A candid retrospective of the A candid retrospective of the monoclonal revolutionmonoclonal revolution

Have the dreams come through ?Have the dreams come through ?

George JanossyGeorge JanossyUniversity College LondonUniversity College London

A candid retrospective of the A candid retrospective of the monoclonal revolutionmonoclonal revolution

Have the dreams come through ?Have the dreams come through ?

George JanossyGeorge JanossyUniversity College LondonUniversity College London

Nancy, 24Nancy, 24thth October 2008 October 2008Nancy, 24Nancy, 24thth October 2008 October 2008

A candid retrospective of A candid retrospective of the monoclonal revolutionthe monoclonal revolution

Have the dreams come Have the dreams come through ?through ?

This talk is about these seven wonderous dream-diamonds.This talk is about these seven wonderous dream-diamonds.

The shepperd falls asleep at the camp-fire The shepperd falls asleep at the camp-fire -- with fabolous consequences. -- with fabolous consequences. He has the dream of the dreams: He has the dream of the dreams: dozens of diamonds are sparkling up in dozens of diamonds are sparkling up in the flames.the flames.When he wakes up many of the these When he wakes up many of the these dream-diamonds vanish -- but …. dream-diamonds vanish -- but …. But miracolously, seven of these gems But miracolously, seven of these gems still remain !still remain !

Dream No.1Dream No.1

Flow cytometry can be used for clinical Flow cytometry can be used for clinical leukaemia diagnosis on the ‘immunophenotyping’ leukaemia diagnosis on the ‘immunophenotyping’ platformplatform

anti-ALL (24 absorptions!)anti-ALL (24 absorptions!)

fetal development: fetal development:

BM is a lymphoid organBM is a lymphoid organ

Flow cytometry can be used for clinical Flow cytometry can be used for clinical leukaemia diagnosis on the ‘immunophenotyping’ leukaemia diagnosis on the ‘immunophenotyping’ platformplatform

anti-ALL (24 absorptions!)anti-ALL (24 absorptions!)

fetal development: fetal development:

BM is a lymphoid organBM is a lymphoid organ

Melvyn Greaves, Melvyn Greaves, Geoff Brown and Geoff Brown and George JanossyGeorge Janossy

Melvyn Greaves, Melvyn Greaves, Geoff Brown and Geoff Brown and George JanossyGeorge Janossy

TdT+,TdT+,cALL cALL

(CD10)+(CD10)+lymphoid lymphoid

blast crisisblast crisisof Ph’+ of Ph’+ chronic chronic myeloid myeloid

leukaemialeukaemia

(Lancet ii: 1058, (Lancet ii: 1058, 1976)1976)

The first clinical use of sorting in The first clinical use of sorting in EuropeEurope

Reviewed in: G. Janossy: Clinical Flow Cytometry, a Hypothesis-Driven Discipline of Modern Reviewed in: G. Janossy: Clinical Flow Cytometry, a Hypothesis-Driven Discipline of Modern Cytomics Cytomics Cytometry Part ACytometry Part A 58A58A:87–97 (2004). :87–97 (2004). Janossy G, Francis GE, Capellaro D, Goldstone Janossy G, Francis GE, Capellaro D, Goldstone AH, Greaves MF. Cell sorter analysis of leukaemia-associated antigens on human myeloid AH, Greaves MF. Cell sorter analysis of leukaemia-associated antigens on human myeloid precursors. precursors. NatureNature 1978; 1978; 276276(5684)(5684): : 176-8.176-8.

Reviewed in: G. Janossy: Clinical Flow Cytometry, a Hypothesis-Driven Discipline of Modern Reviewed in: G. Janossy: Clinical Flow Cytometry, a Hypothesis-Driven Discipline of Modern Cytomics Cytomics Cytometry Part ACytometry Part A 58A58A:87–97 (2004). :87–97 (2004). Janossy G, Francis GE, Capellaro D, Goldstone Janossy G, Francis GE, Capellaro D, Goldstone AH, Greaves MF. Cell sorter analysis of leukaemia-associated antigens on human myeloid AH, Greaves MF. Cell sorter analysis of leukaemia-associated antigens on human myeloid precursors. precursors. NatureNature 1978; 1978; 276276(5684)(5684): : 176-8.176-8.

The extraordinary hypothesis The extraordinary hypothesis by by

M.F.GreavesM.F.Greaves leading to the immuno-leading to the immuno-phenotypingphenotyping

Acute lymphoid leukaemia (ALL) must come from an Acute lymphoid leukaemia (ALL) must come from an early stem cell – early stem cell – which will which will have surface markershave surface markers

these will not be leukaemia specific: these will not be leukaemia specific: it will be the first anti-it will be the first anti- precursor (stem) cell antiserumprecursor (stem) cell antiserum

balanced experimental designbalanced experimental design T heterologous antiserum (absorbed, specific)T heterologous antiserum (absorbed, specific) B surface Ig and Class-II (absorbed, specific)B surface Ig and Class-II (absorbed, specific) myeloid markersmyeloid markers all this on leukaemias and normal cellsall this on leukaemias and normal cells

Terminal transferase (Hoffbrand, Bollum) and Terminal transferase (Hoffbrand, Bollum) and immuno-histochemistry (Catovsky, immuno-histochemistry (Catovsky, D.Y.Mason)D.Y.Mason)

Cell lines (Minowada) Cell lines (Minowada)

Fluorescence-activated cell analysis Fluorescence-activated cell analysis and sortingand sorting (FACS) (FACS)

THE PLATFORM WAS BORNE; see GEIL, EuroFlow, etc. THE PLATFORM WAS BORNE; see GEIL, EuroFlow, etc. reviewed by Keating P Cambrosio A. Biomedical platforms—realigning the normal and the pathological in late-twentieth-century medicine. reviewed by Keating P Cambrosio A. Biomedical platforms—realigning the normal and the pathological in late-twentieth-century medicine. Cambridge: MIT Press; 2003.Cambridge: MIT Press; 2003.

Acute lymphoid leukaemia (ALL) must come from an Acute lymphoid leukaemia (ALL) must come from an early stem cell – early stem cell – which will which will have surface markershave surface markers

these will not be leukaemia specific: these will not be leukaemia specific: it will be the first anti-it will be the first anti- precursor (stem) cell antiserumprecursor (stem) cell antiserum

balanced experimental designbalanced experimental design T heterologous antiserum (absorbed, specific)T heterologous antiserum (absorbed, specific) B surface Ig and Class-II (absorbed, specific)B surface Ig and Class-II (absorbed, specific) myeloid markersmyeloid markers all this on leukaemias and normal cellsall this on leukaemias and normal cells

Terminal transferase (Hoffbrand, Bollum) and Terminal transferase (Hoffbrand, Bollum) and immuno-histochemistry (Catovsky, immuno-histochemistry (Catovsky, D.Y.Mason)D.Y.Mason)

Cell lines (Minowada) Cell lines (Minowada)

Fluorescence-activated cell analysis Fluorescence-activated cell analysis and sortingand sorting (FACS) (FACS)

THE PLATFORM WAS BORNE; see GEIL, EuroFlow, etc. THE PLATFORM WAS BORNE; see GEIL, EuroFlow, etc. reviewed by Keating P Cambrosio A. Biomedical platforms—realigning the normal and the pathological in late-twentieth-century medicine. reviewed by Keating P Cambrosio A. Biomedical platforms—realigning the normal and the pathological in late-twentieth-century medicine. Cambridge: MIT Press; 2003.Cambridge: MIT Press; 2003.

Dream No.2Dream No.2

Monoclonal antibodies can be used for Monoclonal antibodies can be used for visualizing cells in tissues, normal and visualizing cells in tissues, normal and pathological (‘monoclonal immunohistology’ pathological (‘monoclonal immunohistology’ platform)platform)

CD1 (NA1/34) on CD1 (NA1/34) on human thymus:human thymus:The first ever tissue The first ever tissue section stained bysection stained bythe first anti-human the first anti-human monoclonal antibodymonoclonal antibody

CD1 (NA1-34) and CD1 (NA1-34) and CD45 (2D1) for T-ALL CD45 (2D1) for T-ALL diagnosis and thymicdiagnosis and thymicoriginorigin

Monoclonal antibodies can be used for Monoclonal antibodies can be used for visualizing cells in tissues, normal and visualizing cells in tissues, normal and pathological (‘monoclonal immunohistology’ pathological (‘monoclonal immunohistology’ platform)platform)

CD1 (NA1/34) on CD1 (NA1/34) on human thymus:human thymus:The first ever tissue The first ever tissue section stained bysection stained bythe first anti-human the first anti-human monoclonal antibodymonoclonal antibody

CD1 (NA1-34) and CD1 (NA1-34) and CD45 (2D1) for T-ALL CD45 (2D1) for T-ALL diagnosis and thymicdiagnosis and thymicoriginorigin

Ken Bradstock, Gianni PizzoloKen Bradstock, Gianni Pizzolo

Bradstock KF, Janossy G, Bollum FJ, Milstein C. Anomalous phenotype Bradstock KF, Janossy G, Bollum FJ, Milstein C. Anomalous phenotype in thymic acute lymphoblastic leukaemia. in thymic acute lymphoblastic leukaemia. NatureNature 1980; 284(5755): 455- 1980; 284(5755): 455-7.7.

Bradstock KF, Janossy G, Bollum FJ, Milstein C. Anomalous phenotype Bradstock KF, Janossy G, Bollum FJ, Milstein C. Anomalous phenotype in thymic acute lymphoblastic leukaemia. in thymic acute lymphoblastic leukaemia. NatureNature 1980; 284(5755): 455- 1980; 284(5755): 455-7.7.

The very first immuno-histological preparation ever: The very first immuno-histological preparation ever: using monoclonal anti-human antibodies using monoclonal anti-human antibodies Oct.1978Oct.1978

NA1/34 CD1 NA1/34 CD1 monoclonal monoclonal antibody antibody bybyMcMichael & McMichael & MilsteinMilstein

chicken chicken anti-human anti-human

Class IIClass II

Subcapsular cortical medullarySubcapsular cortical medullarycortex thymus thymuscortex thymus thymusSubcapsular cortical medullarySubcapsular cortical medullarycortex thymus thymuscortex thymus thymus

thymocyte – T lymphocyte maturationthymocyte – T lymphocyte maturationthymocyte – T lymphocyte maturationthymocyte – T lymphocyte maturation

IgAIgAIgAIgAIntraepithelial lymphocytesIntraepithelial lymphocytesIntraepithelial lymphocytesIntraepithelial lymphocytes

In the gutIn the gut CD45+ intraepithelial lymphocytesCD45+ intraepithelial lymphocytes ((CD8+; not shownCD8+; not shown) stained in combination with ) stained in combination with anti-anti-IgAIgA (lumen and plasma cells)(lumen and plasma cells)

In the gutIn the gut CD45+ intraepithelial lymphocytesCD45+ intraepithelial lymphocytes ((CD8+; not shownCD8+; not shown) stained in combination with ) stained in combination with anti-anti-IgAIgA (lumen and plasma cells)(lumen and plasma cells)

IgAIgAIgAIgA

IgAIgAIgAIgA

Selby Selby et al.et al.Gut 22:169,1981,Gut 22:169,1981,CEI 44: 453. 1981CEI 44: 453. 1981

Selby Selby et al.et al.Gut 22:169,1981,Gut 22:169,1981,CEI 44: 453. 1981CEI 44: 453. 1981

Janossy, G. et al. Nature 288: 81.1980Janossy, G. et al. Nature 288: 81.1980Janossy, G. et al. Nature 288: 81.1980Janossy, G. et al. Nature 288: 81.1980

This is, however, This is, however, old-fashionedold-fashioned amateur workamateur work

when compared to when compared to the informative the informative

power and clarity of power and clarity of confocal confocal microscopymicroscopy and and

to the elegance of to the elegance of dynamic dynamic intravital intravital imagingimaging ( Jackson ( Jackson Eger; this meeting)Eger; this meeting)

This latter workThis latter work also brings up considerations about lymphocytes also brings up considerations about lymphocytes migrating to sites, and slowing down there, where the migrating to sites, and slowing down there, where the antigens are deposited: for antigens are deposited: for diagnosing active diagnosing active tuberculosistuberculosis by flow cytometryby flow cytometry using sputum-borne lymphocytes using sputum-borne lymphocytes

also brings up considerations about lymphocytes also brings up considerations about lymphocytes migrating to sites, and slowing down there, where the migrating to sites, and slowing down there, where the antigens are deposited: for antigens are deposited: for diagnosing active diagnosing active tuberculosistuberculosis by flow cytometryby flow cytometry using sputum-borne lymphocytes using sputum-borne lymphocytes

Flow cytometry of active tuberculosis

Broncho-alveolar lavage (blood versus lung) -- paper by S.Barry et al. JCI 2004) BAL

PPD

BALNo Ag

24.2%

2.31%

0.78%

1.13%

BloodPPD 0.09%

0.02%

CD

4

IFN-

a b

c d

BALPPD

IFN-

IFN- TNF-

35.1%

2.48%

TB is a lung-disease Analysis of IFN- synthetic capacity of lung-derived lymphocytes:What do you think to be the more informative technology:Flow-cytometry or ELISpot?

TB is a lung-disease Analysis of IFN- synthetic capacity of lung-derived lymphocytes:What do you think to be the more informative technology:Flow-cytometry or ELISpot?Janossy, G. et al.: The role of flow cytometry in the IFN-g based diagnosis of active tuberculosis and its Janossy, G. et al.: The role of flow cytometry in the IFN-g based diagnosis of active tuberculosis and its coinfection with HIV-1 – a technically oriented review. Cytometry Part B 74B (suppl.1) S141-151coinfection with HIV-1 – a technically oriented review. Cytometry Part B 74B (suppl.1) S141-151

Janossy, G. et al.: The role of flow cytometry in the IFN-g based diagnosis of active tuberculosis and its Janossy, G. et al.: The role of flow cytometry in the IFN-g based diagnosis of active tuberculosis and its coinfection with HIV-1 – a technically oriented review. Cytometry Part B 74B (suppl.1) S141-151coinfection with HIV-1 – a technically oriented review. Cytometry Part B 74B (suppl.1) S141-151

Dream No.3 – well, this is an ugly Dream No.3 – well, this is an ugly oneone

Monoclonal Monoclonal CD4CD4 antibodies can be used antibodies can be used to monitor patients: ‘slope’ of progression to monitor patients: ‘slope’ of progression to AIDS and CD4 regeneration on therapy to AIDS and CD4 regeneration on therapy (‘primary CD4 gating’)(‘primary CD4 gating’)

Documenting the natural history of HIV disease in the Royal Documenting the natural history of HIV disease in the Royal Free Haemophilia cohort (from 1982 CD4 counting onwards) Free Haemophilia cohort (from 1982 CD4 counting onwards) Phillips AN, Lee CA, Elford J, Janossy G, Timms A, Bofill M, Kernoff PB. Serial CD4 Phillips AN, Lee CA, Elford J, Janossy G, Timms A, Bofill M, Kernoff PB. Serial CD4 lymphocyte counts and development of AIDS. lymphocyte counts and development of AIDS. LancetLancet 1991; 337(8738): 389-92. 1991; 337(8738): 389-92.

Primary CD4 gating Primary CD4 gating Janossy G, Jani I, Gohde W. Affordable CD4(+) T-cell Janossy G, Jani I, Gohde W. Affordable CD4(+) T-cell counts on 'single-platform' flow cytometers I. Primary CD4 gating. counts on 'single-platform' flow cytometers I. Primary CD4 gating. Br J HaematolBr J Haematol 2000; 111(4): 1198-2082000; 111(4): 1198-208

Panleucogating as the preferred method in resource-poor Panleucogating as the preferred method in resource-poor countries countries Glencross D, Scott LE, Jani IV, Barnett D, Janossy G. CD45-assisted Glencross D, Scott LE, Jani IV, Barnett D, Janossy G. CD45-assisted PanLeucogating for accurate, cost-effective dual-platform CD4+ T-cell enumeration. PanLeucogating for accurate, cost-effective dual-platform CD4+ T-cell enumeration. CytometryCytometry 2002; 50(2): 69-77. 2002; 50(2): 69-77. Also: Also: Glencross et al. Cytometry Part B &4B S40-Glencross et al. Cytometry Part B &4B S40-S51S51

Monoclonal Monoclonal CD4CD4 antibodies can be used antibodies can be used to monitor patients: ‘slope’ of progression to monitor patients: ‘slope’ of progression to AIDS and CD4 regeneration on therapy to AIDS and CD4 regeneration on therapy (‘primary CD4 gating’)(‘primary CD4 gating’)

Documenting the natural history of HIV disease in the Royal Documenting the natural history of HIV disease in the Royal Free Haemophilia cohort (from 1982 CD4 counting onwards) Free Haemophilia cohort (from 1982 CD4 counting onwards) Phillips AN, Lee CA, Elford J, Janossy G, Timms A, Bofill M, Kernoff PB. Serial CD4 Phillips AN, Lee CA, Elford J, Janossy G, Timms A, Bofill M, Kernoff PB. Serial CD4 lymphocyte counts and development of AIDS. lymphocyte counts and development of AIDS. LancetLancet 1991; 337(8738): 389-92. 1991; 337(8738): 389-92.

Primary CD4 gating Primary CD4 gating Janossy G, Jani I, Gohde W. Affordable CD4(+) T-cell Janossy G, Jani I, Gohde W. Affordable CD4(+) T-cell counts on 'single-platform' flow cytometers I. Primary CD4 gating. counts on 'single-platform' flow cytometers I. Primary CD4 gating. Br J HaematolBr J Haematol 2000; 111(4): 1198-2082000; 111(4): 1198-208

Panleucogating as the preferred method in resource-poor Panleucogating as the preferred method in resource-poor countries countries Glencross D, Scott LE, Jani IV, Barnett D, Janossy G. CD45-assisted Glencross D, Scott LE, Jani IV, Barnett D, Janossy G. CD45-assisted PanLeucogating for accurate, cost-effective dual-platform CD4+ T-cell enumeration. PanLeucogating for accurate, cost-effective dual-platform CD4+ T-cell enumeration. CytometryCytometry 2002; 50(2): 69-77. 2002; 50(2): 69-77. Also: Also: Glencross et al. Cytometry Part B &4B S40-Glencross et al. Cytometry Part B &4B S40-S51S51

CD

4 – C

D8

CD

4 – C

D8

CD

4 – C

D8

CD

4 – C

D8

How to cope How to cope with THAT one?with THAT one?

Without going Without going bankrupt?bankrupt?

These samples These samples are from HIV+ are from HIV+ patients waiting patients waiting for or on anti-for or on anti-retroviral retroviral therapytherapy

See special See special supplement supplement Clinical Clinical Cytometry 74B; Cytometry 74B; 2008. 2008.

How to cope How to cope with THAT one?with THAT one?

Without going Without going bankrupt?bankrupt?

These samples These samples are from HIV+ are from HIV+ patients waiting patients waiting for or on anti-for or on anti-retroviral retroviral therapytherapy

See special See special supplement supplement Clinical Clinical Cytometry 74B; Cytometry 74B; 2008. 2008.

Glencross D, Aggett H, Stevens WS and Mandy F.: African Regional external Quality Glencross D, Aggett H, Stevens WS and Mandy F.: African Regional external Quality Assessment for CD4 evaluation, and performance of laboratories. Clinical Cytometry 74B: Assessment for CD4 evaluation, and performance of laboratories. Clinical Cytometry 74B: S69-79. (2008)S69-79. (2008)

Glencross D, Aggett H, Stevens WS and Mandy F.: African Regional external Quality Glencross D, Aggett H, Stevens WS and Mandy F.: African Regional external Quality Assessment for CD4 evaluation, and performance of laboratories. Clinical Cytometry 74B: Assessment for CD4 evaluation, and performance of laboratories. Clinical Cytometry 74B: S69-79. (2008)S69-79. (2008)

Flow cytometry is Flow cytometry is quantitativequantitative

quantitative:quantitative: the optimal method to count, the optimal method to count, precisely and accurately, large numbers of precisely and accurately, large numbers of cellscells in in large numbers of samples (absolute numbers and large numbers of samples (absolute numbers and percentages of cells)percentages of cells)

the next topic is to show that:the next topic is to show that: quantitative:quantitative: the optimal method to count, the optimal method to count,

precisely and accurately, the numbers of precisely and accurately, the numbers of moleculesmolecules on the cell surfaces – in correlation on the cell surfaces – in correlation with the mean fluorescence intensity (MFI)with the mean fluorescence intensity (MFI)

this works best with FITC and PE – and not so this works best with FITC and PE – and not so much with the tandem flourochrome conjugated much with the tandem flourochrome conjugated markersmarkers

flow cytometry can be contained to remain simple flow cytometry can be contained to remain simple and it beats other methods, such as ELISpot, and it beats other methods, such as ELISpot, hands down in terms of quantitative potentialshands down in terms of quantitative potentials

Dream No. 4Dream No. 4

It is so easy to transform the values of It is so easy to transform the values of mean fluorescence intensity (MFI) into mean fluorescence intensity (MFI) into values of expressed molecules per cells values of expressed molecules per cells becausebecause

reliable method such as the QIFI (Quantitative reliable method such as the QIFI (Quantitative Indirect Immuno-Fluorescence) exists andIndirect Immuno-Fluorescence) exists and

the biological controls are so stable the biological controls are so stable CD4 on T cells, CD45 on lymphocytes show CD4 on T cells, CD45 on lymphocytes show

virtually no variations; experienced flow virtually no variations; experienced flow cytometrists are constantly using these normal cytometrists are constantly using these normal controls and many others to standard runs on controls and many others to standard runs on their cytometers (Carleton Steward et al.)their cytometers (Carleton Steward et al.)

Bikoue A, George F, Poncelet P, Mutin M, Janossy G, Sampol J. Quantitative analysis of Bikoue A, George F, Poncelet P, Mutin M, Janossy G, Sampol J. Quantitative analysis of leukocyte membrane antigen expression: normal adult values. leukocyte membrane antigen expression: normal adult values. CytometryCytometry 1996;26:137–147. 1996;26:137–147. Reviewed in:Reviewed in: G. Janossy: Clinical Flow Cytometry, a Hypothesis-Driven Discipline of Modern G. Janossy: Clinical Flow Cytometry, a Hypothesis-Driven Discipline of Modern Cytomics Cytomics Cytometry Part ACytometry Part A 58A:87–97 (2004). 58A:87–97 (2004).

Bikoue A, George F, Poncelet P, Mutin M, Janossy G, Sampol J. Quantitative analysis of Bikoue A, George F, Poncelet P, Mutin M, Janossy G, Sampol J. Quantitative analysis of leukocyte membrane antigen expression: normal adult values. leukocyte membrane antigen expression: normal adult values. CytometryCytometry 1996;26:137–147. 1996;26:137–147. Reviewed in:Reviewed in: G. Janossy: Clinical Flow Cytometry, a Hypothesis-Driven Discipline of Modern G. Janossy: Clinical Flow Cytometry, a Hypothesis-Driven Discipline of Modern Cytomics Cytomics Cytometry Part ACytometry Part A 58A:87–97 (2004). 58A:87–97 (2004).

A range of standard values A range of standard values (QIFI)(QIFI)

The clinical use of quantitative The clinical use of quantitative values for MFIvalues for MFI

unusual populationsunusual populations clear subsets clear subsets definition of cut-off points to define definition of cut-off points to define

subsetssubsets aberrant aberrant combinations combinations of markersof markers

leukaemias, leukaemias, lymphomaslymphomas alterations alterations due to immuno-due to immuno-therapy therapy

-, +/-, +, ++, +++ or negative, duller, dull, very weak,weak,a bit, medium, strong, stronger, bright, brighter, mindboggling-, +/-, +, ++, +++ or negative, duller, dull, very weak,weak,a bit, medium, strong, stronger, bright, brighter, mindboggling

Dream No. 5Dream No. 5

Monoclonal antibodies, humanized, Monoclonal antibodies, humanized, chimeric, complement-fixing or toxin-chimeric, complement-fixing or toxin-labelled, are going to be therapeutically labelled, are going to be therapeutically usefuluseful

in collaboration with Sandoz/Novartis; the in collaboration with Sandoz/Novartis; the third successful therapeutic agent were made third successful therapeutic agent were made (Janossy, Amlot: basilimaximab – (Janossy, Amlot: basilimaximab – SimulectSimulect: : anti-CD25 to ameliorate organ rejection given anti-CD25 to ameliorate organ rejection given together with Cyclosporin)together with Cyclosporin)

Ricin-A chain conjugated CD22 (in Ricin-A chain conjugated CD22 (in collaboration with E. Vitetta and P. Amlot)collaboration with E. Vitetta and P. Amlot)

Integrated collaborationIntegrated collaboration

Requires strong Requires strong leadership and a leadership and a long time-scalelong time-scale

(10-15 yrs)(10-15 yrs)

Requires strong Requires strong leadership and a leadership and a long time-scalelong time-scale

(10-15 yrs)(10-15 yrs)

Dream No. 6: “one-stop Dream No. 6: “one-stop shop”shop”

An emerging technology is the multiplexed immunoassays read by flow cytometry, for the diagnosis of

infectious diseases. In these assays, multiple fluorescent microspheres, conjugated to different antigens or

antibodies, constitute the solid phase for detecting antibodies or antigens in

biological samples. These flow cyto-meters as field instruments could

replace ELISA and RIA tests and are also be capable of doing cellular

immunological tests such as CD4+ T-cell enumeration and Plasmodium

falciparum detection in whole blood.

An emerging technology is the multiplexed immunoassays read by flow cytometry, for the diagnosis of

infectious diseases. In these assays, multiple fluorescent microspheres, conjugated to different antigens or

antibodies, constitute the solid phase for detecting antibodies or antigens in

biological samples. These flow cyto-meters as field instruments could

replace ELISA and RIA tests and are also be capable of doing cellular

immunological tests such as CD4+ T-cell enumeration and Plasmodium

falciparum detection in whole blood.

WBCC + CD4WBCC + CD4T cell countsT cell counts

5-plex5-pleximmunoassaysimmunoassays

ParasitaemiaParasitaemiain malariain malaria

Small clinicalSmall clinicalflow cytometerflow cytometer

volumetricvolumetricbattery operatedbattery operated

red-diode laser operatedred-diode laser operated3 parameter ( 1 side scatter)3 parameter ( 1 side scatter)

(2 (2 fluorochromesfluorochromes))IBM compatibleIBM compatible

digital processing digital processinge-mail linke-mail link

Fig.2. The various applications of small, portable battery operatedclinical flow cytometers that operate on red diode laser includehaematological white blood cell counts and absolute CD4 counts [93], 5-plex multiparameter analysis of immunoassays [this review] and theanalysis of parasitaemia during Plasmodium falciparum infection [94].These instruments may establish a new field of flow cytometry that canreach a wider range of laboratories than the current models.

Jani IV, Janossy G, Brown DW, Mandy F. Multiplexed Jani IV, Janossy G, Brown DW, Mandy F. Multiplexed immunoassays by flow cytometry for diagnosis and immunoassays by flow cytometry for diagnosis and surveillance of infectious diseases in resource-poor surveillance of infectious diseases in resource-poor settings. settings. Lancet Infect DisLancet Infect Dis 2002; 2(4): 243-50. 2002; 2(4): 243-50.

Jani IV, Janossy G, Brown DW, Mandy F. Multiplexed Jani IV, Janossy G, Brown DW, Mandy F. Multiplexed immunoassays by flow cytometry for diagnosis and immunoassays by flow cytometry for diagnosis and surveillance of infectious diseases in resource-poor surveillance of infectious diseases in resource-poor settings. settings. Lancet Infect DisLancet Infect Dis 2002; 2(4): 243-50. 2002; 2(4): 243-50.

Luminex-100; FACSArray with 96-well Luminex-100; FACSArray with 96-well microplate readermicroplate readerLuminex-100; FACSArray with 96-well Luminex-100; FACSArray with 96-well microplate readermicroplate reader

The first study The first study to show the to show the importance of importance of detecting detecting minimal minimal residual residual leukaemialeukaemia

Salamanca Salamanca A.OrfaoA.Orfao

Karolinska Karolinska A.Porwit-A.Porwit-

MacDonaldMacDonald

St.Jude, St.Jude, Memphis Memphis D.Campana & D.Campana & E. Coustan-E. Coustan-SmithSmithD

ream

No

.7D

ream

No

.7D

ream

No

.7D

ream

No

.7

On therapy, normal B-cell On therapy, normal B-cell precursors are temporarily absentprecursors are temporarily absent

Aberrant combinations of Aberrant combinations of markers can identify malignant markers can identify malignant cells, even if present in very low cells, even if present in very low proportions in the sample.proportions in the sample.

The induction-therapy produced The induction-therapy produced effects on the normal populations effects on the normal populations provides chances for simplifying provides chances for simplifying the assaysthe assays

This arrangement is taking the This arrangement is taking the the parameter of ‘blast decrease the parameter of ‘blast decrease rate’ directly relevant to patient rate’ directly relevant to patient management management

bone marrow samples 18-22 bone marrow samples 18-22 days post-induction therapydays post-induction therapy

This was developed by Campana and Coustan-Smith at St.Jude, Memphis, and then formed the diagnostic basis for ‘out-sourcing’ from St.Jude to Brasil, the poor area of the country such as Recife. T.Eden and others called attention that many ALL-sufferer children were lost to therapy by toxicity, abandonment and secondary infections. So, good responders get less drugs. Sandlund et al. Blood 2002: 100: 43-47.Sandlund et al. Blood 2002: 100: 43-47.Coustan-Smith, E. Ribiero RC et al. Blood 2006; 97-102.Coustan-Smith, E. Ribiero RC et al. Blood 2006; 97-102.Howard S C, Pedrosa M et al. JAMA 2004; 291: 2451-75. Howard SC, Campana D et al. Leukemia 2005; Howard S C, Pedrosa M et al. JAMA 2004; 291: 2451-75. Howard SC, Campana D et al. Leukemia 2005; 19: 323-5.19: 323-5.Reviewed by Janossy G. The changing pattern of smart flow cytometry Biotechnology J. 3: 32-42. 2008.Reviewed by Janossy G. The changing pattern of smart flow cytometry Biotechnology J. 3: 32-42. 2008.

Minimal Residual Leukaemic Minimal Residual Leukaemic Disease (MRD) ‘lite’ versionDisease (MRD) ‘lite’ versionMinimal Residual Leukaemic Minimal Residual Leukaemic Disease (MRD) ‘lite’ versionDisease (MRD) ‘lite’ version

MRD “Light” MRD “Light” vsvs Standard MRD by Flow Standard MRD by Flow Cytometry and PCRCytometry and PCR

Coustan-Smith, E. Ribiero RC et al. Blood 2006; 97-102Coustan-Smith, E. Ribiero RC et al. Blood 2006; 97-102Coustan-Smith, E. Ribiero RC et al. Blood 2006; 97-102Coustan-Smith, E. Ribiero RC et al. Blood 2006; 97-102

Prognostic significance of CD19+ Prognostic significance of CD19+ CD10+ and/or CD34+ at Day 19CD10+ and/or CD34+ at Day 19

Prognostic factorPrognostic factor P value*P value*CD19CD19++ CD10 CD10++ and/or CD34 and/or CD34+ (+ (Present vs absent)Present vs absent) 0.0030.003

Age (<1 vs 1-9 vs >10 yrs)Age (<1 vs 1-9 vs >10 yrs) 0.9060.906

WBC(<50WBC(<50 x 10x 1099/L vs ≥ 50 x 10/L vs ≥ 50 x 1099/L)/L) 0.0950.095

DNA Index (≥1.16 vs others)DNA Index (≥1.16 vs others) 0.2600.260

BCR-ABLBCR-ABL 0.0040.004

TEL-AML1TEL-AML1 0.9440.944

MLL-AF4MLL-AF4 0.3430.343

NCI Risk (Standard vs High)NCI Risk (Standard vs High) 0.3420.342

*Univariate analysis; by multiple regression analysis MRD was the only significant independent prognostic factor (P = 0.025)

Recife Pilot Study #1Recife Pilot Study #1

Traditional Traditional risk features risk features

Day 19 Day 19 CD19+ CD10+ and/or CD19+ CD10+ and/or

CD34+ CD34+

Risk Risk GroupGroup

GoodGood <0.01%<0.01% LowLow

GoodGood >0.01%>0.01% StandardStandard

PoorPoor AnyAny HighHigh

Poor = T-lineage ALL, or B-lineage ALL with WBC ≥ 50K, age 10-15 yrs, testicular/CNS leukemia, and/or adverse genotypes (BCR-ABL, MLL rearrangements, hypodiploidy <45 chromosomes)

Results presented at SIOP, Results presented at SIOP, 20082008

REFINING AND REDUCING INTENSITY OF REFINING AND REDUCING INTENSITY OF THERAPY FOR PEDIATRIC ACUTE THERAPY FOR PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) IN LYMPHOBLASTIC LEUKEMIA (ALL) IN DEVELOPING COUNTRIES: DEVELOPING COUNTRIES:

THE RECIFE-ALL-2005 STUDYTHE RECIFE-ALL-2005 STUDY

Pedrosa F Pedrosa F 11, Rivera GK, Rivera GK22, Campana D, Campana D22, , Coustan-Smith ECoustan-Smith E22 , Pedrosa M , Pedrosa M11, Lins M, Lins M11, , and Ribeiro RCand Ribeiro RC22

11Instituto Materno Infantil de Pernambuco, Instituto Materno Infantil de Pernambuco, Recife, Brazil and Recife, Brazil and 22 St. Jude Children’s St. Jude Children’s Research Hospital, Memphis, TN, USAResearch Hospital, Memphis, TN, USA

Brilliance, dedication Brilliance, dedication and artand art

That is flow cytometry – a collaborative, That is flow cytometry – a collaborative, constantly developing practical science constantly developing practical science with academic involvement with academic involvement (could be stronger)(could be stronger) and loyal and efficient industry support and loyal and efficient industry support (who (who are not charitable institutions and are entitled to their hard-are not charitable institutions and are entitled to their hard-earned profits)earned profits)

It is a quantitative method to count cells and It is a quantitative method to count cells and quantitative to count moleculesquantitative to count molecules

this lecturer is a happy person (xtatic)this lecturer is a happy person (xtatic)

is then there anything to grumble about ?is then there anything to grumble about ?

well, well, if you insist,if you insist, there are two problems there are two problems

Grumbling aboutGrumbling about

Flow cytometry could be far more powerful; it Flow cytometry could be far more powerful; it could reach areas other techniques can not could reach areas other techniques can not (Heineken beer effect)(Heineken beer effect)

Methods could be introduced fasterMethods could be introduced faster How long are we going to wait to introduce CD45 for quality How long are we going to wait to introduce CD45 for quality

controlling haematological differential counts?controlling haematological differential counts? What is the case with paediatric infectious disease What is the case with paediatric infectious disease

diagnosis ? diagnosis ? Why “dream 6 (one-stop shop)” is not coming along when Why “dream 6 (one-stop shop)” is not coming along when

the best instruments such as the Luminex-100 and the FACS-the best instruments such as the Luminex-100 and the FACS-Array are all there ready to be used ?Array are all there ready to be used ?

How is it possible the ELISpot for tuberculosis diagnosis is How is it possible the ELISpot for tuberculosis diagnosis is spreading (non-quantitative and inflexible method) and flow spreading (non-quantitative and inflexible method) and flow cytometry fails to make a proper impact ? cytometry fails to make a proper impact ?

Grumbling aboutGrumbling about

The flow cytometry with its enthusiastic The flow cytometry with its enthusiastic supporting science provides the supporting science provides the impression to the ‘uninitiated’ (impression to the ‘uninitiated’ (who arewho are…) …) that it is over-complicated and frequently that it is over-complicated and frequently simply incomprehensiblesimply incomprehensible

please, once a method is introduced and please, once a method is introduced and works, commit yourself to a final stage of works, commit yourself to a final stage of simplification and explain this to the outside simplification and explain this to the outside crowdcrowd

please, trust that you technology is simply please, trust that you technology is simply unbeatable even if it is explained wellunbeatable even if it is explained well

please, do not over-spend – hard times are please, do not over-spend – hard times are here and are still coming here and are still coming

Special issue of resource-restricted flow cytometr in Clinical Cytometry Special issue of resource-restricted flow cytometr in Clinical Cytometry Supplement 74B; 2008Supplement 74B; 2008Special issue of resource-restricted flow cytometr in Clinical Cytometry Special issue of resource-restricted flow cytometr in Clinical Cytometry Supplement 74B; 2008Supplement 74B; 2008

Two models:

Central lab with QA Spreading its

influence to daughter laboratories

Simple method in

the ‘bushes’

?Non-starter, only good for wasting

the cash

Millions of $-s

Can be complicated and relatively

expensive,

Price tag $50-80/test

FDA regulations may ‘secure’ complicated

protocols

Difficult to train the beginners

Results can be suboptimal, even in the

West

Simpler

Less expensive

No FDA, only QA

Easy to train

Better results than the West

high volume flow cytometry for

huge numbers

Larsen,C.H: The fragile environments of inexpensive

CD4+ T cell counting in the least developed countries

Clin.Cytometry, 73B: Jan. 2008

‘‘smart’ flow cytometry smart’ flow cytometry (Janossy, G. Biotechn. J. 2008; 3: 32-42(Janossy, G. Biotechn. J. 2008; 3: 32-42

Organized quality Organized quality assessment (QA) assessment (QA) schemes are essential schemes are essential for training and for training and education (QASI, education (QASI, NEQAS and External NEQAS and External QA in the Netherlands)QA in the Netherlands)

Flow Cytometry for the Global Flow Cytometry for the Global Village:Village:

The best news for a while The best news for a while (EWGCCA)(EWGCCA)