Flow cytometry is gradually replacing,in immuno-hematology laboratories thefluorescence microscope to performquickly analyzes on a large number ofcells in suspension.
Flow cytometry is the study of a suspension monocellular, colored by a fluorochrome,flowing in a liquid vein and passing in front a light beam (most often a laser source)thanks to an optical system studying light transmitted and the light diffracted and or re-emitted by a fluorochrome (immunofluorescence).
DESCRIPTION OF THE DEVICE
We take, as an example of description,the Coulter XL device. This device is composedof three parts:
- The cytometer: allows the physical passage of the sample in the flow cell or chamber analysis and collection of light signals from diffraction and fluorescence.
- The computer station: manages all signals from the cytometer and allows the data processing.
- Pneumatic and electrical supply:contains laser power supplies and the cytometer as well as the compressor necessary for pneumatic circuits.
The cytometer includes:
- A monochromatic laser light source:Argon laser which constitutes the source of excitationat 488 nm (blue line).
- An analysis chamber or Flow cell, inwhich passes the analyzed sample and where thecells are illuminated by the laser beam.
- Size and structure detectors whichmeasure the deflected or scattered light emittedby cells when passing through the beamlaser.
- Size and structure detectors whichmeasure the deflected or scattered light emittedby cells when passing through the beam laser.
- Fluorescence detectors that measurethe fluorescence emitted by the cells during passage through the laser beam.
The suspension of previously marked cellsis injected into the center of the vein fluid by a200 µm internal diameter needle. The flow ofcells is driven by the liquid vein whosediameter gradually decreases to reach20 µm.
The elements line up behind each otherothers and parade at an imposed speed of passageby the velocity of the vein fluid. The passage of sample flow diameter from 200µm to one 20 µm diameter is called hydrofocusing.
Analysis of the light scattered towardsfront: (Forward Scatter: FS)
Under the effect of the light excitation of the beamlaser, the cell diffuses part of the lightreceived in all directions. The lightforward diffracted, i.e. in the axisoptical, is proportional to the size of the cell.This is the FS signal.
This signal is measured by a detector called photodiode which converts energy bright in electrical energy.
Analysis of the light scattered at 90 degrees or at wide angles (side Scatter: SS)
This is the light from the laser that penetratesinside the cell and which is refracted in a transparent medium like the cytoplasm. This phenomenon of refraction or reflection, in some case, depends on the intrinsic properties of the cell like the cytoplasm, the presence of granulations more or less abundant and of the ratio nucleocytoplasmic.
This signal is measured by a photodiode.
The cell is marked by a fluorochrome. The one-ci absorbs the light energy provided by the laser and restores part of it in the form ofphotons of wavelength characteristic offluorochrome.
Fluorescence signals, wellthat issued in all directions, are measured at90 degrees by special detectors: thephotomultipliers whose main role isto amplify the photons received.In multiple marking, it is necessary to separatethe signals between them to measure themindividually.
For this, there are filtersinterference, tilted at 45 degrees, called mirrorsdichroic, which allow transmissioneach wavelength towards its detectorrespective.
A fluorochrome is a molecule which, in the state ofrest, has the property of absorbing the energy emitted bya light source to pass theelectrons of its atoms from a sublayer A to aother upper B layer, corresponding to astate of excitement.
The wavelengths that are absorbedconstitute the excitation spectrum of a molecule.The return of these electrons to their initial layeris accompanied by a release of energycharacterized by the emission of photons. It’s here fluorescence . The emission wavelengthsof these photons are a property of eachfluorescent molecule, and they depend onatoms of the molecule.
Wave lengths emitted by a molecule constitute its spectrumof emission .In practice, the excitation wavelength offluorochromes used in CMF is optimal at 488nm and the source is an Argon laser.The characteristics of fluorochromes allowa triple and quadruple marking analysis inmono-laser argon configuration emitting at 488nm.
ANALYSIS OF CELLS INCYTOMETRY
The principle of cell analysis in CMF is based on the immunofluorescence reaction. This reaction combines the specificity of antibodies monoclonals and the properties of fluorochromes. It is a method of choice for revelation of cellular antigens.
The techniques of marking are of two types:- Direct immunofluorescence where the antibody employee is conjugated directly to a fluorochrome.- Indirect immunofluorescence where the antibody engaged in the reaction is revealed by a second antibody itself fluorescent.
This technique is carried out in two stages:
- antigen -1 st antibody interaction
- 1st antibody – 2nd antibody interaction(fluorescent conjugate).
Study of membrane antigens
Membrane antigens can be studied sure :
- blood or bone marrow cellsisolated on density gradient (Ficoll)
- blood or bone marrow cells after lysis of red blood cells
- bronchoalveolar lavage cells.
Whole blood labeling is a techniques implified more suited to routine and large series of samples. It involves a lysis of red blood cells with a lysis reagent.
Detection of intracellular antigens
This detection requires permeabilizationadvance of cells to ensure entry ofreagentsinvolvedinthereactionimmunofluorescence. The interest of this methodis that certain antigens canbe detected at the most intracytoplasmic stageearly differentiation such as CD3 inLAL type T and CD22 in LAL typepre-
THE DIFFERENT APPLICATIONS OFCMF
One of the most common clinical applications of flow cytometry is the determination of antigens cell phones. heisof immunopheno typing of normal cells and pathological. The use of monoclonal antibodies specific differentiation classes (CD) of human leukocytes will allow recognition from a number of cell populations. AT to date more than 200 CDs have been defined.
Some are restricted to lines, others correspond to stages of differentiation or to functional aspects. Antibodies are available who recognize:- Cell lines: T lymphocytes,B lymphocytes, Natural Killers (NK) cells,cellsMyeloids, MonocytesandMacrophages, from the stem cell tothe mature cell…- Membership or activation markerscytokine receptors- Functionally defined populations:cytotoxic, secretory, auxiliary cells,suppressive, memories …
Diagnosis and classification of hematopoietic proliferations
The CMF is very useful for the classification ofhematopoietic tumors involving blood,marrow, body fluids or other tissuesince cell lines and markers ofdifferentiation have been very intensively studiedin these tumors.
PHENOTYPING OF LEUKEMIA
The phenotyping of acute leukemias is delicate. hecan be made from a direct debitbone marrow or from a sampleblood. Some leukemias may beassigned to type T lines or linestype B, others belong to lineagesmyeloblastic or monocytic.
In somenumber of cases, leukemia may appearmultiphenotypics with markersbelonging to several lines, phenotypes thatusually not found on cellsnormal. In other cases, a complete absence ofcell line markers can lead to conclusionto undifferentiated type leukemia.
The analysis of an ALL of line B mustinclude at least the use of CD19, CD20,CD22 possibly supplemented by researchof intracytoplasmic µ chain, of CD22intracytoplasmicandofthe expressionmembrane immunoglobulins.
Membrane markers of the T line aremajor to define a proliferationtype T lymphoblastic: CD2, CD5, CD7 andthe search for the intracytoplasmic expression of theCD3The proliferation of the myeloid lineage can beidentified by CD13, CD14, CD15, CD33,Myeloperoxidase, CD68 as well as CD41, CD42,CD61 (platelet line) and Glycophorin A(CD235) and B (CD236) (erythroid line).
SYNDROME PHENOTYPING LYMPHO PROLIFERATIVE
Phenotyping of lymphoproliferative syndromeshas become essential.A wider range of antibodies is required tothis phenotyping. The first step is toidentify the cell line involved(proliferation B or T or NK). The second step is to use more markers specific.
The concept of “residual illness” meansusually identification, usingsensitive techniques, pathological cellsin a patient in complete clinical remission.
Thedetection of rare CD 10+ LAL cells in theperipheral blood by CMF was an initial modelof a technique that is potentially applicable toothers.Early relapse detection trialsmalignant tumors by marker analysiscells were focused on the bloodperipheral or marrow, since these tissuesare easy to retrieve and study in singlecell suspension in CMF.
CD34 + cell count in theframework of the bone marrow transplant
Clinically, stem cell transplantshematopoietic are part of the arsenaltherapy made available tohematologists and oncologists. Initially,bone marrow allografts (MO) represented almost all of the transplants performedand, for several years, cell transplantsperipheral strains (auto or allografts) constitute the vast majority of cases.
Besides, stem cell transplant iseven faster with blood progenitors peripheral. Mobilization protocols andobtaining these cells largely rely on thecytometry.The CD34 marker can predict the ability toregeneration from stem cellshematopoietic. We currently know that”Good” bone marrow transplant contains 3 to 5 10 6CD34 + cells per kg of recipient.For the count of progenitorshematopoietic. we often use an anti-AcCD45 and a specific anti-CD34 monoclonal Ab.The use of absolute value balls allowsto establish the precise count and not thepercentage.
Diagnosis of immunodeficiency states
Exploring immunocompetent cellsspecific and non-specific involves immunophenotypic analyzes (sub-populations,adhesion molecules, activation markers …) andfunctional tests (cell proliferation in vitro, secretion immunoglobulins or interleukins, phagocytosis, NK activity …)
Acquired immunodeficiency states (infectionby the HIV virus)
HIV targets the CD4 receptor of CD4 + lymphocytes leading to deficit immune system acquired by destroying these cells. The constant progressive depletion ofCD4 + T cells, (both in percentage and absolute number) was clearly associated with these verity of HIV-related illness. The people negative tend to have a count of CD4 + T cell base that stays at long term without much fluctuation.
From the six months after seroconversion, the number ofCD4 + drops and, within two years, 30 to50% of the initial number of CD4 + T lymphocytesare destroyed.
A big number of people asymptomatic HIV positive people keep for many years stable levels about 600 / mm 3 .
A rapid decrease in CD4 + T cell count is a sign un favorable prognosis and can announce the appearance of opportunistic infections. After the AIDS diagnosis, the number of T lymphocytes CD4 + remains very low and may drop to levels not measurable before death. The population of cytotoxic CD8 lymphocytes is frequently increased in absolute number in the initial stage of HIV infection.
The percentage of CD4 + T cells, theabsolute number of CD4 + T cells and theCD4 + / CD8 + ratio all have excellent valueprognosis for the prediction of AIDS and arestrongly correlated with each other.
Cell phenotype in LBA (bronchoalveolar lavage)
Certain pathologies such as sarcoidosis andallergic alveolitis may haverelatively close clinical pictures donot allowing an accurate diagnosis while thetherapy will not be the same at all. Thephenotyping of bronchoalveolar fluid willthen a precious diagnostic tool- The increase of percentage of CD4 lymphocytes or increased ratio CD4 / CD8 points to sarcoidosis.
- The increase in the percentage of CD8 lymphocytes or decreased ratioCD4 / CD8 points to an allergic alveolitis.
- CD8 hyperlymphocytosis is observed in HIV infection.
HLA B 27 phenotyping
The HLA B27 phenotype is often associated withankylosing spondylitis. The technique ofmicrolymphocytotoxicity is long, uses Acpolyclonal that produce cross reactionswith other antigens.
In flow cytometry, allthese disadvantages are erased: no reactionsinterpretation, the interpretation cannot lead todiscussion and the results can beexpressed quantitatively.
CMF is able to identify proteins fromplatelet surface. Deficits inplatelet glycoproteins have been identified intwo congenital functional diseases ofplatelets: gpIIb / IIIa (CD41) in theGlanzman’s thrombasthenia and gpIb (CD42)in Bernard-Soulier’s disease.
The count of reticulocytes
Reticulocytes are red blood cells that havebeen in circulation for less than one to twodays. Their count is one of the mostuseful for the diagnosis of anemia.
The normal percentage of reticulocytes is only from 0.5 to 1.5% and so, for precisionsatisfactory, it takes at least 1000cells. Which is very time consuming, if it isdone manually. Reticulocyte counting can be done byCMF most often using orange thiazole(emission at 530 nm), but also acridine orange as an RNA marker.
The cell cycle and CMF
The CMF makes it possible to study the different phases of thecell cycle in tumors to judge their proliferation potential and detect the presence possible cellular clones with abnormal content in genetic material.We classically distinguish in a cell cycle4 phases, G 1 , S, G 2 and M, in relation to the state function of cells and their material content genetics (DNA).
In addition to these 4 cell-specific phases cycling, we distinguish the so-called G 0 phase during which the cell has a fixed DNA level at 2n. This phase corresponds to a post-mitotic state,out of cycle, transient or permanent.
Cells in transient G 0 can be stem cells or differentiated recruitable cells, remained able to proliferate. Cells in G Opermanent are often active cells of thepoint of view of their protein synthesis and theirspecific activity but become unable toproliferate.In flow cytometry, the staining techniqueuses propidium iodide which will be insertedbetween the strands of DNA.
And so the CMF puts inevidence three phases which are commonlycalled: G O / G 1 , S, G 2 + M.A cell cycle histogram of cellsnormal has good characteristics known.
In broad terms we can cite mainly:
- A relatively small proliferation, therefore thepart corresponding to phase S and phaseG2 + M is very unimportant compared topeak represented by cells in the G0 / G1 phase.
- A single population and therefore a single peak of cells in G0 / G1.
The interpretation of a cell cycle in CMF mustbe done with care. At first, we canextract from the histogram obtained informationqualitative: presence of one or morepopulations, ploidy of the Go / G 1 peak (s) by relation to an internal or external standard, populationin phase S important or not, and quantitative information: percentage of each of the populations, percentage of different phases of the cell cycle for eachof populations.
Control of leuko reduced preparations
Leukocytation of labile blood products(CGR red blood cell concentrate,CPS standard platelets, platelet concentrateCPA apheresis) helps minimizemaximum risk of viral transmission,mainly intraleukocytic viruses, as wellthan prevent anti-HLA alloimmunization.
The quality control of this leukoreduction was difficult because only achievable by counting manual. The CMF brings us an answer interesting given its speed and precision. Theresidual leukocytes are labeled with iodidepropidium after treatment with detergent,allowing the penetration of it insidethe cell, and RNAse to eliminate RNAreticulocytary and platelet. The use ofautofluorescent beads of known concentrationallows us to calculate the number of globuleswhites per µl and therefore deduct the total number ofresidual leukocytes in the blood productanalysis.
Cell sorting is the ability to separatephysicallydifferentsubpopulationscell phones. The cells are sorted according toproperties observed during the analysis.
Theparticles of interest are chargedelectrically deflected at the output by a fieldelectric and collected in two tubes (thepositive on one side and negative on the other).
Theuncharged particles are removed.Sorting results in a homogeneous populationof cells in a state of purity which allows themre-cultivation and maintains their maincharacteristics.
The CMF has several advantages:
- Detection and quantification of numerous subcell populations simultaneously
- Count hundreds of times faster than for the techniques manual immunofluorescence
- Reproducibility of results and goodsensitivity
- Precise quantification of signal strengthallowing cell sortingThis CMF has, however, limitations:
- High cost
- Sufficient training requirement forusers
- ossible production of unwanted results if the operator collects a fluorescence signalfrom an unaffected population