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HelpOrder Code DHRP Dihydrorhodamine Flow Cytometric Phorbol Myristate Acetate Test, Blood
Useful For
Evaluating chronic granulomatous disease (CGD), X-linked and autosomal recessive forms, complete myeloperoxidase deficiency
Monitoring chimerism and nicotinamide adenine dinucleotide phosphate oxidase (NOX) function post-hematopoietic cell transplantation
Assessing residual NOX activity pretransplant
Identifying of female carriers for X-linked CGD
Assessing changes in lyonization with age in female carriers
Reporting Name
DHR Flow PMA, BSpecimen Type
WB Sodium HeparinShipping Instructions
Testing is performed Monday through Friday. Specimens not received by 4 p.m. Central time on Friday may be canceled.
Collect and package specimen as close to shipping time as possible. Ship specimen overnight in an Ambient Shipping Box-Critical Specimens Only (T668) following the instructions in the box. It is recommended that specimens arrive within 24 hours of collection.
Specimens arriving on the weekend and observed holidays may be canceled.
Necessary Information
Ordering healthcare professional name and phone number are required.
Specimen Required
Two whole-blood sodium heparin specimens are required, one from the testing patient and the other from an unrelated healthy donor as a control.
Supplies: Ambient Shipping Box-Critical Specimens Only (T668)
Patient:
Container/Tube: Green top (sodium heparin)
Specimen Volume: 5 mL
Collection Instructions: Send whole blood specimen in original tube. Do not aliquot.
Normal Control:
Container/Tube: Green top (sodium heparin)
Specimen Volume: 5 mL
Collection Instructions:
1. Collect a control specimen from the unrelated healthy donor within an hour of the patient's specimen collection time.
2. Label clearly as Normal Control and the corresponding patient information.
3. Send the whole blood specimen in the original tube. Do not aliquot.
4. Rubber band patient specimen and control vial together.
Specimen Minimum Volume
1 mL
Specimen Stability Information
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| WB Sodium Heparin | Ambient | 48 hours | GREEN TOP/HEP |
Reject Due To
| Gross hemolysis | Reject |
| Gross lipemia | Reject |
Reference Values
|
Result Name |
Unit |
Cutoff for defining normal |
|
% PMA ox-DHR+ |
% |
≥95% |
|
MFI PMA ox-DHR+ |
MFI |
≥60 |
|
Control % PMA ox-DHR+ |
% |
≥95% |
|
Control MFI PMA ox-DHR+ |
MFI |
≥60 |
MFI = mean fluorescence intensity
PMA = phorbol myristate acetate
DHR = dihydrorhodamine
The appropriate age-related reference values for Absolute Neutrophil Count will be provided on the report.
Method Description
A sodium heparin whole blood specimen is incubated at 37° C in the presence of DHR123. Phorbol myristate acetate (PMA) stimulant is added and mixed with the whole blood specimen for additional incubation at 37° C. The sample is then centrifuged, and the cell pellet is subsequently lysed with ammonium chloride at room temperature. Lysed samples are then washed with azide-free phosphate buffered saline prior to staining with LIVE/DEAD viability marker and CD15 at room temperature. Finally, cells are washed, centrifuged, and resuspended in 1% paraformaldehyde prior to analysis. Viable neutrophils are identified by the use of the viability dye and further confirmed by the presence of CD15. Approximately 20,000 viable neutrophil events in the unstimulated sample are used to set the limits for number of events collected for flow cytometry. The results are derived as delta % DHR+ neutrophils after PMA stimulation and mean fluorescence intensity.(O'Gorman MR, Corrochano V. Rapid whole-blood flow cytometry assay for diagnosis of chronic granulomatous disease. Clin Diagn Lab Immunol. 1995;2[2]:227-232; Kuhns DB: Diagnostic testing for chronic granulomatous disease. Methods Mol Biol. 2019;1982:543-571)
Day(s) Performed
Monday through Friday
Performing Laboratory
Mayo Clinic Laboratories in Rochester
CPT Code Information
86352
Method Name
Flow Cytometry
Genetics Test Information
Approximately 70% of chronic granulomatous disease cases are X-linked and are due to disease-causing variants in the CYBB gene, encoding the gp91phox protein. The following genes may have genetic variants inherited in an autosomal recessive pattern: NCF1 (p47phox), NCF2 (p67phox), CYBA (p22phox), and NCF4 (p40phox). Disease-causing variants in NCF1 account for 25% of cases, while variants in NCF2 and CYBA account for 5% of cases each. Disease-causing variants in the NCF4 gene have been described but are rare.