20
Kit para Detecção de Kit para Detecção de Infecção/Sepse Infecção/Sepse Fabricado por: Trillium Diagnostics - U.S.A. Distribuído no Brasil por: DPM Diagnóstica – Rio de Janeiro
Kit para Detecção de Kit para Detecção de Infecção/SepseInfecção/Sepse
Fabricado por:Trillium Diagnostics -
U.S.A.
Distribuído no Brasil por: DPM Diagnóstica – Rio de
Janeiro
Leuko64 Assay: Leuko64 Assay: Quantitative Flow Cytometric Assay Kit for Quantitative Flow Cytometric Assay Kit for
Infection/Sepsis DetectionInfection/Sepsis Detection
Bruce H. Davis, M.D.Trillium Diagnostics, LLC
Scarborough, Maine U.S.A.www.trilliumdx.com
Severe Sepsis: Severe Sepsis: Comparison With Other DiseasesComparison With Other Diseases
†National Center for Health Statistics, 2001. §American Cancer Society, 2001. *American Heart
Association. 2000. ‡Angus DC et al. Crit Care Med.Crit Care Med. 29:1303, 2001. 29:1303, 2001.
0
50
100
150
200
250
300
AIDS* Colon BreastCancer§
CHF† Severe Sepsis‡
Cas
es/1
00,0
00
Incidence of Severe Sepsis Mortality of Severe Sepsis
0
50,000
100,000
150,000
200,000
250,000
Dea
ths/
Yea
r
AIDS* SevereSepsis‡
AMI†Breast Cancer§
Estimated annual 12 million cases with suspectedSepsis in North America, UK, and Europe
18 million cases annually worldwide with 1,400 deaths per day
Healthcare cost of sepsis ~$16 billion/year
Laboratory Indicators of Clinical Acute Laboratory Indicators of Clinical Acute Inflammation Response (Sepsis)Inflammation Response (Sepsis)
Standard of Care Diagnostic Assays of InfectionStandard of Care Diagnostic Assays of InfectionLeukocyte Counts (neutrophilia) - CBCLeukocyte Counts (neutrophilia) - CBC– absolute countsabsolute counts– band counts or left shift (immaturity index)band counts or left shift (immaturity index)
Cultures for suspected infectionCultures for suspected infectionSedimentation RateSedimentation RateC-Reactive Protein (CRP)C-Reactive Protein (CRP)
New Generation Assays of SepsisNew Generation Assays of SepsisGranulocyte CD64 Granulocyte CD64 Cytokine and receptor levels (intracellular or plasma, Cytokine and receptor levels (intracellular or plasma, TNF-a, IL-6, IL-12, CD14, CD16, etc.)TNF-a, IL-6, IL-12, CD14, CD16, etc.)Procalcitonin plasma levelsProcalcitonin plasma levelsEndotoxin Activity AssayEndotoxin Activity Assay
Scientific Basis for Quantitative PMN CD64 as an Scientific Basis for Quantitative PMN CD64 as an Improved Diagnostic Test of Infection/SepsisImproved Diagnostic Test of Infection/Sepsis
PMN CD64 expression is negligible in the healthy state (<2,000 PMN CD64 expression is negligible in the healthy state (<2,000 molecules per cell) - low false positive ratemolecules per cell) - low false positive ratePMN CD64 expression becomes elevated under the influence of the PMN CD64 expression becomes elevated under the influence of the inflammatory related cytokines, such as interferon-inflammatory related cytokines, such as interferon-(3-4 hrs), G-(3-4 hrs), G-CSF (4-6 hrs), IL-12 CSF (4-6 hrs), IL-12 Increased PMN CD64 expression results in enhanced antibody-Increased PMN CD64 expression results in enhanced antibody-mediated functional responses (phagocytosis, oxidative burst, mediated functional responses (phagocytosis, oxidative burst, bactericidal activity) in PMNs – pathophysiologically significant bactericidal activity) in PMNs – pathophysiologically significant changechangePMN CD64 becomes elevated in the presence of infection/sepsisPMN CD64 becomes elevated in the presence of infection/sepsis– Final cytokine pathway effect at cellular level – barometer of “sickness”Final cytokine pathway effect at cellular level – barometer of “sickness”– Better performance than cell counts, left shift, and CRPBetter performance than cell counts, left shift, and CRP
High specificity - PMN CD64 expression is not elevated in:High specificity - PMN CD64 expression is not elevated in:– Malignancy of myeloid cells (CML, MPD, MDS)Malignancy of myeloid cells (CML, MPD, MDS)– Any drug therapy (other than cytokines) Any drug therapy (other than cytokines) – Clinical conditions with localized tissue damage (myocardial ischemia, Clinical conditions with localized tissue damage (myocardial ischemia,
uncomplicated surgery, and exercise injury)uncomplicated surgery, and exercise injury)– pregnancypregnancy
PMN CD64: PMN CD64: Publications indicating utility as an Publications indicating utility as an inflammatory marker in sepsis and infection detectioninflammatory marker in sepsis and infection detection
• Guyre et al: J Clin Invest 86:1892-96, 1990• Davis BH et al: Laboratory Hematology, 1:3-12, 1995 • Herrara et al: J. Med. Micro. 12:34 , 1996• Quayle JA et al: J Immunol 91: 266-73, 1997• Leino et al. Clin Exp Immunol 107:37-43, 1997• Fjaertoft et al, Pediatr Res 45:871-76, 1999• Moallem HJ et al: Scand J Immunol 52:184-89, 2000• Bakke AC et al: Clin Appl Immunol Rev 1:267-75, 2001 • Barth E et al: Cytokine 14: 299-302, 2001• Qureshi et al , Clin Exp Immunol 125:258, 2001• Fisher et al, Intensive Care Med. 27: 1848-52, 2001• Hirsch et al, Shock 16: 103-8, 2001• Layseca-Espinosa et al,, Pediatr Allergy Immunol 13: 319-27, 2002• Ng et al, Pediatr Res 51: 296-303, 2002• Allen E et al: Ann Rheum Dis 61:522-5, 2002• Wagner et al, Shock 19:5-12, 2003• Briggs et al, Lab Hematol 9:117-124, 2003• Ng PC et al, Pediatr Res 56: 796-803, 2004• Davis BH, Expert Rev Mol Diag, 5:193-207, 2005• Davis BH and Bigelow NC, Lab Hematol, 11:137-147, 2005• Fjaertoft G et al, Acta Paediatr 94:295-302, 2005• Davis BH et al, Arch Path Lab Med, 130(5):654-61, 2006
Leuko64 Assay: Leuko64 Assay: Simple and objective method Simple and objective method for quantitation of PMN CD64 in bloodfor quantitation of PMN CD64 in blood
Monoclonal Antibody reagent “cocktail”: anti-CD64 FITC Monoclonal Antibody reagent “cocktail”: anti-CD64 FITC clones (Moabs 22 and 32.2) and anti-CD163 PE clone clones (Moabs 22 and 32.2) and anti-CD163 PE clone (monocyte marker for gating)(monocyte marker for gating)Simple lyse, no wash staining method requiring only 50 Simple lyse, no wash staining method requiring only 50 L L blood blood Addition of fluorescent calibration bead (FITC and starfire Addition of fluorescent calibration bead (FITC and starfire red) suspension (used for instrument set-up and calibration) red) suspension (used for instrument set-up and calibration) – traceable to NIST standard SRM 1932– traceable to NIST standard SRM 1932Flow cytometric analysis collecting 3 colors, Flow cytometric analysis collecting 3 colors, uncompensated data, on 50,000 cells following instrument uncompensated data, on 50,000 cells following instrument set up on beads set up on beads Automated data analysis of listmode files (iterative cluster Automated data analysis of listmode files (iterative cluster finding gating to find cell populations) using the Leuko64 finding gating to find cell populations) using the Leuko64 software reporting results as PMN CD64 index.software reporting results as PMN CD64 index.Total assay time less than 45 minutesTotal assay time less than 45 minutes
Leuko64 Assay Kit* from Trillium Diagnostics, LLCLeuko64 Assay Kit* from Trillium Diagnostics, LLC
Components:
Reagent A: Cocktail of Anti-CD64 FITC and Anti-CD163 PE monoclonal antibodies
Reagent B: 10X Red Cell Lysis Buffer
Reagent C: Fluorescent microsphere suspension for instrument set-up and quantitation (traceable to NIST SRM 8640)
Software DVD: automated data analysis and reporting; allows for bead value assignment; package insert; instrument acquisition protocols
250 and 75 test sizes available
www.Trilliumdx.com
*Patent pending
Trillium Diagnostics Leuko64 Assay: Trillium Diagnostics Leuko64 Assay: Standardized Instrument Set-up Standardized Instrument Set-up
with NIST Traceable Beads (reagent C)with NIST Traceable Beads (reagent C)
100 101 102 103 104
Side Scatter
02
00
40
06
00
80
01
00
0
Fo
rwa
rd S
ca
tte
r
100 101 102 103 104
CD64 FITC
10
01
01
10
21
03
10
4
FL
3-H
eig
ht
100 101 102 103 104
CD163 PE
10
01
01
10
21
03
10
4
FL
3-H
eig
ht
100 101 102 103 104
CD64 FITC
02
04
06
08
01
00
Nu
mb
er
100 101 102 103 104
CD163 PE
02
04
06
08
0
Nu
mb
er
100 101 102 103 104
FL3-Height
05
01
00
15
02
00
25
03
00
35
0
Nu
mb
er
Trillium Diagnostics Leuko64 Assay: Trillium Diagnostics Leuko64 Assay: Performance CriteriaPerformance Criteria
Kit stability of 3 years – long outdateKit stability of 3 years – long outdateExcellent precisionExcellent precision– <5% CV intra-assay reproducibility<5% CV intra-assay reproducibility– <10% CV inter-assay or inter-instrument reproducibility<10% CV inter-assay or inter-instrument reproducibility
Total assay time less than 1 hourTotal assay time less than 1 hourStable results on blood samples stored up to 30 Stable results on blood samples stored up to 30 hours at room temperaturehours at room temperatureSoftware requiring minimal user interaction, Software requiring minimal user interaction, databasing capability, and editable report databasing capability, and editable report generationgenerationWorks on all models of clinical 3-6 color flow Works on all models of clinical 3-6 color flow cytometerscytometers
Leuko64 Software: Automated cluster Leuko64 Software: Automated cluster finding gating of calibration beadsfinding gating of calibration beads
Trillium Diagnostics, LLC *** Leuko64 AssayBead Calibration
Auto
0.1 1 10 100CD64 FITC (CD64 Index)
Aut
o
1010
010
0010
000
CD
163
PE
(C
D16
3 In
dex
)
BHD.FACS.01 2-6-03.01.0011443
2.00100.00
Gated Events CD64 FITC Index CD163 PE Index
Gate1: Beads only
10 100 1000 10000CD163 PE (CD163 Index)
110
100
1000
1000
0F
L3-
Hei
gh
t
Gate2: Bead Singlets
1 10 100 1000 10000FL3-Height
0.1
110
100
Sid
e S
catt
er (
Lo
g)
Calibration Values for Lot Specific CD64 andCD163 Index Units with Data Collected on aBecton Dickinson FACScan or FACSCaliburInstrument.
Leuko64 Calibration Beads Lot # 03-001 Expiration: 31 January 2004
CalibrationBead
Gating
Purpose of calibration beads:
• Instrument set-up
• CD64 Index
• CD163 Index
• Lot to lot correlations
Software uses cluster finding algorithms to locate lymphocytes, monocytes, and granulocytes.
Lymphocytes serve as an internal negative control (CD64 negative) and software will alert user if lymphocyte gate has a CD64 index greater than 1.00
Monocytes serve as an internal positive control (monocytes normally have moderate levels of CD64) and software will alert user if monocyte gate has a CD64 index less than 3.00, which may indicate need for gate adjustment or failure to add proper antibody volume (reagent A).
PMN CD64 Index reported with values of < 1.00 typical of healthy individuals
Software has database function that can imported into Microsoft Excel
PMN CD64 Index reported with values of >1.50 indicative of infection
Leuko64 Software: Summary Leuko64 Software: Summary ReportReport
Neonatal Study: Neonatal Study: PMN CD64 Index correlates PMN CD64 Index correlates best with C-Reactive Protein and presence of best with C-Reactive Protein and presence of
sepsissepsisPMN CD64 in Neonate Samples
0.00
1.00
2.00
3.00
4.00
5.00
6.00
0 1 2 3 4 5 6
No Infection Treated for Infection
PM
N C
D64 I
nd
ex
Infection Detection Standard Lab Tests Leuko64 Assay Sensitivity 47.6% 90.5% Specificity 92.6% 96.3% Pos. Predictive Value 83.3% 95.0% Neg. Predictive Value 69.4% 92.9% Likelihood Ratio 6.43 24.46
Neonatal Samples: Correlation of CRP and PMN CD64
y = 1.0404x - 1.068r = 0.6732
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00PMN CD64 Index
C re
activ
e Pr
otei
n (m
g/dL
)
Y=1.0404X – 1.068r = 0.6732
Leuko64 Measurements on Abbott Cell Dyn Leuko64 Measurements on Abbott Cell Dyn Instruments: Correlation with Flow CytometryInstruments: Correlation with Flow Cytometry
Neutrophil CD64 Index (Trillium Leuko64 Assay)
y = 0.986x + 0.203
r2 = 0.9177N = 45
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
0.00 2.00 4.00 6.00 8.00 10.00
PMN CD64 Index (FACScan flow cytometry)
PM
N C
D6
4 I
nd
ex
(A
bb
ott
Sa
pp
hir
e)
Cell Dyn 4000 Assays
y = 0.9496x + 0.0723
r2 = 0.963
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
CD64 Bead Ratio (FACScan)
CD
64
Be
ad
Ra
tio
(D
ell
Dy
n 4
00
0)
SapphireSapphire CD-4000
Anticipated Clinical Utility of Leuko64 Assay of Anticipated Clinical Utility of Leuko64 Assay of PMN CD64 ExpressionPMN CD64 Expression
Screening for infection/sepsis or illness severity in outpatients and Screening for infection/sepsis or illness severity in outpatients and hospitalized patients - triage rolehospitalized patients - triage roleTherapeutic monitor of antibiotic response in infection Therapeutic monitor of antibiotic response in infection – potential indicator for conversion of I.V. to oral therapypotential indicator for conversion of I.V. to oral therapy– benefit of reduction in antibiotic use and subsequent development of benefit of reduction in antibiotic use and subsequent development of
resistant organismsresistant organismsInfection screening of post-operative and post-chemotherapy patients, Infection screening of post-operative and post-chemotherapy patients, HIV+ patients, and others at risk for infection/sepsisHIV+ patients, and others at risk for infection/sepsisDistinction between inflammatory leukemoid reaction and Distinction between inflammatory leukemoid reaction and myeloproliferative disorder in patients with unexplained neutrophiliamyeloproliferative disorder in patients with unexplained neutrophiliaAdjunct test with blood culturesAdjunct test with blood cultures– Earlier indicator of patients with sepsis prior to culture result availabilityEarlier indicator of patients with sepsis prior to culture result availability– Interpretation of false positive blood cultures with contaminate bacteriaInterpretation of false positive blood cultures with contaminate bacteria
Bruce H. Davis, M.D.Trillium Diagnostics, LLC
Scarborough, Maine U.S.A.www.trilliumdx.com
DPM DiagnósticaNiterói, Rio de Janeiro
m.brTelefax: (21) 2711-0009
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