High sensitivity PNH testing by Flowcytometry

Prof. Heba Raslan.National Cancer Institute- Cairo University

Consultant Hematopathology -Flowcytometry section head.

King Fahad Specialty hospital-Dammam.Secretary General of the PAF-WG

Paroxysmal Nocturnal Hemoglobinuria (PNH)• Paroxysmal nocturnal hemoglobinuria (PNH) is a rare

hematopoietic stem cell defect, life-threatening disease caused by nonmalignant clonal expansion of one or more hematopoietic stem cells that have a somatic mutation on the X-chromosome phosphatidylinositol glycan complementation class A gene.

• As a result, the complex biosynthetic pathway required for the synthesis of the Glycophosphatidylinositol (GPI) moiety that anchors a variety of cell surface proteins is disrupted, causing the progeny of affected stem cells to be deficient in all GPI anchored proteins.

• Activity of the enzymatic C3 &C5 convertases continues unchecked, leads to generation of terminal complement complex, deposited on cell surface of erythrocytes, resulting in lysis and release of hemoglobin and other cell contents.

Paroxysmal Nocturnal Hemoglobinuria (PNH)This deficiency can be seen in both the WBC and RBC ; WBC are not affected by the GPI-deficiency however RBCs are vulnerable to complement-mediated lysis

It’s not paroxysmal: Even in the absence of symptoms, destructive progression of hemolysis is ongoing.

It’s not nocturnal; Hemolysis in PNH is constant, 24 hours a day.

Hemoglobinuria:Less commonly seen complication ¾ patients present without hemoglobinuria.

PNH RBCs lack TCC (terminal complement inhibitor)

Complement attack PNH RBCs are lysed and contents are released into the plasma

Working classification developed for PNH; it includes three syndromes or sets of symptoms:

1. Classical PNH.2. PNH in the setting of another specified bone marrow disorder (e.g.

PNH/aplastic anemia or PNH/refractory anemia- myelodysplastic syndrome [MDS])

3. Subclinical PNH (PNH-sc).

Clinical indications for PNH testing:1. PNH patients have major symptoms;• Intravascular hemolysis evidenced by hemoglobinuria and signs of

hemolytic anemia.• Thrombosis with unusual features and sites.• Evidence of bone marrow failure. 2. 35% of PNH patients dye within 5 years of diagnosis even with supportive care.3. Early detection may impact treatment regimen and outcomes for patients with PNH and Bone Marrow failure.4. The Availability of an Effective PNH Treatment Eculizumab “Solaris” Warrants Testing in High-risk Patient Populations

• Since 1990 - implementation of FCM. • Since 1996 - method of choice.

• 2010 - ICCS guidelines provides suggested approaches with recommended MoAb combinations.

• 2012 - Practical guidelines specific reagent cocktails, detailed analytical strategies.

• 2013 - Harmonization and Standardization.

• 2014 - New validated approaches. Implementation of CD157.

• 2016 – New international guidelines– in preparation

TimeLine for development of FCM PNH assays

Practical guidelines for PNH and High-Sensitivity PNH testing from 2010 - 2016 by the International Clinical

Cytometry Society

Target of ICCS in PNH testing

Use gating antibodies on ≥2 different cell populations (not lymphocytes) to test with ≥2

different antibodies against ≥2 different surface GPI-linked

proteins (or GPI by FLAER)

Important considerations for optimization and Validation of PNH testing

Step 1: Pre-analytical considerations:1.Specimen recommendations and Limitations.2. Selection of reagents and panels; to produce interpretable histograms.a. Reagent and Panel Selection:– It is important to select the most specific reagents with the best signal/noise ratio, e.g. -CD59 is preferred over CD55 for RBCs. -FLAER/CD24 for WBC Granulocytes.

-FLAER/CD14 for WBC-Monocytes. -Antibodies should tittered.b. Lineage specific gating:- Sensitivity increase by increasing the number of lineage specific markers,e.g. - GPA (CD235a) for RBCs. - CD15 for granulocytes/neutrophils.

- CD64 or CD33 for monocytes.3. Instrument optimization:a. Appropriate Voltage adjustmento Cells positive for the antibody should show bright signalo Cells negative for the antibody need to be “on scale”b. Optimize compensation settingso WBC: setting may be similar to Leukemia/lymphoma, but need to

be tweaked if using FLAER-Alexa488o RBC: need separation compensation setting

Step 2: Assay quality control:1.Validation of PNH assay.Several normal peripheral blood samples should be run, to verify adequate staining of antibodies in normal cells, and to determine the background and sensitivity of the assay.2. PNH Surveysa. NEQAS (UK)b. CAP RBC and WBC Surveyc. Inter-laboratory comparisons of PNH+ samples

(containing larger and smaller PNH clones) may help to improve confidence levels of detection.

Step 3: Analytical considerations (ICCS Guidelines):• Procedure (Wash vs No wash in RBCs).• Lineage-specific gating.• Interpretation of results.Step 4: Clear reporting (ICCS Guidelines):• Avoid misleading terminology (Note here negative

finding for GPI-linked antibody means positive for PNH clone).

FCM as a ‘gold standard’ for PNH diagnosisSpecimen recommendations and Limitations:

• Sample type: Peripheral blood is the preferred specimen. Bone marrow is not the sample of choice as immature myeloid precursors doesn’t express, or may have very low level of GPI-anchored proteins.

• Anticoagulant: No specific anticoagulant; though most experience has been with EDTA

• WBC analysis: Neutrophils and Monocytes best performed in 24 – 48 hrs because of degradation.

• Be aware that very low numbers of neutrophils and monocytes difficult to be reported, number of events for the target population (negative for both markers) should be 50 -100 in order that a desirable specificity is achieved.

• RBC analysis: May be done up to 7 days if kept at 4C.• Store the sample Horizontally, so the cells will not sediment

on each other and create weight, that destroy faster.• Run the normal sample after the patient sample, not

before.

Reagent performance requirements:

Monoclonal antibody titration: Serial dilutions with decreasing volumes.

Monoclonal antibody titration: Reagent performance

requirements:

Antibody A: the plateau is not reached even when using 20 µL using less antibody is not recommendedAntibody B: as low as 0.5µL of Ab yields 85% of the maximum staining intensity.

Selection of reagents & panels; to produce interpretable histograms

Four - Color Panel:RBCs: CD235a / CD59.Granulocytes: FLAER / CD24 / CD15 / CD45.Monocytes (reflex): FLAER / CD14 / CD64 (or CD33) / CD45.Five - Color panel:Red Blood Cells: CD253a / CD59.Granulocytes and Monocytes: FLAER / CD24 / CD14 / CD15 / CD45.Monocytes only (Reflex): FLAER / CD33 / CD14 / CD64 / CD45.Six - Color Panel:RBCs: CD253a/ CD59.Granulocytes: Monocytes: FLAER /CD24/ CD14/ CD15/ CD45/ CD64 or CD33

Paroxysmal Nocturnal Hemoglobinuria

RBCs Testing on ICCS Guidelines

RBC assay: Reagents and Conjugates• GPI-linked antibody selection:- CD59: • It shows best signal/noise ratio. • PE conjugate works well. • MEM43 Clone is the best. (CD55 is not used any more

– too dim).• Gating antibody: - Anti-GPA (CD235a): The only choice. • FITC flurochromes is the most widely used

conjugate as it give little aggregation, it is used for lineage-specific gating on RBCs.

• KC16 and 10F7MN most tested and validated clones Note: That PNH cocktails can be stored up to 3-6 months.

RBCs Testing Procedure1. Make 1:100 Dilution of peripheral blood (EDTA).

2. Pipette 50-100 µl of this dilution into bottom of the test tube, don’t pipette the last drop use reverse pipetting (make sure no blood is smeared on the side of the tube, as they do not get stained adequately with the antibody)

3. Add appropriately tittered CD59-PE.4. Add appropriately tittered GPA-FITC (CD235a), and

vortex gently.5. Incubate in the dark at RT for 20 minutes ( up to 60

min.)6. Wash twice with PBS; to achieve good separation

between Type I, II and III RBCs.. (1200 rpm/ 5 min).7. Resuspend in 0.5-1ml PBS.8. Rack vigorously; dragging the tube across a sample

rack 3-4 times to break up aggregates.9. Analyze within 15 min to avoid fading of CD235a-FITC

staining.10.Run on the flow cytometer using your PNH-RBC panel.

• Most cases: 50,000 cells are enough (Poisson statistics suggests that if you find zero PNH RBCs in 50,000 events, the probability is 99% that the level of PNH RBCs will be <0.01%).

• Limit Of Detection LOD: smallest number of events required to reproducibly detect a PNH clone = 20 events.

• Limit Of Quantification LOQ: smallest number of events

required to reproducibly quantify a PNH clone = 50 events.

• If the percentage of PNH cells reads 0.01%, a reflex tube is set up to collect 500,000 events to visualize a cluster of PNH cells in the expected location (CD59 negative and high CD235a expression). Ideally we are looking for the acquisition of 50-100 PNH cells.

Events count:

Validation of high-sensitivity RBC assayMeasure assay sensitivity using ‘Spiking Experiment’

Serial dilutions of PNH sample in normalBased on 2-6,000,000 RBCs collected in list-mode data

files

Linear decrease in PNH clone size.

Alternate Options for PNH QC in RBCs

Type III Type II Type I

Step 1: Run normal RBCs

with GPA and with CD59 to determine the position of normal RBCs (Type I cells)

Step 2: Run normal RBCs with

GPA and without CD59 to determine the position of RBCs with complete CD59 Deficiency (Type III cells)Step 3:

Run suspected PNH patient with GPA /CD59.

1. RBC gate: to gate out debris.2.GPA+ gate: to gate out GPA-negative cells.

RBC - Normal Control (PB)

3.Dot Plot GPA-CD59 is gated on GPA+ RBCs

4. Single Parameter histogram is also gated on GPA+ RBCs

RBCs showing PNH Type III Clone- Dot Plot the blue gate represent the PNH Type III cells, which show complete absence for CD59, cells show same level of GPA staining as normal cells (red).

-Single parameter histogram of CD59 expression (gated on GPA+ RBCs) can be used together with dot plot to establish cursor setting (for Type I, II and III RBCs)

-(Aqua) gate for aggregates should be checked as clumping may affect the distribution of type I, II and III RBCs; there mustn't be more than 1-2% aggregates.

Normal Expression of CD59 (Type I) and Abnormal Expression of CD59 (Type II and III) in RBCs

Normal RBC’s with normal CD59 expression (Type I cells)Life span:120days.PNH clone with complete CD59 deficiency (Type III cells)Life span:10-15days.PNH clone with complete CD59 deficiency (Type III cells) &partial CD59 deficiency (Type II cells) Intermediate.

RBC analysis – pitfallsImportance of “Racking”

Tubes were vortexed lightly: 29% Aggregates

Same tubes were racked vigorously: 0.5% Aggregates

Why CD55 was excluded?

CD59 shows great separation between Type I, II and III RBCs.

CD55 has been proven to show poor separation between normal and PNH RBCs and also does not separate Types II and III.

MEM43 is the CD59 clone of choice, regardless of vendor

How much is the importance of Clone / Conjugates in this assay?

Clone MEM43 with PE

Clone P282 with PE

Optimizing compensation is very important

Poorly compensated case

Properly compensated case

Optimizing compensation is very important

The gate did not include all negative events; it is one channel from the baseline that excludes a large portion of the negative events; sample also had an overcompensation issue, which pushed the negative events further to the left. 

Adjusted gate 64.0% Type III PNH

The Importance of double Washing step of the RBCs

The patient RBCs was initially not washed; a shoulder can be seen in the Type II area.

After washing this sample twice the CD59-negative Type III PNH clone can easily be identified.

Missing of the twice washing step may not allow for the identification of a PNH clone.

Value of staining of the RBCs with CD235a (GPA)• Addition of CD235a

enables the lab to gate on true RBCs only resulting in a clean PNH assay (as it is important to ensure that the CD59-negative events are truly RBCs.

• Identifies staining artifacts in the type II area, which may otherwise be misinterpreted as Type II PNH cells.

• Staining of RBCs with CD235a (GPA) vs CD59 provides Quality Control of the test.

• CD235a-FITC/CD59 –PE is the preferred antibody combination with best signal/noise ratio– select most sensitive and specific clone (e.g. MEM43 and

p282)– use CD59-PE preferably– addition of GPA (preferably FITC conjugate) results in

higher sensitivities and cleaner RBC assays • Washing twice and “racking” is important!• Analysis of 50,000 RBCs can result in sensitivity of 0.1%

(50 PNH cells) in a clean assay• Difference in Clone size between RBC and WBC is

important to determine hemolysis and/ or transfusion(RBC clone usually lower than WBC clone)

• Report both Type II and Type III as the total PNH Clone if there is a separate Type II RBC population present

RBCs assay: summary

PNH Testing – WBC Panel

• Granulocytes :- Most typically used to assess PNH clone size in WBC; as they also have the GPI deficiency but do not seem to be functionally affected by this. - Occasionally, Type II granulocytes can be seen.• Monocytes:- Should also be analyzed to confirm the granulocyte PNH clone.- Monocyte clone size often larger than granulocyte clone (reason

unknown), however the precision is lower due to lower cell number.

- Occasionally, Type II monocytes can be seen.• Lymphocytes:- Not a suitable target population for testing due to long lifespan,

and the clone small size.- Excellent Antibody performance as Internal Quality Control as B

cells have strong expression of FLAER and CD24, T cells positive for FLEAR negative for CD24.

- Looking at the lymphocyte populations and making sure they are visible populations (‘on-scale’) and not crushed or bleeding into other channels also serves as an indicator to verify optimal Voltage and compensation settings.

Detection of PNH clones in WBC

• FLAER-Alexafluor488- conjugated inactive pro-aerolysin from Aeromonas hydrophila.

• Binds to GPI-anchor, not the protein moiety and it does not cause lysis.

• It is the strongest discrimination of normal population from GPI-deficient myeloid populations.

• However FLAER cannot be used for detecting PNH red blood cells as; it do not possess surface–bound proteolytic enzymes required to process the pro-aerolysin.

NB. Protect from light and from prolonged exposure to temperatures above 2-8C˚.

What is FLEAR?

WBC assay: reagents For Granulocytes:- FLAER / CD24 appears to be the preferred antibody combination to detect PNH clones in granulocytes.- CD59 is not recommended as it can give false positives on the

granulocytes. - CD16 may be lost from granulocytes in MDS or due to CD16

polymorphisms. .For Monocytes: - FLAER / CD14 is the most tested combination to detect PNH clones

in monocytes.FLAER/CD157 looks promising to assess both monocytes and neutrophils.Lineage-specific gating: results in higher sensitivities and cleaner WBC assays • CD15 for granulocytes (not CD33)• CD64 for monocytes (CD33 is second choice)• CD45 can be very useful to gate out CD45 Negative debris, pattern

recognition (back gating to see what the populations are e.g. blasts, platelets, other immature cells)

Sample preparation:Stain-then-lyse method may better preserve the light scatter characteristics for clearer gating. 1. Pipette 50-100 µL of peripheral blood (EDTA) into test tube.2. Add appropriately tittered antibodies (rinse out antibody

thoroughly).3. Incubate in the dark at room temperature for 30 min.4. Lyse with your laboratory’s lysing reagent (e.g. Immunoprep, FACS

Lyse, ammonium chloride).5. Wash once with PBA (1200 rpm/5 min.).6. Resuspend in 0.5–1 mL of PBA.Analysis: On the flow cytometer using your PNH-WBC panels.

WBC assay: testing procedure

Event count:• Most cases: 50,000 cells are enough for most assays, the

acquisition of 50,000 granulocytes is sufficient to achieve a 0.1% sensitivity (50 PNH events).

• LOD: smallest number of events required to reproducibly detect a PNH clone = 20 events.

• LOQ: smallest number of events required to reproducibly quantify a PNH clone = 50 events

Cells 1 2 3 4 5 6

4 colors G FLEAR CD24 CD15 CD45

4 colors M FLEAR CD14 CD64/CD33

CD45

4 colors G+M FLEAR CD24 CD14 CD33

5 colors G+M FLEAR CD24 CD14 CD15 CD45

5 colors G+M FLEAR CD24 CD14 CD15 CD64/CD33

5 colors G+M FLEAR CD157 CD15 CD45 CD64

6 colors G+M FLEAR CD24 CD14 CD15 CD45 CD64

6 colors G+M FLEAR CD24 CD14 CD15 CD45 CD33

ICCS- Recommended reagents for WBC analysis

Beckman Coulter recommended 4 colors panels

Beckman Coulter recommended 6 colors panels

Granulocytes FLEAR Alexa Fluor 488

CD24-PESN3(eBio)ALB9(BC)

CD15-PC580H5(BC)

CD45-PC7J33(BC)

monocytes FLEAR Alexa Fluor 488

CD14-PE61D3 (eBio)RMO52 (BC)

CD64-PC522(BC)

CD45-PC7J33(BC)

Granulocytes+ Monocytes

FLEAR Alexa Fluor 488

CD24-PESN3(eBio)ALB9(BC)

CD15-PCy580H5(BC)

CD64-PCy522(BC)

CD14APC700RMO52(BC)

CD45-KOJ33(BC)

Beckman Coulter recommended 5 colors panels

Granulocytes+ Monocytes

FLEAR Alexa Fluor 488

CD157-PESY11B5(eBio,BC)

CD64-ECD22(BC)

CD15-PC580H5(BC)

CD45-PC7J33(BC)

• CD157 : promising GPI-specific reagent work for both neutrophils and monocytes.

• Non FLAER based assay following the current guidelines? CD14 and CD24 in combination with CD157.

PNH WBC assay without FLAER ?

Granulocytes + Monocytes

CD15- FITCH198(BD)

CD157-PESY11B5(eBio, BC)

CD45 PerCP-C5.52D1(BD)

CD64 PE10.10 (BD)

CD24-PESN3(eBio)

CD14-APC-H761D3(BD)

Normal Peripheral Blood sample: WBC -Granulocytes/Neutrophils

Step 1: Gating out debris

Step 2: Gating on CD15++ Granulocytes(Excluding the intermediate CD15+Eosinophils)

Step 3: No PNH Clone detected in CD15++ Granulocytes

Importance of Gating on CD15++ Granulocytes

Query PNH clone??

Right gating No PNH clone

False positive PNH clone

Normal Peripheral Blood sample: WBC - Monocytes

•Accurate assessment of PNH monocytes, lineage-specific gating on CD64+ or CD33+ monocytes is preferred

Gating on CD45 vs SS allows for determination of PNH clone in Monocytes but size of the PNH clone will not be accurate (78.9%)

Lineage-specific gating on CD64 vs SS allows for a more accurate assessment of the size of the PNH clone in Monocytes (83.3%)

Importance of Gating on CD64+ Monocytes

PNH QC options in WBCs

Step 1: Run normal WBCs with gating antibodies and

with GPI-linked antibodies to determine the position of normal WBCs.

Step 2: Run normal WBCs with gating antibodies and without GPI-linked antibodies to determine the position of WBCs with complete GPI-Deficiency.

Step 3: Run suspected PNH patient with gating antibodies and GPI-linked antibodies.

e.g. Presence of 54.4% PNH Granulocytes

Type II granulocytes in PNH with FLAER staining

In the current patient:* PNH Type II granulocytes: 6.2%Type II cells can be only detected with the FLAER/CD24 combination.

* PNH Type III granulocytes: 54.7%* Total PNH clone size: 60.9%

-PNH patients with Type II granulocytes tend to be more thrombocytopenic than patients without Type II granulocytes.

Document the presence of Type II cells if they are clearly clustered, (as seen in this example) but you have to combine Types II and III if present.

Type III (Green) and Type III (Blue) PNH clone in granulocytes

Type II monocytes in PNH with FLAER staining

- Normal Type I monocytes: 28.5% - PNH Type II monocytes: 5.5%-PNH Type III monocytes: 66.0% -Total PNH clone size: 71.5%

CD14

SSC

Analytical considerations- Resolution

More Colors More Independent Information

Analytical considerations- ResolutionA. If the negative population

has low background: Populations well resolved.

B. If the negative population has high background: Populations NOT resolved.

C. If the negative population has low background, high CV: Populations NOT resolved.

The ability to resolve populations is a function of both background and spread of

the negative population

Analytical considerations- ResolutionResolution

Sensitivity

Background Spread

- Non/specific staining - Cell Autofluorescence - Unbound fluorochrome

Spillover- Detection efficiency* (laser power, laser alignment, optical efficiency, optics, PMT sensitivity, poor PMT, dirty flow cell, or filters) - Electronic noise

Sample Instrument

Analytical considerations- Resolution

T cells, no PE stain

MIF (PE)

- Resolution for a given fluorescence parameter is decreased by increased spread due to spillover from other fluorochromes.

- This spread is NOT eliminated by compensation.

- More colors = more spillover.

Spillover increases Spread (CV)

Compensation

• In multicolor FCM each fluorochrome emits into a number of another detectors.

• We have to correct spillover in both directions.• Compensation still accounts for the most

frequent errors in implementation of multicolor FCM.

Computer Assisted Compensation BC and BD platforms 1. Set optimal PMT voltages using unstained particles or

lymphocytes: a) BC platform: 0.3± 0.01b) BD platform: > 2.5 rSD of the electronic noise as

defined from baseline c) EuroFlow: target values using 8th peak from Rainbow

beads.

2. Acquire single stained compensation tubes for each fluorochrome used in the assay: DO NOT CHANGE PMTv.

3. Generate computer assisted compensation matrix: a) BC platform: Auto Setup Scheduler or Kaluza software.b) BD platform: CS&T module and DIVA.

Internal control populations to assess optimal cytometer settings

Lymphocyte is the Biological Quality Control for the Antibody and the Instrument performance.

Compensation check on lymphocytesGood Compensation Poor Compensation

B lymphocytes are positive to FLEAR and CD24, T lymphocytes are positive to FLEAR and negative to CD24.

Validation of high-sensitivity WBC assays using ‘spiking

4-colour granulocyte assay sensitivity

4-colour monocyte assay sensitivity

-FLAER / CD24 appears to be the preferred antibody combination to detect PNH clones in granulocytes-FLAER / CD14 is the most tested combination to detect PNH clones in monocytes.

-Lineage-specific gating results in higher sensitivities WBC assays

CD15 for granulocytes CD64 and / or CD33CD45 can be very useful for pattern recognition (back-gating to see type of the populations.

-Analysis of 50,000 granulocytes result in sensitivity of 0.05–0.1%-(25–50 PNH cells) in a clean assay-Report both Type II and Type III granulocytes and monocytes as the total PNH clone if they are present-

WBCs assay: summary

Reporting methods of flowcytometry assays Qualitative assays:• Lack relativity to the amount of analyte tested.• Definite data is reported (Positive vs Negative).e.g. leukemia / lymphoma Immunophenotyping.

Semi quantitative assays:• Does not use calibration standard; numerical data is

reported: % of gated cells.• There is definite cut-off or threshold for disease

detection. e.g. PNH assay, MRD assays.

Quantitative assays:• We have to use calibration standard to estimate the

absolute quantitative values for unknown samples. e.g. CD34 stem cell assay, lymphocyte subset assessment.

Reporting of PNH ResultsPNH Important terms:

If the PNH population detected is >20% -------Major PNH Clone.If the PNH population detected is >1% -------PNH Clone.If the PNH population detected is 0.1% - 1% --------Minor PNH Clone.If the PNH population detected is ≤ 0.1% ------ Rare cells with GPI deficiency.

- It is important not to over-interpret small PNH clones as clinical PNH.- Report size and Type; Type II and Type III RBCs, as well as

Type II and Type III granulocytes (even though their significance is not established). Than report Total PNH clone size.

- Recommend Repeat samples every six months on same patient, and comment on change in size of PNH clone.

- Provide histograms if possible.

Reporting – ICCS PNH Positive Case

References:1. Rother RP et al. Nat Biotechnol 2007;25,11:1256-64; 2. Parker C et al. Blood 2005;106:3699-7093. Sugimori C et al. Blood 2006;107:1306-1314 4. McKeage K. Drugs. 2011;71(17):2327-2345. 5. Adapté de : Johnson RJ et al. J Clin Pathol: Mol Pathol.

2002;55:145-152.6. Brodsky R. Paroxysmal Nocturnal Hemoglobinuria. dans :

Hematology - Basic Principles and Practices. 4th ed. R Hoffman; EJ Benz; S Shattil et al. eds. Philadelphie, PA: Elsevier Churchill Livingstone; 2005;419-427.

7. International PNH Interest Group. Blood. 2005;106:3699-3709.8. Rother RP et al. JAMA. 2005;293:1653-1662.9. Hill A et al. Br J Haematol. 2007;137:181-192.10.Lee JW et al. Hematologica. 2010;95(s2): Abstract #505 and 506.11.Hill A et al. Br J Haematol. 2010; May;149(3):414-425.12.Hillmen P et al. Am J Hematol. 2010;85: 553-559.13.Borowitz et al.;Cytometry 2010;178B;211-230.14.Sutherland et al. ;Cytometry 2012; 82B;195-208.- Sutherland et

al. Cytometry 2014.15.Marinov et al.;Cytometry 2013;84B;229-236.- Marinov et

al.;IJLH:2014 accepted16.Marinov et al.;IJLH:2015.17.Marinov et al.; Clin HemLab Med, 2013; 51: 2133-2139.18.Marinov et al.; ClinLab, 2014;60:;217-224.19.Marinov et al. 2016; Cytomtery, Part B- accepted20.Barnett D et al.;Cytometry 2013;84B;309-314.21.Wood B et al.; Cytometry 2013;84B;315-323

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