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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


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


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


Monocytes CD14

Granulocytes CD24

0 256 512 768 1024


0 256 512 768 1024


Granulocytes CD66abce

Granulocytes CD66c

S. Richards

SELECTION OF ANTIBODIES

0 256 512 768 1024


0 256 512 768 1024


0 256 512 768 1024


0 256 512 768 1024


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


M1 1056 22.40

File: J0101817.LMD



M2 1573 33.45

File: J0101818.LMD


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


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



Quad Events % Gated

UL 0 0.00UR 23 0.16

LL 17 0.12LR 14252 99.72



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


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


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


Quad Events % GatedUL 0 0.00

UR 1844 92.38LL 1 0.05

LR 151 7.57



Quad % Gated

UL 0.00UR 1.06LL 1.05

LR 97.89



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


Quad % GatedUL 0.01UR 0.10LL 96.64LR 3.26


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



UR 46 2.32LL 1668 83.99

LR 208 10.47



Quad % Gated

UL 0.00UR 0.46LL 0.99

LR 98.55



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



UR 8235 76.78LL 344 3.21

LR 978 9.12



Quad % Gated

UL 0.00UR 0.02LL 0.08

LR 99.90



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?)


Gate: G2

Gated Events: 1253


UR 168 13.41

LL 304 24.26

LR 781 62.33


Gate: G3

Gated Events: 11415

Quad % GatedUL 0.00

UR 0.04

LL 0.66

LR 99.31


Gate: G4

Gated Events: 4453


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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