Ficoll-Gradient Method of Separation

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.

Detection of membrane antigens

Multicolour flow cytometry:

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.

Rationale for choosing antibody panels

There are different strategies for choosing which antibody panel to use, such as: (1) using a screening panel of McAbs followed by a more targeted panel according to the screening results; (2) choosing an acute leukaemia or a lymphoproliferative panel according to the suspected/referral diagnosis; (3) deciding on a specific panel of McAbs following morphological assessment of a film of the specimen after staining with May–Grünwald–Giemsa.

Detection of Surface Immunoglobulin

Lymphoproliferative disorders of mature B cells are distinguished from their normal counterparts by the identifications of two main types of phenotypic abnormalities: surface immunoglobulin light chain restriction and aberrant B-cell antigen expression (Fig. 16.2).

Figure 16.2 Surface immunoglobulin light chain staining. (A) Dot plot showing strong expression of lambda light chain in a case of follicular lymphoma. (B) By contrast, weak/dim expression of lambda light chain in a case of CLL (mixture of CLL cells and T lymphocytes). Cells are negative with anti-kappa in both cases.

The method of detection of surface heavy and light chain immunoglobulins by flow cytometry differs from the one used to detect other surface antigens. This is because the interpretation of staining for kappa/lambda and heavy chains can be made more difficult by the presence of nonspecific staining giving rise to either false positivity or negativity which can be misleading. This non-specific staining may be due to cytophilic antibodies binding to Fc receptors (monocytes and some lymphocytes) or to coating of antibodies to cell membranes of damaged or dying cells.

To overcome this problem, there are several options: the specimen can be washed with an isotonic solution prior to staining for the surface immunoglobulins. Non-specific staining can also be minimized by incubating the cells with serum prior to staining.

This phenomenon can be excluded by gating on B cells during data analysis, for instance assessing the surface immunoglobulins on a CD19+/CD45+ gate.

Finally, some B-cell lymphoproliferative disorders such as chronic lymphocytic leukaemia (CLL) may express surface immunoglobulin very weakly. It is preferable to use polyclonal antibodies to detect light chain restriction in these cases

There are two methods suitable for detecting surface Ig in blood and bone marrow cells, according to whether a PBS wash or a lysing procedure is used as the first step.

Detection of Intracellular Antigens

This method is applied to the identification of antigens that are expressed within the cell, i.e. in the cytoplasm or nucleus. For example, intracellular immunoglobulins, MPO, lysozyme, CD3, CD79a, BCL2, TdT and Ki67 can all be detected by this method.

There are several commercially available kits containing solutions to fix and permeabilize cells to detect cytoplasmic or nuclear antigens. Overall, these reagents have little or no effect on the light scatter pattern, although their reliability and consistency for detecting particular nuclear and cytoplasmic antigens may vary.^2,3

The kits contain two solutions: solution A is the fixing agent based on a paraformaldehyde solution and solution B is a lysing agent based on a combination of a lysing solution and a detergent.

The methods follow the manufacturer’s kit instructions. Details that follow are for the method using Fix and Perm (Invitrogen).⁴

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.

Similarly in cases of B-cell disorders, it is more informative to gate on CD19-positive cells and side scatter and for T-cell disorders to apply a gate on CD3-positive cells and side scatter. Multicolour flow cytometry gating of plasma cells is performed to differentiate between clonal and normal plasma cells by combining sequential gating on the CD45-negative/CD138-positive cells and then to look specifically at other antigen expression on these cells.

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: