Rapid Determination of Chimerism Status Using Dihydrorhodamine Assay in a Patient with X-linked Chronic Granulomatous Disease Following Hematopoietic Stem Cell Transplantation
A 3-month-old male infant presented with fever. His leukocyte count was 19.4×109/L; neutrophils, 12.086×109/L; Hb, 8.6 g/dL; platelet, 287×109/L; C-reactive protein, 6.57 mg/dL. Liver function tests showed AST activity of 158 U/L and ALT activity of 119 U/L. Computed tomography showed a hepatic mass in segment IV with a diameter of 50 mm, multiple satellite hepatic and splenic nodules, hepatosplenomegaly, and necrotizing mediastinal lymphadenopathy. Open liver biopsy revealed suppurative inflammation with abscess, and Serratia marcescens was identified from a culture of liver tissue. A liver abscess with S. marcescens and necrotizing mediastinal lymphadenopathy suspected of disseminated tuberculosis due to BCG injection raised concerns for CGD. Subsequently, treatment with antibiotics as well as antifungal and antituberculous medications was initiated.\nThe neutrophil oxidative burst test was performed using the DHR assay before and after stimulation of neutrophils with phorbol myristate acetate (PMA) (). No DHR fluorescence was detected after stimulation (0.2%), consistent with a phenotype observed in X-linked CGD. Molecular genetic analysis of the CYBB gene encoding the gp91phox mutation, was performed using direct DNA sequencing to confirm the diagnosis of X-linked CGD. A novel frameshift mutation in exon 9 of the CYBB gene was identified (). The DHR assay and genetic tests performed on both the mother and brother of the patient revealed that the mother was a carrier of X-linked CGD but his elder brother was not. The family history was reviewed, and we found that the maternal uncle of the patient had died with pneumonia 2 months after birth, and he was, therefore, presumed to have been affected with CGD.\nThe patient received granulocyte transfusions 9 times at 4-5 months of age. Left hemihepatectomy was performed to remove a liver abscess during the same period; this surgery demonstrated chronic granulomatous inflammation. At the age of 7 months, multiple BCG granulomas, detected to be Mycobacterium tuberculosis by real-time PCR, were developed on the left upper arm as well as the neck and axillary area. Incision and drainage were subsequently performed.\nAt the age of 13 months, HLA-C 1 allele mismatched (11/12), allogeneic-unrelated, peripheral blood stem cell transplantation (uPBSCT) was performed under a conditioning regimen of busulfan (1.1 mg/kg), fludarabine (40 mg/m2), and rabbit anti-thymocyte (2.5 mg/kg). The blood group of the recipient and donor was A/Rh+ and B/Rh+, respectively. Dose of graft mononuclear cells, CD34+ cells, and CD3+ cells were 7.83×108/kg, 2.64×106/kg, and 2.38×108/kg, respectively. Engraftment of neutrophils and platelets occurred on day 13 and day 43, respectively, after HSCT.\nEighteen days after HSCT, preemptive treatment with ganciclovir for cytomegalovirus (CMV) viremia was administered. On day 27, amphotericin B treatment for probable invasive aspergillosis infection was initiated. On day 69 after HSCT, the patient developed grade II acute graft versus host disease of the gut.\nChimerism status following HSCT was monitored in peripheral blood or bone marrow mononuclear cells using STR-PCR analysis. At 2, 4, 8, 12, and 19 weeks after HSCT, donor cell chimerism was 68.3%, 94.1%, 84.7%, 40.9%, and 0%, respectively. The DHR assay, which was performed simultaneously, revealed 93.7%, 98.8%, 80.1%, 49.3%, and 0% of reactivity following PMA stimulation (). The fraction of the cell population, which previously showed a complete lack of DHR fluorescence, reappeared at 8 weeks after HSCT, and it gradually increased until defective neutrophils replaced the entire neutrophil population (). Finally, engraftment failure was determined by the absence of both donor cell chimerism and normal neutrophil population.\nThe patient underwent a second HSCT from the same allogeneic donor after a conditioning regimen of total body irradiation (300 cGy), melphalan (45 mg/m2), and rabbit anti-thymocytes (2.5 mg/kg). Graft mononuclear cell, CD34+ cell, and CD3+ cell doses were 29.57×108/kg, 5.69×106/kg, and 18.53×108/kg, respectively. Engraftment of neutrophils and platelets occurred on day 14 and day 46 after HSCT, respectively. One hundred and twenty days after the second HSCT, the donor chimerism level monitored by STR-PCR was 100%, and defective neutrophil activity was not identified through DHR assay.