Flow Cytometry - definitions

(2) Flow cytometry - definitions.

Affordable flow cytometry

I. At the Durban AIDS conference the issue of inexpensive (affordable) flow cytometry was raised in order to back up HIV related services in an economical manner [1]. This is an integrated approach leading to a new phase in the development of clinical flow cytometry.

History of flow cytometry: (very) "lucky" constellations [2]

II. The grey box called a flow-cytometer accomodates the end-result of an astonishing multidisciplinary collaboration among engineers, biophysicists, biochemists, histo- and molecular pathologists, cytologist, haematologists, immunologists, HIV physicians and medical epidemiologists [2]. The concepts have gradually developed as follows:

° Flow cytometry, investigating cells in a flow system instead of on a static microscope, was put into practice by Coulter. where he used impedance to count red cells.
° 'Rapid Cell Spectroscopy' was introduced by Kamentsky to leucocyte differential counting with the help of 2-parameter histograms and computer assisted displays;
° Optical cell counters, dark background with fluorescence dies, lasers and photomultiplier tubes were all added by Fulwyler;
° The importance of immunological reagents was advocated by the Herzenbergs;
° The precision engineering of flow cells was applied on the first commercial flow cytometers made in Germany by Goehde;
° Well engineered cytometers with increasing sensitivity, complexity, computerisation and practicality appeared in the forms of Cytofluorograph (Ortho), FACS (Becton-Dickinson), Spectrum III (Ortho), EPICS (Coulter), etc.

III. In summary, flow cytometry is a kaleidoscopically colourful discipline that has just become available at the appropriate time for various clinical applications - rewarding the imaginative scientists who had the vision to foresee these needs. These brilliant contributions have been:

° Optical filter technology [3],
° Fluorescent dies to construct molecular probes [4],
° Laser technology,

° Precision syringes for volumetric measurements,
° Monoclonal antibodies to identify >200 clusters of antigens on the surface and inside

the cells,

° Fixatives to preserve cellular antigens,

° An extraordinary increase in the speed and storage capacity of computers,

° The clinical need for using flow cytometry in as a diagnostic tool,

° Clinical protocols by international committees,

° Quality control organizations and lastly,

° The internet

Current state-of-the-art

Antibodies have been primarily tested in fluorescein-isothiocyanate (FITC; green) conjugated form; 10-12 years ago phycoerythrin (PE; orange) and third-red colour conjugated tandem reagents were also added to construct triple colour antibody combinations elicited with a single 488nm laser (Fig.2; top). At that time research- and clinical protocols were still similar. Since then these two areas have diverged.

Cytometers used for research have become more complex by using two or more lasers and at least 4-colour immunofluorescence (but without direct absolute cell counting facility). An example has been assembled at the Stanford Shared FACS facility [5] capable of exciting the following 10 antibody conjugates covering the whole spectrum: Cascade blue (excited by UV laser), FITC, PE, Cy5-PE, Cy5.5-PE and Cy7-PE (excited by a 488-nm laser) and Cy5 or APC, Alexa-595, Cy5.5-APC and Cy7-APC (excited by a 600-nm dye-laser) [5].
Cytometers in the clinical service were mostly required to count absolute numbers of cells and such dedicated equipment has been developed [6].

Legend to above: Routine three-colour FCM mostly operates with a single laser source at the lower range of visible spectrum (green to red; top bar). At the top level of research [5], the whole spectrum may be used with the help of multiple lasers (middle bar). This sophisticated technology paves the way for new applications: new reagents in the deep red range are now available. These Cy5- and/or APC-conjugated antibodies can be applied with inexpensive red diode lasers (bottom bar; long advocated by Shapiro), to introduce a new chapter in flow cytometry. [2].

The main aims of clinical flow cytometry

Three major areas of clinical flow cytometry include (i) CD4 counts in HIV infection, (ii) CD34+ precursor cell analysis and (iii) the analysis of leuco-depleted blood products. All of the latter require an absolute counting facility. The fourth important area is in the area of leukaemia/ lymphoma diagnosis and the assessment of minimal (residual) disease. here less emphasis is placed on exact absolute counts. In the first 3 fields special service facilities have been developed for absolute counts (see below), while in leukaemia laboratories multiparameter analysis (e.g. FACS with Paint-a-gate software; not discussed here) is used.

Different approaches to absolute counting

"Initial scientific improvisations"

These early heroic attempts used isolated washed blood mononuclear cells, indirect immunofluoresce (IF) staining and two machines in an uncoordinated fashion, one for lymphocyte count (mostly in Haematology departments) and one for % IF positive cells among lymphocytes (mostly in Immunology departments). Thereafter calculated values were generated with a 30-60% error. Such an improvised "service" was, however, already good enough to rapidly document in the first cases of adult immunodeficiency syndrome and that a selective CD4 lymphopenia was the pathological basis of a new type of suspected infection.

Double Platform Systems

"Double platform" systems (reviewed in ref. 6) employ two well-harmonized machines, a haematological counter plus a flow cytometer. These run parallel tubes of the same whole blood sample with no-wash technology. One of these tubes, analysed on the flow cytometer, is prestained with monoclonal antibodies. The crucial issue is, as explained below, that the two machines need to count a common parameter to precisely correlate the results obtained. This extra step is, really, an unwanted complication. The common parameter can be lymphocytes - to make your life difficult. We suggest that the common parameter should be the leucocytes (PanLeucogating) - to make your life easy and the test less expensive, see [12]. Precise absolute CD4 counts are thus calculated from the leucocyte counts (provided by the haematological analyser) and the CD4% values (among leucocytes, provided by the flow cytometer).

Single Platform Systems

These are machines/platforms especially designed to count the absolute numbers of antibody labelled cells, equipped with multiple sample loader, programming facility and computer support. These are of two major types:

1. Volumetric flow cytometers count absolute numbers of cells in a given volume (ORTHO Cytoron and DAKO Galaxy)

2. Bead based systems that have no direct counting/ volumetric facility (Trucount/ Flow Count) counted on FACS Calibur or Coulter XL (reviewed in ref. 6,7)

Overkill" is undesirable

Severe "overkill" in the system is elegantly shown in [8]. In a candid account how investigations can be simplified, Shapiro describes (in [2]. pages 61, 215-6, 236) that an intracellular IF staining method for total cellular protein has lost its commercial viability when it had been shown that the total protein content and the (orthogonal) side scatter gave identical information on the various blood cell types. Why to go complicated when a simple scatter analysis would do? He asks:"how much is enough/too much"? There are many similar questions. Sometimes the budgets available influence the answer. We should ask: Is side scatter enough without analysing forward scatter? Do we need to perform CD3 T cell staining together with the staining for CD4 and CD8? And so on.

Legend to b: Flow cells and detectors can be constructed to recognize 3 parameters such as (orthogonal) side scatter for morphologic discrimination, and two fluorescence channels (for double labelling with 2 antibodies). Such an arrangement is likely to be sufficient for routine service work. (Modified from the Purdue university website).

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