Burst test using FagoFlow in patients with CGD and hematopeotic stem cell transplantation
Andrea Poloučková, M.D.Department of Immunology, Charles University, 2nd Medical Faculty and University Hospital Motol, Prague
Phagocytosis Is Always the Best Time of Day
CGD diagnosis
• NBT (nitro-blue tetrazolium chlorid)
• Chemiluminiscence• Flow cytometry – Fago Flow test is based on the
measurement of respiratory (oxidative) burst of granulocytes after their stimulation with E. coli bacteria. After the ingestion of bacteria, phagocytes activate the NADP oxidase producing reactive oxidative intermediates (respiratory burst). Reactive intermediates inside phagocytes oxidize dihydrorhodamine 123 (DHR123) into fluorescent rhodamine123 which is detected by a flow cytometer
• Enzyme activity (MPO)
• Molecular - genetic analysis of the defect genes
CGD therapy• Antibiotic and antifungal profylaxis and
therapy
• Neutrophil infusions
• Ɣ-INF
• Hematopoetic stem cell transplantation – Goudemand J, Anssens R, Delmas-Marsalet Y, Farriaux JP, Fontaine G: [Attempt to treat
a case of chronic familial granulomatous disease by allogenic bone marrow transplantation]. Arch Fr Pediatr 1976, 33(2):121-129.
– HLA identical family donor, complicated course of the disease
• Gene therapy– Ott, M. G. et al. Correction of X-linked chronic granulomatous disease by gene therapy,
augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nature Med. 2 Apr 2006
Patient 1(PB, 2006)
• FH:negative
• from 9 months – respiratory infections
• 18 months - multiple liver foci – susp. on malignancy, transfer to our hospital
- laparotomy – multiple liver abscesses (Staph. epidermidis)
Patient 1- CGDLab. findings:• NBT: 0
Molecular-genetic analysis: • gp91-phox mutation (CYBB, exon 4 – genotype 334:T>C, S112P)• mother - carrier
Treatment:• ATB: cefotaxim, oxacilin, amikacin
meropenem (24 days), teicoplanin (76), ciprofloxacin (59)• corticosteroids, γ – interferon• granulocyte infusions
Before SCT:•21 months – without clinical and laboratory signs of infection,
USG – significant regression of liver abscesses
Patient 1- SCT• 7.11.2007 (aged 21 months)• donor: MUD, 10/10, BM
• conditioning: Fludarabine, Melfalan, MabCampath• GVHD prophylaxis: CsA, Methylprednisolone 1mg/kg/day
• engraftment: ANC D+12, Plt D+16
• D+16: liver USG - without focal finding
• complications: bilateral interstitial pneumonitis increasing mixed chimerism
•D+61 – NBT 26
• aGVHD: gr II (gut) D+63 - corticosteroids
• discharge from BMT unit: D+83 (aged 24 months)
Patient 1- 16 months after SCT
• without IS treatment, no signs of GVHD• permanent increase of the mixed chimerism (D+70 29% autolougous chimerism, 1 year after Tx 75% autologous chimerism)• decrease in NBT and FagoFlow • NBT test: 12 - 7 – 6 - 4 (normal range > 9)• FagoFlow test: 65% - 77% - 22% (normal range > 76)• without clinical problems• therapy: penicilin
Hemopoetic chimerism
0
20
40
60
80
100
14 42 70 98 126 154 182
days after SCT
% o
f al
loge
neic
hem
opoi
esis
withdrawal of MP D+109
withdrawal of CsA D+147
Granulocytes
66% .. 41%
Patient 1 - FagoFlow
Patient 2(LK, 2006)
FH: negative
• 6 months – cervical lymphadenopathy, fever, hepatomegaly, splenomegaly – therapy with ATB
• 7 months – abscess of soft tissue of the head with osteomyelitis and intracranial penetration (Serratia marcescens)
Patient 2 - CGDLab. findings:• NBT: 0
Molecular- genetic analysis: • gp91-phox mutation (CYBB, exon 4 – genotype 91:CGA>TGA, R91X)• mother - carrier
Treatment:• ATB: amikacin, ciprofloxacin, linezolid + itraconazol, trimetoprim• corticosteroids • γ – interferon
Before SCT:•11 months – without clinical and laboratory signs of infection,• MRI of CNS - mild residual changes
Patient 2 - HSCT
• 19.9.2007 (aged 12 months)• donor: MUD, 9/10 (Cw), BM
• conditioning: Busulfan, Cyclophosphamide, Thymoglobulin• GVHD prophylaxis: CsA, Methotrexate
• engraftment: ANC D+19, Plt D+42
• complications: ascites, hepatopathy and nefropathy D+15
(imunopathological ?) – corticosteroids
bilateral interstitial pneumonitis
CMV infection • aGVHD: gr II (gut) D+62 - corticosteroids, MMF
• discharge from BMT unit: D+110 (aged 15 months)
Patient 2 - 19 months after SCT
• 10 months after Tx: no immunosupression, no signs of GVHD• stable mixed chimerism (9O% allogeneic) • 11 month after Tx – viral infection – aneamia (Coombs +++) – AIHA• Therapy: Mabthera (7 months), prednison, IVIG
• Nowadays: autologous chimerism 10-15%• NBT test: 16 (n. 9)• flow cytometry (FagoFlow): 61%• therapy: Prednison, clotrimoxazol, penicilin,
Sporanox
Patient 2 - FagoFlow
ConclusionFagoFlow – very rapid and easy test, recognize
the defects of NADPH system
Mixed chimerism
Dynamics, interpretation of the results
Greek mythology:Chimera – the creature belching the flame with the head of the lion, the body of the goat, the tail of the snake (Bader et al., 2005).
Thanks
Patients and their families
Prof. Anna Šedivá, M.D., PhD.
Renata Formánková, M.D., PhD.
Doc. Petr Sedláček, M.D., PhD.
Prof. Jan Starý, M.D., PhD.
Aleš Janda, M.D., PhD.
Jarmila Grecová