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EPIC CODE: LAB5320 Dihydrorhodamine Flow Cytometric Test, Blood

Additional Codes

Sunquest: DHRM
Mayo:        DHR

Useful For

Evaluation of chronic granulomatous disease (CGD), X-linked and autosomal recessive forms, RAC2 deficiency, complete myeloperoxidase  deficiency


Monitoring chimerism and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function post-hematopoietic cell transplantation


Assessing residual NADPH oxidase activity pretransplant


Identifying female carriers for X-linked CGD


Assessing changes in lyonization with age in female carriers

Reporting Name

DHR Flow, B

Specimen Type

WB Sodium Heparin

Shipping Instructions

Specimens are required to be received in the laboratory weekdays and by 4 p.m. on Friday. 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.


Samples arriving on the weekend and observed holidays may be canceled.

Necessary Information

Ordering physician name and phone number are required.

Specimen Required

Both a whole blood sodium heparin specimen and a whole blood sodium heparin control specimen from an unrelated, healthy donor are required.


Supplies: Ambient Shipping Box-Critical Specimens Only (T668)



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 a normal (healthy), unrelated person within an hour of the patient's specimen collection time.

2. Label clearly on outermost label normal control.

3. Send whole blood specimen in original tube. Do not aliquot.

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

Clinical Information

Chronic granulomatous disease (CGD) is caused by genetic alterations in the gene components that encode the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex. These alterations result in an inability to produce superoxide anions required for killing bacterial and fungal organisms. Other clinical features include a predisposition to systemic granulomatous complications and autoimmunity.(1) There are 6 known genes associated with the clinical phenotype of CGD.(2) The gene defects include disease-causing variants in the CYBB gene, encoding the gp91phox protein, which is X-linked and accounts for approximately 70% of CGD cases. Other genetic causes are autosomal recessive in inheritance and occur in one of the following genes: NCF1 (p47phox), NCF2 (p67phox), CYBA (p22phox), NCF4 (p40phox) and CYBC1.(3) Typically, patients with X-linked CGD have the most severe disease, while patients with p47phox defects tend to have the best outcomes. Disease-causing variants s in NCF4 and CYBC1 have been the most recently described rare causes of disease(3,4). There is significant clinical variability even among individuals with similar variants, in terms of NADPH oxidase function, indicating that there can be several modulating factors including the genetic alteration, infection history, and granulomatous and autoimmune complications. There appears to be a correlation between very low NADPH superoxide production and worse outcomes. CGD can be treated with hematopoietic cell transplantation, which can be effective for the inflammatory and autoimmune manifestations.


It has been shown that survival of patients with CGD was strongly associated with residual reactive oxygen intermediate (ROI) production, independent of the specific gene alteration.(5) Measurement of NADPH oxidase activity through the dihydrorhodamine (DHR) flow cytometry assay contributed to the assessment of ROI. The diagnostic laboratory assessment for CGD includes evaluation of NADPH oxidase function in neutrophils, using historically the nitroblue tetrazolium test or currently the more analytically sensitive DHR test, as described here. Activation of neutrophils with phorbol myristate acetate (PMA) results in oxidation of DHR to a fluorescent compound, rhodamine 123, which can be measured by flow cytometry. Flow cytometry can distinguish between the different genetic forms of CGD.(6,7) Complete myeloperoxidase (MPO) deficiency can cause a false-positive result for CGD in the DHR flow cytometric assay (8); however, there is a difference between the percent DHR+ neutrophils and the mean fluorescence intensity after PMA stimulation that allows discrimination between true X-linked CGD and complete MPO deficiency. Further, the addition of recombinant human MPO enhances the DHR signal in MPO-deficient neutrophils but not in CGD neutrophils.(8)


It is important to have quantitative measures in the DHR flow cytometry assay to effectively use the test for diagnosis of the different forms of CGD as well as for monitoring chimerism and NADPH oxidase activity post- hematopoietic cell transplantation. These quantitative measures include assessment of the relative proportion (%) of neutrophils that are positive for DHR fluorescence after PMA stimulation and the relative fluorescence intensity of DHR  on neutrophils after activation.


This assay can also be used for the diagnostic evaluation of RAC2 deficiency, which is a neutrophil defect that causes profound neutrophil dysfunction with decreased chemotaxis, polarization, superoxide anion production, azurophilic granule secretion. This disease is caused by inhibitory variants in the RAC2 gene, which encodes a Rho family GTPase essential to neutrophil activation and NADPH oxidase function.(9) Patients with RAC2 deficiency have been shown to have normal neutrophil oxidative burst when stimulated with PMA, indicating normal NADPH oxidase activity, but abnormal neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP), which is a physiological activator of neutrophils. The defective oxidative burst to fMLP, but not to PMA, is consistent with RAC2 deficiency.(10) By contrast, gain of function variants in RAC2 would lead to a an exaggerated response to fMLP.(11)


Female carriers of X-linked CGD can become symptomatic for CGD due to skewed lyonization (X chromosome inactivation).(12) Age-related acquired skewing of lyonization can also cause increased susceptibility to infections in carriers of X-linked CGD.(13) While inherited pathogenic variants are more common in CGD, there have been reports of de novo variants in the CYBB gene, causing X-linked CGD in male patients whose mothers are not carriers for the affected allele. Additionally, somatic mosaicism has been reported in patients with X-linked CGD who have small populations of normal cells.(14) There are also reports of triple somatic mosaicism in female carriers (15,16) as well as late-onset disease in an adult female who was a somatic mosaic for a novel variant in the CYBB gene.(17)


Therefore, the clinical, genetic, and age spectrum of CGD is varied and laboratory assessment of NADPH oxidase activity after neutrophil stimulation, coupled with appropriate interpretation, is critical to achieving an accurate diagnosis or for monitoring patients posttransplant.

Reference Values

Result name


Cutoff for defining normal

% PMA ox-DHR+






% fMLP ox-DHR+






Control % PMA ox-DHR+



Control MFI PMA ox-DHR+



Control % fMLP ox-DHR+



Control MFI fMLP ox-DHR+




PMA = phorbol myristate acetate

DHR = dihydrorhodamine

MFI = mean fluorescence intensity

fMLP = N-formyl-methionyl-leucyl-phenylalanine


The appropriate age-related reference values for Absolute Neutrophil Count will be provided on the report.


An interpretive report will be provided, in addition to the quantitative values.


Interpretation of the results of the quantitative dihydrorhodamine (DHR) flow cytometric assay has to include both the proportion of positive neutrophils for DHR after phorbol myristate acetate and/or N-formyl-methionyl-leucyl-phenylalanine stimulation, and the mean fluorescence intensity .Additionally, visual assessment of the pattern of DHR fluorescence is helpful in discriminating between the various genetic defects associated with chronic granulomatous disease and complete myeloperoxidase deficiency.


An interpretive report will be provided, in addition to the quantitative values.


Interpretation of the results of the quantitative dihydrorhodamine (DHR) flow cytometric assay has to include both the proportion of positive neutrophils for DHR after phorbol myristate acetate and/or N-formyl-methionyl-leucyl-phenylalanine stimulation, and the mean fluorescence intensity .Additionally, visual assessment of the pattern of DHR fluorescence is helpful in discriminating between the various genetic defects associated with chronic granulomatous disease and complete myeloperoxidase deficiency.

Supportive Data

Dihydrorhodamine  analysis was performed to assess neutrophil oxidative burst in 157 healthy donors, 74 children and 83 adults.

Clinical Reference

1. Kang EM, Marciano BE, DeRavin SS, et al: Chronic granulomatous disease: Overview and hematopoietic stem cell transplantation. J Allergy Clin Immunol. 2011 Jun;127(6):1319-1326

2. Segal BH, DeCarlo ES, Kwon-Chung KJ, et al: Aspergillus nidulans infection in chronic granulomatous disease. Medicine. 1998 Sep;77(5):345-354

3. Arnadottir GA, Norddahl GL, Gudmundsdottir S, et al. A homozygous loss-of-function mutation leading to CYBC1 deficiency causes chronic granulomatous disease. Nat Commun. 2018 Oct 25;9(1):4447

4.van de Geer A, Nieto-Patlan A, Kuhns DB, et al. Inherited p40phox deficiency differs from classic chronic granulomatous disease. J Clin Invest. 2018 Aug 31;128(9):3957-3975. doi:10.1172/JCI97116

5. Kuhns DB, Alvord WG, Heller T, et al: Residual NADPH oxidase and survival in chronic granulomatous disease. N Engl J Med. 2010 Dec 30;363(27):2600-2610

6. Vowells SJ, Fleisher TA, Sekhsaria S, et al: Genotype-dependent variability in flow cytometric evaluation of reduced NADPH oxidase function in patients with chronic granulomatous disease. J Pediatr. 1996 Jan;128(1):104-107

7. Vowells SJ, Sekhsaria S, Malech H, et al: Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes. J Immunol Methods. 1995 Jan;178(1):89-97

8. Mauch L, Lun A, O'Gorman MRG, et al: Chronic granulomatous disease (CGD)and complete myeloperoxidase deficiency both yield strongly reduced DHR 123 test signals but can be easily discerned in routine testing for CGD. Clin Chem. 2007 May;53(5):890-896

9. Ambruso DR, Knall C, Abell AN, et al: Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation. Proc Natl Acad Sci U S A 2000 Apr;97(9):4654-4659

10. Accetta D, Syverson G, Bonacci B, et al: Human phagocyte defect caused by a RAC2 mutation detected by means of neonatal screening for T cell lymphopenia. J Allergy Clin Immunol. 2011 Feb;127(2):535-538

11. Hsu AP, Donko A, Arrington ME, et al: Dominant activating RAC2 mutation with lymphopenia, immunodeficiency, and cytoskeletal defects. Blood. 2019 May 2;133(18):1977-1988

12. Roesler J: Carriers of X-linked chronic granulomatous disease at risk. Clin Immunol 2009 Feb;130(2):233. doi: 10.1016/j.clim.2008.09.013

13. Rosen-Wolff A, Soldan W, Heyne K, et al: Increased susceptibility of a carrier of X-linked chronic granulomatous disease (CGD) to Aspergillus fumigatus infection associated with age-related skewing of lyonization. Ann Hematol.. 2001 Feb:80(2):113-115

14. Yamada M, Okura Y, Suzuki Y, et al: Somatic mosaicism in two unrelated patients with X-linked chronic granulomatous disease characterized by the presence of a small population of normal cells. Gene. 2012 Apr 10:497(1):110-115

15. de Boer M, Bakker E, Van Lierde S, et al: Somatic triple mosaicism in a carrier of X-linked chronic granulomatous disease. Blood. 1998 Jan 1;91(1):252-257

16. Noack D, Heyworth PG, Kyono W, Cross AR: A second case of somatic triple mosaicism in the CYBB gene causing chronic granulomatous disease. Hum Genet. 2001 Aug;109(2):234-238

17. Wolach B, Scharf Y, Gavrieli R, et al: Unusual late presentation of X-linked chronic granulomatous disease in an adult female with a somatic mosaic for a novel mutation in CYBB. Blood. 2005 Jan 1;105:61-66

18. Kuhns DB: Diagnostic testing for chronic granulomatous disease. Methods Mol Biol. 2019;1982:543-571.

19. Delmonte OM, Fleisher TA: Flow cytometry: Surface markers and beyond. J Allergy Clin Immunol. 2019 Feb;143(2):528-537

20. Knight V, Heimall JR, Chong H, et al: A toolkit and framework for optimal laboratory evaluation of individuals with suspected primary immunodeficiency. J Allergy Clin Immunol Pract. 2021 Sep;9(9):3293-3307.e6

Method Description

A sodium heparin whole blood specimen is incubated at 37° C in the presence of DHR123. Phorbol myristate acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) stimulant is added and mixed with the whole blood specimen for additional incubation at 37° C. The specimen is then centrifuged, and the cell pellet is subsequently lysed with ammonium chloride at ambient temperature. Lysed specimens are then washed with azide-free phosphate buffered saline prior to staining with LIVE/DEAD viability marker and CD15 at ambient 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 specimen are used to set the limits for number of events collected for flow cytometry. The results are derived as delta % DHR-positive neutrophils after PMA or fMLP stimulation and mean fluorescence intensity for each stimulant for DHR flow cytometry.(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

Report Available

3 to 4 days

Specimen Retention Time

4 days

Performing Laboratory

Mayo Clinic Laboratories in Rochester

CPT Code Information

86352 x2

LOINC Code Information

Test ID Test Order Name Order LOINC Value
DHR DHR Flow, B 98122-5


Result ID Test Result Name Result LOINC Value
ANC Absolute Neutrophil Count 751-8
PMAP % PMA ox-DHR+ 85376-2
PMAM MFI PMA ox-DHR+ 85374-7
FMPPP % FMLP ox-DHR+ 85373-9
FMPM MFI fMLP ox-DHR+ 85370-5
ANCC Control Absolute Neutrophil Count 85369-7
PMAPC Control % PMA ox-DHR+ 85377-0
PMAMC Control MFI PMA ox-DHR+ 85375-4
FMPPC Control % fMLP ox-DHR+ 85372-1
FMPMC Control MFI fMLP ox-DHR+ 85371-3
DHRI Interpretation 69052-9

NY State Approved


Method Name

Flow Cytometry

Test Classification

This test was developed using an analyte specific reagent. Its performance characteristics were determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

Secondary ID


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 and CYBC1 genes have been described but are rare.