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Immunocytochemistry, or immunohistochemistry, is the method by which antibodies are used to detect cellular antigens in clinical samples. This can be performed on all routine diagnostic samples, including blood and bone marrow smears and bone marrow trephine (BMT) biopsies, in the assessment of hematological malignancies. The methodology enables simultaneous assessment of antigen expression and cell morphology so that cells of interest can be identified by their appearance and their phenotype determined. The major role of immunocytochemistry is in the diagnosis and classification of hematological malignancies, but it also has applications in lymphoma staging, determining prognosis, detecting potential immuno-therapeutic targets and for disease monitoring. Immunocytochemistry is used routinely to determine the phenotype (lineage and stage of differentiation) of neoplastic cells and is essential for the classification of hematological malignancies according to WHO criteria. This chapter describes the principles and applications of immunocytochemistry in the analysis of hematological malignancies. It includes discussion of monoclonal antibodies, hematopoietic differentiation and technical considerations, and illustrates some applications with clinical examples.
The majority of antibodies used in immunocytochemistry are monoclonal antibodies. These were first generated in 1975 by fusing an immortal myeloma cell line with splenic B-cells from an animal immunized with the desired antigen. The resulting hybridoma (or hybrid cell) produces antibodies with unique heavy and light chains and hence binding site. They are therefore clonal, have a single specificity and are termed “monoclonal” antibodies. Many thousands of monoclonal antibodies are available covering a wide range of cellular antigens.
The diagnosis of hematological malignancies has benefited enormously from recent scientific advances. In spite of this, morphology remains critically important and is the key front-line diagnostic technique which must not be overlooked. The light microscopy appearances may be diagnostic but, in addition, they are the foundation upon which decisions about further scientific assessment are based. In the modern era that utilizes a multi-parameter approach to disease classification, morphology is the screening test that determines the further investigations necessary. This opening chapter describes the principles of morphological assessment of the blood and bone marrow in the diagnosis, staging and monitoring of hematological malignancies. It will guide the reader through the process of blood and bone marrow microscopy and demonstrate how morphology alone can often give an indication of the underlying diagnosis. The chapter is not intended as a textbook or atlas of hematological neoplasms; for this, the reader is referred to one of numerous excellent monographs and atlases.
Abnormalities on a blood count, be they quantitative or qualitative, may be the first indication of a hematological malignancy and will generally lead to a blood film being examined. Whereas currently this is most commonly performed by light microscopy, automated image capture methods are increasingly being used. It is recommended for the assessment of neoplastic cells that morphological review be performed manually as a well-trained pair of eyes is more discriminating than a programmed computer.
The diagnosis of hematological malignancies is complex, expensive and evolving rapidly. There is a myriad of tests available and these are of increasing importance in the diagnosis and ongoing assessment of hematological malignancies. Optimal test utilization requires knowledge of the many individual diseases and the range of tests available to investigate them. Morphology, cell phenotyping, cytogenetics and molecular genetics are all essential and these must be used in a structured approach with the results integrated to give an overall diagnosis. To use these tests appropriately requires an understanding of the principles and roles of each of these test types and how they supplement traditional morphological assessment. As director of a large hemato-oncology diagnostic service and supervisor of hematologists-in-training, I have seen the difficulties trainees have experienced, and the time, effort and resources wasted on poorly focused testing due to lack of knowledge in these areas. As a consequence, and in the absence of texts on this subject, I was motivated to write a book to explain the diagnostic techniques and how they should optimally be applied to hematological malignancies.
This multi-authored book by an international panel of experts gives a state-of-the-art account of the principles and applications of the laboratory investigations available in the analysis of hematological malignancies in blood and bone marrow. The first five chapters provide a succinct review of the diagnostic techniques covering morphology, immunocytochemistry, flow cytometry, cytogenetics and molecular genetics.
The preceding chapters have illustrated that the analysis of hematological malignancies utilizes a range of cellular and genetic techniques of increasing sophistication and sensitivity. These generate bio-information of greater complexity than ever before, thereby requiring extensive data interpretation and integration. Only when the results of all test parameters are viewed together can the data facilitate an accurate and timely diagnosis, give prognostic indices, and be useful for monitoring treatment and/or disease progression. This approach necessitates a centralized laboratory facility staffed with a multi-skilled medical and scientific workforce focused on hemato-oncology diagnostics. They must be capable of carrying out a wide repertoire of diagnostic investigations and have the ability to interpret the data. This integrated approach to hemato-oncology diagnostics has proven advantages for clinicians and their patients. These comprehensive diagnostic centers are now being established in a number of countries. This chapter describes the structure of and role for integrated hemato-oncology diagnostic services.
What is an integrated hemato-oncology diagnostic service?
An integrated hematological malignancy (or hemato-oncology) diagnostic service is a comprehensive diagnostic facility for the processing and analysis of pathology samples from patients with, or suspected of having, a hematological neoplasm (Figure 6.1). Multiple test modalities are completed on a single sample, the results integrated and interpreted within the clinical context, a single report generated, and the information communicated to the requesting clinician. The concept is of “one patient, one diagnosis, one report” and this is only achievable with a multi-disciplinary laboratory performing all the relevant and up-to-date investigations.
The diagnosis and monitoring of hematological malignancies is complex and requires a systematic approach. Morphology, cell phenotyping, cytogenetics and molecular genetics are essential, and the results must be integrated. Diagnostic Techniques in Hematological Malignancies details the principles and applications of each of these test types in the diagnosis of hematological malignancies in blood and bone marrow. The first section describes the test modalities – including methodological principles, data interpretation and limitations – and is illustrated by clinical examples. The second section focuses on the clinical entities, detailing the most appropriate tests for diagnosis, staging and monitoring of different hematological malignancies and includes test utilization to identify prognostic markers and potential therapeutic targets. With contributions from multiple international experts, this illustrated book is an essential resource for qualified and trainee hematologists, oncologists, and pathologists. It's a practical and useful guide, providing a rational and structured approach to the laboratory assessment of hematological malignancies.
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