Modern hematology emphasizes a multiparametric diagnostic approach and the basic parameters, beside history of the disease and clinical examination, are morphological, immunophenotypic and genetic evaluation. Flow cytometry plays an important role in diagnosis of a large group of hematological diseases. This article reviews the basic principles of flow cytometry and its use in hematology diagnosis, with emphasis on chronic lymphoproliferations.
by Dr Nataša Lazić
Introduction
In modern diagnostics, flow cytometry has an important place as one of the basic and irreplaceable tools for diagnosis, classification, monitoring and prediction of malignant hematological disease [1]. The extreme complexity of these diseases, on one hand, and the availability of the different therapeutic protocols for the different types of these diseases on the other, makes accurate and precise diagnosis imperative. Contributing to this is the fact that the World Health Organization (WHO), in the Classification of Tumours of Hemopoietic and Lymphoid Tissues, suggests a multiparametric approach in diagnosing these diseases; basic parameters required are morphological, immunophenotypic and genetic analysis for each entity of the disease, in addition to a detailed history of the disease and clinical examination [2, 3]. The clinical picture and cell morphology, as a well-known and traditionally-used means of examination, are insufficient in many cases; quite often, because of a similar clinical presentation and cell morphology, it is not possible to draw a diagnostic conclusion based on these findings or a wrong diagnosis may be reached in some cases.
Coulter’s principle of measuring the change in the electrical impedance of the individual cells flowing through the measuring cell, in the late 1940s, was the basis for construction of the first hematologic counter and later for the flow cytometer. Later inventions added new detection capabilities, such as light scatter and fluorescence detection. Fluorescent activated cell sorting (FACS) was invented in the late 1960s by Herzenberg, Bonner, Sweet and Hullet. Introduced as a commercial machine in the early 1970s, this is the class of instruments now commonly referred to as flow cytometer [4]. The invention of monoclonal antibodies by Milstein and colleagues in 1977 opened new perspective for flow cytometry. Further developments, especially in electronics, led to modern cytometers with multiple lasers, detectors, better performance characteristics, and the ability to measure larger amounts of data.
Flow cytometry principles
Flow cytometry is a powerful technology that simultaneously measures many aspects of single particles, usually cells. Any suspended particle or cell from 0.2–150 μm is suitable for analysis. However, it can also measure soluble molecules if trapped onto a particulate surface and bound by fluorochromes. Virtually any component or function of a cell can be measured if the fluorescent probe can be made to detect it.
Sample preparation should provide a homogeneous suspension of cells with monoclonal antibodies conjugated with fluorochromes of a different emission spectrum. Depending on the sample, it most often includes incubation, erythrocyte lysis, centrifugation, washing and fixation.
The cytometer needs to be adjusted to have the appropriate performance characteristics (linearity, sensitivity, CV, electronic and optical background noise, fluorescence detector efficiency, etc). This is achieved by adjusting voltages on the detectors and by spectral overlap compensation (Fig. 1).
The three main systems of flow cytometer are fluidics, optics and electronics (Fig. 2). Parameters measured include forward scatter (FSC) corresponding to cell size, side scatter (SSC) depending on internal complexity and fluorescence intensity for different fluorochromes.
Becoming more available in clinical laboratories, a wide range of clinical applications of flow cytometry are constantly expanding and the most common among them are in, for example, lymphoma and leukemia diagnosis, stem cell enumeration for transplantation, estimation of minimal residual disease, paroxysmal nocturnal hemoglobinuria diagnosis, immunodeficiencies, HIV infection.
Flow cytometry in hematology
Flow cytometric immunophenotyping enables examination of the phenotype of the separate cells in the suspension and summarizing of the results, which gives data about the presence or absence of antigen expression as well as the expression intensity [5]. Hence, an immunophenotypic pattern is obtained on the cell population of interest for the examined disease. Meanwhile, there are no separate antigens specific for the particular disease. Instead, their mutual relation is observed and analysed, which makes the analysis of the flow cytometry results very demanding and complex, but usually very useful and precise owing to the huge amount of data that can be collected from the cells [6]. Therefore, flow cytometry helps with determining the cell line, the degree of cell maturity, abnormal patterns of expression and provides a detailed immunophenotype of the pathological cell population [7]. From information on all the aforementioned factors, a diagnostic conclusion is drawn if there is a phenotype characteristic for some disease. In the case of an atypical phenotype, the disease is assigned to the appropriate group and additional tests should be done to gain a precise diagnosis (such as immunohistochemical, FISH, molecular tests).
CD markers (clusters of differentiation) are blood cell antigens that enable their characterization. CD nomenclature was developed and reviewed by HLDA (Human Leukocyte Differentiation Antigen) workshops started in 1982. There were 10 such workshops and the nomenclature now encompasses about 400 CD markers. Monoclonal antibodies against those antigens are used for immunophenotype characterization.
The antibody panel for the analysis of the sample to be tested by flow cytometry depends, to a large extent, on the available information of other findings made for that patient. According to the Bethesda Group recommendations from 2006, which were aimed at regulating a more systematic approach in this field (and are still valid today), before sending a sample to flow cytometry, a detailed history of the disease, clinical examination, microscopic examination of cell morphology, and other laboratory tests should be carried out, and based on this, diagnosis or differential diagnosis determined. In this way significant rationalization and cost reduction can be achieved [8].
Immunophenotype characterization for chronic lymphoproliferative disorders
For both of the two major groups of malignant hematologic diseases, those derived from mature and from immature cells, flow cytometry is of a great importance. Neoplasms of mature lymphoid cells, according to the WHO Classification, include chronic lymphoid leukemia and non-Hodgkin’s lymphoma. Their basic characteristic is that they have an immunophenotype similar to mature lymphoid cells and, accordingly, they show an absence of immaturity indicators (CD34, TdT). According to the origin, in relation to the cell line, they can be divided into T, B and NK neoplasms. [7]
Mature B-cell lymphoproliferations make up most of the malignant blood diseases: 90 % of the total lymphoid malignancies, according to WHO data. They present 4 % of the newly discovered carcinomas per year. As already known, the malignant cell derived from B-cell lineage in most cases imitates the normal B-cells stopped at a certain maturity level. The classification of this disease group mostly relies on this fact. The most common in this group are chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), follicular lymphoma, splenic marginal zone lymphoma, mantle cell lymphoma (MCL), plasma cell leukemia [12]. Immunophenotype characterization in the diagnosis of B-cell chronic lymphoproliferative diseases is an irreplaceable method and, together with morphology, it presents the essential search that should be undertaken in the diagnosis of these diseases[2, 9]. Based on the finding of the immunophenotype characterization it is possible to discover aberrant expression patterns and establish the phenotypic characteristics related to particular diseases. The application of a scoring system as an additional tool is the result of a need for some standardization and quantification in the diagnosis of B-cell chronic lymphoproliferative diseases. In order to increase the precision of the scoring system, different studies with different CD markers are taken [10–12]. The most common scoring system of 5 points includes CD5, CD23, FMC7, CD79b and surface immunoglobulin chains with an accuracy of 96.6 % if a three-point cut-off is used [10].
In most cases of CLL, cell morphology is characteristic and typical for this disease. However, in a number of cases, flow cytometry has a huge and decisive significance for diagnosis (Fig. 3) [13]. CLL and MCL share many morphological and immunophenotypic features [14]. As a result of their partial overlap, a differential diagnosis of MCL is most considered when making a diagnosis of CLL. Because of the different therapeutic approach and prognoses of the diseases, their diagnostic differentiation is very important. For that purpose cyclin D1 testing is recommended [15, 16]. Unlike the other chronic lymphoproliferations, HCL cells do not match any stage of the normal lymphoid cells development. Morphologically typical HCL cells have fine, hair-like, cytoplasmic projections, which are sometimes difficult to find in the peripheral blood smear. Because of this and a very specific immunophenotype, flow cytometry is essential for HCL diagnosis [14, 17].
Advantages
The possibility of combining more antibodies in the same tube and analysing their interactions on the population of interest for the given disease is the greatest advantage of multiparametric flow cytometry, which involves simultaneously collecting and analysing a large amount of data from cells or particles.
Considerations
Comprehensive analysis involves considering possible causes of false-positive or false-negative results, thus avoiding an incomplete or incorrect interpretation of flow cytometry data (Fig. 4).
Other difficulties, such as non-standardized methods, particularly the issue of regulation in cytometry, different antibody panels, cut-off values, analysis subjectivity – recommended visual approach, result analysis complexity, report form, etc., are the subject of work by various associations dealing with cytometry in order to achieve harmonization in this area [13].
References
1. Paiva A, Alves GVA, Sales VSF, Silva ASJ, Silva DGKC, Alves E, Bahia F, Freitas RV, De Oliveira Paiva HD, Cavalcanti GB, Jr. Utility of flow cytometry immunophenotyping and hematological profile in chronic lymphoproliferative disorders. Blood 2017; 130: 5326 [poster abstract].
2. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman J (eds). WHO classification of tumors of haematopoietic and lymphoid tissues. IARC 2008; Chapters 1, 8, 10. ISBN 978-9283224310.
3. Boyd SD, Natkunam Y, Allen JR, Warnke R. Selective immunophenotyping for diagnosis of B-cell neoplasms: immunohistochemistry and flow cytometry strategies and results. Appl Immunohistochem Mol Morphol 2013; 21: 116–131.
4. Herzenberg LA, Parks D, Sahaf B, Perez O, Roederer M, Herzenberg LA. The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem 2002; 48: 1819–1827.
5. Braylan RC. Impact of flow cytometry on the diagnosis and characterization of lymphomas, chronic lymphoproliferative disorders and plasma cell neoplasias. Cytometry A 2004; 58: 57–61.
6. Brown M, Wittwer C. Flow cytometry: principles and clinical applications in hematology. Clin Chem 2000; 4: 1221–1229.
7. Craig FE, Foon FA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood 2008; 111: 3941–3967.
8. Oberley MJ, Fitzgerald S, Yang DT, Morgan A, Johnson J, Leith C. Value-based flow testing of chronic lymphoproliferative disorders: a quality improvement project to develop an algorithm to streamline testing and reduce costs. Am J Clin Pathol 2014; 142: 411–418.
9. D’Arena G, Keating MJ, Carotenuto M. Chronic lymphoproliferative disorders: an integrated point of view for the differential diagnosis. Leuk Lymphoma 2000; 36: 225–237.
10. Matutes E, Wotherspoon A, Catovsky D. Differential diagnosis in chronic lymphocytic leukemia. Best Pract Res Clin Haematol 2007; 20: 367–384.
11. Matutes E, Owusu-Ankomah K, Morilla R, Garcia Marco J, Houlihan A, Que TH, Catovsky D. The immunological profile of B cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia 1994; 8: 1640–1645.
12. Moreau EJ, Matutes E, A’Hern RP, Morilla AM, Morilla RM, Owusu-Ankomah KA, Seon BK, Catovsky D. Improvement of the chronic lymphocytic leukemia scoring system with the monoclonal antibody SN8 (CD79b). Am J Clin Pathol 1997; 108: 378–382.
13. Rawstron AC, at al. Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: an European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project. Cytometry B Clin Cytom 2018; 9: 121–128.
14. Asaad NY, Abd El-Wahed MM, Dawoud MM. Diagnosis and prognosis of B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL) and Mantle cell lymphoma (MCL). J Egypt Natl Canc Inst 2005; 17: 279–290.
15. Matutes E, Polliack A. Morphological and immunophenotypic features of chronic lymphocytic leukemia. Rev Clin Exp Hematol 2000; 4: 22–47.
16. Vose JM. Mantle cell lymphoma; update on diagnosis, risk stratification and clinical management. Am J Hematol 2015; 90: 739–745.
17. Bacal NS, Mantovani E, Grossl S, Nozawa ST, Kanayama RH, Brito ACM, Albers CEM, de Campos Guerra JC, Mangueira CLP. Flow cytometry: immunophenotyping in 48 hairy cell leukemia cases and relevance of fluorescence intensity in CDs expression for diagnosis. Einstein 2007; 5: 123–128.
The authors
Nataša Lazić MD
Institute for Clinical Laboratory Diagnostics, University Clinical Centre of the Republika Srpska, Republika Srpska, Bosnia and Herzegovina
*Corresponding author
E-mail: natasa.lazic.bl@gmail.com
New RESIST kit: RESIST-4 O.K.N.V.
, /in Featured Articles /by 3wmediaThe Largest Parameter Menu in Clinical Mass Spectrometry
, /in Featured Articles /by 3wmediaValveless Dispensers & Metering Pumps
, /in Featured Articles /by 3wmediaEmerging Biomarker in Atherosclerotic Risk Assessment
, /in Featured Articles /by 3wmediaDiagnostics for infectious diseases
, /in Featured Articles /by 3wmediaAutomation and integration of LC-MS/MS services into the clinical laboratory workflow
, /in Featured Articles /by 3wmediaDespite significant inherent advantages of liquid chromatography-tandem mass spectrometry (LC-MS/MS) over immunoassay techniques in clinical laboratory applications, its adoption into routine practice has been slower than might have been expected. The barriers to more widespread uptake are a function of issues in the laboratory workflow. This article analyses those issues and discusses how they can be overcome by improved automation and integration with the laboratory information management system, drawing on examples from the North West London Pathology (NWLP) clinical laboratories at Imperial College Healthcare NHS Trust.
by Dr Emma L. Williams
Introduction
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen over two decades of use in specialist clinical laboratories in the UK, offering a number of significant advantages over immunoassay techniques. These advantages include increased specificity, sensitivity and accuracy, as well as the detection of multiple analytes within a single assay. There is no need for an antibody for analyte detection and the method is not susceptible to the antibody-based interferences that plague immunoassays [1]. LC-MS/MS is suitable for multiple sample matrices and avoids the need for radioactive tracers. LC-MS/MS assays also have a wider dynamic measurement range and have improved between-method bias when compared to immunoassays.
LC-MS/MS initially played a role in specialist clinical laboratories in areas such as newborn screening, inborn errors of metabolism, toxicology and in immunosuppressant and therapeutic drug monitoring. More recently LC-MS/MS has established a role in diagnostic endocrinology, with the first appearance of LC/MS-MS for the measurement of vitamin D in the international vitamin D external quality assurance scheme (DEQAS) in 2005. There are now over 150 labs registered in this scheme using LC/MS-MS for the measurement of vitamin D. However, automated immunoassay still dominates and represents 69% of participants registered in the DEQAS scheme. Why has there not been more widespread adoption?
A number of issues have inhibited wider adoption and routine use of LC/MS-MS in the clinical laboratory. First among these is the use of labour-intensive manual workflows, which result in lower throughput, decreased productivity and longer turnaround time. Furthermore, a high level of technical expertise is needed, not only for method development, but also for troubleshooting assay and equipment failures. In addition to the high initial capital costs of purchasing the equipment, ongoing personnel costs are higher because of the need for more technically competent staff. With a clear understanding of where the bottlenecks in the process arise, these barriers can be overcome.
Figure 1 depicts the six main steps of a typical LC/MS-MS workflow, from sample receipt and extraction, separation in the LC, MS/MS analysis, data review and reporting of the results [2]. Of these steps it is the pre- and post-analytical stages that are the most time consuming and therefore if there is a focus on streamlining these, maximum benefit can be achieved. A number of steps can be taken to streamline the workflow, and these come under three broad headings of reduced manual processes, increased throughput and improved integration. Dependence on manual processes can be reduced by the automation of liquid handling and extraction, use of barcode reading for worklist generation and implementation of automated data analysis. Throughput can be increased with strategic column and sample management and by analyte multiplexing. Integration can be improved by bi-directional interfacing of the LC/MS-MS system to the laboratory information management system (LIMS) allowing automatic worklist upload and results download. These three strategic areas will be discussed in more detail below.
Reduced manual processes
Unlike the case with immunoassay, samples for LC-MS/MS usually require extraction prior to analysis. Historically this extraction step utilized liquid–liquid extraction or protein precipitation, these being carried out after the addition of internal standard to the calibrators, quality controls and patient samples. All of these steps involved manual pipetting and were very slow and time consuming. Use of an automated liquid-handling platform for the pipetting of samples and addition of internal standard allows some of the steps of liquid–liquid extraction and protein-precipitation methods to be automated. These liquid-handling platforms are available from a number of suppliers including Hamilton and Tecan.
With the advent of 96-well plate technology it became possible to carry out fully automated off-line solid phase extraction (SPE) using platforms such as the Freedom Evo (Tecan) and the Biomek NX (Beckman Coulter). More recently, supported liquid extraction (SLE), which allows solvent extraction to occur on a diatomaceous earth inert support, has also become available in a 96-well plate format. The Extrahera system (Biotage) enables automation of SLE by carrying out all of the pipetting and extraction steps required. In the NWLP laboratory, this system is used for the extraction of patient samples for vitamin D measurement by LC-MS/MS. A sample throughput of up to 50,000 samples per annum is achieved with capacity remaining for additional extractions for use in other LC-MS/MS applications. The system is robust and reliable with good pipetting precision and uses disposable pipette tips, thus avoiding sample carry over. Figure 2 depicts the Tecan Freedom Evo 200 and Biotage Extrahera liquid handlers in use in the NWLP laboratory.
In some manufacturers’ LC-MS/MS systems, on-line sample preparation and extraction is enabled by use of turbo flow or 2D chromatography. On-line protein precipitation and SPE is also now available using the Clinical Laboratory Automated sample preparation Module (CLAM)-2000 (Shimadzu Corporation) [3] and the Rapidfire 365 MS system (Agilent) [4] respectively. These latter examples most closely resemble the immunoassay workflow, whereby samples are introduced into the analytical system without any sample preparation or pre-treatment.
Increased throughput
Increased throughput can be achieved through the use of column and sample managers, allowing multiple assay batches to be queued up for overnight analysis of different LC-MS/MS assays. LC multiplexing enables multiple columns to be coupled to one tandem mass spectrometry system, maximizing the MS detection capability. In this approach, the use of quaternary solvent pumps in the LC enables column switching between different columns using different mobile phases. Finally there is analyte multiplexing, which can use manufacturers’ kits or in-house laboratory developed tests (LDTs). This approach enables multiple analytes to be detected in a single chromatographic separation by the use of multiple reaction monitoring for MS/MS detection. Perkin Elmer and Chromsystems both provide kits enabling the simultaneous measurement of multiple steroid hormones within a single assay panel. In the NWLP laboratory an in-house LDT steroid panel for the simultaneous measurement of androstenedione, 17-hydroxyprogesterone and testosterone has been implemented. This multiplexed assay has replaced the previous stand-alone assays for these analytes, thus increasing throughput and offering faster turnaround time. The assay utilizes off-line SPE using Waters Oasis PRiME HLB 96-well plates and the Tecan Freedom Evo 200 automated liquid handler [5].
Improved integration
Improved integration can be achieved by the use of bi-directional interfacing between the LIMS and the LC-MS/MS instrument software. Nowadays, manufacturers of LC-MS/MS systems offer customer support to allow their systems to be interfaced to the LIMS. One example is the MassLynx LIMS interface (Waters), which enables both worklist download and results upload. The MassLynx LIMS interface is accessed via the LC-MS/MS system software allowing sample worklists, created by barcode scanning of the patient samples, to be imported directly. Following peak integration and analyte quantitation the results are directly transmitted from the LC-MS/MS to the LIMS via an HL7 interface. This avoids the need for manual transcription thus saving a great deal of staff time and eliminating transcription errors.
The ultimate aim of LC-MS/MS integration is to achieve complete integration of LC-MS/MS instruments into the automated workflow of high-throughput routine clinical laboratories. With the recent introduction of the Cascadion LC-MS/MS analyser (Thermo Fisher Scientific) this ultimate aim has now been achieved [6]. This analyser offers a complete LC-MS/MS solution including primary blood tube sampling, on-board sample extraction, LIMS connectivity and a random access workflow enabling the provision of a 24/7 service. Traceable manufacturer’s kits are offered for the measurement of a panel of immunosuppressant drugs, testosterone and vitamin D with further assay kits in the development pipeline. The Cascadion analyser is shown in Figure 3.
Summary
LC/MS-MS automation and integration is now a reality, allowing faster sample processing and improved turnaround time, as well as offering increased staff productivity, improved quality and reduced error rate. Staff time is liberated for further service development, allowing the more rapid introduction of validated in-house LDTs into the assay repertoire. Finally there is the possibility of complete analyser integration allowing routine, high-throughput analysis, as is already the standard approach for the common immunoassay platforms. This exciting development will support the more widespread adoption of LC-MS/MS in the routine clinical laboratory by offering complete automation and integration, overcoming the barriers discussed in this article and enabling the inherent advantages of LC/MS-MS in clinical laboratory practice to be more fully realized.
References
1. Jones AM, Honour JW. Unusual results from immunoassays and the role of the clinical endocrinologist. Clin Endocrinol Oxf 2006; 64: 234–244.
2. Zhang YV, Rockwood A. Impact of automation on mass spectrometry. Clin Chim Acta 2015; 450: 298–303.
3. Shimadzu. CLAM-2000. Fully automated sample preparation module for LCMS. (https://www.shimadzu.com/an/lcms/clam/index.html).
4. Jannetto PJ, Langman LJ. High-throughput online solid-phase extraction tandem mass spectrometry: Is it right for your clinical laboratory? Clin Biochem 2016; 49: 1032–1034.
5. Williams EL. LC-MS/MS measurement of serum steroids in the clinical laboratory. Clinical Laboratory International 2017; Sept: 18–20.
6. ThermoFisher Scientific. Cascadion SM Clinical Analyzer (www.thermofisher.com/cascadion).
The author
Emma L. Williams PhD, FRCPath
North West London Pathology, Imperial College Healthcare NHS Trust, London, UK
E-mail: emma.walker15@nhs.net
Inflammation: a newly identified risk of depression?
, /in Featured Articles /by 3wmediaAccording to the World Health Organization, depression affects more than 300 million people and is the leading cause of ill health and disability worldwide. Currently, diagnosis of depression involves the use of questionnaires about the patient’s general health, the way they are feeling and how this is affecting them. Blood tests are carried out during diagnosis, but are for the purpose of excluding other conditions, such as thyroid disease or vitamin D deficiency, that can give rise to symptoms similar to depression. There is no physical test for depression per se. Treatment for depression ranges from ‘wait and see’ and exercise for very mild forms through to self-help groups, talking therapies, such as counselling and cognitive behavioural therapy, for mild to moderate depression, as well as antidepressant medication for the more severe end of the spectrum. There are several classes of antidepressant drugs and treatment is largely through a process of trial and error in order to determine what does or does not work for certain patients, as it is recognized that there is a large variation in the way individuals respond to the different medications. Additionally, although numbers vary, conservative estimates suggest at least 30% of patients do not respond to antidepressant medication, and suffer from what is termed treatment-resistant depression.
However, recently, a line of research about one cause of depression has been gaining traction: the role of inflammation. Recent work suggests that an overactive immune system causing higher levels of inflammation results in an increased risk of depression and that these patients are less likely to respond to antidepressants; perhaps, therefore, the cause of treatment-resistant depression. It has also been noticed that patients taking anti-inflammatory medication for rheumatoid arthritis experience improvements in mood that are more profound than just feeling happier because of reduced pain; changes that have been confirmed by brain scans. Professor Ed Bullmore, Head of the Department of Psychiatry at the University of Cambridge, is certain that inflammation can cause depression and his new book, The Inflamed Mind: A radical new approach to depression, is about to bring these ideas to the attention of a much more general audience. The exciting relevance of this research for clinical lab diagnostics is the thought that a blood test for biomarkers of inflammation will help in an objective diagnosis of a certain type of depression and that treatment will be much better tailored to the individual – perhaps the individuals who fail to respond to current antidepressants. Even if this benefits only a small proportion of people with depression, because of the prevalence of the condition a large number of people will benefit.
Flow cytometry and immunophenotyping for chronic lymphoproliferative disorders
, /in Featured Articles /by 3wmediaModern hematology emphasizes a multiparametric diagnostic approach and the basic parameters, beside history of the disease and clinical examination, are morphological, immunophenotypic and genetic evaluation. Flow cytometry plays an important role in diagnosis of a large group of hematological diseases. This article reviews the basic principles of flow cytometry and its use in hematology diagnosis, with emphasis on chronic lymphoproliferations.
by Dr Nataša Lazić
Introduction
In modern diagnostics, flow cytometry has an important place as one of the basic and irreplaceable tools for diagnosis, classification, monitoring and prediction of malignant hematological disease [1]. The extreme complexity of these diseases, on one hand, and the availability of the different therapeutic protocols for the different types of these diseases on the other, makes accurate and precise diagnosis imperative. Contributing to this is the fact that the World Health Organization (WHO), in the Classification of Tumours of Hemopoietic and Lymphoid Tissues, suggests a multiparametric approach in diagnosing these diseases; basic parameters required are morphological, immunophenotypic and genetic analysis for each entity of the disease, in addition to a detailed history of the disease and clinical examination [2, 3]. The clinical picture and cell morphology, as a well-known and traditionally-used means of examination, are insufficient in many cases; quite often, because of a similar clinical presentation and cell morphology, it is not possible to draw a diagnostic conclusion based on these findings or a wrong diagnosis may be reached in some cases.
Coulter’s principle of measuring the change in the electrical impedance of the individual cells flowing through the measuring cell, in the late 1940s, was the basis for construction of the first hematologic counter and later for the flow cytometer. Later inventions added new detection capabilities, such as light scatter and fluorescence detection. Fluorescent activated cell sorting (FACS) was invented in the late 1960s by Herzenberg, Bonner, Sweet and Hullet. Introduced as a commercial machine in the early 1970s, this is the class of instruments now commonly referred to as flow cytometer [4]. The invention of monoclonal antibodies by Milstein and colleagues in 1977 opened new perspective for flow cytometry. Further developments, especially in electronics, led to modern cytometers with multiple lasers, detectors, better performance characteristics, and the ability to measure larger amounts of data.
Flow cytometry principles
Flow cytometry is a powerful technology that simultaneously measures many aspects of single particles, usually cells. Any suspended particle or cell from 0.2–150 μm is suitable for analysis. However, it can also measure soluble molecules if trapped onto a particulate surface and bound by fluorochromes. Virtually any component or function of a cell can be measured if the fluorescent probe can be made to detect it.
Sample preparation should provide a homogeneous suspension of cells with monoclonal antibodies conjugated with fluorochromes of a different emission spectrum. Depending on the sample, it most often includes incubation, erythrocyte lysis, centrifugation, washing and fixation.
The cytometer needs to be adjusted to have the appropriate performance characteristics (linearity, sensitivity, CV, electronic and optical background noise, fluorescence detector efficiency, etc). This is achieved by adjusting voltages on the detectors and by spectral overlap compensation (Fig. 1).
The three main systems of flow cytometer are fluidics, optics and electronics (Fig. 2). Parameters measured include forward scatter (FSC) corresponding to cell size, side scatter (SSC) depending on internal complexity and fluorescence intensity for different fluorochromes.
Becoming more available in clinical laboratories, a wide range of clinical applications of flow cytometry are constantly expanding and the most common among them are in, for example, lymphoma and leukemia diagnosis, stem cell enumeration for transplantation, estimation of minimal residual disease, paroxysmal nocturnal hemoglobinuria diagnosis, immunodeficiencies, HIV infection.
Flow cytometry in hematology
Flow cytometric immunophenotyping enables examination of the phenotype of the separate cells in the suspension and summarizing of the results, which gives data about the presence or absence of antigen expression as well as the expression intensity [5]. Hence, an immunophenotypic pattern is obtained on the cell population of interest for the examined disease. Meanwhile, there are no separate antigens specific for the particular disease. Instead, their mutual relation is observed and analysed, which makes the analysis of the flow cytometry results very demanding and complex, but usually very useful and precise owing to the huge amount of data that can be collected from the cells [6]. Therefore, flow cytometry helps with determining the cell line, the degree of cell maturity, abnormal patterns of expression and provides a detailed immunophenotype of the pathological cell population [7]. From information on all the aforementioned factors, a diagnostic conclusion is drawn if there is a phenotype characteristic for some disease. In the case of an atypical phenotype, the disease is assigned to the appropriate group and additional tests should be done to gain a precise diagnosis (such as immunohistochemical, FISH, molecular tests).
CD markers (clusters of differentiation) are blood cell antigens that enable their characterization. CD nomenclature was developed and reviewed by HLDA (Human Leukocyte Differentiation Antigen) workshops started in 1982. There were 10 such workshops and the nomenclature now encompasses about 400 CD markers. Monoclonal antibodies against those antigens are used for immunophenotype characterization.
The antibody panel for the analysis of the sample to be tested by flow cytometry depends, to a large extent, on the available information of other findings made for that patient. According to the Bethesda Group recommendations from 2006, which were aimed at regulating a more systematic approach in this field (and are still valid today), before sending a sample to flow cytometry, a detailed history of the disease, clinical examination, microscopic examination of cell morphology, and other laboratory tests should be carried out, and based on this, diagnosis or differential diagnosis determined. In this way significant rationalization and cost reduction can be achieved [8].
Immunophenotype characterization for chronic lymphoproliferative disorders
For both of the two major groups of malignant hematologic diseases, those derived from mature and from immature cells, flow cytometry is of a great importance. Neoplasms of mature lymphoid cells, according to the WHO Classification, include chronic lymphoid leukemia and non-Hodgkin’s lymphoma. Their basic characteristic is that they have an immunophenotype similar to mature lymphoid cells and, accordingly, they show an absence of immaturity indicators (CD34, TdT). According to the origin, in relation to the cell line, they can be divided into T, B and NK neoplasms. [7]
Mature B-cell lymphoproliferations make up most of the malignant blood diseases: 90 % of the total lymphoid malignancies, according to WHO data. They present 4 % of the newly discovered carcinomas per year. As already known, the malignant cell derived from B-cell lineage in most cases imitates the normal B-cells stopped at a certain maturity level. The classification of this disease group mostly relies on this fact. The most common in this group are chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), follicular lymphoma, splenic marginal zone lymphoma, mantle cell lymphoma (MCL), plasma cell leukemia [12]. Immunophenotype characterization in the diagnosis of B-cell chronic lymphoproliferative diseases is an irreplaceable method and, together with morphology, it presents the essential search that should be undertaken in the diagnosis of these diseases[2, 9]. Based on the finding of the immunophenotype characterization it is possible to discover aberrant expression patterns and establish the phenotypic characteristics related to particular diseases. The application of a scoring system as an additional tool is the result of a need for some standardization and quantification in the diagnosis of B-cell chronic lymphoproliferative diseases. In order to increase the precision of the scoring system, different studies with different CD markers are taken [10–12]. The most common scoring system of 5 points includes CD5, CD23, FMC7, CD79b and surface immunoglobulin chains with an accuracy of 96.6 % if a three-point cut-off is used [10].
In most cases of CLL, cell morphology is characteristic and typical for this disease. However, in a number of cases, flow cytometry has a huge and decisive significance for diagnosis (Fig. 3) [13]. CLL and MCL share many morphological and immunophenotypic features [14]. As a result of their partial overlap, a differential diagnosis of MCL is most considered when making a diagnosis of CLL. Because of the different therapeutic approach and prognoses of the diseases, their diagnostic differentiation is very important. For that purpose cyclin D1 testing is recommended [15, 16]. Unlike the other chronic lymphoproliferations, HCL cells do not match any stage of the normal lymphoid cells development. Morphologically typical HCL cells have fine, hair-like, cytoplasmic projections, which are sometimes difficult to find in the peripheral blood smear. Because of this and a very specific immunophenotype, flow cytometry is essential for HCL diagnosis [14, 17].
Advantages
The possibility of combining more antibodies in the same tube and analysing their interactions on the population of interest for the given disease is the greatest advantage of multiparametric flow cytometry, which involves simultaneously collecting and analysing a large amount of data from cells or particles.
Considerations
Comprehensive analysis involves considering possible causes of false-positive or false-negative results, thus avoiding an incomplete or incorrect interpretation of flow cytometry data (Fig. 4).
Other difficulties, such as non-standardized methods, particularly the issue of regulation in cytometry, different antibody panels, cut-off values, analysis subjectivity – recommended visual approach, result analysis complexity, report form, etc., are the subject of work by various associations dealing with cytometry in order to achieve harmonization in this area [13].
References
1. Paiva A, Alves GVA, Sales VSF, Silva ASJ, Silva DGKC, Alves E, Bahia F, Freitas RV, De Oliveira Paiva HD, Cavalcanti GB, Jr. Utility of flow cytometry immunophenotyping and hematological profile in chronic lymphoproliferative disorders. Blood 2017; 130: 5326 [poster abstract].
2. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman J (eds). WHO classification of tumors of haematopoietic and lymphoid tissues. IARC 2008; Chapters 1, 8, 10. ISBN 978-9283224310.
3. Boyd SD, Natkunam Y, Allen JR, Warnke R. Selective immunophenotyping for diagnosis of B-cell neoplasms: immunohistochemistry and flow cytometry strategies and results. Appl Immunohistochem Mol Morphol 2013; 21: 116–131.
4. Herzenberg LA, Parks D, Sahaf B, Perez O, Roederer M, Herzenberg LA. The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem 2002; 48: 1819–1827.
5. Braylan RC. Impact of flow cytometry on the diagnosis and characterization of lymphomas, chronic lymphoproliferative disorders and plasma cell neoplasias. Cytometry A 2004; 58: 57–61.
6. Brown M, Wittwer C. Flow cytometry: principles and clinical applications in hematology. Clin Chem 2000; 4: 1221–1229.
7. Craig FE, Foon FA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood 2008; 111: 3941–3967.
8. Oberley MJ, Fitzgerald S, Yang DT, Morgan A, Johnson J, Leith C. Value-based flow testing of chronic lymphoproliferative disorders: a quality improvement project to develop an algorithm to streamline testing and reduce costs. Am J Clin Pathol 2014; 142: 411–418.
9. D’Arena G, Keating MJ, Carotenuto M. Chronic lymphoproliferative disorders: an integrated point of view for the differential diagnosis. Leuk Lymphoma 2000; 36: 225–237.
10. Matutes E, Wotherspoon A, Catovsky D. Differential diagnosis in chronic lymphocytic leukemia. Best Pract Res Clin Haematol 2007; 20: 367–384.
11. Matutes E, Owusu-Ankomah K, Morilla R, Garcia Marco J, Houlihan A, Que TH, Catovsky D. The immunological profile of B cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia 1994; 8: 1640–1645.
12. Moreau EJ, Matutes E, A’Hern RP, Morilla AM, Morilla RM, Owusu-Ankomah KA, Seon BK, Catovsky D. Improvement of the chronic lymphocytic leukemia scoring system with the monoclonal antibody SN8 (CD79b). Am J Clin Pathol 1997; 108: 378–382.
13. Rawstron AC, at al. Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: an European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project. Cytometry B Clin Cytom 2018; 9: 121–128.
14. Asaad NY, Abd El-Wahed MM, Dawoud MM. Diagnosis and prognosis of B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL) and Mantle cell lymphoma (MCL). J Egypt Natl Canc Inst 2005; 17: 279–290.
15. Matutes E, Polliack A. Morphological and immunophenotypic features of chronic lymphocytic leukemia. Rev Clin Exp Hematol 2000; 4: 22–47.
16. Vose JM. Mantle cell lymphoma; update on diagnosis, risk stratification and clinical management. Am J Hematol 2015; 90: 739–745.
17. Bacal NS, Mantovani E, Grossl S, Nozawa ST, Kanayama RH, Brito ACM, Albers CEM, de Campos Guerra JC, Mangueira CLP. Flow cytometry: immunophenotyping in 48 hairy cell leukemia cases and relevance of fluorescence intensity in CDs expression for diagnosis. Einstein 2007; 5: 123–128.
The authors
Nataša Lazić MD
Institute for Clinical Laboratory Diagnostics, University Clinical Centre of the Republika Srpska, Republika Srpska, Bosnia and Herzegovina
*Corresponding author
E-mail: natasa.lazic.bl@gmail.com
Establishing flow cytometry as a primary diagnostic method for the investigation of suspected platelet function disorders
, /in Featured Articles /by 3wmediaAlthough considerable progress has been made in our understanding of the role of platelets in hemostasis, the analytical methods clinically available for investigating platelet function defects remain limited. Herein, we describe an initiative at Linköping University Hospital, Sweden, to use flow cytometry for measuring platelet function in patients with a suspected bleeding disorder.
by Dr Niklas Boknäs, Dr Sofia Ramström and Prof. Tomas Lindahl
Introduction
Although many patients seek professional help for bleeding problems, very few end up receiving an informative diagnosis, even when the presenting symptoms are clearly abnormal [1]. At present, our diagnostic tools for the investigation of bleeding symptoms are tailored for identifying serious disorders with dramatic symptoms such as hemophilia and Glanzmann’s thrombastenia, but often fail to identify the underlying defect in mild bleeding disorders (MBD) [2]. Ironically, the reverse is also often true, as the clinical significance of many tests performed during conventional laboratory investigations of MBDs is ill-defined [3].
Platelet function disorders (PFDs) represent a subcategory of MBDs where the underlying hemostatic defect is caused by abnormally low platelet pro-hemostatic activity. As PFDs produce virtually identical clinical symptoms to many other conditions causing bleeding problems, diagnosing PFDs necessitates access to reliable laboratory testing of platelet function. Ideally, such tests could provide important guidance in a number of clinical situations, such as when deciding on whether to give pharmaceutical prophylaxis in the event of frequent bleeding or surgery and when assessing the risks associated with the use of thromboprophylaxis after thrombosis and surgery in the individual patient.
Unfortunately, clinical tests evaluating platelet function have evolved poorly during recent decades, despite the introduction of new promising techniques. Light transmission aggregometry (LTA), the method currently considered gold standard for evaluating platelet function, has been used for more than five decades and comprises continuous measurement of the optical density of stirred platelet-rich plasma after stimulation with agonists. LTA gives information about how platelets aggregate upon stimulation, but does not enable measurement of other aspects of platelet pro-hemostatic activity such as platelet adhesion, granule secretion and alterations of platelet membrane structure to accelerate coagulation. From our experience, the clinical value of LTA in terms of explaining patient symptoms is limited, and this is supported by studies failing to show an association between results from LTA and the severity of bleeding problems among patients with MBD [1, 4]. In addition to this limitation, LTA remains poorly standardized and labour-intensive, making performance of LTA only feasible in specialized hemostasis laboratories.
Flow cytometry for the diagnosis of PFD in patients with MBD
In an effort to overcome these problems with the methods currently used for diagnosing PFD, we and others have switched to employing whole-blood flow cytometry for the diagnosis of PFD among patients with MBD. Whole-blood flow cytometry for platelet function testing (FC-PFT) was developed in the 1980s [5, 6]. A description of the analytical principle behind flow cytometry is outside the scope of this article, but in this context, the technique can extremely briefly be described as a powerful method to quantify the presence of different epitopes on the surface of platelets after platelet activation by the use of fluorescent probes that bind to the cell surface. Compared to LTA, FC-PFT confers the following practical advantages [7]:
In addition to these practical benefits with FC-PFT, the method confers several other advantages. For example, it produces numerical results that are easy to interpret, and can give information about several different aspects of platelet activation by the employment of different fluorescent probes detecting distinct events during platelet activation [9]. The ability to measure different aspects of platelet function also allows the direct diagnosis of rare disorders, such as Bernard-Soulier syndrome, Glanzmann’s thrombastenia and Scott syndrome, without the need for sequential testing [10].
Unfortunately, until recently no studies had addressed the clinical utility of FC-PFT for diagnosing clinically relevant PFDs. To address this issue, we recently published a clinical study comparing the results from FC-PFT with symptom severity in a cohort of bleeders [11]. The study was performed on 105 patients referred to Linköping University for evaluation of platelet function. Only patients wherein a complete diagnostic work-up including a full blood cell count, APTT (activated partial thromboplastin time), PT (prothrombin time), FVIII (factor 8) and von Willebrand factor (antigen and ristocetin cofactor activity) had excluded the presence of von Willebrand disease or a coagulation disorder were included in the study. Bleeding symptoms were assessed by a single experienced clinician blinded to the laboratory results of the study. In our panel for FC-PFT, we included analysis of fibrinogen binding (indicating activation of the fibrinogen receptor glycoprotein (GP)IIb/IIIa responsible for platelet aggregations) as well as P-selectin exposure (indicating release of platelet alpha granules) after platelet stimulation with a panel of four different agonists that specifically activate the most important platelet receptors: P2Y12 and P2Y1 (ADP); the thrombin receptors PAR1 and PAR4 [PAR1-activating peptide (AP), PAR4-AP]; and the collagen receptor GPVI (CRP-XL). To assess the contribution of dense granules to platelet activation, we designed an indirect test wherein the effects of pre-incubation with apyrase (which degrades ADP) was used as a measure of functional dense granule release. A flow chart illustrating the flow cytometry protocol is provided in Figure 1.
Our results clearly demonstrate that abnormal test results using FC-PFT are associated with a more severe bleeding phenotype in patients with MBDs. In fact, a high symptom burden was 5–8 times more common among patients with more than two abnormal test results in our study as compared to patients with two or fewer abnormal test results (Fig. 2), depending on which method that was used for calculating the reference range for the different tests. When results pertaining to the fifth percentile of the patient material was classified as abnormal and more than two abnormal test results were used as a predictor for bleeding symptom severity, a high symptom burden was predicted with as specificity of 95 % and a positive predictive value of 80 %. It should be noted however, that the clinical material was insufficient to allow for a prospective validation of these estimates in a separate patient cohort.
Discussion
In our opinion, FC-PFT for clinical use should as a minimum comprise: (a) testing of platelet integrin activation, either directly by the use of the anti-PAC-1 antibody (recognizing GPIIb/IIIa) or indirectly by measuring fibrinogen binding or microaggregate formation; (b) a marker of alpha granule secretion, preferably by using an antibody directed towards P-selectin; and (c) a test of dense granule secretion to accurately assess the clinically most important hemostatic functions of platelets. Ideally, a clinical protocol for FC-PFT should also include a marker of platelet procoagulant platelet activity and a fluorescent marker binding to GPIbα, in order to provide a more complete assessment of the platelet hemostatic repertoire and diagnose the rare hereditary disorders Scott syndrome and Bernard-Soulier syndrome. In our own protocol, we have recently incorporated these two additional functionalities. We have also improved our protocol by incorporating the use of fixatives and pre-preparation of frozen reagents in order to improve reproducibility and increase the time- and cost-efficiency of the protocol. Recently, very promising methodological improvements have been made by other researchers, such as the use of fluorescent beads as an internal control for standardizing results and facilitating comparisons between different instruments [12] and the use of a modular diagnostic algorithm to ensure efficient and exact diagnosis [13]. Thus, continuous efforts are being made to firmly establish FC-PFT as an attractive alternative for platelet function testing in the setting of MBDs.
References
1. Quiroga T, Goycoolea M, Panes O, Aranda E, Martínez C, Belmont S, Muñoz B, Zúñiga P, Pereira J, Mezzano D. High prevalence of bleeders of unknown cause among patients with inherited mucocutaneous bleeding. A prospective study of 280 patients and 299 controls. Haematologica 2007; 92(3): 357–365.
2. Quiroga T, Mezzano D. Is my patient a bleeder? A diagnostic framework for mild bleeding disorders. ASH Educ Progr B 2012; 2012(1): 466–474.
3. Harrison P. Platelet function analysis. Blood Rev 2005; 19(2): 111–123.
4. Lowe GC, Lordkipanidzé M, Watson SP, UK GAPP study group. Utility of the ISTH bleeding assessment tool in predicting platelet defects in participants with suspected inherited platelet function disorders. J Thromb Haemost 2013; 11(9): 1663–1668.
5. Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987; 70(1): 307–315.
6. Lindahl TL, Festin R, Larsson A. Studies of fibrinogen binding to platelets by flow cytometry: an improved method for studies of platelet activation. Thromb Haemost 1992; 68(2): 221–225.
7. Michelson A. Flow cytometry: a clinical test of platelet function. Blood 1996; 87: 4925–4936.
8. Frelinger AL, 3rd, Grace RF, Gerrits AJ, Berny-Lang MA, Brown T, Carmichael SL, Neufeld EJ, Michelson AD. Platelet function tests, independent of platelet count, are associated with bleeding severity in ITP. Blood 2015; 126(7): 873–880.
9. Ramström S, Södergren AL, Tynngård N, Lindahl TL. Platelet function determined by flow cytometry: new perspectives? Semin Thromb Hemost 2016; 42(3): 268–281.
10. Rubak P, Nissen PH, Kristensen SD, Hvas A-M. Investigation of platelet function and platelet disorders using flow cytometry. Platelets 2015; 27(1): 66–74.
11. Boknäs N, Ramström S, Faxälv L, Lindahl TL. Flow cytometry-based platelet function testing is predictive of symptom burden in a cohort of bleeders. Platelets 2017; doi: https://doi.org/10.1080/09537104.2017.1349305
12. Huskens D, Sang Y, Konings J, van der Vorm L, de Laat B, Kelchtermans H, Roest M. Standardization and reference ranges for whole blood platelet function measurements using a flow cytometric platelet activation test. PLoS One 2018; 13(2): 1–16.
13. Andres O, Henning K, Strauß G, Pflug A, Manukjan G, Schulze H. Diagnosis of platelet function disorders: a standardized, rational, and modular flow cytometric approach. Platelets 2017; doi: 10.1080/09537104.2017.1386297.
The authors
Niklas Boknäs*1,2 MD, PhD; Sofia Ramström3,4 PhD; Tomas Lindahl3 MD, PhD
1Department of Hematology and Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
2Australian Centre for Blood Diseases, Monash University, Melbourne, Australia
3Department of Clinical Chemistry and Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
4School of Medical Sciences, Örebro University, Örebro, Sweden
*Corresponding author
E-mail: niklas.boknas@gmail.com
Ber-EP4 (CD326) testing by flow cytometry: a rationalized algorithm-based approach
, /in Featured Articles /by 3wmediaFlow cytometry has traditionally been used to identify hemato-lymphoid neoplasms. However, the flow cytometry laboratories that deal with tissues would often receive samples that have an epithelial neoplasm. In our laboratory, we use flow cytometry to identify cells with epithelial differentiation using Ber-EP4 antibody that targets CD326 (EpCAM). We have formulated an algorithm-based approach for the application of this marker. This approach has been elaborated in this article.
by Dr Pranav Dorwal and Dr Helen Moore
Introduction
The use of flow cytometry in the laboratory has traditionally been applied for diagnosing lymphomas and leukemias. The biggest advantage that flow cytometry has over histopathology is a much quicker turn-around-time, as most of the samples are fresh and can be processed right away, unlike a histopathology sample which needs to undergo fixation and processing before it is ready to be examined. Any additional testing on flow cytometry samples can be performed instantly, whereas the same usually requires another day in the histopathology lab. Many of the lymph node malignancies (primary lymphomas versus metastatic involvement) can appear undifferentiated. In these cases, the histopathologist needs the help of a plethora of immunohistochemical markers to reach a diagnosis. The ability to identify samples where non-hematological malignancies are present can be helpful for the treating physician as well as the reporting histopathologist, who can then test with a more dedicated panel. The ultimate aim of this testing is to get an early diagnosis so that patient’s treatment is not delayed.
A large number of markers have been used to identify epithelial differentiation in tumours by immunohistochemistry (IHC), including cytokeratin (CK), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125) as well as epitopes recognized by the antibodies LeuM1 (anti-CD15 antibody), and MOC-31 and Ber-EP4 antibodies, both of which recognize epitopes on EpCAM (the epithelial cell adhesion molecule). However, most of these are not available for use by flow cytometry. EpCAM (also known as CD326) was first discovered in 1979 and at that time thought to be specific for colonic carcinoma [1]. Ber-EP4 is, therefore, an anti-CD326 antibody which binds to a cell membrane glycoprotein on human epithelia. There is a comprehensive list of tumours that are Ber-EP4 positive, as described by Went et al. and Spizzo et al. [2, 3]. The traditional use of Ber-EP4 in histopathology has been limited essentially for differentiation between adenocarcinoma and malignant mesothelioma [4]. This could be due to the fact that other epithelial markers (such as CK) are expressed more often than the CD326 (EpCAM) in epithelial malignancies and thus are more helpful in lineage determination.
We use an algorithmic approach to decide the flow cytometry panel to be applied (Fig. 1). When the clinical details or radiological findings are indicative of a non-hematopoietic malignancy, we apply the CD326 panel. This panel is composed of CD326, CD56 and CD45. CD56 was included in the panel to identify myeloma cells (which may be present in the CD45-negative region) and cells with neuroendocrine differentiation. If, on analysis, there is no CD45-negative population and the sample is composed of predominantly lymphoid cells, a lymphoid screening panel is then used. Samples that are received with diagnosis of suspected lymphoma are initially processed with a routine lymphoid screening panel. In these cases, Ber-EP4 antibody is tested only if large numbers of CD45-negative events are identified.
Method for Ber-EP4 testing
The tissue and fine-needle aspirate (FNA) samples are received fresh in RPMI medium. The tissues are placed on the metal sieve and ground using a glass pestle to form a cell suspension using 2 % PBS-FCS. This suspension is subsequently filtered, which is then washed and lysed. The cell count is ascertained by the cell counter only in cases of larger tissues, where we may have to dilute the sample to adjust the cell count to approximately 10×109/L. FNA and core biopsies are usually paucicellular and do not need a cell count.
The sample is stained with 5 µl of CD45-PC5 [Immunotech SAS (Beckman Coulter)], 20 µl of CD56-PE (Immunotech SAS) and 10 µl of monoclonal mouse anti-human epithelial antigen-FITC conjugated antibody (Clone: Ber-EP4) (Dako Denmark A/S). The sample is then incubated at 4 °C for 30 minutes, followed by a washing step and is ready to be run on the flow cytometer (Beckman Coulter Life Sciences). A total of 10 000 events are acquired with the time threshold set at 300 seconds for the acquisition.
Flow cytometric analysis
The flow cytometric analysis is performed using Navios and Kaluza softwares (Beckman Coulter Life Sciences). The various populations of interest are gated with the focus on identifying the expression of CD326 (with or without CD56) in the CD45-negative population.
Discussion
In our experience of testing for CD326 by flow cytometry, we have been able to comment on the presence or absence of CD326 expression in CD45-negative populations (Figs 2(a, b) and 3). The various carcinomas where we have identified CD326 positivity are: adenocarcinoma, small cell carcinoma, Merkel cell carcinoma, renal cell carcinoma, squamous cell carcinoma, prostate carcinoma, germ cell tumour of testis, and myxoma. We have observed that the expression of CD326 in melanomas can be variable, but they more frequently express CD56. The co-expression of CD326 and CD56 usually indicates a neuroendocrine tumour. Our concordance rate with histopathology using CD326 testing was found to be 97.6 %, which we have published previously [5].
CD326 expression has also been reported to be a prognostic marker with poor outcomes in epithelial ovarian and gall bladder carcinomas [6, 7]. Another important role of this testing could be application in decision making for use of monoclonal antibodies for targeted therapy. The first EpCAM targeting antibody, Catumaxomab (trade name Removab, Fresenius Biotech GmbH) received European market approval in EpCAM-positive carcinomas for the treatment of malignant ascites. Another modification that could be useful in diagnosing epithelial malignancies is to apply Ki67 testing using flow cytometry. This could be done in the same tube as CD326, and thus more information could be obtained with the same amount of sample [8].
There has been considerable data describing the use of the Ber-EP4 antibody in malignant effusions [9–11]. The literature mentions that the presence of epithelial cells in the body fluid should raise the suspicion of metastatic epithelial malignancy, as the reactive body fluids may be composed of lymphocytes and reactive mesothelial cells in varying proportions. There have been multiple studies in the past where flow cytometric CD326 testing has been applied for identifying epithelial cells in body fluid effusions. We have found that our results have a very good concordance with histopathology results. This is in keeping with the findings of Davidson et al., although their study looked at the detection of malignant cells in effusions [12].
The disadvantage of using Ber-EP4 for identifying epithelial differentiation is that there are many epithelial malignancies that do not express CD326 (EpCAM). As mentioned earlier, the use of a broader antibody like cytokeratin (pan-CK) may solve this problem. But unfortunately, such an antibody is not currently available for clinical use by flow cytometry, to the best of our knowledge. Meanwhile, Ber-EP4 should give us the answer in most of the cases. Another disadvantage is that CD326 will be negative in cases of neoplasms of mesenchymal origin, such as sarcomas.
Most flow cytometry laboratories across the world will liaise with histopathology departments for the diagnosis of non-Hodgkin lymphomas. The use of Ber-EP4-testing flow cytometry may play an important role even in epithelial malignancies. The antibody used by us is a CE-marked antibody for in vitro diagnostics and, thus, requires a limited verification process. We followed the method recommended by the manufacturer. The rapid turn-around-time of flow cytometry results makes it a useful screening tool. Our experience shows that flow cytometric testing for CD326 (EpCAM) can be a useful method for diagnosing non-lymphoid malignancies that are poorly differentiated. We suggest that this method would be more useful if the protocol for its application is set up in consultation with the histopathology department, along with setting up a channel of bilateral communication. The histopathologist, based on the flow cytometry information provided, can then set up a more directed immunohistochemical panel. We would like to emphasize at this stage that the aim of the flow cytometric CD326 testing is not to formally diagnose carcinomas, but to highlight the presence of epithelial cells which may lead to the diagnosis of carcinoma. Final classification obviously remains the role of the histopathologist.
References
1. Patriarca C, Macchi RM, Marschner AK, Mellstedt H. Epithelial cell adhesion molecule expression (CD326) in cancer: a short review. Cancer Treat Rev 2012; 38(1): 68–75.
2. Went PT, Lugli A, Meier S, Bundi M, Mirlacher M, Sauter G, Dirnhofer S. Frequent EpCam protein expression in human carcinomas. Hum Pathol 2004; 35(1): 122–128.
3. Spizzo G, Fong D, Wurm M, Ensinger C, Obrist P, Hofer C, Mazzoleni G, Gastl G, Went P. EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis. J Clin Pathol 2011; 64(5): 415–420.
4. Sheibani K, Shin SS, Kezirian J, Weiss LM. Ber-EP4 antibody as a discriminant in the differential diagnosis of malignant mesothelioma versus adenocarcinoma. Am J Surg Pathol 1991; 15(8): 779–784.
5. Dorwal P, Moore H, Stewart P, Harrison B, Monaghan J. CD326 (EpCAM) testing by flow cytometric BerEP4 antibody is a useful and rapid adjunct to histopathology. Cytometry B Clin Cytom 2017; doi: 10.1002/cyto.b.21543.
6. Spizzo G, Went P, Dirnhofer S, Obrist P, Moch H, Baeuerle PA, Mueller-Holzner E, Marth C, Gastl G, Zeimet AG. Overexpression of epithelial cell adhesion molecule (Ep-CAM) is an independent prognostic marker for reduced survival of patients with epithelial ovarian cancer. Gynecol Oncol 2006; 103(2): 483–488.
7. Varga M, Obrist P, Schneeberger S, Mühlmann G, Felgel-Farnholz C, Fong D, Zitt M, Brunhuber T, Schäfer G, et al. Overexpression of epithelial cell adhesion molecule antigen in gallbladder carcinoma is an independent marker for poor survival. Clin Cancer Res 2004; 10(9): 3131–3136.
8. Sikora J, Dworacki G, Zeromski J. DNA ploidy, S-phase, and Ki-67 antigen expression in the evaluation of cell content of pleural effusions. Lung 1996; 174: 303-313.
9. Pillai V, Cibas ES, Dorfman DM. A simplified flow cytometric immunophenotyping procedure for the diagnosis of effusions caused by epithelial malignancies. A J Clin Pathol 2013; 139(5): 672–681.
10. Krishan A, Ganjei‐Azar P, Hamelik R, Sharma D, Reis I, Nadji M. Flow immunocytochemistry of marker expression in cells from body cavity fluids. Cytometry A 2010; 77(2): 132–143.
11. Risberg B, Davidson B, Dong HP, Nesland JM, Berner A. Flow cytometric immunophenotyping of serous effusions and peritoneal washings: comparison with immunocytochemistry and morphological findings. J Clin Pathol 2000; 53(7): 513–517.
12. Davidson B, Dong HP, Berner A, Christensen J, Nielsen S, Johansen P, Bryne M, Asschenfeldt P, Risberg B. Detection of malignant epithelial cells in effusions using flow cytometric immunophenotyping. Am J Clin Pathol 2002; 118(1): 85–92.
The authors
Pranav Dorwal* MBBS, DCP, DNB; Helen Moore MBChB, FRACP, FRCPA
Waikato Hospital, Pembroke St, Hamilton 3204, New Zealand
*Corresponding author
E-mail: Pranav.dorwal@waikatodhb.health.nz