flow cytometry: what you need to know to comply 2012 lap ... · • identifyygg the most...

Flow Cytometry: What You Need to Know to ComplyFlow Cytometry: What You Need to Know to Comply2012 LAP Audioconferences and WebinarsMichael Keeney, ART, FIMLS, FCSMLS(D)Jeannine T Holden MDJeannine T. Holden, MD

October 17, 2012www.cap.org v. 1.0

Presenter Biography

Michael Keeney, ART, FIMLS, FCSMLS(D)

Presenter Biography

Michael Keeney is the Coordinator of Hematology/Flow Cytometry at the London Health Sciences Centre and Associate Scientist with the Lawson Health Research Institute in London, Ontario. Mike is Past Chairman of the Hematology and Professional Advisory Committee for QMP-LS and a Hematology and Professional Advisory Committee for QMP LS and a consultant for the College of American Pathologists, Diagnostic Resource Committee from 2000-2012.

In 2006, Mike was awarded the Wallace H . Coulter Distinguished Lecturer Award in recognition of outstanding contribution to the field of clinical cytometry and in 2009 Mike was awarded a Distinguished Fellowship from the Canadian Society of Medical Laboratory Sciences.

Recent activities have focused on the identification and characterization of Recent activities have focused on the identification and characterization of circulating tumor cells, PNH testing and polychromatic flow cytometry in the diagnosis and monitoring of leukemia and lymphoma.

© 2012 College of American Pathologists. All rights reserved. 2

Presenter BiographyPresenter Biography

Jeannine T. Holden, MD

Jeannine Holden is Associate Professor of Pathology & Laboratory Medicine at Emory University School of Medicine in Atlanta, Georgia, and Director of Hematopatholgy & Flow Cytometry at Emory Medical Laboratories. She has taught in the International Clinical Cytometry Society's Clinical Cytometrytaught in the International Clinical Cytometry Society s Clinical CytometryCourse since 1995 and in 2010 assumed directorship of the course. She has served on ICCS Council in multiple capacities and is currently Vice President.

She has participated in numerous consensus activities, including the 2006 Bethesda Internation Consensus, and drafted the Clinical Indications document.

Jeannine’s interests are primarily in the areas of flow cytometricimmunophenotyping of hematolymphoid malignancies and patient immunophenotyping of hematolymphoid malignancies and patient safety/quality improvement.


ObjectivesObjectives

• Identify the most challenging Flow Cytometryy g g y yChecklist requirements.

• Describe practical approaches for demonstrating compliance with Flow Cytometry requirements.

• Develop and apply practical strategies to maintain contin o s compliance ith Flo C tometrcontinuous compliance with Flow Cytometryrequirements.


Most Commonly Asked Question TopicsMost Commonly Asked Question Topics

• Proficiency Testingy g• Specimen Collection and Handling• Quality ControlQuality Control• Reagents• CD34 Stem Cells• CD34 Stem Cells• Leukemia/Lymphoma Panels

PNH Testing• PNH Testing


Proficiency TestingProficiency Testing

• PT required for all testingq go Enrollment in a CAP-accepted PT program is required for

the following analytes: − CD 34+ enumeration− Fetal red blood cell, flow− HLA B27 typing flow cytometry− Lymphocyte subsets− Reticulocyte count, flow cytometry

o Alternate PT assessment is required for all other flow testing f d b th l b tperformed by the laboratory.


Proficiency Testing FLO 18385Proficiency Testing – FLO.18385

FLO.18385 – For laboratories that perform only interpretations of flow immunophenotyping data for leukemias and lymphomas, the laboratory participates in a peer education program in interpretive flow cytometry of hematolymphoid neoplasia.

• Our laboratory performs only the interpretation of flow cytometry data The technical component is performed by cytometry data. The technical component is performed by one of the other laboratories in our healthcare system. Are we required to perform PT?


Proficiency Testing FLO 18385Proficiency Testing – FLO.18385

• The short answer is yes!

• By far the most complex activity in leukemia and lymphoma immunophenotyping is correct interpretation of data.

Th t t d it i ff ti f th • The current move toward monitoring effectiveness of therapy and detection of residual disease has increased the complexity of analysis exponentially.


Proficiency Testing FLO 18385 (cont )Proficiency Testing – FLO.18385 (cont.)

• Approaches:o Participation in either a peer education program such as the CAP

Survey FL5 or a laboratory-developed program is required. o An internal or laboratory developed program may include

i l ti t i l ith th l b t i ithi th circulating case material with other laboratories or within the laboratory’s own practice with documentation of peer review or other educational options.

o Partnering with another laboratory is also an excellent idea as it o Partnering with another laboratory is also an excellent idea as it allows for discussion on cases rarely seen in an individual laboratory.

o For those laboratories performing Minimal Residual Disease Detection, comparison of results and gating strategies should be compared to a national laboratory experienced in this work.


Proficiency Testing COM 01500Proficiency Testing – COM.01500

COM.01500 – For tests for which CAP does not require PT, the laboratory at least semi-annually: 1)participates in external PT, or 2) exercises an alternative performance assessment system for determining the reliability of analytic testing.

• We are enrolled in the CAP PT Survey FL3 – ImmunophenotypicCharacterization of Leukemia/Lymphoma. Regarding COM.01500, is it required to perform alternative performance assessment for antibodies not included in a particular Survey?


Proficiency Testing COM 01500Proficiency Testing – COM.01500

• For diagnostic panels, it is currently not required that every antibody be tested within a panel – only those deemed appropriate by the clinical history and morphology.

• If the laboratory is enrolled in the CAP Survey FL3 –Leukemia/Lymphoma Immunophenotyping, it is not required to perform external PT for every antibody used.

• However, quality control is required for every antibody used within a panel. o Evaluation of reactivity with normal cell populations within each

sample is probably the best quality control procedure available to assess correct antibody staining to assess correct antibody staining.


Proficiency Testing COM 01500 (cont )Proficiency Testing – COM.01500 (cont.)

• Our stem cell lab participates in the FL4 CD34 Survey. At a recent CAP inspection, we received a deficiency because we were not doing proficiency testing for viability. The Stem Cell Processing Survey offers PT for viability, but that Survey is not

i d d d t t t ll i it I it i d t d required and we do not want to enroll in it. Is it required to do some type of alternate proficiency testing for viability testing?



• Although CAP includes viability testing under the Transfusion Medicine Survey – CBT, SCP Cord Blood and Stem Cell Processing, it is not required to perform proficiency testing for viability testing.

• Viability is an assessment for the integrity of the sample and nonviable cells may nonspecifically bind to many antibodies and interfere with accurate immunophenotyping.p yp g



• For stem cell analysis in products that are known to contain a large number of dead and dying cells (eg, cord blood) a viability assessment should be performed.

• For fresh products such as apheresis or peripheral blood, For fresh products such as apheresis or peripheral blood, laboratories should document that the length of time between sample collection and processing does not have a significant impact on cell viability.g p yo Of note, total cell viability is less useful than the viability of

the actual CD34+ cells.o Kits for the assessment of viable CD34+ cells are readily

available from most major manufacturers.


Specimen Collection and HandlingSpecimen Collection and Handling

• Potential variables that may affect specimen y pintegrityo Handling at collection

T f ti l to Type of anticoagulanto Time of collection/age of sampleo Transportationo Transportationo Temperature


Specimen Collection and Handling FLO 22050Specimen Collection and Handling – FLO.22050

FLO.22050 – There are documented criteria for the rejection of unacceptable specimens or the special handling of sub-optimal specimens.

• When a sub-optimal bone marrow sample is submitted for a full diagnostic panel, what is the best approach for handling this sample while providing as much information to the this sample while providing as much information to the clinician?



• For replaceable samples reject with a note to the referring physician on reason for rejection.

• Irreplaceable samples should be analyzed whenever possible.o Samples of bone marrow, body fluids, and tissues fall into this o Samples of bone marrow, body fluids, and tissues fall into this

group.

• Note should be made of the reason the sample is sub-optimal:o Clotted – Note if correct anticoagulant was used Processing the o Clotted Note if correct anticoagulant was used. Processing the

sample after first filtering through a gauze mesh may yield enough viable cells to attain a diagnostic result.

o Hemolyzed – Note unusual handling/transport conditions. o Specimen hot or cold to touch? Check viability and process.o Specimen exceeds stability limit. Check viability and process if

sample is irreplaceable.


Specimen Collection and Handling FLO 22050 (cont )Specimen Collection and Handling – FLO.22050 (cont.)

• The report issued on an irreplaceable sample should note the issue leading to a possibly compromised result; provide viability information when necessary to aid in interpretation and suggest mechanisms to reduce the possibility of

h iblrecurrence where possible.

• Any compromised specimen in which no abnormal population • Any compromised specimen in which no abnormal population is found should clearly state that this does not rule out disease.



FLO.22000 – Procedures are adequate to verify sample identity and integrity(includes specimens of blood, body fluids, and tissues).

• Do preserved samples need to be checked for viability?

• Response: No. However, if the laboratory standard procedure requires a viability dye in every sample, this can be performed on a control cell In this case all cells “live and dead” should on a control cell. In this case, all cells live and dead should be included in the viability gate. See next slide.



NNonviable


Quality Control

• Procedures for the continual assessment of all test

Quality Control

activities to ensure a specified quality of product is achieved and maintained.o Instrument QCo Instrument QCo Reagent QCo Procedural/Method /

QC(Quantitative/Qualitative)o Patient QC(internal)


Quality Control FLO 30260Quality Control – FLO.30260

FLO.30260 – Procedures are established for determining appropriate color compensation settings.

I it t bl t l th i t t’ t t d • Is it acceptable to only use the instrument’s automated spectral compensation software or is a manual procedure still required? If so, please provide examples when a manual procedure would be necessaryprocedure would be necessary.



• Most manufacturers of flow cytometer instruments include detailed daily start-up procedures that include calibration, optical alignment check, and voltage and compensation validation to ensure proper instrument performance.

• Depending on the type of instrument, this may be performed electronically by the instrument or manually by the user.

• Where feasible manufacturer instructions should be followed • Where feasible, manufacturer instructions should be followed and results obtained must fall within the expected ranges.



• For compensation specifically, manufacturers’ recommendations may be used initially to determine instrument setup. However, validation of appropriate compensation should be performed by analyzing control samples with known and expected results for staining with the antibodies under considerationstaining with the antibodies under consideration.

• For example, in the analysis of the CD3 subsets CD4 and CD8, it would be expected to see mutually exclusive populations of the CD4 and CD8 populations if gated on CD3CD4 and CD8 populations if gated on CD3.

• In leukemia and lymphoma immunophenotyping and in high level polychromatic systems (5-10 color and above), it is essential to define compensation matrix for each tube under analysis. Once compensation matrix for each tube under analysis. Once established, compensation for this type of analysis should not be adjusted unless there is a major change to the instrument (new laser) or post service affecting instrument performance.



FLO.23737 – The performance of reagents and staining procedures are verified by the use of positive controls.

• Could you clarify the frequency for performing quality control • Could you clarify the frequency for performing quality control for leukemia/lymphoma immunophenotyping? o Is the QC frequency requirement the same when antigen

positive cells are not readily available though commercial positive cells are not readily available though commercial controls or patient materials?

o Are there any recommendations for verifying the performance of CD103?performance of CD103?



• Percentage positive or negative for individual antibodies are rarely useful in leukemias and lymphoma immunophenotyping.

• For antibodies used in more qualitative assays, whole blood For antibodies used in more qualitative assays, whole blood cells can be used for virtually all antibodies, targeting selective subpopulations (eg, lymphs, RBCs, platelets, monocytes, neutrophils).p )

• Use normal reactivity patterns (determined by lab or peer-reviewed literature)o lymphocytes (FMC7 CD25) o lymphocytes (FMC7, CD25) o monocyte (CD11C, CD14, CD36) o neutrophil (CD10, CD16), RBC (CD71, CD235a) and

l t l t (CD41 CD42 CD61)o platelets (CD41, CD42, CD61)



• Common problem antibodies are TdT, CD34, CD103, and CD117, which are typically not found in any significant number in peripheral blood.

• Commercial controls (eg, for TdT and CD34) are readily Commercial controls (eg, for TdT and CD34) are readily available.

• CD1a, CD103, and CD117 are not. Reactive/normal bone marrows (determined by morphology and chart review) can marrows (determined by morphology and chart review) can be used to define the expected range of CD117+ (and CD34+) myeloblasts.



• In cases where hairy cell leukemia is suspected by immunophenotype (monoclonal population with CD19+,CD20+, CD11c+,CD25+, CD103+), correlation with histological findings can be documented as proof of

ti itreactivity.

• Split sample testing with another laboratory in the region if feasible is an excellent way to confirm reactivity of y yuncommon antibodies.

• In 2013, CAP will be offering two new Surveys – Rare Flow Antigen Validation (RFAV) 1 and 2 – one for CD1a and one for Antigen Validation (RFAV) 1 and 2 one for CD1a and one for CD103.


Quality Control FLO 23737 (cont )Quality Control – FLO.23737 (cont.)

• What controls are appropriate for ZAP 70?

• ZAP-70 is a prognostic marker in B-cells in identifying different forms of Chronic Lymphocytic Leukemia (CLL).

N l l h t l ti ithi th l t t d • Normal lymphocyte populations within the sample tested are often used as an internal control. ZAP-70 is expressed most highly in NK cells, followed by T cells and weakest in normal B cell populationscell populations.

• ZAP-70 positivity can be expressed as a ratio of the abnormal cells to the normal populations within the test.


Quality Control FLO 23737 (cont )Quality Control – FLO.23737 (cont.)


Quality Control FLO 30730 30760Quality Control – FLO.30730, .30760

FLO.30730 – There are procedures established for distinguishing abnormal cells of interest from normal cells based on their light scatter and fluorescence properties.

FLO.30760 – There is a procedure to distinguish fluorescence-FLO.30760 There is a procedure to distinguish fluorescencenegative and fluorescence-positive cell populations.

• Could you provide some best practice examples to clarify the intent of these requirements?


Quality Control FLO 30730 30760Quality Control – FLO.30730, .30760

• Positive controls are required to confirm that the methods used to prepare target cells, the immunologic reagents, and the staining procedures are performing optimally.

• Positive reagent controls and procedural or process controls perform this function.

• Most of the monoclonal antibodies included in leukemia and lymphoma typing panels react with subpopulations of normal WBC.

• Under some circumstances, a sample drawn from a normal donor that is drawn, prepared, and stained in parallel with the patient specimen may serve as a positive procedural control.

• It is not required to analyze this type of procedural control sample weekly if the laboratory is active and if within run positive and negative control results on a neoplastic hematolymphoid specimen

i i idemonstrate appropriate reactivity.


Reagents

• The recommendations of the manufacturer for the

Reagents

proper use of reagents must be followed.o Sample preparation

St i i t lo Staining protocols


Reagents COM 30450Reagents – COM.30450

COM. 30450 – New reagent lots and/or shipments are checked against old reagent lots or with suitable reference material before or concurrently with being placed in service.

• What is the best approach for validating a new lot of reagent and/or antibodies?



• The intent of the requirement is to ensure that the new reagent lot/shipment provides a clinically comparable result to the old reagent and that the antibodies are working effectively.

• Comparing the results of the old and new reagent run in parallel on the same fresh control (patient or normal) is recommended.

• When validating antibody cocktail new lots, it is recommended to compare each individual antibody by using a side scatter versus fluorescence plot and having defined acceptance criteria from old lot to new lot (for example, must be within +/- 0.5 decade log fluorescence).

• Only if there is agreement between old and new lots should the lab make the cocktail and then test that against the old cocktail. In this way, if a single titration needs adjusted, only one antibody in the cocktail needs to be adjusted and then the new cocktail made. cocktail needs to be adjusted and then the new cocktail made.



• Additionally, simply by looking at all of the populations in a normal sample, it is possible to verify the reactivity is as expected. For example, CD4 will be bright on lymphs, dimmer on monos, and mainly negative on the granulocyte

l ti population.

• Once a new lot has been verified, it is possible to use the patient’s own normal cells to verify the activity of most p y yantibodies. As an example, CD10 (CALLA), used in leukemia/lymphoma typing, is negative in mature normal lymphocytes but positive on mature neutrophils.


Reagents COM 40300 Reagents – COM.40300

• COM.40300 – The laboratory verifies or establishes analytic accuracy and precision for each test.

A th d ti f f i i iti l lid ti • Are there recommendations for performing an initial validation for antibody cocktails?


Reagents COM 40300 Reagents – COM.40300

• Once each individual antibody has been titrated, a small batch (5-10 test) of the antibody combination should be prepared and tested against several samples to ensure there are no unexpected interactions between the fluorochromes

h i d t thwhen mixed together.

• Theoretical issues are steric hindrance between two fluorochromes with epitopes close together on the cell surface p p g(rare in the speakers experience) and changes in fluorescence intensity of one or more of the fluorochromeswhen combined into a larger volume.


Reagents FLO 30720Reagents – FLO.30720

FLO.30720 – Methods are established to ensure that immunoglobulin staining is intrinsic and not extrinsic (cytophilic).

• Is it required that both lambda and kappa light chain reagents be included in the same tube or can the laboratory perform an alternate procedure that also provides acceptable results?an alternate procedure that also provides acceptable results?



• When staining for kappa or lambda light chains, both antibodies can be included in the same tube on different fluorochromes or alternatively kappa can be present in one tube and lambda in another.

• The advantages of having kappa and lambda in the same tube are potential for reduced cost and analysis of exactly the same population for both light chains.

• The disadvantage of this method is that for very dimly stained cells, as can be seen for example in some cases of CLL, it is not always possible to determine clonality because the background on dissimilar fluorochromes may be different.fluorochromes may be different.

• Note also that monoclonal kappa and lambda reagents will not recognize all B-cells, whether normal or malignant, so if you’re using them you need to be aware of that issue. them you need to be aware of that issue.



• For example, by using kappa PE and lambda PE in different tubes on the same fluorochrome, it may be possible to overlay the histograms from one tube to the other, revealing which is dimly positive. We have used this in the following combination: o CD19/kappa/CD5 o CD19/lambda/CD5

• And here’s another combination that offers the additional And here s another combination that offers the additional advantage of even more gating antibodies, enabling the analyst to drill down on suspicious B-cell subsets:o Kappa/CD20/CD45/CD19ppo Lambda/CD20/CD45/CD19



Cannot overlay as K/L are on different fluorochromes

Can overlay as K/L are on

lambda

kappa Can overlay as K/L are on the same fluorochromes

pp


CD34 Stem CellsCD34 Stem Cells

• Enumeration of CD34+ Hematopoietic Stem and pProgenitor Cellso Specimen collection and handling

S l tio Sample preparationo Specimen integrityo Specimen age and storage conditionso Specimen age and storage conditions


CD34 Stem Cells FLO 30564CD34 Stem Cells – FLO.30564

FLO.30564 – The laboratory has a procedure to document the viability of CD34 positive cells in apheresis samples immediately before freezing (autologous donors) or infusion (allogeneic donors).

• This requirement has been revised in the 2012 edition – why is it no longer required to sample an aliquot of the product for no longer required to sample an aliquot of the product for apheresis samples stored more than 4 hours?



• When the laboratory has documented proof that the method of storage does not have an adverse effect on the viability of an apheresis product, they do not necessarily have to perform viability assessment after storage.

• However, should there be a deviation from the standard process (eg, pack left at room temperature overnight instead of in the fridge, fridge failure) then a viability assessment g g ) yshould be performed.



FLO.30585 - A statistically valid number of CD34+ events are collected to ensure clinically relevant precision and accuracy.

Wh i it t bt i i i f 100 CD34 t • Why is it necessary to obtain a minimum of 100 CD34+ events and what would you do if there is an extremely low count?



• 100 CD34+ cells was defined in the ISHAGE guidelines as the minimum number of events required to give a clinically acceptable coefficient of variation (C.V.) which was defined by the authors as 10%.

• For very low counts, the laboratory can either increase the total number of events to reach this number, or as is most relevant in the clinical lab, report the value as below a certain pthreshold eg, < 2 CD34 x106/L.

• Note, it is acceptable to add the results of two separate analyses to reach 100 CD34+ events analyses to reach 100 CD34+ events.


Leukemia/Lymphoma Panels OverviewLeukemia/Lymphoma Panels Overview

• Antibody panels must be sufficiently comprehensivey p y p• Appropriate utilization

o Confirmation of clinical or morphologic impression?

o Primary diagnostic modality?


Leukemia/Lymphoma Panels OverviewLeukemia/Lymphoma Panels Overview

• Stainingo Consider specimen type, clinical history, morphologic

impression in choice of antibodies choosing combinations informative for both normal and abnormal populations

• Acquisitiono Gated acquisition should be performed with caution; when

in doubt acquire all eventsin doubt acquire all events

• Analysiso Familiarity with normal and common malignant

phenotypesphenotypes

• Interpretationo Inspection of plots/histograms in ALL cases


Leukemia/Lymphoma OverviewLeukemia/Lymphoma Overview

• Reporting of resultso “Percentages” for individual antigens (“CDs”) not informative

- Waste of time and effort- Potential for error (transcription, re-interpretation by non-

qualified individuals)qualified individuals)o Provide differential diagnosis; be as specific as possibleo Bethesda International Consensus Recommendations

− descriptions of antibody fluorescence intensity and distribution desc p o s o a body uo esce ce e s y a d d s bu o should be included

− antibody distribution: “Negative;” “Positive;” or “Partially expressed” and relative to appropriate negative control

i f i i i i− antibody fluorescence intensity: “Dim;” “Bright;” or “Heterogeneous” with intensity relative to closest normal hematolymphoid population



• When reporting using the Bethesda guidelines, could you provide an example of how to report Antibody Fluorescence Intensity (AFI) for the neoplastic population relative to the closest normal differentiation population?



• Antibody Fluorescence Intensity (AFI) need only be reported when it provides relevant information provides relevant information.

• For example, over expression of CD20 when compared to a normal mature B cell is often seen in hairy cell leukemia.

• Under expression of CD20 or surface kappa or lambda in CLL compared to a mature B cell is also common.

• In acute leukemia, the under or over expression of an early B-cell population marker may aid in monitoring disease progression or remission. An example of this would be over expression of CD58 in acute lymphoblastic leukemia cells when compared to normal hematogones. hematogones.

• The key point is that not all antigens need to be described compared to their normal counterparts – only those that provide additional information to the clinician (or another laboratory professional in information to the clinician (or another laboratory professional in order to compare an earlier result from an outside lab with a new result).


T Cell Lymphoproliferative DisorderT Cell Lymphoproliferative DisorderNormal

Abnormal

Abnormal T cell population with Normal CD3 and CD8 expression and dim CD2 and CD5 as comapred to residual normal cells

PNH TestingPNH Testing

• Flow Cytometry has become the method of choice to screen for PNHo More sensitive and specific than Ham’s and Sucrose

Hemolysis test

• Specimen recommendationo Peripheral blood

• Selection of Antibodies

• CD55 analysis no longer recommended for red blood cells

FLAER t i i • FLAER staining


PNH TestingPNH Testing

Are there standardized protocols for routine PNH testing?• Yes! The following two recent documents:1: Borowitz MJ, Craig FE, Digiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ;

Clinical Cytometry Society. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry Cytometry B Clin Cytom 2010 Jul;78(4):211 30 hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom. 2010 Jul;78(4):211-30.

o Developed as a consensus document by the ICCS; provides an overview and general criteria for PNH testing in routine and in high sensitivity assays.

2: Sutherland DR, Keeney M, Illingworth A. Practical guidelines for the high-sensitivity detection and 2: Sutherland DR, Keeney M, Illingworth A. Practical guidelines for the high sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry B Clin Cytom. 2012 Jul;82(4):195-208.

o Highly technical document which outlines exactly how to achieve accurate results in both red and white cell analysis in PNH. A standardized method is detailed and ypotential pitfalls and caveats are documented.

Additionally, there is a CLSI document (H52-A2) currently undergoing review which is written by the second group and should be available early 2013.


i C i d i *PNH Testing – Current Testing Recommendations*

• Red cellsCD235 ( l h i ) ith CD59 o CD235a (glycophorin) with CD59

o CD235a used to gate red cell population, CD59 GPI linked

• Neutrophilso FLAER/CD24/CD15/CD45 o CD15 used to gate mature neutrophils, CD24 and FLAER detect

absence of GPI proteins

• Monocyteso FLAER/CD14/CD64/CD45o CD64 preferred over CD33 to separate neutrophils and basophils

from monocytes.o CD14 and FLAER detect absence of GPI proteins.

*Other antibodies may provide similar or better performance. Those Other antibodies may provide similar or better performance. Those listed have been validated in external QA studies


PNH TESTING ON RBC QUALITY ASSURANCE MATERIALPNH TESTING ON RBC QUALITY ASSURANCE MATERIAL


PNH TESTING ON GRANULOCYTES QUALITY ASSURANCE MATERIALPNH TESTING ON GRANULOCYTES QUALITY ASSURANCE MATERIAL


ResourcesResources

• Wood BL, Arroz M, Barnett D, DiGiuseppe J, Greig B, Kussick SJ, et.al. Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasis by flow cytometry: optimal reagents and reporting for the flow cytometricdiagnosis of hematopoietic neoplasia The Cytometry Part B; Clinical diagnosis of hematopoietic neoplasia. The Cytometry Part B; Clinical Cytometry Supplement 72B: S14-S22, 2007.

• Clinical and Laboratory Standards Institute (CSLI). Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Immunologically Defined Cell Populations by Flow Cytometry; Approved Guideline-Second Edition. CLSI document H42-A2, 2007.

• Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline – Second Edition. CLSI guideline H43-A2, 2007.


ResourcesResources

• Borowitz MJ, Craig FE, Digiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ; Clinical Cytometry Society. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. CytometryB Clin Cytom 2010 Jul;78(4):211-30 B Clin Cytom. 2010 Jul;78(4):211 30.

• Sutherland DR, Keeney M, Illingworth A. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry Cytometry B Clin Cytom hemoglobinuria clones by flow cytometry. Cytometry B Clin Cytom. 2012 Jul;82(4):195-208.


Thank You!Thank You!

Questions?Questions?


flow cytometry: what you need to know to comply 2012 lap ... · • identifyygg the most...

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