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QuantiBRITE CD20

Monoclonal mouse anti-human reagent foridentification of cells expressing CD20

antigen

23-5544-018/2010

Form Catalog No.

PE 347220

BD, BD Logo and all other trademarks are property ofBecton, Dickinson and Company. © 2010 BD

1. INTENDED USEQuantiBRITE™ CD20 is intended for invitro diagnostic use in the identification ofcells expressing CD20 antigen, using aFACS™ brand flow cytometer.

�e flow cytometer must be equipped todetect light scatter and the appropriatefluorescence, and be equipped withappropriate analysis software (such asCellQuest™ or LYSYS™ II) for dataacquisition and analysis. Refer to yourinstrument user’s guide for instructions.

ApplicationsExpression of CD20 in thecharacterization of hematologic neoplasia1

2. COMPOSITIONCD20, clone L27,2 is derived fromhybridization of mouse Sp2/0 myelomacells with spleen cells from BALB/c miceimmunized with the LB lymphoblastoidcell line. CD20 is composed of mouseIgG1 heavy chains and kappa light chains.CD20 PE is supplied in phosphate-bu�ered saline (PBS) containing bovineserum albumin (BSA) and 0.1% sodiumazide. Concentrations are listed in Table 1.

WARNING: Sodium azide is harmful ifswall owed (R22). Keep out of reach ofchildren (S2). Keep away from food, drink,and animal feedingstu� (S13). Wear

Table 1. Bottling concentrations

PE 12.5 µg in 1.0 mL of PBS 12.5 µg/mL

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suitable protective clothing (S36). Ifswallowed, seek medical adviceimmediately and show this container orlabel (S46). Contact with acids liberatesvery toxic gas (R32). Azide compoundsshould be flushed with large volumes ofwater during disposal to avoid deposits inlead or copper plumbing where explosiveconditions can develop.Antibody purity is as follows.PE: ≥95% 1:1 PE:mAb ratio, as measuredby size-exclusion chromatography (SEC)

3. STORAGE AND HANDLING�e antibody reagent is stable until theexpiration date shown on the label whenstored at 2° to 8°C. Do not use after theexpiration date. Do not freeze the reagentor expose it to direct light during storageor incubation with cells. Keep the outsideof the reagent vial dry.Do not use the reagent if you observe anychange in appearance. Precipitation ordiscoloration indicates instability ordeterioration.

4. REAGENTS OR MATERIALS REQUIRED BUT NOT PROVIDED

1. Falcon™ disposable 12 x 75-mmcapped polystyrene test tubes (BDCatalog No. 352058) or equivalent

2. Micropipettor with tips (BDElectronic Pipette, BD Catalog No.646539) or equivalent

3. Vortex mixer 4. FACS Lysing Solution (10X) (BD

Catalog No. 349202). For dilution

instructions and warnings, refer tothe product insert.

5. QuantiBRITE PE PhycoerythrinFluorescence Quantitation Kit(BD Catalog No. 340495). Refer tothe product data sheet forinstructions.

6. Centrifuge 7. CellWASH™ (BD Catalog N o.

349524) or a wash bu�er ofphosphate-bu�ered saline (PBS) with0.1% sodium azide.

8. CellFIX™ (BD Catalog N o.340181) or 1% paraformaldehydesolution in PBS with 0.1% sodiumazide. Store at 2° to 8°C in amberglass for up to 1 week. WARNING: Formaldehyde is harm-ful by inhalation, in contact with skin,and if swallowed (R20/21/22). It isirritating to eyes and skin (R36/38).Exposure can cause cancer. Possiblerisks of irreversible e�ects (R68). Cancause sensitization by skin contact(R43). Keep locked up and out of thereach of children (S1/2). Wear suit-able protective clothing and gloves(S36/37). If swallowed, seek medicaladvice immediately and show thecontainer or label (S46). Dispose ofaccording to federal, state, and localregulations.

9. FACS brand flow cytometer. Refer tothe appropriate instrument user’sguide for information.

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5. SPECIMEN(S)

Reagents can be used forimmunophenotyping by flow cytometrywith a variety of specimen types,including peripheral blood, bone marrowaspirates or biopsies, and other body fluidsor tissues. Each type of specimen can havedifferent storage conditions andlimitations that should be consideredprior to collection and analysis.3,4

Samples with large numbers of nonviablecells can give erroneous results due toselective loss of populations and toincreased nonspecific binding ofantibodies to nonviable cells. Viability ofsamples should be assessed and a cut-offvalue established. A cut-off value of atleast 80% viable cells has been suggested.3

WARNING: All biological specimensand materials coming in contact withthem are considered biohazards. Handleas if capable of transmitting infection5,6

and dispose of with proper precautions inaccordance with federal, state, and localregulations. Never pipette by mouth.Wear suitable protective clothing andgloves. Fixation has been reported toinactivate HIV.7

6. PROCEDURE

1. Add the appropriate volume of CD20fluorochrome-conjugated monoclonalantibody to 100 µL of whole blood ina 12 x 75-mm tube. Refer to theappropriate vial label for volume.

2. Vortex gently and incubate 15 to 30minutes in the dark at roomtemperature (20° to 25°C).

3. Add 2 mL of 1X FACS LysingSolution.

4. Vortex gently and incubate for 10minutes in the dark at roomtemperature.

5. Centrifuge at 500 x g for 5 minutes.Remove the supernatant.

6. Add 2 to 3 mL of CellWASH (orwash buffer) and centrifuge at 500 x gfor 5 minutes. Remove thesupernatant.

7. Add 0.5 mL of CellFIX (or 1%paraformaldehyde solution) and mixthoroughly. Store at 2° to 8°C untilanalyzed. BD Biosciences recommendsanalyzing within 24 hours of staining.

Analytical Results

Abnormal numbers of cells expressing thisantigen or aberrant expression levels of theantigen can be expected in some diseasestates. It is important to understand thenormal expression pattern for this antigenand its relationship to expression of otherrelevant antigens in order to performappropriate analysis.

Flow Cytometry

Vortex the cells thoroughly at low speed toreduce aggregation before running themon the flow cytometer.8 When ready toread samples, dilute a tube of freshQuantiBRITE beads according to packageinstructions. Set up the instrument usingFACSComp™ software, version 4.2, lyse/no-wash (LNW) setting and run thesamples and the QuantiBRITE tube usingthe same settings. Before acquiringsamples, adjust the threshold to minimize

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debris and ensure populations of interestare included. Figure 1 displaysrepresentative data performed onperipheral blood and gated onlymphocytes. Laser excitation is at 488 nm.

Figure 1 Representative data analyzed with a FACS brand flow cytometer

Analyze your data using Attractors™,version 3.1 or higher, or QuantiCALC™,automated quantitation analysis softwarethat converts the FL2 axis toPE-conjugated–antibodies bound per cell(ABC) and reports statistics in CD20 PEABC. Alternatively, you can performmanual quantitation using CellQuest andMicrosoft® Excel software (refer to theQuantiBRITE PE data sheet).

Internal Quality Control

BD recommends using CaliBRITE™beads and FACSComp™ software to setphotomultiplier tube (PMT) voltages,

fluorescence compensation, and to checkinstrument sensitivity prior to use. Referto the CaliBRITE Beads package insertand the FACSComp Software User’s Guide.

BD recommends running a controlsample daily from a normal adult subjector a commercially available whole bloodcontrol to optimize instrument settingsand as a quality control check of thesystem.9

7. PERFORMANCE CHARACTERISTICS

Specificity

CD20 antigen is a phosphoprotein with amolecular weight of 35 or 37 kilodaltons(kd), depending on the degree ofphosphorylation.10 The antigen is notglycosylated.10

CD20 antigen is expressed onB lymphocytes synchronous with theexpression of surface IgM.10,11 Theantigen is present on both resting andactivated B lymphocytes but is lost beforedifferentiation into plasma cells.10 TheCD20 antigen is found in both mantle-zone and germinal-center areas ofsecondary follicles of lymphoid tissue andcan be expressed on follicular dendriticcells (FDCs) in germinal centers.10

Low-level expression of the CD20 antigenhas been detected on a subpopulation ofT lymphocytes.12

Sensitivity

Sensitivity is defined as the minimumnumber of ABC that can be reliablydetected. The ABC that represent noise ornon-specific staining for the CD20antigen was based on monocytes, which

010

00

100 104FL2-H

SS

C-H

R4

050

100 104FL2-H

Cou

nts

M1

gated on B cells

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are known to be negative for CD20. Theminimum ABC was determined to be 745(average plus three standard deviations).

Reproducibility

CD20 was submitted to the ThirdInternational Workshop and Conferenceon Human Leucocyte DifferentiationAntigens. Participating laboratoriesevaluated clone L27 as part of a blindpanel of antibodies and reportedconsistent results.2

Repeatability

To determine the repeatability of stainingwith each reagent, samples were stainedwith multiple lots of reagents. Thedifferent samples used in the evaluationprovided an average mean fluorescenceintensity (MFI) value as shown in Table 2.For each sample, two different lots ofreagents generated a pair of results.Individual SDs were determined from thepaired results for each sample. IndividualSDs were combined to derive a pooled SDfor each reagent that provides an estimateof within-sample repeatability.

8. LIMITATIONS

Conjugates with brighter fluorochromes(PE, APC) will give greater separationthan those with other dyes (FITC,PerCP). When populations overlap,calculation of the percentage of cellspositive for the marker can be affected bythe choice of fluorochrome.

Use of monoclonal antibodies in patienttreatment can interfere with recognitionof target antigens by this reagent. Thisshould be considered when analyzingsamples from patients treated in thisfashion. BD Biosciences has notcharacterized the effect of the presence oftherapeutic antibodies on theperformance of this reagent.

Single reagents can provide only limitedinformation in the analysis of leukemiasand lymphomas. Using combinations ofreagents can provide more informationthan using the reagents individually.Multicolor analysis using relevantcombinations of reagents is highlyrecommended.

As reagents can be used in differentcombinations, laboratories need tobecome familiar with the properties ofeach antibody in conjunction with othermarkers in normal and abnormal samples.

Reagent data performance was collectedtypically with EDTA-treated blood.Reagent performance can be affected bythe use of other anticoagulants.

Table 2. Repeatability of mean fluorescence intensity (MFI) of B lymphocytes across different lots and across

multiple donors (N)

Na

a. N = number of samples

Average MFI Pooled SD Pooled %CV

PE 8 3,698 224.1 6.06

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WARRANTY

The product sold hereunder is warranted onlyto conform to the quantity and contents statedon the label at the time of delivery to thecustomer. There are no warranties, expressed orimplied, that extend beyond the description onthe label of the product. BD’s sole liability islimited to either replacement of the products orrefund of the purchase price. BD is not liablefor property damage, personal injury, oreconomic loss caused by the product.

TROUBLESHOOTING

REFERENCES1. Marti GE, Faguet G, Bertin P, et al. CD20 and

CD5 expression in B-chronic lymphocyticleukemia. Ann NY Acad Sci. 1992;651:480-483.

2. Ling NR, Maclennan ICM, Mason DY. B-celland plasma cell antigens: new and previouslydefined clusters. In: McMichael AJ, ed. LeucocyteTyping III: White Cell Differentiation Antigens.New York, NY: Oxford University Press;1987:303-334.

3. Rothe G, Schmitz G. Consensus protocol for theflow cytometric immunophenotyping ofhematopoietic malignancies. Leukemia.1996;10:877-895.

4. Stelzer GT, Marti G, Hurley A, McCoy P, Jr.,Lovett EJ, Schwartz A. U.S.-Canadian consensusrecommendations on the immunophenotypicanalysis of hematologic neoplasia by flow cytometry:standardization and validation of laboratoryprocedures. Cytometry. 1997;30:214-230.

5. Protection of Laboratory Workers from InfectiousDisease Transmitted by Blood, Body Fluids, andTissue: Tentative Guideline. Villanova, PA:National Committee for Clinical LaboratoryStandards; 1991. NCCLS document M29-T2.

6. Centers for Disease Control. Update: universalprecautions for prevention of transmission ofhuman immunodeficiency virus, hepatitisB virus, and other bloodborne pathogens inhealth-care settings. MMWR. 1988;37:377-388.

7. Nicholson JK, Browning SW, Orloff SL,McDougal JS. Inactivation of HIV-infected H9cells in whole blood preparations by lysing/fixingreagents used in flow cytometry. J ImmunolMethods. 1993;160:215-218.

8. Jackson AL, Warner NL. Preparation, staining,and analysis by flow cytometry of peripheralblood leukocytes. In: Rose NR, Friedman H,Fahey JL, eds. Manual of Clinical LaboratoryImmunology. 3rd ed. Washington, DC: AmericanSociety for Microbiology; 1986:226-235.

9. Clinical Applications of Flow Cytometry: QualityAssurance and Immunophenotyping of PeripheralBlood Lymphocytes; Tentative Guideline. Wayne,PA: National Committee for Clinical LaboratoryStandards; 1992. NCCLS document H42-T.

Problem Possible Cause Solution

Poor resolution between debris and lymphocytes

Cell interaction with other cells and platelets

Prepare and stain another sample.

Rough handling of cell preparation

Check cell viability; centrifuge cells at lower speed.

Inappropriate instrument settings

Follow proper instrument setup procedures; optimize instrument settings as required.

Staining dim or fading

Cell concentration too high at staining step

Check and adjust cell concentration or sample volume; stain with fresh sample.

Insufficient reagent

Repeat staining with increased amount of antibody.

Cells not analyzed within 24 hours of staining

Repeat staining with fresh sample; analyze promptly.

Improper medium preparation (azide omitted)

Use azide in staining medium and washing steps.

Few or no cells Cell concentration too low

Resuspend fresh sample at a higher concentration; repeat staining and analysis.

Cytometer malfunctioning

Troubleshoot instrument.

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10. Dörken B, Möller P, Pezzutto A, Schwartz-Albiez R, Moldenhauer G. B-cell antigens:CD20. In: Knapp W, Dörken B, Gilks WR, etal, eds. Leucocyte Typing IV: White CellDifferentiation Antigens. 1 ed. New York, NY:Oxford University Press; 1989:46-48.

11. Loken MR, Shah VO, Dattilio KL, Civin CI.Flow cytometric analysis of human bone marrow.II. Normal B-lymphocyte development. Blood.1987;70:1316-1324.

12. Hultin LE, Hausner MA, Hultin PM, Giorgi JV.CD20 (Pan-B cell) antigen is expressed at a lowlevel on a subpopulation of human T lymphocytes.Cytometry. 1993;14:196-204.

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