get downgraded by epithelial and other processes leading to mesenchymal change (epithelial–mesenchymal transition, EMT). Thus CTCs heterogeneity is an enormous challenge and always demands more enriching the CTCs resulting in reduction in specificity. It is also evident that even when the cells isolated meet CTC phenotypic criteria, a large number of the cell are not cancer cells. The quantity of CTC in a sample is heavily dependent on what the detection platform considers to be CTC. CTC also fails to support long-term bio-marking since, a large number of CTC platforms need the blood used to be processed immediately after collection24. Besides, CTCs are fragile and have a high rate of degeneration when the blood is collected through the standard evacuation blood collection tubes23. However, CTC tests have show efficiency at detecting a number of tumor cells and metastasis. For since CellSave can provide CTC enumeration although it cannot facilitate tumor cell molecular genetics analysis. It can test for breast, prostate and colorectal cancer and help determine the survival rate of the patients. CTC can also serve as an early indicator of lung cancers. Common CTC detection approaches include protein-based approaches and mRNA-based approaches25. cell free DNA (cfDNA) Cancer patients have a high serum concentration of cfDNA than health individuals. The concentration has a relation with malignancy. Most cfDNA in the plasma is fragment about 150-180 bp length. Fragment bellow 150 bp are associated with mutations. There is a lot of wild DNA in the plasma which has nothing to do with cancer or tumors. The wild DNA usually results in technical challenge in the effectiveness of the various platforms to detect cfDNA in the liquid biopsies23. For instance it can lead to false results. Also despite the ability of cfDNA to analyze genetic and epigenetic various present in tumor DNA the fall short when it comes to analyze messenger RNA. On the other hand, a notable advantage of cfDNA is its potential for analysis for bio-banked body fluids such as frozen plasma. Also cfDNA is usually available in large quantities compared to CTCs from the individual. This cfDNA has a higher efficiency of testing for mutations compared to CTCs. Despite being promising, the challenges associated with cfDNA cannot be downplayed. The approach cannot characterize a tumors mutation status. In addition, there may be low volume of mutant alleles since cfDNA has a half-life of about 15 minutes to a few hours23. Another challenge is the extreme variations in the cfDNA concentration in different patients. The variance is also tumor specific and presents a challenge in achieving a maximum yield of cfDNA as well as pairing with a platform that can detect the limited tumor DNA in the background of wild DNA. cfDNA also has limited ability to make out mutation within plasma since it can only work with patients with a large number of circulating copies of the mutation. All platforms the soles use allelic form but do not take the mutations number into consideration have enormous limitations. This because allelic frequency suffers enormous interference from wild DNA that is not related to tumors26. This challenge results in the need to determine and take advantage of factors that affect wild DNA circulation such as exercises and blood collection techniques. Thus control parameters are necessary in the pre analytical phase to control daily activities. Also, blood has to be processed immediately. Factors such as delayed processing and storage temperatures can lead to release of wild DNA and affect the allelic frequency. Alternative, plasma is preferable of serum since it can also help avoid cell lysis that occurs when coagulating blood. Exosomes Exosomes are vesicles that contain DNA, RNA and proteins. They are actively released in the body from cell. Exosomes carry a lot of cell information since they are the intercellular messengers. Although exosome vesicles types are yet to be established, a majority of them have a diameter of 30-200 nanometers and can be isolated from body fluids including plasma, urines, saliva, serum and cerebrospinal fluid23. Exosomes can be reliable as biomarkers because of the stability with which they carry proteins and genetic materials such as RNA and proteins for their cells of origin. Exosomes can stimulate the growth of tumor cells, encourage angiogenesis and inhibit immune response. They also participate in metastatic processes. Tumor cells shed thousands of exosomes every day and thus a patient can have high as hundreds of billions of them in every mL of plasma. Exosome with tumor specific RNA mutation can be isolated for body fluids and can also be stored frozen for years. DNA test of exosomes can reveal mutations and gene amplification. A single exosome carries limited information but since they are abundant it is easy to conduct comparison extract vesicle and get the messenger RNA molecules23. Analysis and characterization of the surface proteins promises the ability to make out, segregate, differentiate and enrich exosomes from different cells. However, there is no reliable method yet for analyzing the proteins on the surface or exosomes although there are a few emerging approaches. The emerging approaches have been able to pinpoint ovarian, pancreases, and lung cancers susceptibility in the bloods of healthy blood donors. A combination of surface-based proteins methods, extraction of RNA from exosomes and qPCR assays shows urgency and sensitivity that can support monitoring of therapeutic responses. They also show resistance when using exosomes from brain tumors. Advancement in techniques also indicates a possibility of separating exosome form plasma and them discretely. Last the lipids in exosome also facilitate isolation and classification of the exosomes based on protein-to lipid ration26. Therefore, despite the promise of effective non-invasive testing for cancers and tumors, and the promise of patient specific therapies, liquid biopsies face enormous hurdles. More hurdles include absence of consensus on suitable technical approaches to adopt26. These include methodologies, right sample types, storage mechanisms, target molecules and appropriate detection techniques. Technical errors can arise with cell, molecules or sample contaminations resulting in statistical errors and wrong interpretations. Thus there is need for standardization of procedures, tools and techniques. 4. Conclusion In conclusion liquid biopsies and traditional histopatholical approaches both serve the same function. They facilitate detection of cancers and monitoring a patient response to therapy. While the both may overlap in some advantages, their technical aspects make them different and each has its own advantage and disadvantages. Although many point out to the superiority of liquid biopsies over the traditional histopathology approaches the two should not be considered as competitors but as complementary approaches that can be combined to limit the disadvantage of each and thus result in improve cancer diagnosis and monitoring of therapy responses. References 1. Allen DC, Cameron AI. Histopathology specimens: Clinical, Pathological and Laboratory Aspects Second Edition. London: Springer, 2013. 2. Gupta E, Bhalla P, Khurana N, Singh T. Histopathology for the diagnosis of infectious diseases. Indian Journal of Medical Microbiology.27(2):100-6 3. Rosen S. World Market for Cancer Diagnostics, 5th Edition, Precision and Personalized Medicine Arrives. Kalorama Diagnostics. New York: 2013 4. Kris MG, Johnson BE, Berry LD, et al. Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs.JAMA.2014;311:1998–2006.doi:10. 1001/jama.2014.3741PMID:24846037. 5. Amir E, Miller N, Geddie W, et al. Prospective study evaluating the impact of tissue confirmation of metastatic disease in patients with breast cancer. J Clin Oncol. 2012;30:587–92.doi:10.1200/JCO.2010.33.5232PMID:22124102 6. Heitzer E, Auer M, Ulz P, Geigl JB, Speicher MR. Circulating tumor cells and DNA as liquid biopsies. Genome medicine. 2013;5(8):73. 7. Cree IA. Liquid biopsy for cancer patients: Principles and practice. Pathogenesis. 2015;2(1–2):1-4. 8. Esposito A, Criscitiello C, Locatelli M, Milano M, Curigliano G. Liquid biopsies for solid tumors: Understanding tumor heterogeneity and real time monitoring of early resistance to targeted therapies. Pharmacology & Therapeutics. 2016;157:120-4 9. Connolly I, Li Y, Gephart M, Nagpal S. The “Liquid Biopsy”: the Role of Circulating DNA and RNA in Central Nervous System Tumors. Current Neurology and Neuroscience Reports.16 (3):1-8. 10. Zandberga E, Kozirovskis V, Abols A, Andrejeva D, Purkalne G, Line A. Cell-free microRNAs as diagnostic, prognostic, and predictive biomarkers for lung cancer Genes. Chromosomes Cancer. 2013; 52(4): 356–369. 11. Ulivi P, Foschi G,Mengozzi M, Scarpi E, Silvestrini R, Amadori D. Peripheral blood miR-328 expression as a potential biomarker for the early diagnosis of NSCLC. Int J Mol Sci. 2013; 14(5): 10332–10342. 12. Sanfiorenzo C, Ilie MI, Belaid A, Barlesi F, Mouroux J, Marquette CH. Two panels of plasma microRNAs as non-invasive biomarkers for prediction of recurrence in resectable NSCLC. PloS One. 2013; 8(1): p. e54596. 13. Adyanthaya S, Jose M. Quality and safety aspects in histopathology laboratory. J Oral Maxillofac Pathol. 2013; 17(3): 402–407. 14. Slaoui M. Histopathology Procedures: From Tissue Sampling to Histopathological Evaluation. Methods in Molecular Biology. 2011; 691:69-82. 15. Duraiyan J, Govindarajan R, Kaliyappan K, Palanisamy M. Applications of immunohistochemistry. J Pharm Bioallied Sci. 2012; 4(Suppl 2): S307–S309. 16. De Matos LL, Trufelli DC, De Matos MGL, Pinhal, MA.Immunohistochemistry as an Important Tool in Biomarkers Detection and Clinical Practice. Biomark Insights. 2010; 5: 9–20. 17. Bunea M, Zarnescu O. New Current Aspects of the Imunohitochemical Techniques. RoumBiotechnol Let. 2001; 6(3): 177-206. 18. Brown C. In situ hybridization with riboprobes: an overview for veterinary pathologists. Vet Pathol. 1998; 35(3):159-67. 19. Richard KR, Kall BK, Corn A, Foucar MK, Hozier J. Automated analysis of fluorescence in situ hybridization on fixed, paraffin-embedded whole tissue sections in B-cell lymphoma. Modern Pathology. 2006; 19:1027-1033. 20. Kumar A. In Situ Hybridization. International Journal of Applied Biology and Pharmaceutical Technology. 2010; 1(2): 418-430. 21. Gurcan MN, Boucheron L, Can A, Madabhushi A, Rajpoot N, Yener B. Histopathological Image Analysis: A Review. IEEE Rev Biomed Eng. 2009; 2: 147–171. 22. Fox H. Is H&E morphology coming to an end? British Medical Journal. 2000;53:38. 23. Brock G, Castellanos-Rizaldos E, Coticchia C, Hu L, Skog J. Liquid biopsy for cancer screening, patient stratification and monitoring. Transl Cancer Res. 2015;4(3):280-290 24. Lie M, Hofman V, Long E, Bordone O, Selva E, Washetine K, Marquette C, Hofman, P. Current challenges for detection of circulating tumor cells and cell-free circulating nucleic acids, and their characterization in non-small cell lung carcinoma patients. What is the best blood substrate for personalized medicine? Ann Transl Med. 2014 Nov; 2(11): 107. 25. Alix-Panabieres C, Pantel K. Circulating Tumor Cells: Liquid Biopsy of Cancer. Clinical Chemistry.2013; 59(1):110-118. 26. Karachaliou N, Mayo-de-las-Casas C, Molina-Vila MA, Rosell R. Real-time liquid biopsies become a reality in cancer treatment. Ann Transl Med. 2015; 3(3): 36. Read More

Thus tissue availability becomes an enormous challenge for the dependability of traditional biopsies. Literature is full of examples of limitation of the traditional biopsies. For instance, according to the estimations as reported by Rosen on 57% of biopsies on lung andenocarcinoma had adequate genes for pathology staining and diagnosis3. Similarly Kris et al. reporting the findings of their study noted that only 66 percent of specimens tested positive for gene mutations, yet all the specimens had oncogenic indicators of mutations4.

Also Amir et al reporting findings of a study noted that molecular testing for bone biopsy sample from sites commonly affected by breast cancer metastasis was only 36 percent accurate5. The traditional histological procedures relay of tissue samples that provide limited information about a patient’s response to threapy6. When collected tissue do not contain adequate tumor cells to facilitate genomic testing they fail to represent the fail reveal the prevailing genomic status of the cancer or tumor.

However, liquid biopsies provide a solution to these challenges. The greatest problem that liquid biopsies solve is the problem of tissue availability. Liquid biopsy can detect tumor material for small samples of body fluids such as raw blood. The process involves an evaluation of circulation tumor cells (CTC) or circulating tumor DNA5,6. The approaches improve the efficiency with which the doctors can interrogate the tumor materials. Both method can be complimentary to each other and as result can facilitate determine whether a patient is eligible for target therapy.

The greatest challenge with tumor material in the blood is that they are in small quantities. Separating and evaluating such small components has always been a challenge, however new developments have proved that it is possible to generate massive information about the patients’ status of from the little components. The components also provide an effective non invasive way of checking the patient’s health status7. At the moment clinical oncologists have adopted the use of liquid biopsy to gain quick real time data of cancer patients.

Unlike the traditional biopsies, liquid biopsies have consistence in generating personalized data and can facilitate personalized treatment8. Liquid biopsy is not limited to blood alone but can use other body fluids9. Current studies indicate the cerebral spinal fluid may be effective at testing proteins from CNS tumors9. Liquid biopsies may also use miRNA profiles in plasma10,11,12. 3. Technical aspects, their advantages and disadvantages 3.1. Methods and techniques in traditional Biopsies For safe and effective functioning, all aspects of histopathology laboratory activities and procedures require accurate evaluation and monitoring.

Quality control must cover pre-analytical, analytical and post-analytical activities13. The safety measures should cover the personal and the environment as well. This is because there are potential biohazards, chemical hazards, chemical hazards, equipment related accidents, as well as risk of fire and electrical hazards13. 3.1.1. Histological procedures There are key standard procedures used in performing histopathology. Histological procedures are an example of basic histopathology techniques.

The procedure can provide quality samples that allow effective use of a light microscope to interrogated changes in animal or human tissues. Tissues collected from an individual are routinely fixed using 10% natural formalin and embedded in paraffin. The tissues are manually sectioned using microtome to yield 4–5 µm-thick paraffin sections14. The sections are dewaxed and subsequently stained using hematoxylin and eosin (H&E). Conversely, the dewaxed section can be put to task for other process such including special stain, in situ hybridization or immunohistochemistry among others.

The process must yield standard, interpretable sections. Histopatholgy procedures have evolved over centuries along with the microscope and by the end of the nineteenth century most of the histology techniques had been documented.

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