Rapid assay for infection in neonates

• US 6,077,665 A
• Filed: 04/08/1997
• Issued: 06/20/2000
• Est. Priority Date: 05/07/1996
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Rabin, Ronald L., Weirich, Erica E., Maldonado, Yvonne, Benitz, William E., Herzenberg, Leonore A., Herzenberg, Leonard A.
• Primary Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/833,677
• Time in Patent Office: 1,169 Days
• Field of Search: 435/7.21, 435/7.2, 435/7.1, 435/6, 530/387.1
• US Class Current: 435/6.11
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G01N 33/56988 HIV or HTLV

Abstract

A rapid assay for infection in immunodeficient patients such as neonates or immunocompromised patients (e.g. HIV or transplant patients) allows diagnosis at initial evaluation, such that antibiotic treatment and confinement to an intensive care unit can be avoided for uninfected patients. The assay can be used for detecting bacterial, viral, or fungal colonization of the blood stream, cerebrospinal fluid (CSF), or urinary tract. The method is particularly useful for sepsis diagnosis. Polymorphonuclear leukocyte (PMN, neutrophil) CD11b (Mac-1, CR3) levels are measured by flow cytometry or laser scanning microscopy in low volume (0.1 ml) whole blood samples. A dual-laser FACS identifies neutrophils by FITC-conjugated anti-CD15 fluorescent antibodies, and identifies surface neutrophil CD11b marked with PE-conjugated anti-CD11b antibodies. Spontaneous upregulation of CD11b is prevented by handling samples at 4° C. or adding a stabilizing compound such as anti-CD14 antibody or adenosine to the samples. A kit comprises calibration markers such as CD11b-coated microspheres for calibrating the device used for analysis according to diagnosis threshold(s).

Claims

1. A method of testing a neonate for infection, the method comprising:

measuring neutrophil cell surface levels of CD11b in a sample from said neonate; and

comparing said cell surface levels of CD11b to an infection diagnosis threshold;

wherein cell surface levels of CD11b that exceed said infection diagnosis threshold is indicative of infection.

2. The method of claim 1, wherein said method of testing is performed within four hours of an initial evaluation of said neonate.

3. The method of claim 1 wherein said measuring is performed by flow cytometry.

4. The method of claim 1 wherein said sample is a whole blood sample of less than 0.2 ml volume.

5. The method of claim 1 wherein said measuring step comprises direct binding of a labeled marker to neutrophil CD11b.

6. The method of claim 1, wherein said sample is exposed to a condition inhibiting spontaneous upregulation of CD11b.

7. The method of claim 6 wherein said condition comprises a temperature substantially equivalent to 4°

C.

8. The method of claim 6, wherein said condition comprises a stabilizing compound.

9. The method of claim 1 wherein said infection comprises a bloodstream infection.

10. The method of claim 1, wherein neutrophils are identified by detecting the specific binding of a neutrophil identification marker.

16. The method of claim 1, wherein said step of measuring neutrophil cell surface level of CD11b comprises:

binding of a labeled CD11b-specific antibody to said neutrophil; and

detecting the level of label bound to said neutrophil.

17. The method of claim 16 wherein said labeled CD-11b-specific antibody is a fluorescent antibody.

18. The method of claim 1 wherein said step of measuring neutrophil expression of CD11b comprises:

measuring intensity of an optical signal from a marker bound to CD11b.

11. A method of testing a population of neonates for infection, the method comprising:

measuring neutrophil cell surface levels of CD11b in samples from said population of neonates;

determining the presence of infection in said neonates;

correlating the cell surface level of CD11b to the presence of infection;

wherein an infection diagnosis threshold is determined to be the cell surface level of CD11b that is present in infected samples.

12. The method of claim 11 wherein samples having a neutrophil cell surface level of CD11b above said infection diagnosis threshold comprises substantially all infected neonates in said population.

13. The method of claim 12 wherein a sensitivity of said method is substantially 100%.

14. The method of claim 13, wherein a specificity of said method is higher than 90%.

15. The method of claim 11 wherein said samples having a neutrophil cell surface level of CD11b below said infection diagnosis threshold consist substantially of uninfected neonates.

19. A kit for testing a neonate for infection, the kit comprising:

a calibration marker for determining the infection diagnosis threshold of neutrophil expression of CD11b, wherein said calibration marker comprises the amount of CD11b that is representative of the amount of CD11b on a neutrophil from an infected sample.

20. The kit of claim 19, wherein said calibration marker comprises a CD11b-coated microparticle.

21. The kit of claim 19, further comprising a marker that specifically detects CD11b.

22. The kit of claim 21 wherein said marker that specifically detects CD11b is a fluorescent anti-CD11b antibody.

23. The kit of claim 21 further comprising a cell type identification marker for identifying neutrophils.

24. The kit of claim 23, wherein said marker for identifying neutrophils comprises a fluorescent antibody.

25. The kit of claim 19, further comprising a stabilizing compound for inhibiting a spontaneous upregulation of CD11b in said sample.

26. The kit of claim 25, wherein said stabilizing compound is selected from the group consisting of adenosine and anti-CD14 antibody.

27. The kit of claim 19, further comprising:

a) a marker that specifically detects CD11b;

b) a neutrophil identification marker; and

c) a stabilizing compound for inhibiting a spontaneous upregulation of CD11b in a sample.

28. The kit of claim 27, wherein:

a) said marker that specifically detects CD11b comprises a first fluorescent antibody;

b) said neutrophil identification marker comprises a second fluorescent antibody spectrally distinguishable from said first fluorescent antibody; and

c) said stabilizing compound is selected from the group consisting of adenosine and anti-CD14 antibody.