Burst test using FagoFlow in patients with CGD and hematopeotic stem cell transplantation

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 allo

gene

ic h

emop

oies

is

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 ?) – corticosteroidsbilateral 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 familiesProf. 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