CD34/CD133 Enumeration Kit

For in vitro diagnostic use. REF 170-070-709

7 50 Sufficient for 50 tests with 10 cells

Miltenyi Biotec B.V. & Co. KG Friedrich-Ebert-Str. 68 51429 Bergisch Gladbach Germany +49 2204 8306-8484 www.miltenyibiotec.com

Contents

1. General information 2 1.1 Intended use 2 1.2 Reagents and contents 2 1.3 Materials required but not provided 3 1.4 Background information 4 1.5 Principle of the CD34/CD133 Enumeration Kit 5 2. Warnings and precautions 6 3. Protocol 7 3.1 Sample requirements 7 3.2 Important notes 8 3.3 Preparation of solutions 9 3.4 Staining of CD34+/CD133+ cells 9 4. Flow cytometric data acquisition and analysis 11 4.1 General description 11 4.2 Description of the detailed gating strategy 13 4.3 Data acquisition and analysis 21 4.4 Analysis of results using counting particles 22 5. Quality control 27 6. Reverse pipetting technique 27 7. Performance data 28 8. Limitations 31 9. References 31 10. Glossary of symbols 34

33513v03 | 2020-05 1 General information

1. General information

1.1 Intended use Determination of the absolute cell number of CD34+ and CD34+/CD133+ hematopoietic progenitor cells by flow cytometry in various human products, such as whole blood, leukapheresis harvest, , cord + + blood and all fractions of CD34 and CD133 cells separated using a CliniMACS System.

1.2 Reagents and contents Components 1 mL CD34/CD133 Staining Cocktail: cocktail consisting of anti-CD45-FITC (clone: 5B1; isotype: mouse IgG2a), anti-CD34-APC (clone: AC136; isotype: mouse IgG2a), and anti-CD133/2-PE (clone: 293C3; isotype: mouse IgG2b). 1 mL CD133 Control: cocktail consisting of anti-CD45-FITC (clone: 5B1; isotype: mouse IgG2a), anti-CD34-APC (clone: AC136; isotype: mouse IgG2a), anti-CD133/2-PE, and anti-CD133/2 pure (clone: 293C3; isotype: mouse IgG2b). 2×10 mL 10× Red Blood Cell Lysis Solution ammonium chloride based lysis solution 2 mL 7-AAD Solution (52.5 µg/mL)

50 Sufficient for 50 tests with 710 cells

Product format All components, except 10× Red Blood Cell Lysis Solution, are supplied in buffer containing stabilizer and 0.05% sodium azide.

2 33513v03 | 2020-05 General information

Store protected from at +2 °C to +8 °C. Do not freeze. The use-by date is indicated on the vial label. For in-use stability at +2 °C to +8 °C storage temperature refer to the use-by date indicated on the vial label. Do not use the reagent after the use-by date. Disposal Chemical residues and remains should be routinely handled as special waste. This must be disposed in compliance with anti-polution and other laws of the country concerned. To ensure compliance contact relevant authorities or an approved waste-disposal company.

1.3 Materials required but not provided • Micropipettes with disposable tips: variable micropipettes with volume ranges of 10–100 µL and 100–1000 µL. • Flow cytometer able to detect 4-color fluorescence (488 nm argon laser, 635 nm red diode laser) and equipped with appropriate computer hardware and software. • Vortex mixer • Double-distilled water • BD Trucount™ tubes (BD cat. no. 340334) • Buffer for optional dilution steps: Prepare a PEB (PBS/EDTA/BSA) buffer containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA) or human serum albumin (HSA), and 2 mM EDTA, e.g., by diluting MACS BSA Stock Solution (# 130‑091‑376) 1:20 with autoMACS® Rinsing Solution (# 130‑091‑222). Keep buffer cold +2( °C to +8 °C). Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced

33513v03 | 2020-05 3 General information

by other , such as human serum albumin, human serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not recommended for use.

1.4 Background information The CD34 antigen is a single-chain transmembrane , expressed on human hematopoietic stem and progenitor cells constituting a small subpopulation of bone marrow cells and peripheral blood cells. The antigen is absent on fully differentiated hematopoietic cells, such as normal peripheral blood lymphocytes, monocytes, granulocytes, erythrocytes, and platelets. After severe damage, for example, after myeloablative conditioning, the hematopoietic system can be reconstituted by transplantation of allogenic or autologous CD34+ hematopoietic progenitor cells. Clone AC136 recognizes a class III epitope of the CD34 antigen. The CD133 antigen is a 5-transmembrane cell surface antigen with a molecular weight of 117 kDa. It is expressed on a subset of CD34 bright stem and progenitor cells in human fetal liver, bone marrow, cord blood, and peripheral blood but is not found on mature blood cells1. In contrast to the CD34 antigen, CD133 is not expressed by late progenitors, such as pre-B-cells, CFU-E, and CFU-G2,3. CD133 has also been found to be expressed on circulating endothelial progenitor cells and fetal neural stem cells as well as on other tissue-specific stem cells, such as renal, prostate, and corneal stem cells4-6. Clone 239C3 recognizes epitope 2 of the CD133 antigen. CD34+ and CD34+/CD133+ cells may have various therapeutic uses. CD34 and CD133 enriched grafts have been employed in autologous7,8 and allogenic transplantation both in the haploidentical9-11 as well as the HLA- matched setting12. The high potential for hematopoietic engraftment of isolated CD133+ cells has been shown in NOD/SCID repopulation assays13,14. CD34 and CD133 enriched cell fractions have also been used as starting fraction for ex vivo expansion of hematopoietic progenitor cells from cord blood15-18.

4 33513v03 | 2020-05 General information

CD133+ stem cells can also be utilized in non-hematological applications, such as regenerative medicine. CD133 is expressed on stem/progenitor cells of different tissues including bone marrow, peripheral blood, cord blood, and liver. CD133+ stem cells have been shown to harbor the capability to differentiate into cell types of various tissues, for example, endothelial cells, neural cells, and hepatocytes. CD133+ stem cells have come into focus in non- hematological applications especially in ischemic heart diseases19-26.

1.5 Principle of the CD34/CD133 Enumeration Kit The gating strategy of the CD34/CD133 Enumeration Kit is based on the ISHAGE guidelines27 for simple and standardized enumeration of CD34+ cells. By employing anti-CD45-FITC, anti-CD34-APC, and anti-CD133/2-PE the allows the identification of CD45+ leucocytes, of CD34+ hematopoietic progenitor cells, which are dim for CD45+ fluorescence and show a low side scatter and of CD34+/CD133+ hematopoietic progenitor cells, which are also CD45dim with a low side scatter. Additionally, for each sample a control sample is stained with the CD133 control containing anti-CD45-FITC, anti-CD34-APC, anti-CD133/2-PE and anti-CD133/2 pure antibody to block any CD133 staining. This is required for the proper setting of the relevant gate during analysis. The kit is suitable for single platform technique²⁸, enabling the absolute cell count determination by analysis of commercially available counting particles present in BD Trucount™ tubes. The CD34/CD133 Enumeration Kit has been designed for use with flow cytometers able to detect 4-color fluorescence (488 nm argon laser, 635 nm red diode laser) and equipped with appropriate computer hardware and software. Optionally, dead cells can be excluded from the analysis by addition of the DNA stain 7-aminoactinomycin D (7-AAD), which is also included in the kit. 7-AAD diffuses through the cell membrane of dead cells and intercalates with their DNA.

33513v03 | 2020-05 5 Warnings and precautions

2. Warnings and precautions • Analysis results obtained by the use of the CD34/CD133 Enumeration Kit shall never be the sole basis for diagnosis and/or therapy of patients with hematological malignancies. • The interpretation of the results is under the full responsibility of the user. • To verify the analysis results every determination should be repeated. • In case of unexpected results repeat the measurement or contact Miltenyi Technical Support +49 2204 8306-8484. • For all handling, consideration of good laboratory practice (GLP) regulations is recommended. • The use of the CD34/CD133 Enumeration Kit is restricted to trained and qualified personnel only. • The reagent should not be used if signs of leakage are observed. • All biological specimens and all materials that come into contact with blood and blood products must be treated as infectious material. Regulations for the treatment and disposal of infectious material must be followed. • The staining cocktails of the CD34/CD133 Enumeration Kit contain sodium

azide (NaN3), a chemical highly toxic in pure form. However, at product concentrations, it is not classified as hazardous. Sodium azide may react with lead and copper plumbing to form highly explosive buildups of metal azides. Upon disposal, flush with large volumes of water to prevent metal azide build-up in plumbing. Safety guidelines must be observed. • The CD34/CD133 Enumeration Kit contains a vial of 7-AAD solution, a chemical highly toxic in pure form. However, at product concentrations, it is not classified as hazardous. Pure 7-AAD is potential carcinogen. Although this component is highly deluted, we recommend to avoid contact with skin and eyes, to wear suitable protective clothing and gloves and appropriate eye/face protection.

6 33513v03 | 2020-05 Protocol

• Do not inhale vapors or dusts. Avoid contact with substance. Ensure adequate supply of fresh air. • For material required but not provided the manufacturers recommendations and safety regulations must be followed.

• The NH4Cl based Red Blood Cell Lysis Solution is 10× concentrated. Although this mixture is not classified as dangerous we recommend to avoid contact with skin and eyes, to wear suitable protective clothing and gloves and appropriate eye/face protection.

3. Protocol

3.1 Sample requirements The CD34/CD133 Enumeration Kit can be used for the enumeration of CD34+ and CD34+/CD133+ cells by flow cytometry from a variety of human cell sources, including whole blood, leukapheresis harvest, bone marrow, cord blood, and all fractions of CD34+ and CD133+ cells separated using a CliniMACS System. Each cell source can have different storage conditions and limitations that should be considered prior to collection and analysis. For the collection of patient samples European and national legislation must be followed. • The samples must be stained within 24 hours. Whole blood samples must be stained within 12 hours. • The viability of the cells should be at least 90%. • For the cell staining procedure a minimum of 200 µL sample is required. • The cell concentration of white blood cells (WBC) or positive enriched cells must not exceed 5×107 cells/mL. If necessary dilute the sample with PEB buffer. Record the pre-dilution factor for the calculation of the final cell concentrations.

33513v03 | 2020-05 7 Protocol

• Due to potential aggregates in bone marrow or cord blood, samples may have to be filtrated before staining and analysis. Filtration can be performed e.g. with Miltenyi Biotec Pre-Separation Filters (REF: 130-095-823).

Note: Filtration may cause cell loss. If possible, request fresh sample material and filtrate only in cases of need.

3.2 Important notes • For all steps pipetting blood samples or Red Blood Cell Lysis Solution use the reverse pipetting technique to assure accurate determination of absolute cell counts. (For more information see chapter 6) • Make sure to use the correct particle count (printed on pouch label) for cell count calculation when using counting particle tubes for analysis. • Make sure to analyze stained and lysed samples within one hour after staining and store the samples on ice until analysis. • Under some conditions red blood cells may not lyse within 10 minutes. In this case extend lysis time to 12 minutes before storing the samples on ice. • Make sure to use the gating strategy described in chapter 4 of this package insert. • Set the gates properly, as improper setting of the gates may create erroneous results.

8 33513v03 | 2020-05 Protocol

3.3 Preparation of solutions Preparation of 1× Red Blood Cell Lysis Solution 1. Dilute 10× Red Blood Cell Lysis Solution 1:10 with double-distilled water

(ddH2O), for example, dilute 1 mL of 10× Red Blood Cell Lysis Solution with

9 mL of ddH2O. Note: Do not dilute with deionized water.

2. Store the prepared 1× Red Blood Cell Lysis Solution at room temperature. Discard unused solution at the end of the day.

3.4 Staining of CD34+/CD133+ cells 1. For each sample label two BD Trucount tubes with A (CD133 Control) and B (CD34/CD133 Staining Cocktail) 2. For each sample tube use a fresh micropipette tip. 3. Add 20 µL of CD133 Control into tube A and 20 µL of CD34/CD133 Staining Cocktail into tube B. 4. Add 20 µL of 7-AAD solution into both tubes. 5. Carefully pipette 100 µL of well-mixed sample to the bottom of each tube. Use reverse pipetting (see chapter 6) for all pipetting steps with blood products or 1× RBC Lysis Solution. Immediately vortex thoroughly for 3 seconds and incubate for 10 minutes at 2–8 °C in the dark. 6. Add 1860 µL of 1× RBC Lysis Solution to each tube. Immediately vortex thoroughly for 3 seconds and incubate for 10 minutes at room temperature in the dark. 7. The analysis should be performed within one hour after staining. 8. Store the samples on ice until analysis.

33513v03 | 2020-05 9 Protocol

Short instructions

1. Prepare samples according to the following table.

Sample Name Reagent [µL] Specimen [µL] CD133 CD34/CD133 7-AAD Control Staining Cocktail

A: CD133 20 - 20 100 Control

B: CD34/CD133 - 20 20 100 Staining

2. Vortex thoroughly and incubate for 10 minutes at 2-8°C in the dark.

3. Add 1860 µL of 1× Red Blood Cell Lysis Solution to each tube.

4. Vortex thoroughly and incubate for 10 minutes at room temperature in the dark.

5. Store on ice until analysis. Analyze within one hour after staining.

Table 1: Short instructions

10 33513v03 | 2020-05 Flow cytometric data acquisition and analysis

4. Flow cytometric data acquisition and analysis

4.1 General description The threshold is set on the CD45-FITC channel during data acquisition and analysis. • CD34+ and CD34+/CD133+ progenitor cells express CD45 with lower staining intensity than lymphocytes. Be careful when setting the threshold and do not exclude these events from the analysis. For data acquisition and analysis create the following dot plots (the regions are set according to the examples shown in plots a) to k): a Plot: Forward scatter (FSC) vs. Side scatter (SSC) b Plot: CD45-FITC vs. SSC c Plot: 7-AAD vs. SSC d Plot: CD34-APC vs. SSC e Plot: CD45-FITC vs. SSC f Plot: Forward scatter vs. SSC g Plot: CD133/2-PE vs. CD34-APC h Plot: CD45-FITC vs. SSC i Plot: FSC vs. SSC j Plot: CD133/2-PE vs. CD45-FITC k Plot: Time vs. CD133/2-PE

33513v03 | 2020-05 11 Flow cytometric data acquisition and analysis

Definition of logical gates Define and label the gates as described in the following table:

Gate Target Definition

G1 Debris Not R1

G2 WBCs Not R1*R2

G3 Viable WBCs Not R1*R2*R3

G4 Not R1*R2*R3*R4

G5 Cluster control CD34 Not R1*R2*R3*R4*R5

G6 CD34+ HPCs Not R1*R2*R3*R4*R5*R6

G7 Not R1*R2*R3*R4* R5*R6*R7

G8 Cluster control CD133 Not R1*R2*R3*R4*R5*R6*R7*R8

G9 CD133+ HPCs Not R1*R2*R3*R4*R5*R6*R7*R8*R9

G10 R10

G11 Counting beads R10*R11

Table 2: Definition of logical gates

12 33513v03 | 2020-05 Flow cytometric data acquisition and analysis

4.2 Description of the detailed gating strategy The detailed description of the gating strategy is shown using a bone marrow sample as an example. The regions are defined during data acquisition of the CD133 control sample. a Plot: FSC vs. SSC Activated gate: no gate Region R1 is defined in this plot. R1 includes the SSC low and FSC low events. The events inside this region should be excluded from the further analysis by creating a gate G1=not R1.

Side scatter R1

Forward scatter

33513v03 | 2020-05 13 Flow cytometric data acquisition and analysis b Plot: CD45-FITC vs. SSC Activated gate: G1=not R1 Set R2 thereby excluding all CD45-negative events from the analysis while all CD45+ cells are included. • CD34+ and CD34+/CD133+ progenitor cells express CD45 with lower staining intensity than lymphocytes. Be careful when setting region R2 and do not exclude these events from the analysis.

R2

Side scatter

CD45-FITC c Plot: 7-AAD vs. SSC Activated gate: G2=not R1*R2 Define region R3 thereby including all viable WBCs. Generate a gate statistic of the plot for later calculations.

Side scatter

R3

7-AAD

14 33513v03 | 2020-05 Flow cytometric data acquisition and analysis d Plot: CD34-APC vs. SSC Activated gate: G3=not R1*R2*R3 Define region R4 thereby including all CD34+ cells with a low SSC. The position of R4 is defined by the properties of the stem cells.

Side scatter

R4

CD34-APC e CD34 cluster control: CD45-FITC vs. SSC Activated gate: G4=not R1*R2*R3*R4 The CD34+ cells form a cluster with low SSC and dim CD45 fluorescence and are gated in region R5. Non-specifically stained events are excluded from this region.

R2

R5 Side scatter

CD45-FITC

33513v03 | 2020-05 15 Flow cytometric data acquisition and analysis f CD34+ HPC determination: FSC vs. SSC Activated gate: G5=not R1*R2*R3*R4*R5 Define a region R6 that identifies a cluster of events meeting all the fluorescence and light scatter criteria of CD34+ progenitor cells. Cells clustered in region R6 exhibit slightly higher forward scatter than that of small lymphocytes and uniformly low side scatter. Any events falling outside region R6 are not included in the % viable CD34+ cells determination. The proper setting of this region should be checked by showing this region in the first Dot Plot (a). The region has to enclose all lymphocytes.

Side scatter R6

Forward scatter

16 33513v03 | 2020-05 Flow cytometric data acquisition and analysis g Plot: CD133/2-PE vs. CD34-APC Activated gate: G6=not R1*R2*R3*R4*R5*R6 Define region R7 with the CD133 Control (Tube A) to include all CD133 positive events. It is important to set region R7 as close as possible to the CD133 negative events to include all CD133 positive and CD133dim-positive cells. A maximum of 10 events is allowed in region R7 on the CD133 control sample. CD34/CD133 Staining sample CD133 Control sample

R7 R7 APC APC 4- 4- CD3 CD3

CD133/2 (293C3)-PE CD133/2 (293C3)-PE

33513v03 | 2020-05 17 Flow cytometric data acquisition and analysis h CD133 cluster control: CD45-FITC vs. SSC Activated gate: G7= not R1*R2*R3*R4*R5*R6*R7 The CD34+/CD133+ cells form a cluster with low SSC and dim CD45 fluorescence and are gated in region R8. Non-specifically stained events are excluded from this region.

R2

R8 Side scatter

CD45-FITC

18 33513v03 | 2020-05 Flow cytometric data acquisition and analysis i CD34+/CD133+ HPC determination plot: FSC vs. SSC Activated gate: G8=not R1*R2*R3*R4*R5*R6*R7*R8 Define a region R9 that identifies a cluster of events meeting all the fluorescence and light scatter criteria of CD34+/CD133+ progenitor cells. Cells clustered in region R9 exhibit slightly higher FSC than that of small lymphocytes and uniformly low SSC. Any events falling outside region R9 are not included in the % viable CD133+ cells determination. The proper setting of this region should be checked by showing this region in the first Dot Plot (a). The region has to enclose all lymphocytes.

Side scatter R9

Forward scatter

33513v03 | 2020-05 19 Flow cytometric data acquisition and analysis

After gating of the cells the counting particles have to be gated according to the following two dot plots. j Plot: CD133/2-PE vs. CD45-FITC Activated gate: no gate Define region R10 by including all FITC and PE double bright counting beads.

R10 C CD45-FIT

CD133/2 (293C3)-PE k Plot: Time vs. CD133/2-PE Activated gate: G10=R10 Define region R11 by including all monodisperse- and bidisperse-counting beads.

R11

33/2 (293C3)-PE CD1

Time [sec.]

20 33513v03 | 2020-05 Flow cytometric data acquisition and analysis

4.3 Data acquisition and analysis The samples are analyzed in the following order: 1. CD133 Control (tube A) 2. CD34/CD133 Staining Sample (tube B) Set the collection criteria in the window “acquisition mode” to “event count” of gate G9 and collect at least 100 CD133+ HPC per tube. Use this gate G9 only as collection criteria, but save complete data file containing all events. • Not less than 100,000 CD45+ events per tube and a minimum of 100 CD133+ events should be counted. When analyzing a target cell fraction of a CliniMACS Separation at least 10,000 CD45+ events should be counted. Additionally, a minimum of 1000 counting particles or a maximum of 10 minutes acquisition time should be sufficient.

33513v03 | 2020-05 21 Flow cytometric data acquisition and analysis

4.4 Analysis of results using counting particles For the calculations the gate statistics of plot c are used. Examples of these statistics are shown in table 3.

To calculate the number of viable WBCs in the sample volume of 100 µl, the events of viable WBCs (G3, gate statistic plot C) are multiplied by the total counting particle number per tube as indicated by the manufacturers instructions, and divided by the acquired particle count gated in G11 (statistic plot C). To get the concentration of viable WBCs per mL multiply this result by factor 10. In case of pre-dilution of the sample you have to multiply this result by the pre-dilution factor.

WBC concentration [cells/mL] =

Events of viable WBC (G3) × total counting particles per tube × 10 × pre-dilution Acquired counting particles (G11) factor

To calculate the viability of WBCs [%], the events of viable WBCs (G3) is divided by the events of WBCs (G2) minus bead count (G11) and multiplied by 100.

Events of viable WBC (G3) Viability of WBC [%] = × 100 (Events WBC (G2) – particle count (G11))

22 33513v03 | 2020-05 Flow cytometric data acquisition and analysis

The frequency of CD34+ HPC [%], can be calculated by dividing the event count of viable CD34+ HPC (G6) by the event count of viable WBC (G3) and multiplying by 100.

Events CD34+ HPC (G6) Frequency of CD34+ HPC [%] = × 100 Events viable WBC (G3)

To calculate the number of viable CD34+ HPC in the sample volume of 100 µl, the events of CD34+ HPC (G6, gate statistic plot C) are multiplied by the total counting particle number per tube and divided by the acquired particle count gated in G11 (statistic plot C). To calculate the concentration of viable CD34+ HPC per ml multiply this result by factor 10. In case of pre-dilution of the sample you have to multiply this result by the pre-dilution factor.

Viable CD34+ HPC concentration [cells/mL] =

Events CD34+ HPC (G6) × total counting particles per tube × 10 × pre-dilution Acquired counting particles (G11) factor

33513v03 | 2020-05 23 Flow cytometric data acquisition and analysis

The frequency of CD34+/CD133+ HPC [%] can be calculated by dividing the event count of viable CD133+ HPC (G9) by the event count of viable WBC (G3) and multiplying by 100.

Events CD133+ HPC (G9) Frequency of CD34+/CD133+ HPC [%] = × 100 Events viable WBC (G3)

To calculate the number of viable CD133+ HPC in the sample volume of 100 µl, the events of CD133+ HPC (G9, gate statistic plot C) are multiplied by the total counting particle number per tube as indicated by the manufacturers instructions, and divided by the acquired particle count gated in G11 (statistic plot C). To get the concentration of viable CD133+ HPC per mL multiply this result by factor 10. In case of pre-dilution of the sample you have to multiply this result by the pre-dilution factor.

Viable CD34+/CD133+ HPC [cells/mL] =

Events CD133+ HPC (G9) × total counting particles per tube × 10 × pre-dilution Acquired counting particles (G11) factor

24 33513v03 | 2020-05 Flow cytometric data acquisition and analysis

Sample ID: CD34/CD133 staining Gate: G2 (WBC) Total events: 324691 Gated events: 320265

Gate Events Gated [%]

G1 320265 100.00

G2 (WBCs) 320265 100.00

G3 (viable WBCs) 309303 100.00

G4 332 0.10

G5 (cluster control CD34) 281 0.09

G6 (CD34+ HPC) 270 0.08

G7 219 0.07

G8 (cluster control CD133) 199 0.06

G9 (CD133+ HPC) 199 0.06

G10 5490 1.71

G11 (counting beads) 5458 1.70

Table 3: Statistic of plot c

33513v03 | 2020-05 25 Flow cytometric data acquisition and analysis

Calculation example

Description Source Value

Pre-dilution factor 1

Events viable WBC (G3) Statistic plot C 309303

Total counting particles per tube Manufacturer's instructions 51511

Acquired bead count (G11) Statistic plot C 5458

Events WBC (G2) Statistic plot C 320265

Events CD34+ HPC (G6) Statistic plot C 270

Events CD133+ HPC (G9) Statistic plot C 199

Table 4: Required values Viable WBC concentration [cells/mL] = (309303 × 51511) / 5458 × 10 × 1 = 2.92 × 10⁷ Viability of WBC [%] = 309303 / (320265 – 5458) × 100 = 98.25% Frequency CD34+ HPC = (270 / 309303) × 100 = 0.09% Viable CD34+ HPC concentration [cells/mL] = (270 × 51511) /5458 × 10 × 1 = 2.55 × 10⁴ Frequency CD133+ HPC = (199 / 309303) × 100 = 0.06% Viable CD133+ HPC concentration [cells/mL] = (199 × 51511)/ 5458 × 10 × 1 = 1.88 × 10⁴

26 33513v03 | 2020-05 Quality control

5. Quality control It is recommended to run regularly a commercially available whole blood control to optimize instrument settings and as a quality control of the system. As most commercially available controls consist of fixed cells these samples should be stained without addition of 7-AAD.

6. Reverse pipetting technique • Make sure to use a fresh pipette tip for each sample. • Use calibrated pipettes only. • If using an adjustable pipette, select the desired volume to be transferred. 1. Attach an appropriate tip to the pipette. 2. Press the pipette plunger down to the second stop and hold the plunger in this position. 3. Place the pipette tip into the sample to a depth of approximately 1 cm and slowly release the plunger. Wait 1–2 seconds and withdraw the pipette from the sample, make sure there are no bubbles in the tip. Carefully wipe any excess fluid from the exterior of the tip without touching the bottom of the tip. 4. Dispense the sample into the receiving tube by pressing the plunger down slowly and steadily to the first stop. Wait 1–2 seconds to allow complete dispensing of the sample. This volume is equal to the desired volume. 5. Remove the pipette tip from the receiving vessel without dispensing the remaining fluid.

33513v03 | 2020-05 27 Performance data

7. Performance data Performance characteristics were obtained at Miltenyi Biotec using the CD34/ CD133 Enumeration Kit for determination of absolute CD34+ and CD34+/CD133+ cell counts in fresh whole blood, leukapheresis harvest, cord blood, bone marrow, CliniMACS CD34+ fraction and CliniMACS CD133+ fraction, respectively. The CD34/CD133 Enumeration Kit had a reproducibility of CV < 0.1 for all specimen tested. Precision Interlaboratory testing was performed at one site by two individual operators. Each three donor samples of leukpaheresis harvest, bone marrow and a CliniMACS CD133+ fraction were split into two aliquots and analyzed by each operator in triplicates. Figure 1 shows a regression plot summarizing viable cell counts/mL of CD34+ and CD34+/CD133+ HPC from all samples.

28 33513v03 | 2020-05 Performance data

Reproducibility CD34/CD133 Enumeration Kit

1,20E+05 Results: CD34+ HPC CD133+ HPC CD34 1,00E+05 R2 = 0.96 Slope = 0.96

8,00E+04 CD133 R2 = 0.97 Slope = 0.95

6,00E+04

4,00E+04 Results Operator 2 [HPC/mL] 2 Operator Results

2,00E+04

0,00E+00 0,00E+00 2,00E+04 4,00E+04 6,00E+04 8,00E+04 1,00E+05 1,20E+05

Results Operator 1 [HPC/mL]

Figure 1: Regression plot Reproducibility

33513v03 | 2020-05 29 Performance data

Additional tests comparing the CD34/CD133 Enumeration Kit with a competitor kit* regarding CD34+ HPC count/mL were performed. Figure 2 shows a regression plot combining the results of these tests. Viable CD34+ cells/mL

1,50E+05 Results:

R2 = 0.93 1,25E+05 Slope = 0.995

1,00E+04

7,50E+04

5,00E+04

2,50E+04 Miltenyi CD34/CD133 Enumeration Kit Miltenyi CD34/CD133

0,00E+00 0,00E+00 2,50E+04 5,00E+04 7,50E+04 1,00E+05 1,25E+05 1,50E+05

Competitor kit*

Figure 2: Regression plot of kit comparison

* (BD Stem Cell Enumeration Kit)

30 33513v03 | 2020-05 Limitations

8. Limitations • The samples must be stained within 24 hours. Whole blood samples must be stained within 12 hours. • The viability of the cells should be at least 90%. • For the cell staining procedure a minimum of 200 µL sample is required. • The cell concentration of white blood cells (WBC) or positive enriched cells must not exceed 5×107 cells/mL. If necessary dilute the sample with PEB buffer. Record the pre-dilution factor for the calculation of the final cell concentrations.

9. References 1. Yin, A. H. et al. (1997) AC133, a Novel Marker for Human Hematopoietic Stem and Progenitor Cells. Blood 90: 5002–5012. 2. Freund, D. et al. (2006) Comparative analysis of proliferative potential and clonogenicity of MACS-immunomagnetic isolated CD34+ and CD133+ blood stem cells derived from a single donor. Cell Prolif. 39: 325–332. 3. Takahiro, S., et al. (2006) Highly Efficient Ex Vivo Expansion of Human Hematopoietic Stem Cells Using Delta1-Fc Chimeric . Stem Cells 24: 2456–2465. 4. Gehling, U. et al. (2000) In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood 95: 3106–3112. 5. Peichev, M. et al. (2000) Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors. Blood 95: 952–958. 6. Uchida, N. et al. (2000) Direct isolation of human central nervous system stem cells. Proc. Natl. Acad. Sci. USA 97: 14720–14725. 7. Koehl, U. et al. (2002) Autologous transplantation of CD133 selected hematopoietic progenitor cells in a pediatric patient with relapsed leukemia. Bone Marrow Transplant. 29: 927–930.

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33513v03 | 2020-05 33 Glossary of symbols

10. Glossary of symbols

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34 33513v03 | 2020-05 Glossary of symbols

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Control

50 Sufficient for 50 tests

In-use stability

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