Neutrophil CD64: Quantitative Flow Cytometric Assay for InfectionSepsis Detection

1
Neutrophil CD64 Quantitative Flow Cytometric
Assay for Infection/Sepsis Detection
Bruce H. Davis, M.D. Trillium Diagnostics,
LLC Scarborough, Maine U.S.A. www.trilliumdx.com
2
Severe Sepsis A Growing Healthcare Challenge
Today
Estimated 12 million cases of suspected Sepsis in
North America, UK, and Europe 18 million cases
worldwide with 1,400 deaths per day Healthcare
cost of sepsis 16 billion/year
1.5 million cases of severe sepsis
Estimated ICU restricted cases in OECD
countries, based on 1995 population data
Angus DC. Crit Care Med. 291303, 2001. Martin
et al, NEJM 3481546, 2003
Linde-Zwirble et al. Crit Care Med. 199927A33
Factors driving increased future incidence of
sepsis from Opal and Cohen. Crit Care Med.
1999271608.
3
Severe Sepsis Comparison With Other Diseases
Incidence of Severe Sepsis
Mortality of Severe Sepsis
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.
4
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
• Coagulation clot aPTT waveform

5
Antigenic Markers of Myeloid Activation

• CD45RA - tyrosine phosphatase
• CD16 - low affinity Fc-gamma receptor
• CD11b - C3bi receptor, LFA-1, integrin, CR3
• CD11c - C3d receptor, LFA-1, CR4
• CD35 - C3b receptor, CR1
• CD62L - L selectin
• CD53, CD63 - tetraspanins
• CD64 - high affinity Fc-gamma receptor (FcgR-I)
  
• CD66b - CEA gene family
• 7D5 - cytochrome b558 subunit of NADPH oxidase
• a-4 Integrin
• HLA, CD16, and CD163 expression on monocytes

Only CD64 has minimal PMN expression in healthy
state Davis BH, Expert Rev Mol Diag, 5193-207,
2005 Elghetany, MT and Davis, BH, Cytometry, in
press, 2005
6
Scientific Basis for Quantitative PMN CD64 as an
Improved Diagnostic Test of Infection/Sepsis

• PMN CD64 expression is negligible in the healthy
  state (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
• PMN CD64 becomes elevated in the presence of
  infection/sepsis final cytokine pathway effect
  at cellular level
• Increased PMN CD64 expression results in enhanced
  antibody-mediated functional responses
  (phagocytosis, oxidative burst, bactericidal
  activity) in PMNs pathophysiologically
  significant change
• 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

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Gated Percnt linMeanX linMedianX R4 943 4.72
1006.19 1060 R5 1307 6.54
85106.61 87801 R6 15961 79.81 11474.33
12008. R10 1262 6.31 628.55
6 R11 1176 5.88 2791.00
28 R12 16169 80.83 1479.29 15
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PMN CD64 Quantitation with Quantum FITC Beads
(Bangs, Inc.) to Convert MFI to MESF Units
Mean Channel Fluorescence
MESF Units
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PMN CD64 Expression in Infection Davis BH et al.
Laboratory Hematology, 13-12, 1995
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Comparison of Neutrophil CD64, Manual Myeloid
Immaturity Counts, and Automated Hematology
Analyzer Flags as Indicators of Infection or
Sepsis
Davis BH and Bigelow NC, Lab Hematol, in press
2005
Methods

• 160 patient blood samples selected from hospital
  laboratory based upon blood counter flagging
• Assays performed CBC, manual leukocyte
  differential counts (H20-A), PMN CD64 by flow
  cytometry
• Retrospective blinded chart review with scoring
• 0 No Infection or inflammation
• 1 Localized infection or tissue injury
• 2 moderate suspicion for systemic infection
  and/or tissue injury
• 3 Documented sepsis and/or severe tissue injury

12
PMN CD64 Expression vs. Band Percents Counts
Davis BH and Bigelow NC, Lab Hematol, in press
2005
Result Weak correlation between PMN count and
band, but not PMN CD64 expression Conclusion
Indicates CD64 to be a related, but likely
independent parameter of cell activation and
cytokine functional upregulation
13
PMN CD64 Expression vs. Band Percents Counts
Davis BH and Bigelow NC, Lab Hematol, in press
2005
Result Moderate correlation between PMN CD64
Expression and band, but weaker coorelation with
immature myeloid fraction Conclusion Indicates
CD64 to be a related, but likely independent
parameter of cell activation and cytokine
functional upregulation
14
PMN CD64 Best Correlate with Instrument Flagging
(Coulter STKS)
Davis BH and Bigelow NC, Lab Hematol, in press
2005
15
PMN CD64 Best Correlate with Clinical Sepsis Score
Davis BH and Bigelow NC, Lab Hematol, in press
2005
16
PMN CD64 Best Predicts Presence of
Infection/Sepsis
Davis BH and Bigelow NC, Lab Hematol, in press
2005
Positive predictive value (PPV), negative
predictive value (NPV), positive likelihood ratio
(LR), and the negative likelihood ratio (LR-)
show neutrophil CD64 to have the best diagnostic
performance
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Infection/Sepsis Markers in Emergency Room
Patients
Davis BH et al, Arch Path Lab Med, in press 2005
Methods

• Patients randomly selected from emergency
  department encounters (N100)
• Assays performed CBC, band counts, Westergren
  Sedimentation Rate, C-reactive protein, PMN CD64
• Retrospective blinded chart review with scoring
• 0 No Infection or inflammation
• 1 Localized infection or tissue injury
• 2 moderate suspicion for systemic infection
  and/or tissue injury
• 3 Documented sepsis and/or severe tissue injury

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Infection/Sepsis Markers in Emergency Room
Patients
Davis BH et al, Arch Path Lab Med, in press 2005
19
Infection/Sepsis Markers in Emergency Room
Patients
Davis BH et al, Arch Path Lab Med, in press 2005
Positive predictive value (PPV), negative
predictive value (NPV), positive likelihood ratio
(LR), and the negative likelihood ratio (LR-)
show neutrophil CD64 to have the best diagnostic
performance
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PMN CD64 in the Post-Surgical Patient
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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 of 0.05 mL blood
  for total of 30 minutes
• Addition of fluorescent calibration bead (FITC
  and starfire red) suspension (also used for
  instrument set-up and calibration) traceable to
  NIST 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 with
  embedded bead values reporting results as PMN
  CD64 index.
• Total assay time less than 60 minutes
• 250 test and 75 test assay kits containing
  antibodies, lysis solution, calibration beads,
  and lot-specific software

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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 for automated data analysis and
  reporting allows for bead value assignment

Patent pending
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• Trillium Leuko64 Assay Gating Strategy
• Monocytes
• CD163 used as internal positive control
• Lymphocytes
• Use as internal negative control
• Beads
• instrument set-up reference
• FITC quantitation standard
• PE quantitation standard

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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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Leuko64 Software Automated cluster finding
gating of monocytes and flagging
-------------------------- Leuko64
Alert --------------------------- The following
alerts have occurred On Test1 Pop2 Monocyte
Positive Control Monocyte CD64 5.00 --------------------------- OK
---------------------------
Monocyte Gating and Positive Control Flagging
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Leuko64 Software Automated cluster finding
gating of lymphocytes and flagging
--------------------------- Leuko64
Alert --------------------------- The following
alerts have occurred On Test1 Pop4 Lymphocyte
Negative Control Lymphocyte CD64
1.00 --------------------------- OK
---------------------------
Lymphocyte Gating and Negative Control Flagging
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Leuko64 Software Automated iterative cluster
finding gating of PMNs
PMN Gating
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Leuko64 Software Summary Report
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Leuko64 Software Value assignment of beads
within software allows lot to lot standardization.
Lot comparisons without software
Lot comparisons using software
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Leuko64 Assay of PMN CD64 Summary of Technical
Enhancements

• Lyse no wash method with pre-diluted, pre-mixed
  reagent format for technical simplicity (50
  microliters of each)
• Software with automated cluster gating to reduce
  inter-observer variability of data analysis
  database functionality
• Monocyte immunologic gating with CD163 Improves
  PMN gate purity and possible secondary immune
  monitor in monocyte CD163 Index
• Proprietary calibration bead for rule based
  instrument set-up and uncompensated data
  collection to achieve low inter-instrument
  variability
• Fluorescence reference calibration bead for CD64
  measurement that allows for low Leuko64 lot to
  lot variability using software bead value
  assignment traceable to NIST FITC standard SRM
  1932 and spectrally matched to reagent
• Internal cellular controls allow for single tube
  assay with Leuko64 software alerts to user
• Rapid assay to correspond to clinical needs
  (assay time

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Methodologic Issues for PMN CD64 Assay Leuko64
Kit Solutions
Observations/solutions
Issue

• Precision Intra and Inter assay
• Inter-instrument comparison
• Stability sample and post-stain
• Sample staining procedure
• Gating on cell clusters
• Granulocytes
• Monocytes
• Lymphocytes
• CD64 Expression Measurement
• MCF vs positive
• MESF Beads
• Lymphocytes
• Custom Bead
• Controls

• CV
• CV 0.96
• Stable at
• Lyse, no wash pH 7.4 30 minutes
• Software approach
• CD163 monocyte gate
• Beads for set-up, lot spec.
• Uncompensated data
• Important aspect of assay
• Unimodal peak, isotypes
• Lot to lot variations, cost
• RBC lysis problems
• Optimal approach - traceability
• Internal controls utilized

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PMN CD64 as a Sepsis Marker in Neonates
Fjaertoft et al Pediatr Res 45871-76, 1999
Similar results by Layseca-Espinosa et al.
Pediatr Allergy Immunol, 2002 13(5) 319-27, Ng
et al. Pediatr Res, 2002. 51(3) 296-303, and Ng
et al. Pediatr Res, 2004 56(5) 796-803
33
Leuko64 Assay in Neonates Study Design

• Tested 139 samples from 48 neonates in MMC
  neonatal intensive care unit, randomly selected.
• Measured PMN CD64 with Leuko64 assay.
• Collected standard laboratory data of CBC,
  leukocyte differential (manual) and calculated
  I/T ratio (bands metamyelocytes myelocytes
  divided by total neutrophilic myeloid cells), and
  C Reactive Protein levels
• Blinded clinical chart review and scoring for
  infection by attending neonatologist for
  likelihood of infection/sepsis
• Comparison of sensitivity, specificity,
  predicative value, and likelihood ratio of
  diagnostic tests PMN CD64 vs standard tests

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Neonate Study PMN CD64 Index Correlates with
Immaturity Index, not Myeloid Cell Counts
35
Neonatal Study PMN CD64 Index correlates best
with C-Reactive Protein and presence of sepsis
Y1.0404X 1.068 r 0.6732
36
Leuko64 Assay in Neonates Conclusions

• PMN CD64 expression with the Leuko64 assay was
  found to be a superior than the standard
  laboratory diagnostic indicators of infection in
  neonates.
• As with previous studies, PMN CD64 expression
  best correlated with C Reactive Protein Levels
  (r0.67) and to a lesser degree with a myeloid
  left shift or immaturity in the blood (r0.32).
• Leuko64 Assay shows low inter-instrument
  variability (CV (CV
• Able to successfully perform assay in all cases
  using standard neonatal blood sampling techniques
  (post CBC)

37
Leuko64 Assay Automation to CBC CD64?
77 Blood Samples assayed in parallel on Abbott
Cell Dyn 4000 with anti-CD64 PE-labeled antibody
and flow cytometry using the Trillium Leuko64
Assay
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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 and others at risk for
  infection/sepsis
• Improved sensitivity and specificity over current
  laboratory tests (PMN counts, CRP, Sed Rate) of
  inflammation
• Distinction between inflammatory leukemoid
  reaction and myeloproliferative disorder in
  patients with unexplained neutrophilia or
  autoimmune disorders
• 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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Acknowledgements Kathleen T Davis -
Trillium Nancy C Bigelow Trillium Karen Becker
- Trillium Victoria Kinney and Hematology Lab -
MMC Sam Machin - UCL, U.K. Carol Briggs UCL,
U.K. Vanya Gant - UCL, U.K. Paul Guyre -
Dartmouth Steve Olsen Beaumont Ejaz Ahmed -
Beaumont Bob Kisabeth Mayo Abe Schwartz -
CQC Bruce Bagwell Verity Ben Hunsburger -
Verity Bernie Fallon Genzyme Kate Shapland -
Genzyme Maine Biotechnology Services, Inc. RD
Systems, Inc.