Читать книгу Immunophenotyping for Haematologists - Barbara J. Bain, Irene Roberts - Страница 8

This technique determines cell size, structure (to some extent) and antigen expression. Cells in suspension are first exposed to a combination of fluorochrome‐labelled monoclonal antibodies (or other lectins or ligands) and then pass in a focused stream through a beam of light generated by a laser. Laser‐generated light is coherent (waves of light are parallel) and monochromatic (single wave length/colour). Large multichannel instruments with multiple lasers are used to identify, count, size and otherwise characterise cells that are hydrodynamically focused and pass in a single file through a narrow orifice in a flow cell. The passing of the cell through a light beam leads to both the scattering of light and the excitation of fluorochromes so that they emit a fluorescence signal. Forward scatter (FSC) of light at a narrow angle is detected and measured and is proportional to cell size. Sideways or side scatter (SSC) of light is detected and measured and is proportional to cell granularity and complexity. Antigens expressed on the surface membrane of cells or, with modified techniques, within cells are detected. After ‘permeabilisation’, both cytoplasmic and nuclear antigens can be detected.

Figure 1.1 Diagrammatic representation of the principles of flow cytometric immunophenotyping.

For each fluorochrome, a selected laser emits light of a specified wavelength that will be absorbed by the fluorochrome. This leads to excitation of the fluorochrome with subsequent emission of light of lower energy and a longer wavelength as the fluorochrome returns to its basal state; this property is known as fluorescence. The amount of light emitted (the number of photons) is proportional to the amount of fluorochrome bound to the cell. The mean fluorescence intensity of a population indicates the strength of expression of the relevant antigen. The emitted light passes through dichroic mirrors, that is, mirrors that reflect some wavelengths and transmit others, so that it is possible, for example, to reflect SSC for measurement and transmit fluorescence signals to another detector such as a photomultiplier tube (Figure 1.1). The detector produces an electrical signal that is proportional to the amount of incident light. Some commonly used fluorochromes are shown in Table 1.1.

The cells that are studied must be dispersed. For peripheral blood and bone marrow aspirate specimens, it is necessary to exclude mature and immature red cells. This is most simply done by lysing red cells using an ammonium chloride solution. Otherwise red cells and their precursors will appear in scatter plots and interfere with gating leucocyte populations of interest. If assessment of immunoglobulin expression is required, there must also be a washing step to remove the plasma that contains immunoglobulin, which would neutralise the monoclonal lambda‐ or kappa‐specific antibody.

Table 1.1 Commonly used fluorochromes.

Fluorescein isothiocyanate (FITC)

Phycoerythrin (PE)

Allophycocyanine (APC)

Peridinin chlorophyll (PerCP)

Cyanine 5 (Cy5), cyanine 5.5 (Cy5.5) and cyanine 7 (Cy7)

Texas red

Pacific blue

Brilliant violet

Krome orange

Alexa Fluor 488 (AF488)

Alexa Fluor 647 (AF647)

Phycoerythrin‐Texas Red X (ECD)

Phycoerythrin‐cyanine 5 (PE‐Cy5)

Phycoerythrin‐cyanine 5.5 (PE‐Cy5.5)

Phycoerythrin‐cyanine 7 (PE‐Cy7)

The great majority of monoclonal antibodies used in immunophenotyping have been characterised at a series of international workshops and those with the same specificity have been assigned a cluster of differentiation (CD) number. This number can be used to refer to both the antibody and the antigen it recognises. There are now more than 350 specificities recognised so that a careful selection of antibodies for diagnostic use is important. In addition to fluorochromes conjugated to monoclonal or polyclonal antibodies, it is also possible to use either fluorochromes that can bind directly to cellular constituents, such as DNA, or labelled modified aerolysins that bind to membrane glycosylphosphatidylinositol glycan A (GPI) (used in the diagnosis of paroxysmal nocturnal haemoglobinuria). Propidium iodide binding can be used to identify non‐viable cells and exclude them from analysis. Monoclonal antibodies that are most used in flow cytometric immunophenotyping are detailed in Part 2.

Results of immunophenotyping are usually shown as a two‐dimensional plot in which FSC, SSC and the expression of certain antigens are plotted against each other, permitting the recognition of the probable nature of a cell cluster in a particular position. It is thus possible to gate on a cellular population of interest. A gate is an electronic boundary; it can either be predetermined or drawn by the operator. There are four commonly used approaches to gating of target populations: FSC versus SSC, CD45 versus SSC, CD19 versus SSC and CD34 versus SSC.

FSC versus SSC is a useful way of screening a specimen to identify normal populations and to highlight abnormal cells as illustrated in Figure 1.2.

Forward scatter is increased in relation to increasing cell size whilst SSC is influenced by cytoplasmic granularity and nuclear complexity. It is a useful means of gating on blasts when CD34 is not expressed, for example in monoblastic leukaemias. Such plots are helpful in identifying large activated lymphocytes, an excess of small lymphocytes or monocytes and even the presence of hairy cells (see Chapter 3). Granular blasts show increased SSC and this is reflected in a shift to the right in the scatter plot. This can be an early indication of a possible acute promyelocytic leukaemia.

Figure 1.2 Delineation of peripheral blood leucocyte populations using forward scatter (FSC) and side scatter (SSC) characteristics.

Figure 1.3 Delineation of peripheral blood or bone marrow leucocyte populations using CD45 expression and SSC.

A plot of CD45 expression and SSC is not only useful for separating normal cell populations but also helps identify precursor cell populations, which frequently show only weak CD45 expression (Figure 1.3).

CD19 versus SSC (Figure 1.4) and CD34 versus SSC (Figure 1.5) plots are useful for isolating B cells and blast cells, respectively.

Back gating is a process whereby a target population identified in one approach can be tracked in another. For example, CD34+ myeloblasts can be isolated using CD34 versus SSC, then colour tracked into the FSC versus SSC plot to show cell size and granularity. With modern multichannel instruments it is possible to study 6–8 or more antigens in a single tube. If multiple tubes are studied, several core antibody‐fluorochrome conjugates can be included in each tube analysed so that cross‐comparison between the same cells stained with different antibody panels in different tubes is possible.

Figure 1.4 Delineation of peripheral blood or bone marrow B‐cell populations using CD19 expression and SSC.

Figure 1.5 Delineation of peripheral blood or bone marrow CD34+ blast populations using CD34 expression and SSC.

Flow cytometric immunophenotyping is used particularly in the investigation of haematological neoplasms, but there are other roles (Table 1.2).

Following analysis, the immunophenotyping laboratory will issue a report detailing the characteristics of any abnormal population identified and offering an interpretation. The strength of expression of any antigen is also of relevance. This may be expressed as

Table 1.2 Role of flow cytometric immunophenotyping.

Haematological neoplasms

Diagnosis of haematological neoplasms

Further classification, e.g. of AML, B‐ALL, T‐ALL

Identification of disease spread, e.g. to the central nervous system

Identification of a therapeutic target, e.g. CD19, CD20, CD30, CD33, CD52

Detection of minimal residual disease (which may include identifying a leukaemia‐specific phenotype at diagnosis)

Identification of hypodiploidy and hyperdiploidy in B‐ALL, including the detection of masked hypodiploidy when there has been duplication of a small hypodiploid clone

Investigation of erythrocytes and their disorders

Diagnosis of paroxysmal nocturnal haemoglobinuria (CD15, CD16, CD24, CD55, CD59, CD66b, CD157, FLAER on neutrophils; CD14, CD55, CD157 and FLAER on monocytes; CD55, CD59 and FLAER on erythrocytes) (reviewed in [1])

Identification of a PNH clone in aplastic anaemia (predictive of better prognosis and a response to immunosuppressive therapy)

Diagnosis of hereditary spherocytosis (eosin‐5‐maleimide binding). Binding is also reduced in hereditary pyropoikilocytosis, South‐East Asian ovalocytosis and congenital dyserythropoietic anaemia, type II

Diagnosis of hereditary stomatocytosis due to RHAG mutation (reduced expression of CD47, which is part of the Rh protein complex)

Detection and enumeration of fetal red cells in maternal circulation (using anti‐RhD when mother is RhD‐positive, or using permeabilised erythrocytes and an antibody to haemoglobin F) or using the two techniques in combination

Investigation of platelets and their disorders

Diagnosis of inherited platelet disorders: Glanzmann’s thrombasthenia, deficiency of platelet glycoprotein IIb/IIIa (CD41/CD61 absent or reduced in three quarters of patients); Bernard–Soulier syndrome, deficiency of glycoprotein I/V/IX (CD41 and CD42a/CD42b moderately reduced); Scott syndrome (annexin V not expressed on activated platelets); GFI1B mutation (CD34 expressed on platelets); Wiskott–Aldrich syndrome (deficiency of WAS protein, reduced or defective CD43 on T lymphocytes)

Investigation of leucocytes and their disorders including investigation of immune function

Investigation of suspected primary immunodeficiency syndromes (reviewed in [2])

Diagnosis of autoimmune lymphoproliferative syndrome (CD3+TCRαβ+CD4–CD8– lymphocytes)

Diagnosis of leucocyte adhesion deficiencies type I (CD18 and CD11a, 11b and 11c deficient) and type II (CD15s deficient); reduced expression of CD11b, CD18 or CD15s by phorbol esterase‐stimulated neutrophils is demonstrated

Diagnosis of neutrophil specific granule deficiency (reduced SSC, CD15, CD16, CD66, myeloperoxidase and lactoferrin)

Diagnosis of chronic granulomatous disease using dihydrorhodamine as a marker of H2O2 production after stimulation of neutrophils; carrier detection is also possible

Enumeration of CD4‐positive T cells in HIV infection

Investigation for lymphocytic variant of hypereosinophilic syndrome (aberrant phenotypes such as CD3–CD4+CD8– or CD3+CD4–CD8–)

Diagnosis of haemophagocytic lymphohistiocytosis (HLH) (upregulation of HLA‐DR on T cells; CD57 and perforin can also be upregulated; testing for deficiency of perforin, SAP, XIAP or CD107a is used to screen for various underlying genetic defects [3, 4]

Diagnosis of persistent polyclonal lymphocytosis

Identification of hypersensitivity by upregulation of CD63 and CD300a on exposure of basophils to a specific allergen [5]

Identification of sepsis by CD64 expression on neutrophils

Other

Enumeration and isolation of haemopoietic stem cells (CD45weak, CD34+, SSClow)

Differential leucocyte counting; the Beckman Coulter Hematoflow, for example, can distinguish neutrophils, eosinophils, basophils, CD16– and CD16+ monocytes, B cells, CD16+ cytotoxic T cells and NK cells, CD16– T cells, myeloblasts, monoblasts, B lymphoblasts and T lymphoblasts*

Enumeration and characterisation of reticulocytes or platelets by the binding of a fluorochrome (e.g. a proprietary mixture of polymethine and oxazine in Sysmex instruments) to RNA or the binding of a fluorescence‐labelled CD61 monoclonal antibody to platelets (CellDyn instruments)*

AML, acute myeloid leukaemia; B‐ALL, B‐lineage acute lymphoblastic leukaemia; CD, cluster of differentiation; FLAER, fluorescent aerolysin; HIV, human immunodeficiency virus; HLA‐DR, human leucocyte antigen‐DR; PNH, paroxysmal nocturnal haemoglobinuria; RNA, ribonucleic acid; SAP, SLAM‐associated protein; T‐ALL, T‐lineage acute lymphoblastic leukaemia; XIAP, X‐linked inhibitor of apoptosis

^* This is not part of conventional immunophenotyping but represents a flow cytometric immunophenotyping technique incorporated into an automated instrument for performing blood counts.

1 –, ±, +, ++;

2 negative, weak, moderate, strong;

3 negative, dim, moderate, bright;

4 hi, lo.

It should be noted that ± indicates weak expression whereas +/– indicates that expression may be positive or negative.

An immunophenotyping result will often also be subsequently incorporated into an integrated report that includes the results of other types of investigation, for example, morphological assessment and cell counts, and cytogenetic or molecular genetic analysis.