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.