BD Tritest CD3/CD19/CD45 Reagent Is Provided in 1 Ml of Buffered Saline with Mêéå~Ìíáçåë Bovine Serum Albumin and 0.1% Sodium • for in Vitro Diagnostic Use

NK fkqbkaba=rpb BD Tritest™ CD3 fluorescein _a qêáíÉëí»=àPLàNVL isothiocyanate (FITC)/CD19 àQR phycoerythrin (PE)/CD45 peridinin chlorophyll protein (PerCP) is a cçê=ÇÉíÉêãáåáåÖ=éÉêÅÉåí~ÖÉë=~åÇ=~ÄëçäìíÉ=Åçìåíë=çÑ= three-color direct immunofluorescence Üìã~å=q=~åÇ=_=äóãéÜçÅóíÉë=áå=ÉêóíÜêçÅóíÉJäóëÉÇ= reagent for use with a suitably equipped ïÜçäÉ=ÄäççÇ flow cytometer to identify and determine the percentages and absolute counts of qÉëíë `~í~äçÖ=kçK mature human T (CD3+) and B (CD19+) RM=qÉëíë PQMPUN lymphocytes in erythrocyte-lysed whole RM=qÉëíë=ïáíÜ= PQMQMR _a qêìÅçìåí=qìÄÉë blood. When used with BD Trucount™ tubes, absolute counts of these populations can be enumerated from a single tube.

OLOMNU OPJPMORJMS= BD Tritest reagent and BD Trucount tubes can be used with the BD FACS™ Loader. fsa Rx Only The reagent can be used with or without an isotype control. © 2018 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. OK prjj^ov=^ka=bumi^k^qflk Human lymphocytes can be divided into three major populations based on their _ÉÅíçåI=aáÅâáåëçå=~åÇ=`çãé~åó _a=_áçëÅáÉåÅÉë biologic function and cell-surface antigen OPRM=nìãÉ=aêáîÉ expression: T lymphocytes, p~å=gçëÉI=`^=VRNPN=rp^ B lymphocytes, and NK lymphocytes. ^ìëíê~äá~å=~åÇ=kÉï=wÉ~ä~åÇ=aáëíêáÄìíçêëW `äáåáÅ~ä=^ééäáÅ~íáçåë _ÉÅíçå=aáÅâáåëçå=míó=iíÇK Q=oÉëÉ~êÅÜ=m~êâ=aêáîÉ Total T and B lymphocytes are used j~Åèì~êáÉ=råáîÉêëáíó=oÉëÉ~êÅÜ=m~êâ to characterize and monitor some forms kçêíÜ=oóÇÉ=kpt=ONNPI=^ìëíê~äá~ of immunodeficiency1–3 and autoimmune _ÉÅíçå=aáÅâáåëçå=iáãáíÉÇ diseases.4,5 NQÄ=dÉçêÖÉ=_çìêâÉ=aêáîÉ= jí=tÉääáåÖíçå= ^ìÅâä~åÇ=NMSM= PK mofk`fmibp=lc=qeb=mol`barob kÉï=wÉ~ä~åÇ When whole blood is added to the reagent, the fluorochrome-labeled antibodies in the reagent bind specifically to leucocyte surface antigens. During acquisition, the cells travel past the laser beam and scatter the laser light. The stained cells fluoresce. These scatter and fluorescence signals, detected by the ÄÇÄáçëÅáÉåÅÉëKÅçã instrument, provide information about the `äáåáÅ~ä^ééäáÅ~íáçåë]ÄÇKÅçã

1 cell’s size, internal complexity, and relative antibodies is noncovalently associated fluorescence intensity. BD Tritest reagents with either α/β or γ/δ TCR (70–90 kDa).16 employ fluorescence triggering, allowing CD19 identifies B lymphocytes and direct fluorescence gating of the 6–8 recognizes a 90-kDa antigen that is lymphocyte population to reduce present on human B lymphocytes at all contamination of unlysed or nucleated red stages of maturation but is lost on plasma blood cells in the gate. cells.17 The CD19 antigen might be When BD Trucount tubes are used, a involved in activation and proliferation of known volume of sample is stained B lymphocytes.17 directly in a BD Trucount tube. The CD45 identifies leucocytes and recognizes lyophilized pellet in the tube dissolves, a 180- to 220-kDa human leucocyte releasing a known number of fluorescent antigen that is a member of the leucocyte beads. During analysis, the absolute common antigen (LCA) family.18 number (cells/µL) of positive cells in the sample can be determined by comparing The CD3, CD19, and CD45 antibodies γ cellular events to bead events. If are composed of mouse 1 heavy chains appropriate software such as and kappa light chains. BD Multiset™ software is used, absolute BD Trucount tubes contain a freeze-dried counts will be determined by the software. pellet of fluorescent beads in a single-use If manually performing data analysis tube. Each BD Trucount pouch contains using software such as BD CellQuest™ 25 tubes, sufficient for 25 tests. Pro software, simply divide the number of positive cellular events by the number of Concentration values are listed in the bead events, then multiply by the following table: BD Trucount bead concentration. oÉ~ÖÉåí `çåÅÉåíê~íáçå=E”ÖLãiF CD3 FITC 2.0 QK ob^dbkq CD19 PE 2.0 oÉ~ÖÉåí=mêçîáÇÉÇI=pìÑÑáÅáÉåí=Ñçê=RM qÉëíë CD45 PerCP 6.25 BD Tritest CD3/CD19/CD45 reagent is provided in 1 mL of buffered saline with mêÉÅ~ìíáçåë bovine serum albumin and 0.1% sodium • For In Vitro Diagnostic Use. azide. It contains FITC-labeled CD3, clone SK7;9–11 PE-labeled CD19, clone • Do not use the reagent if you observe SJ25C1;12 and PerCP-labeled CD45, any change in appearance. Precipitation clone 2D1 (HLe-1).13 or discoloration indicates instability or CD3 identifies T lymphocytes and deterioration. recognizes the epsilon chain of the CD3 • The antibody reagent contains sodium antigen/T-cell antigen receptor (TCR) azide as a preservative; however, take complex.14 This complex is composed of care to avoid microbial contamination, at least six proteins witha range in which can cause erroneous results. molecular weight from 20–30 kilodaltons t^okfkd All biological specimens (kDa).15 The antigen recognized by CD3 and materials coming in contact with them are considered biohazards.

2 Handle as if capable of transmitting • Do not freeze the reagent or expose it to infection19,20 and dispose of with direct light during storage or incubation proper precautions in accordance with with cells. Keep the reagent vial dry. federal, state, and local regulations. • Store BD Trucount tubes in their Never pipette by mouth. Wear suitable original foil pouch at 2°C–25°C. To protective clothing, eyewear, and avoid potential condensation, open the gloves. Fixation has been reported to 21 pouch only after it has reached room inactivate HIV. temperature and carefully reseal the • BD FACS™ lysing solution is required pouch immediately after removing a and contains diethylene glycol and tube. Examine the desiccant each time formaldehyde. Refer to the BD FACS you open the pouch. If the desiccant has Lysing Solution instructions for use turned from blue to lavender, discard (IFU) for warnings. the remaining tubes. Use tubes within 1 hour after removal from the foil • If BD Trucount tubes are used, the pouch and do not use beyond the addition of a precise volume of blood is critical to achieving the result. Pipettes expiration date indicated on the must be calibrated to deliver exactly packaging. 50 µL of sample. An electronic pipette that operates in the reverse pipetting RK fkpqorjbkq mode is available through BD. If this or The BD Tritest CD3/CD19/CD45 reagent a similar pipette is not used, perform the and BD Trucount tubes are designed for reverse pipetting technique (see Reverse use on flow cytometers equipped with Pipetting in Section 7 for a brief appropriate computer hardware and description). Refer to the pipette software. We recommend the manufacturer’s instructions for more BD FACSCalibur™, BD FACSort™, or information. BD FACScan™ flow cytometer; however, results can be achieved using other • Bead count varies by lot of BD Trucount platforms. The flow cytometer must be tubes. It is critical to use the bead count equipped with a 488-nm laser capable of shown on the current lot of detecting light scatter (forward and side) BD Trucount tubes when entering this and three-color fluorescence with value in the software or when manually emission detectable in three ranges: 515– calculating absolute counts. Do not mix 545 nm, 562–607 nm, and >650 nm. The multiple lots of tubes in the same assay. instrument must be able to threshold or • BD Trucount tubes are designed for use discriminate using the >650-nm channel. with a specific lyse/no-wash procedure. The BD FACS Loader can also be used Do not attempt to threshold on forward with this product. scatter (FSC) for data collection. We recommend using BD Calibrite™ píçê~ÖÉ=~åÇ=e~åÇäáåÖ beads and BD FACSComp™ software, • Store the reagent at 2°C–8°C. Do not version 2.0 or later, for setting the use after the expiration date shown on photomultiplier tube (PMT) voltages, the label. setting the fluorescence compensation, and checking instrument sensitivity before

3 use. For users of flow cytometers percentages, blood samples held at room manufactured by companies other than temperature can be stained within BD, refer to the manufacturer’s 24 hours of draw and then analyzed instructions for setting up three-color within 24 hours of staining. immunophenotyping. fåíÉêÑÉêáåÖ=`çåÇáíáçåë BD has developed software applications Do not use previously fixed and stored such as BD Multiset that automatically patient specimens. Whole blood samples calculate absolute counts when refrigerated before staining can give BD Trucount tubes are used. However, aberrant results. Samples obtained from other software packages can be used for patients taking immunosuppressive drugs data acquisition and analysis and the can yield poor resolution.24 Blast cells can absolute counts can be calculated interfere with test results. Hemolyzed manually. samples should be rejected.

SK pmb`fjbk=^ka=`liib`qflk= TK mol`barob mobm^o^qflk oÉ~ÖÉåí=mêçîáÇÉÇ Collect blood aseptically by venipuncture22,23 into a sterile EDTA • BD Tritest CD3/CD19/CD45 (Catalog BD Vacutainer® blood collection tube No. 340381), or (lavender top). BD Tritest CD3/CD19/ • BD Tritest CD3/CD19/CD45 with CD45 reagent and BD Trucount tubes BD Trucount tubes (Catalog No. have been validated with both liquid and 340405) dry formulations of EDTA. A minimum of 100 µL of whole blood is required for this oÉ~ÖÉåíë=~åÇ=j~íÉêá~äë=oÉèìáêÉÇ=_ìí=kçí= procedure. Follow the collection tube mêçîáÇÉÇ manufacturer’s guidelines for the • BD Calibrite 3 beads (Catalog No. minimum volume of blood to be collected 340486) to ensure proper specimen dilution; • BD FACS lysing solution (10X), 100 mL especially when determining absolute (Catalog No. 349202). Refer to the counts with BD Trucount beads. BD FACS Lysing Solution IFU for Obtain a white blood cell (WBC) count dilution instructions and warnings. and a differential white cell count from • Reagent-grade (distilled or deionized) the same whole blood sample before water staining to ensure that the WBC count is within the linear range (see Section 11, • BD Vacutainer EDTA blood collection Performance Characteristics: Linearity) or tubes or equivalent to calculate absolute counts from •Falcon®* disposable 12 x 75-mm percentages. polystyrene test tubes or equivalent (if When determining absolute counts, not using BD Trucount tubes) anticoagulated blood stored at room • Vortex mixer temperature (20°C–25°C) must be stained within 6 hours of draw and then analyzed G c~äÅçå=áë=~=êÉÖáëíÉêÉÇ=íê~ÇÉã~êâ=çÑ=`çêåáåÖ= within 6 hours of staining. For fåÅçêéçê~íÉÇK

4 • Micropipettor with tips expelled by pressing to the first stop • Bulk dispenser or pipettor (450 µL) for again. dispensing BD FACS lysing solution • For reverse pipetting, the button is depressed to the second stop. When the • BD FACSFlow™ sheath fluid (Catalog button is released, excess sample is No. 342003) or equivalent drawn up into the tip. A precise volume `^rqflk Use only BD FACSFlow of sample is expelled by pressing the sheath fluid diluent to dilute button to the first stop, leaving excess BD Calibrite beads. sample in the tip. • BD Trucount™ Controls (Catalog No. pí~áåáåÖ 340335), necessary if using BD Trucount tubes 1. For each patient sample, label a 12 x 75-mm tube with the sample • Lysable whole blood control (available identification number. commercially) For absolute counts, label a pí~áåáåÖ=íÜÉ=`Éääë BD Trucount tube in place of the BD Tritest reagents can be used with or 12 x 75-mm tube. without an isotype control to assess the klqb Before use, verify that the amount of nonspecific antibody binding. BD Trucount bead pellet is intact and If you want to use a control, BD Tritest™ within the metal retainer at the IgG1/IgG1/CD45 isotype control reagent bottom of the tube. If this is not the (Catalog No. 340385) is available. case, discard the BD Trucount Tube Lyse red blood cells following staining and replace it with another. using diluted (1X) BD FACS lysing 2. Pipette 20 µL of BD Tritest CD3/ solution. Use care to protect the tubes CD19/CD45 reagent into the bottom from direct light. Perform the procedure of the tube. at room temperature (20°C–25°C). See Precautions in Section 4 and Interfering If using a BD Trucount tube, pipette Conditions in Section 6. just above the stainless steel retainer. Do not touch the pellet. oÉîÉêëÉ=máéÉííáåÖ 3. Pipette 50 µL of well-mixed, If BD Trucount tubes are used, the anticoagulated whole blood into the addition of a precise volume of blood is bottom of the tube. critical to achieving the result. If a BD electronic pipette or a similar pipette that klqb Avoid smearing blood down delivers a precise volume of blood is not the side of the tube. If whole blood used, perform reverse pipetting. This remains on the side of the tube, it will technique takes advantage of two stops in not be stained with the reagent. a pipette. If using a BD Trucount tube, accuracy • For normal pipetting, the button is is critical. Use the reverse pipetting depressed to the first stop. Sample is technique to pipette sample onto the drawn up by releasing the button, then side of the tube just above the retainer.

5 4. Cap the tube and vortex gently to mix. Visually inspect the CD45 vs SSC dot plot. Incubate for 15 minutes in the dark at The lymphocyte population should appear room temperature (20°C–25°C). as a bright, compact cluster with low SSC. 5. Add 450 µL of 1X BD FACS lysing Monocytes and granulocytes should also solution to the tube. appear as distinct clusters. Do not proceed with analysis if populations are diffuse 6. Cap the tube and vortex gently to mix. and there is little or no separation Incubate for 15 minutes in the dark at between clusters. room temperature (20°C–25°C). The See Figure 1, Figure 2, and Figure 3 for sample is now ready to be analyzed on representative data from a the flow cytometer. hematologically normal adult sample cäçï=`óíçãÉíêó stained with CD3/CD19/CD45 in a If samples are not to be analyzed BD Trucount tube. immediately after preparation, store them cáÖìêÉ=N råÖ~íÉÇ=àQR=îë=pp`=Ççí=éäçí=EN=Z=àQRH= in the dark at room temperature (20°C– äóãéÜçÅóíÉëF 25°C). Vortex the cells thoroughly (at low speed) to reduce aggregation before running them on the flow cytometer.25 If using the BD FACS Loader, vortex tubes immediately before placing them into the loader racks. Acquire and analyze list- mode data using the appropriate software such as BD CellQuest Pro or BD Multiset. Before acquiring samples, adjust the threshold to minimize debris and ensure populations of interest are included. nì~äáíó=`çåíêçä cáÖìêÉ=O råÖ~íÉÇ=àP=îë=àNV=Ççí=éäçí=ïáíÜ= Run a control sample daily from a normal _a qêìÅçìåí=ÄÉ~Ç=Ö~íÉ=EN=Z=^ÄëçäìíÉ=Åçìåí=ÄÉ~ÇëF adult subject or a commercially available whole blood control to optimize instrument settings and as a quality control check of the system.23 The BD Tritest isotype control reagent is optional to set fluorescence markers for detecting the presence of nonspecific staining. Use commercial controls providing established values for percent positive and absolute counts with each run to assess system performance.

6 cáÖìêÉ=P ióãéÜçÅóíÉJÖ~íÉÇ=`aP=îë=`aNV=Ççí=éäçí • V is the test volume

VK ifjfq^qflkp • Laboratories must establish their own normal reference ranges for the BD Tritest CD3/CD19/CD45 reagent parameters that can be affected by sex of patient, age of patient, and preparative technique. Race of patient can also have an effect,26 although sufficient data is not available to establish this. Age, sex, clinical characteristics, and race of patients should be known when a reference 27 UK obpriqp range is determined. Reference ranges provided are for information only. Results are reported as the percentage of positive cells per lymphocyte population • BD Tritest CD3/CD19/CD45 reagent or as the number of positive cells per has not been validated for use with microliter of blood (absolute count). heparin or acid citrate dextrose (ACD) liquid anticoagulants in determining `~äÅìä~íáåÖ=^ÄëçäìíÉ=`çìåíë absolute counts with BD Trucount During analysis, the absolute number tubes. (cells/µL) of positive cells in the sample • BD Tritest CD3/CD19/CD45 reagent is can be determined by comparing cellular not intended for screening samples for events to bead events. If BD Multiset the presence of leukemic cells or for use software is used, absolute counts will be in phenotyping samples from leukemia determined automatically. For manual patients. data analysis using BD CellQuest Pro or other software, simply divide the number • Absolute counts are not comparable of positive cellular events by the number between laboratories using different of bead events, then multiply by the manufacturer’s equipment. BD Trucount bead concentration. NMK bumb`qba=s^irbp The absolute count of the cell population (A) can be obtained using the following oÉÑÉêÉåÅÉ=o~åÖÉë equation: The reference ranges for CD3/CD19/ A = X/Y x N/V, where: CD45 shown in Table 1 were determined at BD Biosciences in San Jose, CA, and at • X is number of positive cell events four clinical centers: Cleveland Clinic • Y is the number of bead events Foundation, Cleveland, OH; Johns Hopkins Hospital, Baltimore, MD; • N is the number of beads per test, which Institute of Tropical Medicine, Antwerp, is found on the BD Trucount foil pouch Belgium; and University of North and can vary from lot to lot Carolina Hospital, Chapel Hill, NC.

7 Subjects were hematologically normal NNK mbocloj^k`b=èô^`qbofpqf`p adults between the ages of 18 and Performance of the reagents was 65 years. established by testing at BD Biosciences q~ÄäÉ=N oÉéêÉëÉåí~íáîÉ=êÉÑÉêÉåÅÉ=ê~åÖÉë=Ñçê= laboratories in San Jose, CA, at an àPLàNVLàQR=êÉ~ÖÉåí=é~ê~ãÉíÉêë=áå= external clinical center in the US or ÜÉã~íçäçÖáÅ~ääó=åçêã~ä=~Çìäíë Europe, or at a combination of sites. rééÉê= ^ÅÅìê~Åó içïÉê=OKR= VTKR= pìÄëÉí å jÉ~å= mÉêÅÉåíáäÉ mÉêÅÉåíáäÉ Lymphocyte subset percentage B lymphocytes 516 14 6 25 enumerations with BD Tritest CD3/CD19/ (%) CD45 were compared with results from Total T 516 72 55 84 BD Simultest™ CD3/CD19. Absolute lymphocytes (%) counts were compared to BD Simultest B lymphocytes 516 280 90 660 results and lymphocyte counts obtained (cells/µL)a on a hematology analyzer. Total T 516 1,410 690 2,540 Aliquots of the same blood sample from lymphocytes (cells/µL)a normal and abnormal donors were analyzed. Regression statistics reported in ~K ^ÄëçäìíÉ=Åçìåíë=êçìåÇÉÇ=íç=íÜÉ=åÉ~êÉëí=NM=ÅÉääëL”iK Table 2 indicate that the results are substantially equivalent. These reference ranges are pooled ranges. Refer to the first limitation for more information about reference ranges. q~ÄäÉ=O oÉÖêÉëëáçå=~å~äóëáë

pìÄëÉí= å päçéÉ fåíÉêÅÉéí= ê o~åÖÉ B lymphocytes (%) 167 0.94 1.6%-positive 0.94 0–44 Total T lymphocytes (%) 167 0.91 5.7%-positive 0.96 24–94 B lymphocytes (cells/µL) 166 0.97 24 cells/µL 0.95 0–1,370a Total T lymphocytes (cells/µL) 166 0.93 118 cells/µL 0.95 130–3,710a

~K ^ÄëçäìíÉ=Åçìåíë=êçìåÇÉÇ=íç=íÜÉ=åÉ~êÉëí=NM=ÅÉääëL”iK táíÜáåJpéÉÅáãÉå=oÉéêçÇìÅáÄáäáíó The results for absolute counts are shown Ten aliquots of specimens from three in Table 3. samples representing high, medium, and q~ÄäÉ=P táíÜáåJëéÉÅáãÉå=êÉéêçÇìÅáÄáäáíó=Ñçê= low CD4 counts were assessed. The %- _a qêáíÉëí=àPLàNVLàQR=êÉ~ÖÉåí positive results were as follows (SD = standard deviation): pìÄëÉí iÉîÉä jÉ~å pa `s~=EBF B lymphocytes High 1,197 160 13 • % CD19: mean = 14, pooled SD = 0.8 (cells/µL) Med 253 23 9 • % CD3: mean = 66, pooled SD = 1.1 Low 104 24 23

8 q~ÄäÉ=P táíÜáåJëéÉÅáãÉå=êÉéêçÇìÅáÄáäáíó=Ñçê= 2.4x103 cells/µL) and the CD3+ range _a qêáíÉëí=àPLàNVLàQR=êÉ~ÖÉåí (125 to 11.3 x 103 cells/µL).

pìÄëÉí iÉîÉä jÉ~å pa `s~=EBF t^oo^kqv Total T High 3,202 308 10 Unless otherwise indicated in any applicable BD lymphocytes general conditions of sale for non-US customers, (cells/µL) Med 1,922 157 8 the following warranty applies to the purchase Low 672 122 18 of these products.

~K `s=Z=ÅçÉÑÑáÅáÉåí=çÑ=î~êá~íáçå THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE pí~Äáäáíó CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF A stability study was conducted to assess MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND the effect of time with respect to NONINFRINGEMENT. BD’S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE BD Tritest reagent performance PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY specifications. The study measured: 1) INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT. changes associated with the storage of whole blood before staining, 2) changes as obcbobk`bp a result of time between staining and data acquisition, and 3) the combined effect of 1. Schmidt RE. Monoclonal antibodies for diagnosis of immunodeficiencies. Blut. 1989;59:200-206. the two. 2. Nicholson JKA. Use of flow cytometry in the Based on the results of this study, we evaluation and diagnosis of primary and secondary immunodeficiency diseases. Arch Pathol Lab Med. recommend staining samples within 1989;113:598-605. 6 hours of draw and analyzing within 3. Foucar K, Goeken JA. Clinical application of 6 hours of staining for absolute counts; or immunologic techniques to the diagnosis of staining samples within 24 hours of draw lymphoproliferative and immunodeficiency and analyzing within 24 hours of staining disorders. Lab Med. 1982;13:403-413. for percentages. 4. Cohen SB, Weetman AP. Activated interstitial and intraepithelial thyroid lymphocytes in autoimmune `êçëëJoÉ~Åíáîáíó thyroid disease. Acta Endocrinol. 1988;119:161- 166. CD3 and CD19 have no known cross- 5. Smolen JS, Chused TM, Leiserson WM, Reeves JP, reactivity to nonlymphocyte-formed Alling D, Steinberg AD. Heterogeneity of elements in blood; however, this CD19 immunoregulatory T-cell subsets in systemic lupus clone has been observed to react with erythematosus: correlation with clinical features. Am J Med. 1982;72:783-790. follicular dendritic cells in germinal 6. Nicholson JKA, Jones BM, Hubbard M. CD4 T- centers of lymphoid tissue by lymphocyte determinations on whole blood histochemical staining.28 specimens using a single-tube three-color assay. Cytometry. 1993;14:685-689. iáåÉ~êáíó 7. Nicholson J, Kidd P, Mandy F, Livnat D, Kagan J. The linearity was assessed by testing Three-color supplement to the NIAID DAIDS within a WBC concentration of 2.5 x 103 guideline for flow cytometric immunophenotyping. Cytometry. 1996;26:227-230. to 31 x 103 WBC/µL and a lymphocyte 2 3 8. Nicholson JKA, Hubbard M, Jones BM. Use of concentration of 2.0 x 10 to 16.7 x 10 CD45 fluorescence and side-scatter characteristics lymphocytes/µL. Results were observed to for gating lymphocytes when using the whole blood be linear within the CD19+ range (52 to lysis procedure and flow cytometry. Cytometry. 1996;26: 16-21.

9 9. Haynes BF. Summary of T-cell studies performed Guideline—Third Edition. Wayne, PA: Clinical and during the Second International Workshop and Laboratory Standards Institute; 2005. CLSI Conference on Human Leukocyte Differentiation document M29-A3. Antigens. In: Reinherz EL, Haynes BF, Nadler LM, 21. Nicholson JK, Browning SW, Orloff SL, McDougal Bernstein ID, eds. Leukocyte Typing II: Human T JS. Inactivation of HIV-infected H9 cells in whole Lymphocytes. New York, NY: Springer-Verlag; blood preparations by lysing/fixing reagents used in 1986:3-30. flow cytometry. J Immunol Methods. 1993;160:215- 10. Kan EAR, Wang CY, Wang LC, Evans RL. 218. Noncovalently bonded subunits of 22 and 28 kd are 22. Procedures for the Collection of Diagnostic Blood rapidly internalized by T cells reacted with Anti– Specimens by Venipuncture; Approved Standard— Leu-4 antibody. J Immunol. 1983;131:536-539. Sixth Edition. Wayne, PA: Clinical and Laboratory 11. Knowles RW. Immunochemical analysis of the T- Standards Institute; 2007. CLSI document GP41-A6. cell–specific antigens. In: Reinherz EL, Haynes BF, 23. Enumeration of Immunologically Defined Cell Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Populations by Flow Cytometry; Approved Human T Lymphocytes. New York, NY: Springer- Guideline—Second Edition. Wayne, PA: Clinical and Verlag; 1986;1:259-288. Laboratory Standards Institute; 2007. CLSI 12. Nadler LM. B Cell/Leukemia Panel Workshop: document H42-A2. summary and comments. In: Reinherz EL, Haynes 24. Giorgi JV. Lymphocyte subset measurements: BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing significance in clinical medicine. In: Rose NR, II: Human B Lymphocytes. New York, NY: Friedman H, Fahey JL, eds. Manual of Clinical Springer-Verlag; 1986;2:3-43. Laboratory Immunology. 3rd ed. Washington, DC: 13. Cobbold SP, Hale G, Waldmann H. Non-lineage, American Society for Microbiology; 1986:236-246. LFA-1 family, and leucocyte common antigens: new 25. Jackson AL, Warner NL. Preparation, staining, and and previously defined clusters. In: McMichael AJ, analysis by flow cytometry of peripheral blood ed. Leucocyte Typing III: White Cell Differentiation leukocytes. In: Rose NR, Friedman H, Fahey JL, eds. Antigens. New York, NY: Oxford University Press; Manual of Clinical Laboratory Immunology. 3rd ed. 1987:788-803. Washington, DC: American Society for 14. van Dongen JJM, Krissansen GW, Wolvers-Tettero Microbiology; 1986:226-235. ILM, et al. Cytoplasmic expression of the CD3 26. Prince HE, Hirji K, Waldbeser LS, Plaeger-Marshall antigen as a diagnostic marker for immature T-cell S, Kleinman S, Lanier LL. Influence of racial malignancies. Blood. 1988;71:603-612. background on the distribution of T-cell subsets and 15. Brenner MB, McClean J, Dialynas DP, et al. Leu 11-positive lymphocytes in healthy blood Identification of a putative second T cell receptor. donors. Diagn Immunol. 1985;3(1):33-37. Nature. 1986;322:145-149. 27. Defining, Establishing, and Verifying Reference 16. Clevers H, Alarcón B, Wileman T, Terhorst C. The T Intervals in the Clinical Laboratory; Approved cell receptor/CD3 complex: a dynamic protein Guideline—Third Edition. Wayne, PA: Clinical and ensemble. Annu Rev Immunol. 1988;6:629-662. Laboratory Standards Institute; 2010. CLSI 17. Dörken B, Möller P, Pezzutto A, Schwartz-Albiez R, document EP28-A3c. Moldenhauer G. B-cell antigens: CD19. In: Knapp 28. Berti E, Parravicini C, Cattoretti G, Delia D, de W, Dörken B, Gilks WR, et al, eds. Leucocyte Braud F, Cusini M. Immunohistochemical reactivity Typing IV: White Cell Differentiation Antigens. New of anti-B cell monoclonal antibodies in thymus, York, NY: Oxford University Press; 1989:34-36. lymph node, and normal skin. In: Reinherz EL, 18. Schwinzer R. Cluster report: CD45/CD45R. In: Haynes BF, Nadler LM, Bernstein ID, eds. Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Leukocyte Typing II: Human B Lymphocytes. New Typing IV: White Cell Differentiation Antigens. New York, NY: Springer-Verlag; 1986;2:313-318. York, NY: Oxford University Press; 1989:628-634. 19. Centers for Disease Control. Perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR. 1988;37:377-388. 20. Protection of Laboratory Workers from Occupationally Acquired Infections; Approved