Kit para Detec

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Kit para Detecção de Infecção/Sepse
Fabricado por Trillium Diagnostics - U.S.A.
Distribuído no Brasil por DPM Diagnóstica Rio
de Janeiro
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Leuko64 Assay Quantitative Flow Cytometric
Assay Kit for Infection/Sepsis Detection
Bruce H. Davis, M.D. Trillium Diagnostics,
LLC Scarborough, Maine U.S.A. www.trilliumdx.com
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Severe Sepsis Comparison With Other Diseases
Incidence of Severe Sepsis
Mortality of Severe Sepsis
Estimated annual 12 million cases with
suspected Sepsis in North America, UK, and
Europe 18 million cases annually worldwide with
1,400 deaths per day Healthcare cost of sepsis
16 billion/year
Cases/100,000
Severe Sepsis
AIDS
AMI
Breast Cancer
Breast
AIDS
Colon
CHF
Severe Sepsis
Cancer
National Center for Health Statistics, 2001.
American Cancer Society, 2001. American Heart
Association. 2000. Angus DC et al. Crit Care
Med. 291303, 2001.
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Laboratory Indicators of Clinical Acute
Inflammation Response (Sepsis)

• Standard of Care Diagnostic Assays of Infection
• Leukocyte Counts (neutrophilia) - CBC
• absolute counts
• band counts or left shift (immaturity index)
• Cultures for suspected infection
• Sedimentation Rate
• C-Reactive Protein (CRP)
• New Generation Assays of Sepsis
• Granulocyte CD64
• Cytokine and receptor levels (intracellular or
  plasma, TNF-a, IL-6, IL-12, CD14, CD16, etc.)
• Procalcitonin plasma levels
• Endotoxin Activity Assay

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Scientific Basis for Quantitative PMN CD64 as an
Improved Diagnostic Test of Infection/Sepsis

• PMN CD64 expression is negligible in the healthy
  state (lt2,000 molecules per cell) - low false
  positive rate
• PMN CD64 expression becomes elevated under the
  influence of the inflammatory related cytokines,
  such as interferon-g (3-4 hrs), G-CSF (4-6 hrs),
  IL-12
• Increased PMN CD64 expression results in enhanced
  antibody-mediated functional responses
  (phagocytosis, oxidative burst, bactericidal
  activity) in PMNs pathophysiologically
  significant change
• PMN CD64 becomes elevated in the presence of
  infection/sepsis
• Final cytokine pathway effect at cellular level
  barometer of sickness
• Better performance than cell counts, left shift,
  and CRP
• High specificity - PMN CD64 expression is not
  elevated in
• Malignancy of myeloid cells (CML, MPD, MDS)
• Any drug therapy (other than cytokines)
• Clinical conditions with localized tissue damage
  (myocardial ischemia, uncomplicated surgery, and
  exercise injury)
• pregnancy

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PMN CD64 Publications indicating utility as an
inflammatory marker in sepsis and infection
detection

• Guyre et al J Clin Invest 861892-96, 1990
• Davis BH et al Laboratory Hematology, 13-12,
  1995
• Herrara et al J. Med. Micro. 1234 , 1996
• Quayle JA et al J Immunol 91 266-73, 1997
• Leino et al. Clin Exp Immunol 10737-43, 1997
• Fjaertoft et al, Pediatr Res 45871-76, 1999
• Moallem HJ et al Scand J Immunol 52184-89, 2000
• Bakke AC et al Clin Appl Immunol Rev 1267-75,
  2001
• Barth E et al Cytokine 14 299-302, 2001
• Qureshi et al , Clin Exp Immunol 125258, 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 61522-5, 2002
• Wagner et al, Shock 195-12, 2003
• Briggs et al, Lab Hematol 9117-124, 2003
• Ng PC et al, Pediatr Res 56 796-803, 2004
• Davis BH, Expert Rev Mol Diag, 5193-207, 2005

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Leuko64 Assay Simple and objective method for
quantitation of PMN CD64 in blood

• Monoclonal Antibody reagent cocktail
  anti-CD64 FITC clones (Moabs 22 and 32.2) and
  anti-CD163 PE clone (monocyte marker for gating)
• Simple lyse, no wash staining method requiring
  only 50 mL blood
• Addition of fluorescent calibration bead (FITC
  and starfire red) suspension (used for instrument
  set-up and calibration) traceable to NIST
  standard SRM 1932
• Flow cytometric analysis collecting 3 colors,
  uncompensated data, on 50,000 cells following
  instrument set up on beads
• Automated data analysis of listmode files
  (iterative cluster finding gating to find cell
  populations) using the Leuko64 software reporting
  results as PMN CD64 index.
• Total assay time less than 45 minutes

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Leuko64 Assay Kit from Trillium Diagnostics, LLC
www.Trilliumdx.com

• 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

Patent pending
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Trillium Diagnostics Leuko64 Assay Standardized
Instrument Set-up with NIST Traceable Beads
(reagent C)
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Trillium Diagnostics Leuko64 Assay Performance
Criteria

• Kit stability of 3 years long outdate
• Excellent precision
• lt5 CV intra-assay reproducibility
• lt10 CV inter-assay or inter-instrument
  reproducibility
• Total assay time less than 1 hour
• Stable results on blood samples stored up to 30
  hours at room temperature
• Software requiring minimal user interaction,
  databasing capability, and editable report
  generation
• Works on all models of clinical 3-6 color flow
  cytometers

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Leuko64 Software Automated cluster finding
gating of calibration beads

• Purpose of calibration beads
• Instrument set-up
• CD64 Index
• CD163 Index
• Lot to lot correlations

Calibration Bead Gating
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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
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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).
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PMN CD64 Index reported with values of lt 1.00
typical of healthy individuals
Software has database function that can imported
into Microsoft Excel
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PMN CD64 Index reported with values of gt1.50
indicative of infection
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Leuko64 Software Summary Report
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Neonatal Study PMN CD64 Index correlates best
with C-Reactive Protein and presence of sepsis
Y1.0404X 1.068 r 0.6732
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Leuko64 Measurements on Abbott Cell Dyn
Instruments Correlation with Flow Cytometry
Sapphire
CD-4000
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Anticipated Clinical Utility of Leuko64 Assay of
PMN CD64 Expression

• Screening for infection/sepsis or illness
  severity in outpatients and hospitalized patients
  - triage role
• Therapeutic monitor of antibiotic response in
  infection
• potential indicator for conversion of I.V. to
  oral therapy
• benefit of reduction in antibiotic use and
  subsequent development of resistant organisms
• Infection screening of post-operative and
  post-chemotherapy patients, HIV patients, and
  others at risk for infection/sepsis
• Distinction between inflammatory leukemoid
  reaction and myeloproliferative disorder in
  patients with unexplained neutrophilia
• Adjunct test with blood cultures
• Earlier indicator of patients with sepsis prior
  to culture result availability
• Interpretation of false positive blood cultures
  with contaminate bacteria

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Contatos
DPM Diagnóstica Niterói, Rio de
Janeiro www.dpmdiagnostica.com.br dpmdiagnostica_at_d
pmdiagnostica.com.br Telefax (21) 2711-0009
Bruce H. Davis, M.D. Trillium Diagnostics,
LLC Scarborough, Maine U.S.A. www.trilliumdx.com