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Test ID: DHRP Dihydrorhodamine Flow Cytometric Phorbol Myristate Acetate Test, Blood

Reporting Name


Useful For

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


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


Assessing residual NADPH oxidase activity pretransplant


Identifying of female carriers for X-linked CGD


Assessing changes in lyonization with age in female carriers

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 of 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 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.


Female carriers of X-linked CGD can become symptomatic for CGD due to skewed lyonization (X chromosome inactivation).(9) Age-related acquired skewing of lyonization can also cause increased susceptibility to infections in carriers of X-linked CGD.(10) While inherited disease-causing 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.(11) There are also reports of triple somatic mosaicism in female carriers (12,13) as well as late-onset disease in an adult female who was a somatic mosaic for a novel variant in the CYBB gene.(14)


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.


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


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 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.

Report Available

3 to 4 days

Day(s) Performed

Monday through Friday

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;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;363:2600-2610

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;128:104(1)-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 13;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. Roesler J: Carriers of X-linked chronic granulomatous disease at risk. Clin Immunol. 2009 Feb;130(2):233; author reply 234. doi: 10.1016/j.clim.2008.09.013

10. 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

11. 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

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

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

14. 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(1):61-66

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

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

17. 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 Name

Flow Cytometry

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.

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

Reference Values

Result Name


Cutoff for defining normal

% PMA ox-DHR+






Control % PMA ox-DHR+



Control MFI PMA ox-DHR+




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.

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.

CPT Code Information


LOINC Code Information

Test ID Test Order Name Order LOINC Value
DHRP DHR Flow PMA, B 98124-1


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
ANCC Control Absolute Neutrophil Count 85369-7
PMAPC Control % PMA ox-DHR+ 85377-0
PMAMC Control MFI PMA ox-DHR+ 85375-4
DHRPI Interpretation 69052-9
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