Kit para Detecção de Infecção/Sepse Fabricado por: Trillium Diagnostics - U.S.A. Distribuído no Brasil por: DPM Diagnóstica – Rio de Janeiro

Leuko64 Assay: Quantitative Flow Cytometric Assay Kit for Infection/Sepsis Detection Bruce H. Davis, M.D. Trillium Diagnostics, LLC Scarborough, Maine U.S.A.

Severe Sepsis: Comparison With Other Diseases Crit Care Med. 29:1303, National Center for Health Statistics, § American Cancer Society, *American Heart Association Angus DC et al. Crit Care Med. 29:1303, AIDS*Colon Breast Cancer § CHF Severe Sepsis Cases/100,000 Incidence of Severe SepsisMortality of Severe Sepsis AIDS* Severe Sepsis AMI Breast Cancer § 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

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

Scientific Basis for Quantitative PMN CD64 as an Improved Diagnostic Test of Infection/Sepsis PMN CD64 expression is negligible in the healthy state (<2,000 molecules per cell) - low false positive rate PMN CD64 expression becomes elevated under the influence of the inflammatory related cytokines, such as interferon- (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

PMN CD64: Publications indicating utility as an inflammatory marker in sepsis and infection detection Guyre et al: J Clin Invest 86: , 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: , 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: , 2001 Qureshi et al, Clin Exp Immunol 125:258, 2001 Fisher et al, Intensive Care Med. 27: , 2001 Hirsch et al, Shock 16: 103-8, 2001 Layseca-Espinosa et al,, Pediatr Allergy Immunol 13: , 2002 Ng et al, Pediatr Res 51: , 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: , 2003 Ng PC et al, Pediatr Res 56: , 2004 Davis BH, Expert Rev Mol Diag, 5: , 2005 Davis BH and Bigelow NC, Lab Hematol, 11: , 2005 Fjaertoft G et al, Acta Paediatr 94: , 2005 Davis BH et al, Arch Path Lab Med, 130(5):654-61, 2006

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 L 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

Leuko64 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 *Patent pending

Trillium Diagnostics Leuko64 Assay: Standardized Instrument Set-up with NIST Traceable Beads (reagent C)

Trillium Diagnostics Leuko64 Assay: Performance Criteria Kit stability of 3 years – long outdate Excellent precision –<5% CV intra-assay reproducibility –<10% 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

Leuko64 Software: Automated cluster finding gating of calibration beads Calibration Bead 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 Report

Neonatal Study: PMN CD64 Index correlates best with C-Reactive Protein and presence of sepsis Y=1.0404X – r =

Leuko64 Measurements on Abbott Cell Dyn Instruments: Correlation with Flow Cytometry SapphireCD-4000

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

Bruce H. Davis, M.D. Trillium Diagnostics, LLC Scarborough, Maine U.S.A. DPM Diagnóstica Niterói, Rio de Janeiro Telefax: (21) Contatos