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
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.
Flow Cytometry
Chronic Granulomatous Disease (CGD)
Neutrophil Oxidative Burst (NOXB)
Nitroblue Tetrazolium (NBT) Assay
Chemiluminescence
Dihydrorhodamine (DHR)
Neutrophil Function
WB Sodium Heparin
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.
Ordering healthcare professional name and phone number are 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.
1 mL
| Gross hemolysis | Reject |
| Gross lipemia | Reject |
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| WB Sodium Heparin | Ambient | 48 hours | GREEN TOP/HEP |
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
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.
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 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 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.
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 NOX activity after neutrophil stimulation, coupled with appropriate interpretation, is critical to achieving an accurate diagnosis or for monitoring patients posttransplant.
| Result Name | Unit | Cutoff for defining normal |
| % PMA ox-DHR+ | % | > or =95% |
| MFI PMA ox-DHR+ | MFI | > or =60 |
| Control % PMA ox-DHR+ | % | > or =95% |
| Control MFI PMA ox-DHR+ | MFI | > or =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.
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 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.
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 Shipping Box-Critical Specimens Only (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 (15).
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.
Dihydrorhodamine analysis was performed to assess neutrophil oxidative burst in 157 healthy donors, 74 children, and 83 adults.
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;363:2600-2610
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;128:104(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;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. Roesler J. Carriers of X-linked chronic granulomatous disease at risk. Clin Immunol. 2009;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;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;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;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;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;105(1):61-66
15. 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.e3
16. Kuhns DB. Diagnostic testing for chronic granulomatous disease. Methods Mol Biol. 2019;1982:543-571
17. Delmonte OM, Fleisher TA. Flow cytometry: Surface markers and beyond. J Allergy Clin Immunol. 2019;143(2):528-537
18. 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
A sodium heparin whole blood specimen is incubated at 37 degrees 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 degrees 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)
Monday through Friday
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.
86352
| Test Id | Test Order Name | Order LOINC Value |
|---|---|---|
| DHRP | DHR Flow PMA, B | 98124-1 |
| 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 |
| 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 |