CD64 EXPRESSION IN WHOLE BLOOD
FACSCALIBUR FLOW CYTOMETER

SHORT ABSTRACT
Neutrophil CD64 becomes elevated in response to bacterial antigens.  Precise measurement can be made with the trillium Leuko64 kit which uses a set of control beads to provide a normalized index. This index can be used to determine if a patient is having a systemic immune response to bacterial antigens.

ABSTRACT

Surface expression of CD64 rapidly increases, within hours, of exposure to bacterial antigens and remains elevated as long as the stimulus persists1.  While slight elevations may be seen in some forms of autoimmune vasculitis and viral infection2,3, it is largely unaffected by traumatic tissue injury and autoimmunity4,5.  CD64 levels are useful in evaluating patients for bacterial infection and sepsis6, avoiding most of the common false positives and limitations of other clinical markers like Procalcitonin and C-reactive protein.  As a quantitative flow cytometric assay, CD64 determinations must be conducted with proper controls to account for lot to lot and patient variability.  The Leuko 64 kit (trillium diagnostic, Maine) provides this level of control using a mixture of three monoclonal antibodies with specificities to CD64 clones 22 and 32.2 and CD163 MAC2-158 and specially calibrated beads and software to provide a normalized CD64 index (nCD64).  This index removes much of the variability associated with quantitative flow cytometry assays allowing the use of an otherwise complex assay by any clinical flow cytometry laboratory comparison across platforms and kits.  Here we present the procedure for measuring the nCD64in human patients.

1.) OPTIMAL SPECIMEN

1.1.) Whole blood sample aseptically collected into a sterile EDTA, heparin, ACD, or sodium citrate collection tube.  
 
1.2.) Mix well after collection to ensure mixing of the blood and the anticoagulant.

1.3.)  The WBC count of the sample must be <25 x 109 cells/L to be run.  If the WBC count is >25 x 109 cells/L dilute the blood with normal saline to obtain a count which is <25 x 109 cells/L.

2.) PROCEDURE

Note: Prior to performing the procedure on a patient specimen calibration should be performed.  The calibration protocol can be seen in the addendum

2.1.	Build and print an nCD64 worksheet from the mainframe computer.

2.2.	Mix the whole blood samples on a mechanical rocker for 3-5 minutes, or until the specimens are well mixed.

2.3.	Bring the 1x Trillium Lyse buffer to room temperature.  One mL of 1x Lyse is needed for each patient sample.  Cold lysing reagent may result in poor lysis and suboptimal assay conditions.

2.4.	Check the WBC count of each sample to be tested.  If the WBC count is >25 x 109 cells/L the sample must be diluted with 0.9% NaCl so it has a WBC count <25 x 109 cells/L.

2.5.	For each patient being tested, label a 12 x 75 polystyrene tube with an Immunology accession label bearing the patient’s name, Social Security number, and accession number.

2.6.	Pipette 50 µL of the monoclonal antibody suspension (Reagent A) into the bottom of each tube.

2.7.	Reverse pipette 50 µL of well mixed whole blood (or sample dilution if WBC count is >25 x 109cells/L) into the bottom of the tube containing the antibody.
•	Reversing Pipetting
When the addition of a precise volume of blood is critical to achieving an accurate result, reverse pipetting must be performed.  Reverse pipetting takes advantage of two stops in a pipette.  Use the Rainin Classic 50 µL pipette to perform the reverse pipetting.  Be sure to use the Rainin RT-250 tips for this pipette.  Make sure the tip is securely on the pipettor.

•	To perform reverse pipetting, depress the button to the second stop.  When the button is released, excess sample is drawn up into the tip.  A precise volume of sample is expelled by pressing the button to the first stop, leaving excess sample in the tip, which is expelled with the tip into the trash.

2.8.	Cap and gently vortex the tubes.  Incubate in the dark at room temperature (18-25˚C) for 10 minutes.

2.9.	Add 1 mL of 1x Trillium Lyse to each tube and vortex to thoroughly mix.  Incubate for 15 minutes at room temperature in the dark.  Intermittent vortexing during this 15 minutes enhances lysis.
2.10.	Add 5 µL of the Leuko64 polystyrene beads (Reagent C) to each tube.  Vortex to mix and run on the flow cytometer using the procedure described below.  Stained sample tubes should be held at 2-8˚C and in the dark until run on the cytometer.  Tubes should be run on the cytometer within 4 hours of staining.

2.11.	Run the tubes on the flow cytometer.

a.	To start up the flow cytometer refer to the “FACSCalibur Start-Up Procedure”.

b.	Perform instrument calibration using the Calibrite and APC beads.  Refer to the “FACSComp Quality Control” procedure.

c.	If this is the first use of the CD64 kit lot number, refer to the calibration section of this procedure to set the PMT voltages, save the instrument settings, and load the analysis software.

d.	If this is not the first use of the CD64 kit lot number, the lot has been calibrated and the instrument settings are saved and ready to use.

e.	Double click the CQ Templates icon on the desktop.

f.	Double click the CD64 ACQ Template.  The Acquisition Template appears.

g.	Click Acquire on the top menu bar.  Highlight Connect to Cytometer.  The Acquisition control bar appears.  Move it off the displays on the screen to a white area.

h.	Click Acquire on the top menu bar and highlight Counters.  The Counters bar appears.  Move it to a white area also.

i.	Click Acquire on the top menu bar.  Highlight Parameter Description.  The Parameter Description box appears. Move it to a white area.

j.	In the Parameter Description box, enter the patient’s last name on the Patient ID line then press Return.  Select the data storage location by clicking folder then in the pop-up box: Desktop, File Storage, then select “File Storage”.  The Panel Box should be checked and CD64 should be displayed.  There is only one tube in the panel so just below the panel name (in the Tube line) CD64/163 should be displayed.

k.	Call up the CD64 Instrument Settings.  Click Cytometer on the top menu bar.  Highlight Instrument Settings.  In the pop-up box click, Open, CQ Templates, Open, CD64 Instr Settings, Open, Set, then Done.

l.	Remove the standby tube from the sample injection port and press the RUN button on the cytometer.  Make sure the fluid control button is on HI.

m.	Place the patient tube on the injection port and click Acquire in the Acquisition Control box.  Look at the display screens and make sure the populations are in their proper place.  Look at the FSC vs SSC plot and identify lymphs, monos and grans.  If all three populations are not visible, the instrument settings are not correct and must be reloaded  as in Step K.

n.	When the populations are properly placed, in the acquisition control box, click Pause, Abort, deselect Set-Up by removing the check mark, then finally Acquire.  A total of 50,000 ungated events will be acquired.

o.	Remove the tube from the sample injection port when the collection is complete.

p.	Go to the Acquisition control box and select Set-Up.  A check mark should be in the box.

q.	Go to the Parameter Description and type in the next patient name on the patient ID line.

r.	Place the tube on the injection port, click Acquire, and observe the populations for correct placement.  When in place go to the Acquisition Control box and click Pause, Abort, Set-Up, then Acquire.  Again 50,000 ungated events will be acquired.

s.	Remove the tube from the injection port and follow steps o-r for all subsequent patient samples.

t.	Clean the cytometer by running 5 minutes of bleach then 5 minutes of water as described in the FACSCalibur Shut Down Procedure.

2.12.	Analyze the data files using the CD64 software.

a.	Double Click the Leuko64 icon on the desktop.

b.	Double Click the QuantiCALC Leuko64 icon in the pop-up box.

c.	Click OK on the Registration page.

d.	To open the correct protocol to run the analysis, click the first icon on the upper tool bar (looks like a folder opening).  An Open Protocol pop-up box appears.  Click on Protocol Files at the bottom of the box and select All Files.  The current CD64 kit lot number will appear in the Files:  Name box.  Highlight the line then click Open.

e.	Another pop-up box appears stating that this protocol is specific for running on the FACSCalibur. Click Yes.

f.	Add the files to analyze.  Click the second icon on the upper tool bar (looks like a string of pages hooked together).  In the upper left box of the edit files pop-up box, open Facstation G4/733 and then File Storage.  The files acquired and stored during the acquisition will appear in the lower box.  Single click one file to highlight it then click Add.  The file moves over to the right box.  Follow this procedure to move all the files over to the right box.  Once all files are in the right side box, click OK.

g.	The first file appears on the screen.  To orientate the page for printing, go to the top menu bar and click File.  Highlight page Set-Up.  In the pop-up box, select the second page orientation (on page sideways, not up and down), then click OK.  The screen now has an enlarged page to view.

h.	Print all reports.  Go to File on the top menu bar and select Print All Reports.  From the printer pop-up box select Print.

i.	The next report appears on the screen.  The page orientation will be OK, just print all reports (File, Print All Reports, Print).

j.	Follow this procedure to print all remaining reports.

k.	Do not exit the software until all reports have printed.

l.	When done, go to File on the top menu bar and select Quit.  Are you sure you want to quit? Select Yes.

3.) CALCULATIONS

3.1 The QuantiCALC software automatically calculates the lymphocyte CD64 and CD163 Index, the neutrophil CD64 and CD163 Index, and the monocyte CD64 and CD163 Index values.

RESULTS

1.	Normal patients will exhibit a low nCD64 index (value <1.2), moderate to high monocyte CD64 Index (value >3.0), and low lymphocyte CD64 index (value <1.0).

2.	Patients with a systemic acute inflammatory response (such as that seen in infection, sepsis and certain forms of vasculitis) will exhibit a variable but higher nCD64 index (value >1.2), moderate to high monocyte CD64 index (value >3.0), and a low lymphocyte CD64 Index (value <1.0).  

3.	The normal index ranges for monocyte CD64 and lymphocyte CD64 are set by the manufacturer.  The normal range of <1.2 for the nCD64 Index was determined by simultaneous analysis of 110 paired blood culture and CD64 Index draws.  Blood culture results were compared to nCD64 Index to determine a cutoff value for true positive sepsis.

4.	The nCD64 index is unaffected by uncomplicated viral infection or autoimmune diseases and is minimally affected by immunosuppressive therapy.

5.	The nCD64 index will rapidly normalize in patients receiving adequate antibiotic therapy and parallels their clinical symptomatology.

Discussion

PROCEDURE NOTES

The nCD64 Index increases during an acute inflammatory response to tissue inquiry, such as occurs with infection or sepsis.  The upregulation of CD64 occurs under the influence of inflammatory cytokines, such as G-CSF and interferon-gamma.  Patients receiving exogenous G-CSF as part of their treatment regimens will have elevated CD64.  Patients receiving steroids may have lower elevations in CD64 levels in response to infection.

Indications for use of CD64 Index – Neutrophil CD64 Index should be used as an adjunct to blood cultures in patients with fevers of unknown origin and suspected bacterial infections (e.g. fever, elevated WBC, elevated ESR).  A negative nCD64 is highly predictive of a negative blood culture and can readily identify most false positive cultures.

Known causes of an elevated index – Patients receiving bone marrow stimulating drugs such as G-CSF may have elevated indices as a result of therapy.  Certain types of vasculitis such as Behcet’s disease may also cause elevation of the nCD64 Index.  Surgical procedures, which cause bacteremia (Clean/contaminated and contaminated field surgeries in particular), can also cause transient elevations of the nCD64.  Studies are ongoing to better define the use of nCD64 among surgical patients, however, clean field surgical procedures do not cause significant elevation of the nCD64 in and of themselves (unlike other inflammatory markers such as C-reactive protein and Procalcitonin).

Known causes of a decreased index – Patients with chronic bacterial infection (such as osteomyelitis) which are under therapy may not have a significant have normal indices.  A normal index should not be interpreted as eradication of infection.

References

1.) Leino L., Sorvajarvi K, Katajisto J et al. Febrile Infection Changes the Expression of IgG Fc Receptors and Complement Receptors in Human Neutrophils in vivo. Clin Exp Immunol 1997;107:37-43. 

2.) Ureten K, Ertenli I, Oztürk MA, Kiraz S, Onat AM, Tuncer M, Okur H, Akdogan A, Apras S, Calgüneri M. Neutrophil CD64 expression in Behçet's disease.
J Rheumatol. 2005;32:849-52. 

3.)  Pauksens K, Fjaertoft G, Doughan-Hakansson L. Venge P.
Neutrophil and monocyte receptor expression in uncomplicated and complicated influenza A infection with pneumonia.
Scand J Infect Dis. 2008;40(4):326-37

4.) Fjaertoft G, Håkansson LD, Pauksens K, Sisask G, Venge P.
Neutrophil CD64 (FcgammaRI) expression is a specific marker of bacterial infection: a study on the kinetics and the impact of major surgery.
Scand J Infect Dis. 2007;39:525-35.

5.)  Matsui T, Ohsumi K, Ozawa N, Shimada K, Sumitomo S, Shimane K, Kawakami M, Nakayama H, Sugii S, Ozawa Y,Tohma S. 
CD64 on Neutrophils Is a Sensitive and Specific Marker for Detection of Infection in Patients with Rheumatoid Arthritis. 
J. Rheumatol 2006; 33:2416-24.

6.)  Icardi M, Erickson Y, Kilborn S, Stewart B, Grief B, Schamweber G.
CD64 index provides simple and predictive testing for detection and monitoring of sepsis and bacterial infection in hospital patients.
J Clin Microbiol 2009 Dec; 47(12): 3914-9