Dilute 10 ml of anticoagulated (e.g. heparinized or ethylenediaminetetra-acetic acid, EDTA-anticoagulated) blood with an equal volume of phosphate buffered saline (PBS), pH 7.3 (see p. 622) or Hanks’ solution. Add 10 ml of the diluted blood, drop by drop, to 7.5 ml of Lymphoprep (Nycomed) and then centrifuge for 30 min at 2000 rpm (approx. 500 g). This results in three visible layers: a top layer of plasma; an interphase layer of mononuclear cells; and a layer of red cells and neutrophils at the bottom. After removing the plasma, pipette the mononuclear cell layer into another tube and wash three times with Hanks’ solution or tissue culture medium.

Red Blood Cell Lysing Methods

Blood and bone marrow samples are treated with a hypotonic erythrocyte lysing solution such as ammonium chloride-based (e.g. Easylyse Dako) or other commercially available solutions (e.g. FACS lysing solution, BD Biosciences). The red blood cells can be lysed either before or after incubation with the McAb (see below) without loss of fractions of mononuclear cells.

The time of incubation with the lysing reagent is important because prolonged exposure may alter the forward and side light scatter (FSC/SSC) patterns, whereas exposure that is too brief leaves red cells intact, resulting in excess debris and inaccurate results.

Prior to incubation with the lysing solution, the white cell count of the blood or bone marrow specimen should be estimated and, if necessary, the sample should be diluted to a maximum white cell concentration of 25–30 × 10⁹ cells/l.

Multicolour Flow Cytometry Methods

There have been considerable improvements in flow cytometry instrumentation in recent years with the introduction of more lasers, more powerful computers and novel software for data acquisition and analysis

At the same time, the introduction of a large number of novel fluorochromes and the application of new McAbs has led to multiparametric immunophenotyping of cells, facilitating the accurate identification of normal and abnormal cell populations. Such advances have led to an increase in the complexity of data obtained and a subsequent increase in the comprehensiveness of the knowledge obtained by the flow cytometrist interpreting such data.¹

Detection of membrane antigens

Multicolour flow cytometry:

1. Stain–Lyse–Wash (Fig. 16.1): Label tubes with the name of the patient, type of specimen, laboratory number and the combination of fluorochrome-conjugated McAb to be used including isotypic controls; isotypic controls are mouse immunoglobulin (Ig) of the same isotope as the McAbs but with no antigen specificity.

Pipette 100 μl of the specimen (whole peripheral blood or bone marrow) into a tube.

Add the appropriate volume of McAb combination labelled with the specific fluorochrome conjugates stated in the laboratory protocol. There are more than a dozen fluorochrome conjugates that can be used for immunophenotyping and indeed some instruments and protocols can utilize up to 20–25 colours in the same tube. The limitations on which fluorochrome combination can be employed will depend on the number of lasers and detectors available in the instrument configuration. The volume of McAbs ranges from 5 to 20 μl, according to the manufacturer’s instructions.

Incubate in the dark at room temperature for 15 min.

Add 1 ml of lysing solution (commercially available) and leave for 10 min in the dark at room temperature. Centrifuge for 5 min at 2000 rpm and discard the supernatant.

Add 2 ml of PBS (pH 7.3) containing 0.02% sodium azide, 0.02% bovine serum albumin (BSA) and 0.01% EDTA (PBS-azide-BSA). Centrifuge for 5 min at 2000 rpm and discard supernatant. Repeat this step one more time.

Resuspend the cells in 0.2–0.5 ml of sheath fluid solution and acquire data on the flow cytometer.

2. Stain–Lyse–No Wash: This method utilizes the same procedure as the previous methodology but after incubation with the lysing solution, data from the sample are acquired on the flow cytometer. This method is ideal for samples with paucity of cells because it minimizes cell loss during the centrifugation of the washing step.

3. Lyse–Stain–Wash: Label tube with the patient’s name, laboratory number and specimen. Add 5 to 10 ml of peripheral blood or bone marrow and add the same volume of ammonium chloride-based lysing solution; mix gently and incubate for 10 min at room temperature. After incubation, centrifuge the tube for 5 min at 300 g, discard supernatant after centrifugation and resuspend the cell pellet in 10 ml of PBS-azide-BSA. Repeat this washing procedure once more. If the cell pellet still contains red cells the lysing step can be repeated.

Figure 16.1 Peripheral blood flow cytometry dot plots with a mixture of chronic lymphocytic leukaemia (CLL) and T lymphocytes. P3 shows a gate on CD19-positive lymphocytes (blue). The CD19-positive lymphocytes also express CD5, CD22 and CD23. T lymphocytes (green) show positivity for CD2 and CD5. Red population correspond to granulocytes – they are negative for B and T markers.

Finally, resuspend the cell pellet in 10 ml of PBS-azide-BSA and perform a white cell count. Aliquot a volume of cell suspension containing 1–2 × 10⁶/tube. If the number of cells in the specimen is not enough for the ideal amount of cells per tube, aliquot the specimen equally between all the tubes. Add appropriate volume of McAb, incubate in the dark, repeat washing procedure and resuspend in 0.2–0.5 ml of sheath fluid. Acquire data on the flow cytometer.

Simultaneous Detection of Cytoplasmic/Nuclear and Membrane Antigens

Method

The first step in simultaneous detection of cytoplasmic/nuclear and membrane antigens (Fig. 16.3) involves immunostaining for membrane antigen detection, followed by cytoplasmic or nuclear antigen staining.

Label tubes with the name of the patient, type of specimen, laboratory number and the McAb.

Pipette 100 μl of specimen (whole blood or bone marrow) into a tube.

Add the appropriate fluorochrome-conjugated McAb, to detect the membrane antigen.

Incubate at room temperature for 15 min.

Without washing, add 100 μl of solution A (fixative) and incubate at room temperature for 15 min. Continue with the steps described earlier for cytoplasmic antigen detection.

Figure 16.3 Flow cytometry dot plots showing CD45 gating and intracytoplasmic staining with the blast population (CD45 weak expression) highlighted in red and lymphocytes in green. TdT/CD19 plot showing simultaneous staining for membrane CD19 and intranuclear TdT. Cytoplasmic μ/CD22 plot showing simultaneous staining for intracytoplasmic markers; blasts are cμ-negative/ cCD22-positive. Lysozyme/CD79a plot showing expression of CD79a in blasts.

Data Analysis Strategies with Multiparametric Flow Cytometry

Multiparametric data including staining with several fluorochromes and the scatter properties of cells is currently used to accurately identify different cell populations and distinct disease entities.

Flow cytometry immunophenotyping provides not only a screening for haemato-oncological disorders but also is an indispensable diagnostic tool. It can identify cells from different lineages, it can determine their stage of maturation, it can discriminate normal cells from abnormal by the assessment of antigen expression or the lack of it and it can quantify the tumour infiltration. It can estimate the presence of minimal residual disease by comparing patterns of expression with that seen in normal counterparts.

Traditionally flow cytometry data was analysed in bivariate plots of two- or three-colour analyses with the application of electronic gates based on the scatter characteristic of cells.

As computers became more powerful, other strategies have been developed employing ‘immunological’ and sequential gating associated with forward and side scatter properties. For instance, in cases of acute leukaemia the preferred routine practice is to gate blast populations based on the side scatter properties and CD45 expression rather than gating solely on forward and side scatter plots.

Finally, a very important use of multicolour flow cytometry analysis strategies is in the detection of minimal residual disease of acute and chronic leukaemias. The sensitivity of these methods may be as good as 0.04% acquiring 50 000 events or 50 to 100 events of interest and increases as more events are acquired.

Until recently, advances made in computing capabilities, and the increased availability in fluorochrome conjugates, had not been matched by the developments in analysis software. However, new independent targeted analysis programmes to deal with these limitations have now been developed, which include novel tactics for the analysis of multiparametric flow cytometry data such as the software developed by the Euroflow consortium.⁵

Quantification of Antigens

Fluorescence quantification is defined as the measurement of the intensity of staining of cells by flow cytometry to provide an absolute value for the light intensity it measures.^6,⁷

Quantification of fluorescence is performed by comparing cell fluorescence with an external standard by using different commercially available beads. The following areas will be covered in this section:

1. Staining of cells and beads

2. Acquisition of data

3. Quantification: calculation of the antigen binding capacity (ABC) or molecules of soluble fluorochrome (MESF) values.

Staining of cells and beads

There are three essential requirements for successful quantification: namely (a) the McAb has to be applied at saturating amounts both to the beads and to the cells in the specimen; (b) the same reagent from a specified company and at the same dilution should be used for the test and for any subsequent tests; and (c) the instrument fluorescence setting should be maintained unchanged once the beads have been run and analysis of the unknown sample should be carried out at the same settings.

The type of bead depends on the procedure used for the sample preparation. The beads are commercially available in kits, which usually comprise two tubes. One tube contains four types of beads with four different levels of fluorescence uptake: one very dim, one very bright and two intermediate; the other tube contains blank (non-fluorescent beads) (Fig. 16.4).

Figure 16.4 Beads for antigen quantification. Upper: Gate used for the acquisition of the beads to eliminate doublets. Lower: Beads fluorescence intensity showing five peaks: blank (M1); dim (M2); bright (M5); and intermediate (M3 and M4).

Acquisition of data

The first step involves acquisition of data relating to the beads on a flow cytometer. The data from a single tube with beads are sufficient for quantification with FCSC Quantum Cellular beads (Bangs Laboratories, IN, USA) and QIFIKIT beads (Dako, Glostrup, Denmark). With quantum simply cellular (QSC) beads (Bangs Laboratories, IN, USA), where a tube is run for each McAb, the tube with the beads for that particular McAb should be run first followed by all the relevant tubes with beads for each of the different McAbs.

To bring the beads into the FSC/SSC dot plot, the SSC voltage must be decreased more than that of the cells. There may be some doublets if the tube is not shaken vigorously and these are excluded by placing a tight gate around the beads and acquiring the data only for these gated beads. The instrument should be set up so that the fluorescence signal of the tube with the blank (unlabelled) beads is located in the region between 0 and 10¹ and four other peaks of fluorescence are seen along the axis of the relevant fluorochrome. When the fluorescence voltage is established, these settings should be maintained throughout the rest of the analysis of the unknown samples. With QSC, the appropriate settings for each individual McAb must be used.

The data for the samples are then obtained. With the FCSC Quantum Cellular and QIFIKIT, only one set of beads is required because the same fluorescence standard curve can be used for the different McAbs to be quantified (e.g. CD5, CD19, CD4, CD8). With QSC, one set of fluorescence beads is stained for each McAb. The samples for a particular McAb should be run with the fluorescence settings obtained from beads stained with the corresponding McAb, so that one fluorescence standard curve should be obtained for each McAb. Thus, one curve is required with CD5-stained beads for all CD5-stained samples; one curve is required with CD19-stained beads for CD19-stained samples and so on.

Quantification: calculation of ABC or MESF values

Relevant software is provided with the quantification kits. These programs are user friendly and take into account the make of the instrument, the voltage used for the sample, the fluorochrome used and the source of the McAb. When the data obtained from the flow cytometer are entered, a standard curve is automatically produced. The standard calibration curve is produced when the values of the peak channels of the blank and the other four peaks obtained from the flow cytometer are entered into the program. The known number of molecules of fluorochrome obtained from the supplier of the beads is also entered into the program. The peak values for the unknown sample and one of the negative control samples are obtained by running them at the same fluorescence setting as the beads. When the peak value obtained with the sample is entered into the program, the ABC/MESF value of the unknown sample is calculated and the data are saved. For the final estimation of the ABC or MESF, the ABC or MESF value of the control tube is subtracted from the ABC or MESF value of the marker.

Immunocytochemistry

The most common immunocytochemical techniques are the immunoperoxidase (IP) and the alkaline phosphatase antialkaline phosphatase (APAAP) methods.^8,⁹ These detect both membrane and intracellular antigens prior to fixation of the preparation. The APAAP method is suitable for use on blood and bone marrow films and permits good preservation of cell morphology. IP is simpler than APAAP and is useful for the study of mature and immature lymphoid cells, but bone marrow samples containing myeloid cells with endogenous peroxidase may give a false-positive reaction unless steps are taken to inhibit the endogenous peroxidase activity. Unfortunately, these procedures may affect cell morphology and thus defeat one of the purposes of the test.

Immunoperoxidase

The IP method can be carried out with directly labelled antibodies (e.g. antihuman Ig conjugated with peroxidase) or by indirect methods using two or three layers. The first layer is a McAb (mouse Ig); the second layer is an antimouse Ig antibody conjugated with horseradish peroxidase; a third layer is a complex of peroxidase and antiperoxidase, which binds to the second layer and is used to reinforce the reaction. The reaction is completed by testing for peroxidase, using diaminobenzidine (DAB).

Method

Prepare cytocentrifuge slides and allow them to dry for at least 6–8 h at room temperature. If not used immediately, they should be wrapped in aluminium foil and stored at –20°C. Before testing, frozen material must be thawed at room temperature for 30 min. Make a ring around the chosen area using a diamond pencil.

Fix the slides in pure acetone for 10 min. If they have been kept at room temperature for more than 3 days, fix them for only 5 min.

Dry in air and then surround the marked area with a silicone ring (Dako pen, Dako Cytomation, or Sigmacote, Sigma-Aldrich).

Incubate for 30 min in a moist chamber with 30 μl of McAb diluted in PBS. The dilution of the McAb should be titrated in the individual laboratory for each batch of reagent, using known positive and negative controls.

Wash (flush) carefully with PBS (pH 7.3). Without allowing the slides to dry, add the second-layer antibody immediately after the second wash.

Incubate for 30 min with 30 μl of peroxidase-conjugated rabbit antimouse Ig antibody (Dako) diluted 1:20 in PBS (pH 7.3) containing 2% human AB serum.

Wash (flush) carefully with PBS (pH 7.3) twice as indicated earlier.

Incubate for 30 min in a moist chamber with 30 μl of peroxidase-labelled swine antirabbit antibody (Dako) diluted 1:20 in PBS (pH 7.3) containing 2% human AB serum.

Wash (flush) carefully with PBS (pH 7.3) twice as explained earlier.

Prepare an IP solution of 30 mg of DAB with 30% hydrogen peroxide in 50 ml of PBS; filter and pour into a coupling jar. Immerse the slides in this solution and incubate for 10 min at room temperature in the dark.

Note that the peroxidase substrate (DAB) is carcinogenic and must be handled with safety precautions, using a fume cupboard and gloves. As an alternative safer procedure, tablets of DAB, which are available commercially (Dako), can be dissolved in PBS.

Rinse in distilled water.

Counterstain with Harris haematoxylin for 10–20 s.

Wash in tap water for 2 min.

Wipe off excess water, let the slides dry in the air and mount with DPX (disterene resin dibutylphthalate in xylene).

For assessment of the reactivity with anti-TdT or other rabbit polyclonal antibodies, carry out an additional incubation for 30 min with a mouse anti-rabbit Ig antibody diluted 1:20 in PBS (pH 7.3) with 2% human AB serum prior to the incubation with the second layer of peroxidase-conjugated rabbit antimouse Ig antibody.

Interpretation

A positive reaction is identified by light microscopy as a dark brown deposit.

Immunoalkaline Phosphatase Antialkaline Phosphatase

The APAAP method involves several steps that can be applied to peripheral blood and bone marrow films. The stages include incubation with the McAb, incubation with a rabbit antimouse Ig antibody and incubation with immune complexes of APAAP. The second and third steps can be repeated to reinforce the reaction.

Method

Make films or cytocentrifuge slides and let them dry for at least 6–8 h. If not used immediately, wrap the slides in aluminium foil and store at −20°C. If frozen, thaw at room temperature for 30 min before carrying out the test.

Make a ring around the chosen area using a diamond pencil. To test more than one McAb in the same slide, several rings can be marked.

Fix in pure cold acetone for 10 min. If the slides have been kept at room temperature for more than 72 h, fix them for only 5 min.

Allow to dry in the air.

When using peripheral blood or bone marrow films, wash around the encircled areas with a cotton stick wet with PBS to remove the adjacent red blood cells.

Surround the marked areas with a silicone ring (Dako pen or Sigmacote).

Incubate for 30 min with 30 μl of McAb diluted in Tris-buffered saline (TBS) 0.05 mol/l, pH 7.6. The appropriate dilution of the McAb must be determined by titrating each batch of reagent, using known positive and negative controls.

For all subsequent procedures, the slides must not be allowed to dry and incubation must be carried out in a moist chamber.

Wash (flush) carefully with TBS 0.05 mol/l and, immediately after the wash, add 20 μl of the second layer consisting of a rabbit antimouse Ig (Dako) diluted in TBS 0.05 mol/l with 2% human AB serum.

Incubate for 30 min at room temperature in a moist chamber.

Wash (flush) again with TBS.

Incubate for 45 min with 100 μl of mouse APAAP complexes (Dako) diluted 1:60 in TBS 0.05 mol/l.

Wash (flush) again with TBS.

Cover the circles with the filtered APAAP developing solution for 15–20 min.

Rinse in distilled water.

Counterstain with Harris haematoxylin for 10–20 s.

Wash in tap water for 2 min.

Wipe off excess water and mount with Glycergel (Dako) or another water-soluble mounting medium.

Do not use DPX to cover preparations because the reaction will be faint or will become negative.

For estimation of the reactivity with anti-TdT or other polyclonal rabbit antibody, carry out a further incubation step with a mouse antirabbit Ig antibody diluted 1:20 in TBS prior to the incubation with the second layer.

Preparation of buffers and solutions

TBS 0.05 M, pH 7.6 (to wash and dilute McAb). Make a stock solution with 60.57 g of tris-hydroxymethyl-methylamine in 500 ml of distilled water. Adjust pH to 7.6 with 385 ml of 1 N HCl. Add distilled water to 1 litre and store at 4°C.

To prepare the working solution, dilute the stock solution 1:10 in 9 g/l NaCl.

TBS 0.1 M, pH 8.2 (to dilute the substrate). Make a stock solution with 1.21 g of Tris and 80 ml of water. Adjust pH to 8.2 with 4.8 ml of 1 N HCl. Add water to 100 ml (this solution can be stored for 1 month at 4°C).

Developing solution (substrate). Mix, in the following order, 20 mg of naphthol AS-MX phosphate (Sigma-Aldrich), 2 ml of N,N-dimethylformamide (Merck), 98 ml of Tris buffer 0.1 mol/l and 24 mg of levamisole (Sigma-Aldrich).

Store in glass flasks at −20°C in 5 ml aliquots.

Thaw immediately before use; add 5 mg of fast red TR salt (Sigma-Aldrich) per vial and filter.

The developing solution is also available commercially as a kit.

Immunological Markers in Acute Leukaemia

Panel of McAb Useful for Diagnosis and Classification

Although there are a large number of McAb-recognizing antigens of haemopoietic cells, for practical reasons a well-defined set of reagents needs to be selected for the study of cases of acute leukaemia. The set of markers described here have been largely selected in accordance with the recommendations of the European Group for the Immunological Classification of Leukaemias (EGIL), the British Committee for Standards in Haematology and the World Health Organization (WHO) classification.¹⁰^–¹²

An initial McAb panel should help to distinguish acute myeloid leukaemia (AML) from acute lymphoblastic leukaemia (ALL) and further classify ALL into B- or T-cell lineage (Table 16.1). This panel is constituted as follows:

1. B-lymphoid markers: CD19, CD10, CD20 and cytoplasmic CD22 and CD79a

2. T-lymphoid markers: CD2, CD7 and cytoplasmic CD3

3. Myeloid markers: CD13, CD33, CD117 and cytoplasmic myeloperoxidase MPO

4. Non-lineage-specific markers, which are expressed in haemopoietic progenitor cells: CD34, HLA-Dr and TdT.

Table 16.1 Panel of monoclonal antibodies for the diagnosis of acute leukaemias

Two aspects that need to be considered are the lineage specificity of the antigen and whether it is expressed in the membrane or the cytoplasm. Some markers are highly specific and sensitive for a particular lineage (e.g. CD3 for T cells and anti-MPO for myeloid cells), whereas others (e.g. CD10, CD13 and CD7) are less lineage specific. Nevertheless, the latter may support a lymphoid or myeloid commitment in cases that are negative with the most specific markers or when results are equivocal. The second aspect to take into account is that the most specific markers are either expressed earlier in the cytoplasm than in the membrane during cell differentiation (e.g. CD3),¹³ or they are only detectable in the cytoplasm (e.g. MPO). Markers of haemopoietic precursors such as TdT or CD34, although not essential, are helpful when problems of differential diagnosis arise between acute leukaemias and lymphomas in leukaemia phase.

A second set of McAb is necessary to classify ALL further into the various subtypes and to identify rare cases of AML derived from cells committed to the megakaryocytic and erythroid lineages. This set comprises cμ staining in B-lineage ALL; CD1a, CD4, CD5, CD8 and anti-TCR in T-lineage ALL; and, in AML, antibodies that detect membrane glycoproteins present in platelets and megakaryocytes or glycophorin A expressed by erythroid precursors.

Identification of cell reactivity with other McAb may include CD14, antilysozyme, CD64 and CD36. Although CD14 and antilysozyme are not specific for acute monoblastic leukaemias, both are more frequently expressed during monocytic differentiation. CD36 is often expressed in poorly differentiated erythroid leukaemias. Although this marker is not specific for erythroid precursors, being expressed also in monoblasts and megakaryocytic cells, when considered together with reactivity with other McAb (e.g. negative for HLA-Dr, antiplatelet McAb and MPO), it is highly indicative of erythroid acute leukaemia.

McAbs against non-haemopoietic cells rarely need to be included when performing immunophenotyping for the diagnosis of acute leukaemias. However, rare cases of neuroblastoma or oat cell carcinoma can mimic acute leukaemia in the bone marrow and in such cases anti-neuroblastoma McAb and the pan-leucocyte marker CD45 may help in establishing the correct diagnosis.

Other markers that are useful for the characterization of acute leukaemias, although not routinely used, are the following:

1. A McAb that recognizes the altered distribution of promyelocytic protein (PML) in cases of acute promyelocytic leukaemia with t(15;17). Although PML is expressed in normal myeloid cells and in blasts from other subtypes of AML, the pattern of expression, e.g. multiparticulate or cytoplasmic in the cases with t(15;17), is different from that of normal myeloid cells or myeloblasts of other types of AML. The latter are either negative with anti-PML or have the protein expressed in larger nuclear bodies. The reactivity with anti-PML needs to be assessed under fluorescence microscopy or light microscopy with an immunocytochemical technique.¹⁴

2. The McAbs 7.1/NG2 and NG1 that are preferentially expressed in a subset of pro-B or early B-cell ALL with 11q23 rearrangement ¹⁵ and in a proportion of AML with features of monocytic differentiation (irrespective of the presence of 11q23 rearrangement).¹⁶^–¹⁸

3. The McAb CD56, CD123 and CD94 to identify rare cases of blastic plasmacytoid dendritic cell neoplasm and natural killer (NK) cell leukaemia/lymphoma in cases without evidence of myeloid and lymphoid commitment with specific cell markers.¹²

Immunological Classification of Acute Leukaemias

There are two major differentiation lineages in the lymphoid system, B and T, and lymphoblastic leukaemias arise from B- or T-precursor cells. Table 16.2 illustrates that only a few McAbs react positively with the most immature lymphoblasts; with maturation, however, more McAbs become reactive. Thus, to demonstrate all cases of leukaemia of a particular lineage, it is always important to include in the battery of McAbs those that will detect the most immature cells. B-lineage ALL is defined by the expression of at least two B-cell antigens, CD79a, CD19, CD10 and/or CD22; T-lineage ALL is defined by the expression of nuclear TdT and CD3. CD7 is also consistently positive in T-ALL. However, the expression of CD7 does not by itself define T-ALL because this McAb is positive in about 20% of cases of AML.

Table 16.2 Immunological classification of acute leukaemias ^a

Lymphoblastic leukaemia/lymphomas (ALL) (TdT+)

B-cell precursor (CD19+ and/or CD79a+ and/or CD22+)

pro-B-ALL (no expression of other B-cell markers)

common-ALL (CD10+, cytoplasmic μ−)

pre-B-ALL (cytoplasmic μ+)

T-cell precursor (cytoplasmic CD3+, CD7+)^b

pro-T-ALL (no expression of other T-cell markers)

pre-T-ALL (CD2+ and/or CD5+)

cortical T-ALL (CD1a+)

mature T-ALL (membrane CD3+)

Acute myeloid leukaemias (AML)

AML (French-American-British [FAB] M0–M5) (anti-MPO+ and/or CD13+ and/or CD33+ and/or CD117+)

Pure erythroid leukaemia (anti-glycophorin A+, anti-blood group antigen+, CD36+)

Megakaryoblastic leukaemias (CD41+, CD42+, CD62P+).

Blastic plasmacytoid dendritic cell neoplasm

Miscellaneous

Mixed phenotype acute leukaemias (MPAL) (coexpression of myeloid and lymphoid markers)^c

Myeloid antigen positive ALL

Lymphoid antigen positive AML

Natural killer (NK) cell lymphoblastic leukaemia/lymphoma

Acute undifferentiated leukaemia

TdT, terminal deoxynucleotidyl transferase; MPO, myeloperoxidase.

a Adapted from the European Group for the Immunological Characterization of Leukemias (EGIL) classification.

b CD4 and CD8 are variably expressed.

c Definition of MPAL is described below.

B- and T-lineage ALL can be further subclassified on the basis of cell differentiation or maturation (Table 16.2). Although this subclassification is not essential for diagnosis, it can be useful because of the correlation between certain B-lineage subtypes and molecular cytogenetic and clinical features.

B-lineage ALL can be classified into three subtypes: pro-B-ALL (previously designated null-ALL), common-ALL and pre-B-ALL (Table 16.2). There is some correlation between these immunological subtypes and molecular genetics and prognosis. The majority of infant ALL with t(4;11)(q21;q23) and/or rearrangement of the MLL gene at 11q23 are pro-B-ALL and often express CD15, whereas the common-ALL phenotype is associated with hyperdiploidy or t(12;21) involving the ETV6 (TEL) gene, both associated with a good prognosis. The t(1;19)(q23;p13) is more common in the subset of pre-B-ALL. ‘Mature B-ALL’ (French-American-British, FAB, L3 ALL) is not classified as ALL in the WHO classification but is included in the group of high-grade, non-Hodgkin lymphomas (NHL) because it corresponds to the leukaemic manifestation of Burkitt lymphoma.¹²

T-lineage ALL can also be subdivided into several subgroups according to the stage of differentiation of the lymphoblasts. In the most immature form, or pro-T-ALL, blasts only express CD7 and cytoplasmic CD3; in pre-T-ALL, there is also expression of CD2 or CD5; cortical T-ALL is defined by the expression of CD1a; and in the rare mature T-ALL, blasts express membrane as well as cytoplasmic CD3 (Table 16.2). T-cell-associated antigens such as CD2, CD5, CD4, CD8 and TCR are expressed with variable frequency in the cortical and mature T-ALL; for instance, coexpression of CD4 and CD8, a phenotype characteristic of normal cortical thymocytes, is frequent in cortical T-ALL. In addition, mature T-ALL can be subclassified in two subgroups on the basis of the membrane expression of the T-cell receptor (TCR) complex molecules, αβ or γδ.

AML can be defined immunologically by the expression of two or more myeloid markers: CD13, CD33, CD117 and anti-MPO in the absence of lymphoid markers.¹⁰ The most specific marker for the myeloid lineage is MPO followed by CD117; as a rule, both are negative in ALL.¹⁹ There is no marker that allows the distinction between the various FAB subtypes of AML.²⁰ However, some McAb may be preferentially positive in certain AML subtypes such as CD14, CD64 and antilysozyme in cases with monocytic differentiation, absence of HLA-Dr and CD34 expression in M3 AML or expression of CD19 in M2 AML. Furthermore, immunological markers are essential for the diagnosis of poorly differentiated myeloid leukaemias or M0 AML in which blasts do not show myeloid features by morphology or cytochemistry.²¹

In poorly differentiated leukaemias in which the first panel of lymphoid and myeloid markers does not show positive results, McAb against platelet glycoproteins Ib, the complex IIb/IIIa and IIIa (e.g. CD41, CD42 and CD62P) should be tested to confirm or exclude the diagnosis of acute megakaryoblastic leukaemia,¹⁰ and McAb to glycophorin A or to the red blood cell groups such as Gerbich should be used to confirm or exclude erythroid leukaemias. In addition, the WHO classification now considers a rare form designated blastic plasmacytoid dendritic neoplasm, previously included in the group of blastoid NK neoplasms, under the umbrella of AML. The neoplastic cells are CD4+, CD56+ and CD123+ and 50% of cases are CD68+. Rarely, they express T or myeloid associated markers.¹²

Immunophenotyping facilitates the diagnosis of an unusual form of acute leukaemia designated mixed phenotype acute leukaemia (MPAL). This leukaemia accounts for <5% of cases and is characterized by the coexpression of a constellation of myeloid and lymphoid antigens in the blast cells. The lack of agreement among various workers on the definition of MPAL has made it difficult to establish whether this constitutes a distinct clinicopathological entity. Until recently, the diagnosis of MPAL was based on a scoring system ^22,²³ proposed by the EGIL group,¹⁰ which aimed to distinguish MPAL from cases of ALL or AML with aberrant expression of a marker from another lineage. In the 2008 WHO classification, the definition of this leukaemia was changed. The myeloid component is defined exclusively by positivity with anti-MPO or a positive cytochemical reaction for MPO and/or by clear evidence of a monocytic component by cytochemistry or immunophenotyping and the T-lymphoid component is defined by the expression of CD3 whether cytoplasmic or membrane. Since there is no marker specific for the B-lymphoid lineage, evidence of B-lymphoid differentiation should be based on the expression of CD19 plus another B-cell marker (CD10, CD22 or CD79) or weak/negative CD19 and strong expression of two of the specified B-cell markers. The WHO does not distinguish cases with co-expression of lymphoid and myeloid antigens (‘biphenotypic’) from those with two separate populations (‘bilineal’). Emphasis is made that some AML with recurrent chromosome abnormalities and those with a background of dysplasia should not be diagnosed as MPAL.¹²

In addition to MPAL, the WHO considers natural killer (NK) cell lymphoblastic leukaemia/lymphoma and acute undifferentiated leukaemia under the umbrella of acute leukaemias of ambiguous lineage.¹² NK cell lymphoblastic leukaemia/lymphoma is a provisional entity. The lack of specific NK markers has made establishing a diagnosis difficult in such cases. However, this diagnosis should be considered in cases that are CD56+, CD94+ and CD161+ that may express immature T-cell-associated antigens such as CD7 and even c CD3 epsilon chain but lack Ig and TCR gene rearrangement and provided blastic plasmacytoid dendritic cell neoplasm has been excluded.^12,²⁴ The other rare subgroup is acute undifferentiated leukaemia in which the blasts lack T and myeloid specific markers, do not express B-lymphoid-associated markers and do not have features of other lineages (i.e. megakaryoblastic, erythroid or plasmacytoid dendritic cells). An extensive panel of McAb needs to be used for the diagnosis of this leukaemia. The blasts usually are HLA-Dr+, CD34+ and CD38+ and may be TdT+.

Immunological Markers in Chronic Lymphoproliferative Disorders

Immunophenotyping is essential for the diagnosis and characterization of the lymphoproliferative disorders. Immunological markers enable one to distinguish lymphoblastic leukaemias and lymphoblastic lymphomas, which are usually TdT-positive, from mature or chronic lymphoid neoplasms, which are consistently TdT-negative. Immunophenotyping also demonstrates whether the malignant cells are of B- or T-lymphoid nature and demonstrates clonality in the B-cell cases. Markers may also be useful to confirm or establish the diagnosis of certain entities that show distinct immunological profiles and others may provide prognostic information.

Panel of McAb for Diagnosis and Classification

The diagnosis of a B- or T-cell disorder requires a small but comprehensive battery of McAb. It is convenient to use a two-step procedure with an initial panel applicable to all cases and a second panel based on the results with the first panel and the tentative diagnosis by clinical features and/or cell morphology (Table 16.3).^11,²⁵

Table 16.3 Panel of monoclonal antibodies for the diagnosis of lymphoid disorders

	B cell	T cell
First-line	SmIg (kappa/lambda), CD19, CD23, FMC7, SmCD79b, SmCD22, CD5^a, CD20^b	CD2, CD5^a
Second-line	CD11c, CD25, CD103, CD123, CD38, CD138, cIg	CD3, CD4, CD7, CD8, CD57

a B-cell subset and T-cell marker. Optional markers: CD79a and natural killer associated (e.g. CD16, CD56 and CD11b).

b Not in initial panel but added because of relevance to treatment. c, cytoplasmic; Ig, immunoglobulin; Sm, surface membrane.

The first panel of markers is intended to distinguish B-cell from T-cell disorders, to demonstrate B-cell clonality, to confirm the diagnosis of CLL and to confirm or exclude a non-CLL B-cell neoplasm. It comprises immunostaining with anti-kappa and anti-lambda, CD2 or CD3 (T-cell marker), CD5 (a marker of T cells and a subset of B cells) and four McAbs that detect antigens in subsets of B cells: CD23, FMC7, CD79b against the β chain of the B-cell receptor and membrane CD22. With the two latter reagents, as well as surface Ig, assessment of the fluorescence intensity is important to distinguish between CLL and other B-cell disorders (Fig. 16.4). The results obtained with this set of McAbs can be combined into a scoring system (Table 16.4) to establish the diagnosis of CLL and to distinguish CLL cases with atypical morphology and CLL with increased numbers of prolymphocytes (CLL/PL) from other B-cell diseases such as B-cell prolymphocytic leukaemia (B-PLL) and B-cell lymphomas in leukaemic phase.²⁵^–²⁷ The characteristic profile of CLL is that of a clonal B-cell with weak surface immunoglobulin (SmIg) usually IgM+IgD±, CD5+, CD23+, FMC7− and weak or negative CD79b and CD22.²⁵^–²⁷ FMC7 has been shown to recognize an epitope of CD20 and it has been suggested that this marker could be replaced by CD20 in the diagnostic scoring system for CLL. However, data show that replacement of FMC7 by CD20 results in a decrease in sensitivity of the scoring because most cases of CLL express CD20 weakly.²⁸ Nevertheless, although CD20 is not particularly useful for diagnosis, it should be incorporated into the panel of markers for chronic lymphoid disorders because it is used increasingly as a therapeutic tool in these conditions. Other markers that may be used include CD200, which appears to be useful in the CD5+ B-cell disorders to distinguish cases of CLL (CD200+) from mantle-cell lymphomas (CD200-),²⁹ and Ki-67, which allows the estimation of the proliferative rate in cases of high-grade lymphoma.

Table 16.4 Scoring system for the diagnosis of chronic lymphocytic leukaemia (CLL)^a

Marker	Points
	1	0
CD5	Positive	Negative
CD23	Positive	Negative
FMC7	Negative	Positive
SmIg^b	Weak	Moderate/strong
CD22/CD79b^b	Weak/negative	Moderate/strong

a Scores for CLL range from 3 to 5, whereas in the other B-cell disorders they range from 0 to 2.

b Membrane expression.

When the marker profile using the first-line panel of McAb yields a B-cell phenotype not typical of CLL, a second panel of McAb can be used. This is selected in light of the review of the cell morphology, clinical information or other laboratory features. For example, estimation of the cell reactivity with four McAbs (CD11c, CD25, CD103 and CD123) is useful to distinguish hairy cell leukaemia (HCL) from other disorders with circulating villous cells that may be confused with HCL, such as splenic marginal zone lymphoma (SMZL) and the HCL variant. Cells from the majority of HCL cases coexpress three or four of the markers mentioned earlier, whereas SMZL and cells from HCL variant are positive with one or at most two of these markers.³⁰ Among the four HCL-associated markers, CD123, which recognizes the β chain of the interleukin 3 receptor is the most useful in distinguishing HCL from SMZL and the variant form of HCL. CD123 is consistently expressed in HCL cells, whereas it is negative in the other two conditions.³¹

When the first-line panel of markers suggests a T-cell phenotype (CD2+ or CD3+, CD5±), expression of other T-cell markers such as CD3 (if not already used), CD7, CD4, CD8 and CD57 may need to be investigated. CD25 may be used in cases of suspected adult T-cell leukaemia lymphoma but its expression is not pathognomonic of this disease as it may be positive in other mature T-cell neoplasms. When markers do not indicate either B lineage or T lineage, but cytology is consistent with a lymphoid lineage, testing for NK cell markers should be done.

Unusual situations may occur in the case of plasma cell leukaemia and myeloma, in which the cells are negative with all T-cell and the majority of B-cell markers including surface Ig expression; clonal plasma cells express Ig only in the cytoplasm (with light chain restriction) and are positive with two common antigens, i.e. CD38 bright and CD138 in conjunction with negativity for CD45.³² The best approach to differentiate normal from neoplastic plasma cells is a multicolour methodology using CD45 negativity versus CD138 strong positivity as gating strategy. Multicolour flow cytometry staining using CD45, CD38, CD138, CD19, CD56, CD20 and cytoplasmic Ig light chains provides the most sensitive and specific method to distinguish normal from clonal plasma cells. Normal plasma cells are characterized by the expression of CD45, CD19, CD38 and CD138 with polyclonal expression of light chains. Clonal plasma cells are characterized by the expression of CD38, CD138, CD56 and intracytoplasmic light chain restriction with no detection of surface light chains and lack of expression of CD45 and CD19 and often no expression of CD20.

Multiparametric flow cytometry generally recognizes fewer plasma cells than those found in the specimen films and trephine biopsy sections. This may be explained by patchy infiltration and by loss of plasma cells during sample processing for flow cytometry studies due to plasma cell clumping.

Other markers that have diagnostic and prognostic value in chronic lymphoid disorders are described below.

p53 protein

The p53 protein can be detected in cells by immunocytochemistry and flow cytometry with specific antibodies. There is a good correlation between p53 expression point mutation with or without deletion of TP53 genes. It therefore appears that there is no need for gene sequencing to be done on a routine basis because flow cytometry, fluorescence in situ hybridization (FISH) or both provide accurate information of the p53 status.³³ Although not essential for diagnosis, this test may be useful as a prognostic indicator because of its correlation with resistance to purine analogue therapy and disease progression in patients with B-cell disorders, particularly CLL.³³^–³⁵

CD38 and ZAP70 expression

CD38 and ZAP70 are two markers shown to have a major prognostic impact in CLL. The expression of CD38 has emerged as a prognostic factor independent of the immunoglobulin heavy chain (IgHV) mutational status.^36,³⁷ CD38 should be assessed by a triple colour flow cytometry method to ensure that the expression is evaluated in the leukaemic cells. Although the first reports used thresholds of 20–30% CD38+ cells to consider this marker as positive, it has become apparent that there is intra-clonal diversity and that a threshold of 7% is the most reliable and informative in terms of prognosis.^38,³⁹ ZAP70 encodes a tyrosine kinase that is expressed in normal T cells, NK cells and a few B cells. Microarray studies in CLL have shown that the pattern of gene expression is very similar in cases with mutated or unmutated IgHV genes with only minor differences in a few genes. Among these, ZAP70 is preferentially expressed in the cases with unmutated IgHV; thus it was suggested that ZAP70 could be used as a surrogate marker for the IgHV mutational status. To this end, it is important to estimate its expression in purified B-CLL cells by RNA analysis or by a multicolour flow cytometric method that allows the simultaneous assessment of ZAP70 expression in T, NK and CLL cells (Fig. 16.5).^39,⁴⁰ At present, it is uncertain which is the best threshold (10% or 20%) for ZAP70+ CLL cells to consider that this marker is positive. There is now evidence that ZAP70 is not a surrogate marker for the unmutated IgVH. Discrepant results are seen in up to 30% of cases and the combination of CD38, ZAP70 and IgVH mutations is needed to establish the prognosis of CLL more precisely.³⁹

Figure 16.5 Flow cytometry dot plots using a four-colour method to detect ZAP70 expression in T/NK lymphocytes and CLL lymphocytes. Upper plots: Gate on the whole lymphocyte population (left) and the expression of ZAP70 (right). Middle plots: Gate on CD3/CD56-positive cells, which represent T and natural killer cells (left) and the expression of ZAP70 in this population (right). Lower plots: Gate on CD5/CD19 positive cells (CLL cells) (left) and the expression of ZAP70 in this subset (right).

McAb against the Variable Regions of the β TCR and Killer Immunoglobulin Receptors

There is a set of McAb that identifies T lymphocytes that bear the various variable regions of the TCR β and hence might be useful to demonstrate clonality of the T-cell population, particularly in cases with large granular lymphocyte (LGL) lymphocytosis, when molecular studies (e.g. polymerase chain reaction, PCR) cannot easily be performed. There is also a potential role in identifying T-cell clones in patients with hypereosinophilia. A good correlation between flow cytometry and PCR has been demonstrated with flow cytometry able to predict clonality with a sensitivity of 93% and specificity of 80%.⁴¹ McAb that recognize molecules belonging to the Ig superfamily, essentially the killer immunoglobulin receptors (KiRs) clustered under CD158a, CD158b, CD158c and CD158e, can also potentially recognize clonal LGL disorders and are particularly useful in those derived from NK cells in which clonality cannot be established by molecular tests.⁴²

McAb Against CD20, CD52 and CD33

The expression of the CD20 and CD52 antibodies should be assessed in patients with lymphoid neoplasms and CD33 in AML in whom antibody therapy with rituximab, alemtuzumab or gemtuzumab ozogamicin, respectively, is contemplated.

Immunological Profiles of Chronic Lymphoproliferative Disorders

The most common immunophenotypes of the B- and T-cell disorders are shown in Tables 16.5 and 16.6. CLL has a phenotype that clearly distinguishes this disease from the other B-cell leukaemias. By contrast, there is overlap on the marker expression in the other B-cell malignancies; for this reason, in cases with a B-cell marker profile different from CLL, the immunophenotypic analysis needs to be interpreted in the light of morphology and other clinical and laboratory information such as histology or genetic analysis to establish the precise diagnosis.²⁷

Table 16.5 Membrane markers in mature B-cell disorders (CD2−)

Table 16.6 Immunological markers in mature T-cell disorders (CD2+)

There is no specific immunological profile that distinguishes the various T-cell diseases (Table 16.6). However, expression of CD8 and CD57, with or without expression of NK-associated markers such as CD16 or CD56, is characteristic of T-cell LGL leukaemia, whereas such expression is rarely seen in other conditions.⁴² By contrast, coexpression of CD4 and CD8 is almost exclusively seen in approximately 25% of T-cell prolymphocytic leukaemia (T-PLL) (Table 16.6).^43,⁴⁴ Other markers may also be differentially expressed in various T-cell malignancies. Thus, for example, there is expression of CD25 in adult T-cell leukaemia lymphoma (ATLL); strong reactivity with CD7 in T-PLL;⁴³^–⁴⁵ expression of granzyme B, TIA-1 or perforins in T-cell or NK-cell LGL leukaemias and TCR γδ in hepatosplenic T-cell lymphoma.⁴²

Immunological Markers for the Detection of Minimal Residual Disease

Immunophenotyping can be a useful tool to detect small numbers of residual leukaemic cells in peripheral blood or bone marrow specimens when such cells are not detected by standard morphology or histopathology. Detection of minimal residual disease (MRD) applies to both acute and chronic lymphoid leukaemias; it is likely to have prognostic significance in terms of probability of relapse and in some instances its detection directs further therapy.

This is carried out by multicolour immunofluorescent methods with a combination of McAbs aimed at identifying ‘aberrant’ phenotypes not present in normal haemopoietic cells and/or by considering the different density in normal and leukaemic cells.⁴⁶^–⁴⁸ For example, although normal bone marrows, particularly in infants or when regenerating after therapy, have a minor cell population with a B-cell precursor phenotype (e.g. TdT+, CD10+, CD19+), similar to that of ALL blasts, quantitative studies provide discrimination between the normal precursors (strong TdT and weak CD10 and CD19) and ALL blasts (weak TdT, strong CD10/CD19).^47,⁴⁸ Similarly, there is a small B-cell population in normal blood and bone marrow that coexpresses CD5, a phenotype characteristic of CLL. However, by estimating the proportions of CD5+ cells within the whole B-cell population (CD19+), it is possible to demonstrate whether cells represent residual leukaemia or normal B-lymphocytes. More recently a standardized multiparameter flow cytometry method has been described for CLL minimal residual disease (MRD). This combines sequential gating, i.e. lymphocytes (G1), CD19+ lymphocytes (G2) and CD5+/CD19+ (G3), with a comparison of the pattern of expression of the following McAbs: CD79b, CD20, CD38, CD81 and CD43 between normal and CLL cells (Fig. 16.6); this has been shown to have a sensitivity of 0.01%.⁴⁹