Test Id : DHR
Dihydrorhodamine Flow Cytometric Test, Blood
Useful For
Suggests clinical disorders or settings where the test may be helpful
Evaluation of chronic granulomatous disease (CGD), X-linked and autosomal recessive forms, Rac2 deficiency, complete myeloperoxidase deficiency
Monitoring chimerism and nicotinamide adenine dinucleotide phosphate oxidase (NOX) function post-hematopoietic cell transplantation
Assessing residual NOX activity pretransplant
Identifying female carriers for X-linked CGD
Assessing changes in lyonization with age in female carriers
Genetics Test Information
Provides information that may help with selection of the correct genetic test or proper submission of the test request
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.
Method Name
A short description of the method used to perform the test
Flow Cytometry
NY State Available
Indicates the status of NY State approval and if the test is orderable for NY State clients.
Reporting Name
Lists a shorter or abbreviated version of the Published Name for a test
Aliases
Lists additional common names for a test, as an aid in searching
Chronic Granulomatous Disease (CGD)
Neutrophil Oxidative Burst (NOXB)
Nitroblue Tetrazolium (NBT) Assay
Chemiluminescence
Dihydrorhodamine (DHR)
Neutrophil Function
Rac2 deficiency
Specimen Type
Describes the specimen type validated for testing
WB Sodium Heparin
Shipping 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.
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
Defines the optimal specimen required to perform the test and the preferred volume to complete testing
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. Clearly label as Normal Control on the outermost label.
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
Defines the amount of sample necessary to provide a clinically relevant result as determined by the testing laboratory. The minimum volume is sufficient for one attempt at testing.
1 mL
Reject Due To
Identifies specimen types and conditions that may cause the specimen to be rejected
| Gross hemolysis | Reject |
| Gross lipemia | Reject |
Specimen Stability Information
Provides a description of the temperatures required to transport a specimen to the performing laboratory, alternate acceptable temperatures are also included
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| WB Sodium Heparin | Ambient | 48 hours | GREEN TOP/HEP |
Useful For
Suggests clinical disorders or settings where the test may be helpful
Evaluation of chronic granulomatous disease (CGD), X-linked and autosomal recessive forms, Rac2 deficiency, complete myeloperoxidase deficiency
Monitoring chimerism and nicotinamide adenine dinucleotide phosphate oxidase (NOX) function post-hematopoietic cell transplantation
Assessing residual NOX activity pretransplant
Identifying female carriers for X-linked CGD
Assessing changes in lyonization with age in female carriers
Genetics Test Information
Provides information that may help with selection of the correct genetic test or proper submission of the test request
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.
Clinical Information
Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Chronic granulomatous disease (CGD) is caused by genetic alterations in the gene components that encode the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 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 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 NOX 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 NOX activity through the dihydrorhodamine (DHR) flow cytometry assay contributed to the assessment of ROI. The diagnostic laboratory assessment for CGD includes evaluation of NOX 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 some genetic forms of CGD.(6,7) DHR test may be normal or mildly impaired in patients who are NCF4 (p40phox) deficient.(4) 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 of DHR positive 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 NOX 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 NOX function.(9) Patients with Rac2 deficiency have been shown to have normal neutrophil oxidative burst when stimulated with PMA, indicating normal NOX 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 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 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.(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 NOX activity after neutrophil stimulation, coupled with appropriate interpretation, is critical to achieving an accurate diagnosis or for monitoring patients posttransplant.
Reference Values
Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
| Result name | Unit | Cutoff for defining normal |
| % PMA ox-DHR+ | % | > or =95% |
| MFI PMA ox-DHR+ | MFI | > or =60 |
| % fMLP ox-DHR+ | % | > or =10% |
| MFI fMLP ox-DHR+ | MFI | > or =2 |
| Control % PMA ox-DHR+ | % | > or =95% |
| Control MFI PMA ox-DHR+ | MFI | > or =60 |
| Control % fMLP ox-DHR+ | % | > or =10% |
| Control MFI fMLP ox-DHR+ | MFI | > or =2 |
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.
Interpretation
Provides information to assist in interpretation of the test results
An interpretive report will be provided, in addition to the quantitative values.
Interpretation of the results of the quantitative dihydrorhodamine (DHR) flow cytometric assay must 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.
Cautions
Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
Specimens are optimally tested within 24 hours of blood draw, though the stability of the assay is within 48 hours of collection. Specimens should be collected in sodium heparin and transported under strict ambient conditions. Use of the Ambient Mailer-Critical Specimens Only box (T668) is encouraged to ensure appropriate transportation of the specimen.
Some disease-causing variants in NCF4 cause only a mild atypical form of chronic granulomatous disease (CGD) and may not be detected by this assay. The DHR test may be normal or mildly impaired in patients who are NCF4 (p40phox) deficient.
Severe glucose-6-phosphate dehydrogenase deficiency can be a phenocopy of CGD both in cellular and clinical terms and can be the underlying reason for an abnormal DHR response.(18)
Hemolyzed specimens may interfere with the assay (ie, high background).
Specimens with an absolute neutrophil count less than 200 will not be accepted for this assay.
Complete myeloperoxidase deficiency can yield a false-positive result.
Supportive Data
Dihydrorhodamine analysis was performed to assess neutrophil oxidative burst in 157 healthy donors, 74 children and 83 adults.
Clinical Reference
Recommendations for in-depth reading of a clinical nature
1. Kang EM, Marciano BE, DeRavin SS, et al. Chronic granulomatous disease: Overview and hematopoietic stem cell transplantation. J Allergy Clin Immunol. 2011;127(6):1319-1326
2. Segal BH, DeCarlo ES, Kwon-Chung KJ, et al. Aspergillus nidulans infection in chronic granulomatous disease. Medicine. 1998;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;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(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;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;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;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;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;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;133(18):1977-1988
12. Roesler J. Carriers of X-linked chronic granulomatous disease at risk. Clin Immunol. 2009;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;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;: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;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;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;105:61-66
18. Siler U, Romao S, Tejera E, et al. Severe glucose-6-phosphate dehydrogenase deficiency leads to susceptibility to infection and absent NETosis. J Allergy Clin Immunol. 2017;139(1):212-219
19. Kuhns DB. Diagnostic testing for chronic granulomatous disease. Methods Mol Biol. 2019;1982:543-571
20. Delmonte OM, Fleisher TA. Flow cytometry: Surface markers and beyond. J Allergy Clin Immunol. 2019;143(2):528-537
21. 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;9(9):3293-3307.e6
Method Description
Describes how the test is performed and provides a method-specific reference
A sodium heparin whole blood specimen is incubated at 37 degrees C in the presence of DHR123. Phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulant is added and mixed with the whole blood specimen for additional incubation at 37 degrees 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)
PDF Report
Indicates whether the report includes an additional document with charts, images or other enriched information
Day(s) Performed
Outlines the days the test is performed. This field reflects the day that the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time before the test is performed. Some tests are listed as continuously performed, which means that assays are performed multiple times during the day.
Monday through Friday
Report Available
The interval of time (receipt of sample at Mayo Clinic Laboratories to results available) taking into account standard setup days and weekends. The first day is the time that it typically takes for a result to be available. The last day is the time it might take, accounting for any necessary repeated testing.
Specimen Retention Time
Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
Performing Laboratory Location
Indicates the location of the laboratory that performs the test
Fees :
Several factors determine the fee charged to perform a test. Contact your U.S. or International Regional Manager for information about establishing a fee schedule or to learn more about resources to optimize test selection.
- Authorized users can sign in to Test Prices for detailed fee information.
- Clients without access to Test Prices can contact Customer Service 24 hours a day, seven days a week.
- Prospective clients should contact their account representative. For assistance, contact Customer Service.
Test Classification
Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR) product.
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
Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Clinic Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.
CPT codes are provided by the performing laboratory.
CPT codes are provided by the performing laboratory.
86352 x2
LOINC® Information
Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the order and results codes of this test. LOINC values are provided by the performing laboratory.
| Test Id | Test Order Name | Order LOINC Value |
|---|---|---|
| DHR | DHR Flow, B | 98122-5 |
| Result Id | Test Result Name |
Result LOINC Value
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
|
|---|---|---|
| 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 |