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Papel da citometria de fluxo na Hemoglobinúria
Paroxística Noturna (HPN)
Marciano Reis, MD, FRCPCChief, Dept. of Clinical PathologySunnybrook Health Sciences CentreWomen’s College Hospital Chief, Dept. of Laboratory HematologyUniversity Health NetworkAssociate Professor University of Toronto
HIAE, April 2009HIAE, April 2009
CURRENT UNDERSTANDING OF PNH
Acquired Hematopoietic Stem Cell diseaseclonality established by sequence analysis of PIG-A genesame mutations in all lineages
Rare disease (2 - 6 per million)
PNH cells are deficient in proteins normally attached to cell membrane by a Glyco-Phosphatidyl-Inositol (GPI) structure
Davitz MA et al. Release of decay-accelerating factor (DAF) from the cell membrane by phosphatidylinositol-specific phospholipase C (PIPLC). Selective modification of a complement regulatory protein. J Exp Med 1986; 163:1150
MOLECULAR DEFECT IN PNH
Somatic mutation of the X-linked Phosphatidyl Inositol Glycan complementation class a (PIG-A) gene
The PIG-A gene contains 6 exons, extends over 17kB
Most PIG-A mutations a small insertions/deletions and of about 200 described mutations, few repeats
PIG-A protein involved in 1st stage of GPI anchor biosynthesis so mutation results in partial or absolute failure to synthesize GPI-linked proteins/glycoproteins
Miyata T et al. The cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis. Science 1993; 259:1318
GPIGPI--ANCHORED STRUCTURESANCHORED STRUCTURES
InP
Gluc
Man
Man
Man
Etn
NH2
Cell MembraneCell Membrane
GPIGPI--linked proteinlinked protein
The biochemical defect in PNH occurs at the first stage in GPI synthesis – transfer of N-acetyl glucosamine from UDP N-acetyl glucosamine to ER-associated phosphatidylinositol.
CD14 CD16 CD24 CD48 CD52
CD55 CD58 CD59 CD66 CD73
CD90 CD108 CD109 CD157
From S. Richards
NORMAL CELLS EXPRESSGPI-LINKED STRUCTURES
Two complement defence proteins are GPI-linked� CD55 (Decay Accelerating Factor)
-regulates formation of C3 convertase
� CD59 (Membrane Inhibitor of Reactive Lysisysis)-restricts formation of membrane attack complex
PNH CELLS DO NOT EXPRESS GPI-LINKED STRUCTURES
CD55 = DAF
CD59 = MIRL
While lack of CD55 and CD59 are diagnostic of PNH, hemolysis is due to lack of CD59
PIG-A MUTATIONS NOT LIMITED TO PNH
Aplastic Anemia50% of AA patients exhibit expansion of PNH clones (15% reach clinical PNH)
Myelodysplastic Syndrome20% of MDS patients have PNH clones(Refractory Anemia usually responds to immuno-suppression)
Socie G et al. Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors. French Society of Haematology. Lancet 1996; 348:573
Hillmen P et al. Natural history of paroxysmal nocturnal hemoglobinuria. N.Engl.J.Med. 1995; 333:1253
WE ALL HAVE A LITTLE PNH IN US
PIG-A mutant blood cells also found in healthy adult (very rare; 2 - 4 /100,000 granulocytes) but why no progression to PNH?
The mutations found in the T cells and CFCs from the same individual are not clonal (unlike those in PNH) and likely occur at the progenitor cell level rather than stem cell level
Multi-hit hypothesis (Luzzato-Young)
1. PIG-A mutation occurs
2. Immune attack spares PNH HSCs
3. Further clonal expansion occurs
Araten DJ et al. Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals. Proc Natl AcadSci U S A 1999; 96:5209
Hu et al. PIG-A mutations in normal hematopoiesis. Blood 2005; 105:3848
CLINICAL FEATURES OF PNH
1. INTRAVASCULAR HEMOLYSISAbsence of CD59 (not CD55) from RBCs is responsible for increased sensitivity of RBCs to activated complement and intravascular hemolysis
Degree of GPI-deficiency is variableType I RBCs: normal GPI expressionType II RBCs: partial GPI expression Type III RBCs: no GPI expression
The greater the % of Type III cells, the greater the intravascular hemolysis: RBC lifespan drops to <20 daysCaveat: only 25% of patients present with nocturnal hemoglobinuriaYamashina et al. Inherited complete deficiency of 20-kilodalton homologous restriction factor (CD59) as a cause of paroxysmal nocturnal hemoglobinuria. N Engl J Med 1990; 323:1184
CLINICAL FEATURES OF PNH
2. THROMBOSES IN UNUSUAL SITES particularly hepatic vein (40%), cerebral vein (14%)
Presenting symptom in 5% of patients, 8 year incidence of 28%25% of thromboses are fatal
Thrombosis causes significant morbidity and mortality Thrombosis is the leading cause of death
Frequency of Thrombosis:directly related to PNH clone size
10 year risk if >50% PNH granulocytes = 44%
10 year risk if <50% PNH granulocytes = 6%Hall et al. Primary prophylaxis with warfarin prevents thrombosis in paroxysmal nocturnal hemoglobinuria (PNH). Blood 2003;102:133
CLINICAL FEATURES OF PNH
3. BONE MARROW FAILUREdeath from progressive pan-cytopeniasprogression to other disordersAplastic anemia
most PNH patients have hypoplastic marrow5% of untreated AA progress to PNH
Myelodyplastic syndrome8 year incidence of 5%
Acute Leukemia8 year incidence of 0.7%
Socie GM et al. Paroxysmal nocturnal haemoglobinuria: Long term follow-up and prognostic factors. Lancet 1996; 348:573
LABORATORY DIAGNOSIS OF PNH
HISTORICALAcidified Serum Test (Ham Test 1939)
Acidified serum activates alternative complement pathway resulting in lysis of patient’s RBCsMay be positive in congenital dyserythropoietic anemiaStill in use today
Sucrose Hemolysis Test (1970)10% sucrose provides low ionic strength which promotes complement binding resulting in lysis of patient’s RBCsMay be positive in megaloblastic anemia, autoimmune hemolytic anemia and others
Less specific than Ham test
LABORATORY DIAGNOSIS OF PNH
FLOW CYOMETRY
absence/partial expression of GPI-linked antigens in two lineages is diagnostic for PNH
ability to definitively and rapidly diagnose PNH by flow cytometry has led to improved patient management and prognosis
van der Schoot et al. Deficiency of glycosyl-phosphatidylinositol-linked membrane glycoproteins of leukocytes in paroxysmal nocturnal hemoglobinuria, description of a new diagnostic cytofluorometric assay. Blood 1990; 76:1853
Hall SE, Rosse WF. The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria. Blood 1996; 87:5332
FLOW ASSAYS FOR PNH
CLASSICAL APPROACH
Detection of at least 2 antigens (historically CD55 and CD59)on two lineages (RBCs and neutrophils)
Caveats:
PNH RBCs can be difficult to detect post-RBC transfusion for severe anemia or hemolysis
Severe neutropenia in some patients makes it difficult to identify target population
Only neutrophils affected in small number of patients
Lymphocytes are not a good target population because of longevity – only lymphocytes arising post-disease onset will be deficient in GPI-linked structures
FLOW ASSAYS FOR PNH
RICHARDS AND HILLMEN; Curr Prot Cytom Unit 6.11, 2002
1. RBCs: Use of CD55, CD59 and CD235a in untransfusedpatient provides the clearest definition of Type III (complete deficiency); Type II (partial) and Type I (normal): >20% type III predicts hemolysis
2. Neutrophils: Use of CD66abce, CD55 and CD16 followed by RBC lysis. Detection of PNH clones by simultaneous analysis of 3 GPI-linked antigens on gated neutrophils
3. Monocytes: Use of CD64 and CD14 and light scatter
4. Adds CD33 APC to Protocol #3
SELECTION OF ANTIBODIES
0 256 512 768 1024
CONTROL PNH.005SSC-Height ->
0 256 512 768 1024
CONTROL PNH.007SSC-Height ->
Monocytes CD14
Granulocytes CD24
0 256 512 768 1024
CONTROL PNH.012SSC-Height ->
0 256 512 768 1024
CONTROL PNH.013SSC-Height ->
Granulocytes CD66abce
Granulocytes CD66c
S. Richards
SELECTION OF ANTIBODIES
0 256 512 768 1024
CONTROL PNH.010SSC-Height ->
0 256 512 768 1024
CONTROL PNH.010SSC-Height ->
0 256 512 768 1024
CONTROL PNH.011SSC-Height ->
0 256 512 768 1024
CONTROL PNH.011SSC-Height ->
Granulocytes
Monocytes
CD55 CD59S. Richards
DIAGNOSING PNH BY FLOW @UHN
In 2004 141 tests for PNH; 3 cases of PNH identified
1. Biocytex RedquantTM (for RBCs); 4-tube assay
I α (CD55) beads
II β (CD59) beads
III Diluted whole blood + CD55 (and secondary FITC)
IV Diluted whole blood + CD59 (and secondary FITC)
2. Biocytex CellquantTM (for granulocytes); 3-tube assay
I α (CD55)/β (CD59) beads
II lysed/washed WB + CD55 (and secondary FITC)
III lysed/washed WB + CD59 (and secondary FITC)
0 1000SS
J0101816.LMD
R1
100 101 102 103 104
SS LOG
R4
0 200 400 600 800 1000AUX(SS)
R2
100 101 102 103 104
SS LOG
J0101790.LMD
R3
100 101 102 103 104
FL1 LOG
M1
M2
Marker Events % GatedAll 10001 100.00
M2 2663 26.63
File: J0101785.LMD
Gate: G4Gated Events: 10001
Marker Events % Gated
All 10003 100.00M1 700 7.00
File: J0101791.LMD
Gate: G4
Gated Events: 10003
Marker Events % GatedAll 4715 100.00
M1 1056 22.40
File: J0101817.LMD
Gate: G2Gated Events: 4715
Marker Events % GatedAll 4703 100.00
M2 1573 33.45
File: J0101818.LMD
Gate: G2Gated Events: 4703
100 101 102 103 104
FL1 LOG
M1
100 101 102 103 104
FL1 LOG
M2
100 101 102 103 104
CD55 FITC
M1
100 101 102 103 104
CD59 FITC
M2
100 101 102 103 104
ab BEADS
M1
M2β beads
α beads
α beads
β beads
CD59-
CD55-
CD55-CD59-
REDQUANT
CELLQUANT
R2 R2
R4 R4
In 2004 141 tests for PNH; 3 cases of PNH
LIMITATIONS BIOCYTEX REDQUANT/CELLQUANT KIT
1. Total time 2 - 2.5 hours per patient sample (sample preparation/staining/analysis)
2. Burdensome to set up (2 different cell prep. regimens, Indirect Fluorescence, 7 tubes)
3. Not very sensitive (5 - 10%)
4. Data not always easy to interpret(manual gating/analysis of data required to optimize determination of clone size)
5. Cellquant must be performed within 8 Hr of sample draw
6. Redquant must be performed within 24 Hr of sample draw
DIAGNOSING PNH WITH FLAER - what is it?
Proaerolysin: 52-kDa protein secreted by Aeromonas sp.
After proteolytic nicking at the cell surface Aerolysin is generated that lyses cells (Howard et al, J Bacteriol 1985;163:336)
Aerolysin binds to the GPI moiety of GPI-linked molecules on the cell surface (Diep et al J Biol Chem 1998; 273:2355)
nicking Binding to GPI lysis
DIAGNOSING PNH WITH FLAER
PNH cells not lysed by Aerolysin(Brodsky et al Blood 1999; 93:1749)
Fluorescently-labeled inactive variant of aerolysin (FLAER)
Binds to but does not cause lysis of cells
FLAER is more sensitive than CD59 or other individual mAbs to GPI-linked structures and FLAER gives a more accurate assessment of the GPI anchor deficit in PNH(Brodsky et al (Am J Clin Path 2000;114:459)
(Luider et al,1st in Canada!!!)
FLAER ASSAY at UHN
CD33PE, CD45ECD, CD14PE-Cy5 (Beckman Coulter)
FLAER (Protox Biotech Victoria BC Canada)
Add 0.5mL of Optilyse C (Beckman-Coulter) to 100µL of anti-coagulated (EDTA) PB and lyse at 20oC for 10 minutes.
Add 1mL PBS and incubate for another 10 minutes. Centrifuge, remove supernatant and resuspend cells in 100µL of PBS/2% albumin.
Add 10µL CD33PE, 5µL CD45ECD, 5µL CD14PE-Cy5, and 10 µL of FLAER ‘working’ solution and incubate for 15 minutes @20oC
Add 1ml of PBS and acquire data (FC500 Beckman Coulter)
If BD Biosystems FACSCalibur is in use, a different cocktail of conjugates may be used
File: Normal PB copy
Gate: G2Gated Events: 2059
Quad Events % Gated
UL 0 0.00UR 2031 98.64
LL 7 0.34LR 21 1.02
100 101 102 103 104CD45-ECD
R1
R4
100 101 102 103 104FLAER
File: Normal PB copy
Gate: G3Gated Events: 14292
Quad Events % Gated
UL 0 0.00UR 23 0.16
LL 17 0.12LR 14252 99.72
File: Normal PB copy
Gate: G4Gated Events: 3175
Quad Events % Gated
UL 0 0.00UR 4 0.13
LL 34 1.07LR 3137 98.80
100 101 102 103 104CD33-PE
R2
R3
100 101 102 103 104FLAER
100 101 102 103 104FLAER
R1
Normal PB
monocytes R1+R2
neutrophils R1+R3
lymphocytes R4
Acquisition Date: 08-Jun-05Gate: G3
Gated Events: 6779
Quad Events % Gated
UL 0 0.00UR 4 0.06LL 3468 51.16
LR 3307 48.78
Acquisition Date: 08-Jun-05Gate: G4
Gated Events: 11273
Quad Events % Gated
UL 0 0.00UR 1 0.01LL 238 2.11
LR 11034 97.88
100 101 102 103 104
FLAER
100 101 102 103 104CD33-PE
R2
R3
Acquisition Date: 08-Jun-05Gate: G2
Gated Events: 1514
Quad Events % Gated
UL 1 0.07UR 412 27.21LL 1081 71.40
LR 20 1.32
100 101 102 103 104CD45-ECD
R1
R4
100 101 102 103 104
FLAER100 101 102 103 104
FLAER
R1
monocytes R1+R2
neutrophils R1+R3
PNH PB Fresh
lymphocytes R4
Sensitivity of FLAER Assay
PNH neutophils express slightly higher levels of CD45 compared to non-PNH neutrophils
This can be used to confirm small FLAER-negative events are PNH clones
Sutherland DR et al AJCP In press 2009
QC/PT MATERIAL FOR PNH
UK NEQASStabilized Normal and PNH samples represent suitable material for 4-colour FLAER assay and CD59 RBC assay
R&D SYSTEMSStabilized PNH sample suitable for FLAER assay and CD59 RBC assayStable for at least 12 weeks
Acquisition Date: 04-Oct-05
Gate: G2Gated Events: 1996
Quad Events % GatedUL 0 0.00
UR 1844 92.38LL 1 0.05
LR 151 7.57
Acquisition Date: 04-Oct-05
Gate: G3Gated Events: 22507
Quad % Gated
UL 0.00UR 1.06LL 1.05
LR 97.89
Acquisition Date: 04-Oct-05
Gate: G4Gated Events: 11258
Quad Events % Gated
UL 0 0.00UR 2 0.02LL 614 5.45
LR 10642 94.53
100 101 102 103 104CD45-ECD
NEQAS #4 WBC
R1
R4
100 101 102 103 104FLAER
NEQAS #4 WBC
100 101 102 103 104FLAER
NEQAS #4 WBC
100 101 102 103 104FLAER
NEQAS #4 WBC
100 101 102 103 104CD33-PE
NEQAS #4 WBC
R2
R3R1
monocytes R1+R2
neutrophils R1+R3
UK NEQAS Stabilised Normal PB
lymphocytes R4
Acquisition Date: 04-Oct-05Gate: G2Gated Events: 3549
Quad Events % GatedUL 19 0.54UR 58 1.63LL 3381 95.27LR 91 2.56
Acquisition Date: 04-Oct-05Gate: G3Gated Events: 18639
Quad % GatedUL 0.01UR 0.10LL 96.64LR 3.26
Acquisition Date: 04-Oct-05Gate: G4Gated Events: 6135
Quad Events % GatedUL 0 0.00UR 2 0.03LL 431 7.03LR 5702 92.94
10 0 10 1 10 2 10 3 10 4CD45-ECD
NEQAS #3 WBC
R1
R4
10 0 10 1 10 2 10 3 10 4FLAER
NEQAS #3 WBC
10 0 10 1 10 2 10 3 10 4FLAER
NEQAS #3 WBC
10 0 10 1 10 2 10 3 10 4FLAER
NEQAS #3 WBC
10 0 10 1 10 2 10 3 10 4CD33-PE
NEQAS #3 WBC
R2
R3
lymphocytes R4
R1
monocytes R1+R2
neutrophils R1+R3
UK NEQAS Stabilised PNH PB
Marker % Gated
All 100.00
M3 4.46M2 1.51
M1 93.93
File: NEQAS #4 RbcsAcquisition Date: 01-Sep-05
Gate: G1
Gated Events: 6749X Parameter: CD59-FITC (Log)
Marker % Gated
All 100.00
M1 71.41M3 25.05
M2 3.55
File: NEQAS #3 RbcsAcquisition Date: 01-Sep-05
Gate: G1
Gated Events: 9284X Parameter: CD59-FITC (Log)
100 101 102 103 104CD59-FITC
NEQAS #4 Rbcs
M3
M2
M1
0 200 400 600 800 1000FS Lin
NEQAS #4 Rbcs
R1
0 200 400 600 800 1000FS Lin
NEQAS #3 Rbcs
R1
100 101 102 103 104
CD59-FITC
NEQAS #3 Rbcs
M1M3 M2
PNH
normal
SUMMARY I
FLAER with CD33, CD45 and CD14 allows the simultaneous detection of PNH clones in monocyte and neutrophil lineages
1 tube (45 min), sensitive (<1%), inexpensive, simple data analysis, range of 4-colour instruments
Samples can be assessed up to 48 hours post draw
FLAER assay more reliably quantifies size of PNH cloneQuality Assurance/Proficiency testing material for this assay is readily available from UK NEQAS and others (R&D ?)
The FLAER can detect non-PNH hematologic disorders in samples submitted for PNH testing
Acquisition Date: 31-Mar-05
Gate: G2Gated Events: 1986
Quad Events % GatedUL 64 3.22
UR 46 2.32LL 1668 83.99
LR 208 10.47
Acquisition Date: 31-Mar-05
Gate: G3Gated Events: 10603
Quad % Gated
UL 0.00UR 0.46LL 0.99
LR 98.55
Acquisition Date: 31-Mar-05
Gate: G4Gated Events: 4582
Quad Events % Gated
UL 0 0.00UR 1 0.02LL 461 10.06
LR 4120 89.92
100 101 102 103 104FLAER
AML AP (PB)
100 101 102 103 104FLAER
AML AP (PB)
100 101 102 103 104FLAER
AML AP (PB)
100 101 102 103 104CD33-PE
AML AP (PB)
R2
R3
100 101 102 103 104CD45-ECD
AML AP (PB)
R1
R4
R1
monocytes R1+R2
neutrophils R1+R3
lymphocytes R4
AML with multi-lineage dysplasia
Acquisition Date: 13-May-05
Gate: G2Gated Events: 10726
Quad Events % GatedUL 1169 10.90
UR 8235 76.78LL 344 3.21
LR 978 9.12
Acquisition Date: 13-May-05
Gate: G3Gated Events: 5059
Quad % Gated
UL 0.00UR 0.02LL 0.08
LR 99.90
Acquisition Date: 13-May-05
Gate: G4Gated Events: 4612
Quad Events % Gated
UL 1 0.02UR 8 0.17LL 235 5.10
LR 4368 94.71
100 101 102 103 104FLAER
AML APAYNE
100 101 102 103 104CD45-ECD
AML APAYNE
R1
R4
100 101 102 103 104FLAER
AML APAYNE
100 101 102 103 104FLAER
AML APAYNE
100 101 102 103 104CD33-PE
AML APAYNE
R2
R3R1
monocytes R1+R2
neutrophils R1+R3
lymphocytes R4
AML post-MDS with multi-lineage dysplasia (FAB M4?)
Acquisition Date: 05-May-05
Gate: G2
Gated Events: 1253
Quad Events % GatedUL 0 0.00
UR 168 13.41
LL 304 24.26
LR 781 62.33
Acquisition Date: 05-May-05
Gate: G3
Gated Events: 11415
Quad % GatedUL 0.00
UR 0.04
LL 0.66
LR 99.31
Acquisition Date: 05-May-05
Gate: G4
Gated Events: 4453
Quad Events % GatedUL 0 0.00
UR 4 0.09
LL 153 3.44
LR 4296 96.47
100 101 102 103 104CD45-ECD
AML PA (PB)
R1
R4
100 101 102 103 104CD33-PE
AML PA (PB)
R2
R3
100 101 102 103 104
FLAER
AML PA (PB)
100 101 102 103 104
FLAER
AML PA (PB)
100 101 102 103 104
FLAER
AML PA (PB)
R1
monocytes R1+R2
neutrophils R1+R3
lymphocytes R4
AML with dysplastic nucleated RBCs and multi-lineage dysplasia FAB M6?
DETECTING PNH WBC CLONES WITH FLAER VERSUS CD59- RBCs
2006 114 samples (underestimate)
2007 187 samples2008 222 samples
2009 250 samples?
523 patient samples (2006 - 08) assessed by1. FLAER assay
2. CD59FITC on RBC3. CD59FITC, CD235PE on RBC (from 2008)
4. CD59PE, CD235FITC on RBC (from 2008)
Red cell assaysA. RedQuant continued to be mandated (AAARRGGHH!!)
B. SINGLE COLOUR (CD59FITC)1. Dilute whole blood 1:150 (now 1:500) in PBS/3% albumen aliquot
20µL (now 50µL of 1:500 with reverse pipetting) into 2 tubes.2. Add 10µL of CD59FITC to one tube (tube 2 is unstained)3. Incubate 15 minutes @RT, add 1 ml PBS and acquire.
C. TWO COLOUR ASSAY (CD59FITC/CD235PE)1. As assay B, except in step 2, add 10µL CD59FITC and 5µL of
CD235PE (pre-diluted 1:50)
D. TWO COLOUR ASSAY (CD59PE/CD235FITC)1. As assay B, except in step 2, add 10µL CD59PE and 2µL of
CD235PE (undiluted)
RESULTS60/523 samples contained PNH clones by FLAER
- 32/60 PNH samples contained CD59- RBCs - PNH clone size with CD59 ALWAYS lower
- Clinical diagnosis - PNH
- 28/60 contained no detectable CD59- RBCs
- Clinical diagnosis- 5/28 PNH- 15/28 Aplastic Anemia- 6/28 AA/MDS - 2/28 BM failure
Sensitivity of 4-colour FLAER assay better than 1%
No Date Patient DiagnosisType 1 Type 2 Type 3 Monos Grans
2 15-Feb-06 MuM 80 2 18 55 60 PNH3 7-Mar-06 FG 89 <1 10 62 62 PNH4 29-Mar-06 TD 37 43 19 73 75 PNH5 30-Mar-06 BS 89 6 5 38 31 PNH6 13-Apr-06 LH 18 81 <1 94 93 PNH7 12-Jul-06 MuM 77 2 20 66 65 PNH8 18-Jul-06 VT 64 13 22 84 89 PNH9 1-Sep-06 MaM 46 23 31 89 91 PNH
10 11-Oct-06 MuM 73 26 <1 71 69 PNH11 26-Oct-06 BS 90 1 9 38 50 PNH12 23-Nov-06 BS 88 1 12 37 49 PNH13 6-Dec-06 PK 55 42 3 82 92 PNH14 14-Feb-07 MuM 68 2 30 77 76 PNH15 4-Apr-07 TD 37 60 3 74 79 PNH16 2-May-07 AB 88 11 <1 44 43 PNH17 23-May-07 MH 89 <1 10 78 72 PNH18 13-Jun-07 MH 87 1 12 88 84 PNH19 21-Jun-07 KG 89 11 <1 5 6 PNH20 8-Aug-07 AS 42 22 37 96 89 PNH21 16-Aug-07 AA 64 23 4 77 80 PNH22 5-Sep-07 MuM 83 2 15 86 86 PNH23 3-Oct-07 MuM 76 3 21 86 87 PNH24 12-Oct-07 MH 89 2 9 85 96 PNH25 18-Jan-08 HA 92 7 1 20 18 PNH26 30-Jan-08 JT 50 1 49 90 90 PNH27 31-Mar-08 HA 93 <1 7 16 13 PNH28 27-May-08 TD 36 61 3 81 84 PNH29 8-Jul-08 MaM 25 72 <1 96 95 PNH30 16-Sep-08 VT 74 24 ~1 74 82 PNH
31 18-Sep-08 TVB <1 96 3 99 99 PNH
32 10-Oct-08 YS 71 28 <1 84 85 PNH
CD59 (%) FLAER NEGATIVE (%)
Samples containing PNH clones by FLAER and RBC assays
1 15-Feb-06 AL 99 <1 <1 1 2 AA2 3-May-06 EV 99 <1 <1 4 3 PNH3 8-May-06 RBr 100 <1 <1 2 2 AA4 26-Jul-06 JM 98 <1 1 83 90 AA5 8-Nov-06 AL 99 <1 <1 3 3 AA6 7-Mar-07 PL 98 <1 <1 3 2 PNH7 4-Apr-07 RBr 96 2 1 2 <2 AA8 16-May-07 AL 99 <1 <1 3 2 AA9 22-Jun-07 LL 98 2 <1 4 8 MDS/AA
10 16-Jul-07 LL 99 <1 <1 2 5 MDS/AA11 13-Aug-07 JN 99 1 <1 4 9 MDS/AA12 12-Sep-07 AL 99 <1 <1 4 4 AA13 31-Oct-07 KY 99 <1 <1 2 1 MDS14 28-Nov-07 LL 99 <1 <1 1.4 1.4 MDS/AA15 20-Feb-08 EH 99 <1 1 21 18 PNH16 27-Feb-08 AL 99 <1 <1 3 3 AA17 26-Mar-08 YH 96 <2 <2 12 10 AA18 31-Mar-08 JN 99 <1 <1 89 52 AA19 3-Apr-08 JN 99 <1 <1 88 52 AA20 8-Apr-08 GL 99 <1 <1 2 7 PNH21 23-May-08 JN 99 <1 <1 92 93 AA22 28-May-08 RR 97 <1 <3 22 20 AA23 3-Jun-08 LL 99 <1 <1 1.4 1.4 MDS/AA24 9-Sep-08 IK 99 1 <1 28 40 PNH
25 24-Sep-08 AL 99 1 <1 3 3 AA26 5-Nov-08 CP 99 1 <1 2 2 BM Failure
27 20-Nov-08 JN 99 >1 <1 94 95 AA (BM)28 3-Dec-08 CP 99 1 <1 2 3 BM Failure
Samples containing only PNH WBC clones by FLAER
SUMMARY II
Proportions of PNH versus Normal clones in RBC lineage is lower than that detected in monocyte and/or granulocyte lineages even in non-transfused patients
FLAER assay more reliably quantifies size of PNH clone than CD55/CD59 on RBCs (probably due to hemolysis)
Monocyte and granulocyte lineages show similar proportions of PNH versus normal clones in most samples
Quality Assurance/Proficiency testing material for this assay is readily available from UK NEQAS (CAP soon?)
The FLAER assay occasionally detects non-PNH hematologic disorders in samples submitted for PNH testing
False negatives with CD59CD59 FITC
Approximately 5% of samples contained CD59- events (2 - 4% or more) when FLAER normal
Repeat testing showed CD59 data in errorFalse negatives still detected even with reverse pipetting!
CD59/CD235CD235PE/CD59FITC
CD235PE causes aggregation even after titrationCD235FITC/CD59PE
CD235FITC no aggregation after titrationCD235FITC/CD59PE obviates false negativesCD235FITC/CD59PE assay sensitivity < 0.1%
USE OF FLAER-BASED WHITE BLOOD CELL ASSAY IN THE PRIMARY SCREENING OF PNH CLONES
Sutherland DR, Kuek N, Azcona-Olivera J, Anderson T, Acton E, Barth D, Keeney M.
Am J Clin Path. In press 2009
USING CD59 ON RBCsFOR PRIMARY SCREENING
OF PNH SAMPLES:
TIME TO LET GO!
ACKNOWLEDGMENTSUNIVERSITY HEALTH NETWORK TORONTORob Sutherland Nancy Kuek Tanya Anderson Erica Acton David BarthRakesh Nayyar Menaka Pai Marciano Reis
LONDON HEALTH SCIENCES CENTRE LONDONSylvia Bamford, Ian Chin-YeeMike Keeney
UK NEQAS Sheffield UKDavid Barnett
St James Hospital Leeds UK Steve Richards
R & D SystemsJuan Azcona-Olivera
HemoglobinuriaHemoglobinuria
Impaired Impaired Renal Renal FxnFxn
�� R/O R/O aHUSaHUS�� R/O TTPR/O TTP
AAAA MDSMDS Unexplained Unexplained Venous or Arterial Venous or Arterial
ThrombosisThrombosis
PNHPNH
Any of� Elevated LDH� Elevated Retic� Hemoglobinuria� Low haptoglobin
Diagnostic Pathway for PNH
High Sensitivity High Sensitivity Flow Cytometry:Flow Cytometry:Quantitative ResultsQuantitative Results
WBCs & WBCs & RBCsRBCs
CoombsCoombs --Negative Negative
Hemolytic Hemolytic anemiaanemia
Unexplained Unexplained CytopeniaCytopenia
Abnormal LDH Abnormal LDH +/+/-- low low
haptoglobinhaptoglobin
Any of� Abdominal pain� Dyspnea� Fatigue� Dysphagia
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