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CELL SEPARATION PRODUCTS FORHLA ANALYSIS

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TABLE 1

CELL TYPE SELECTION METHODSEROLOGY (SEE PAGE 4) FCXM (SEE PAGE 6) CHIMERISM (SEE PAGE 8)

PBMC1 BLOOD2 PBMC1 BLOOD2 PBMC1 BLOOD2

TOTALLYMPHOCYTES

Positive 18684HLA3

Negative

15263HLA3

19961HLA

15263HLA

T CELLS

PositiveCD2 18657HLA 18687HLA 18657HLA 18687HLA

CD3 18051HLA4 18081HLA4 18051HLA 18081HLA 18051HLA 18081HLA

Negative

19051HLA15061HLA19951HLA

19051HLA15061HLA19951HLA

Lymphoid(CD3+)

15271HLA

B CELLS

PositiveCD19CD20

CD19/CD20

18054

1808418685HLA5

18054

18454HLA5

18084

18184HLA5

Negative19054HLA

15064HLA19954HLA

19054HLA

15064HLA19954HLA

MYELOID CELLS/GRANULOCYTES

Positive

CD15CD33

CD33/66b

CD66b

18257

18681HLA18287HLA18683HLA

18682

NegativeMyeloid

Granulocyte

15272HLA15121

MONOCYTES Positive CD14 18058 18088

NK CELLS Positive CD56 18055 18085HLA

HEMATOPOIETICPROGENITORS

Positive CD34 18056 18086

1. PBMC= Peripheral Blood Mononuclear Cell ; kit will also work on single cell suspension of bone marrow, spleen, and lymph node

2. Kit also works on other red blood cell containing samples (i.e. cord blood, buffy coat, bone marrow).

3. Red = RosetteSep kits, Purple = EasySep kits

4. Please contact STEMCELL Technologies for compatibilit y with other products.

5. Coming Soon!

PRODUCT LISTING FOR

HLA APPLICATIONS

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CELL SE PARATION PRODUCTS FOR HLA ANALYSIS

3

The role of the HLA (Human Leukocyte Antigen) complex is of critical importance in immune responses and transplant medicine.

Since the late 1960s, research on human organ and tissue transplantation reinforced the importance of HLA matching for graft

survival and transfusion efficiency. Today, HLA testing is a standard procedure that is important in organ and hematopoietic

transplantation.

The specific and varied distribution of HLA antigens requires precise, accurate, and consistent HLA testing. In a busy HLA lab,

finding powerful tools that facilitate accurate HLA testing while processing a high number of samples is important.

At STEMCELL Technologies, we provide highly optimized cell separation solutions for a variety of HLA applications to facilitate

high-volume sample processingandclear,reliable test results. Our HLA cell isolation products have been formulated for:

Two cell isolation systems are available for these HLA tests: RosetteSep(see page 10) and EasySep(see page 11), see Table 1.

In addition, EasySep can be completely automated using RoboSep, The Fully Automated Cell Separator (see pages 12-13).

OUR CELL ISOLATION SYSTEMS ARE PARTICULARLYSUITABLE FOR HLA LABS AS ALL PROCEDURES ARE:

GENTLE ON CELLS

QUICK

EASY

PERFORMED AT ROOM TEMPERATURE

Serology (see pages 4-5)

Flow Cytometry Crossmatch (FCXM) (see pages 6-7)

Chimerism Analysis (see pages 8-9)

CELL SEPARATION SOLUTIONS FOR

HLA ANALYSIS

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FIGURE 1: Cells separated using EasySep HLA reagents andanalysed in a Complement-Dependent Cytotoxicity assay(CDC assay).

ROSETTESEP

Immunodensity cell separation procedure - isolates untouched T and B cells directly from whole blood

simply and quickly (see page 10).

Cell preparations are cleaner and less damaged when analyzed by microscope (see Figure 2).

Desired cells isolated with purities of up to 95% for accurate serological HLA testing.

EASYSEP

Immunomagnetic cell separation procedure - isolates cells with purities of up to 99% (see page 11).

The small magnetic particle size reduces the risk of membrane shearing and damage to the cell

compared with other magnetic isolation systems (see Figure 2).

Clear, simple, and readable serological HLA testing.

For clear and reliable analysis of serological HLA testing, RosetteSep and EasySep kits are the ideal solution for the isolation of

clean cell preparationsthat show bright and uniform fluorescence under the microscope (see Figure 1 and Figure 2). Both systemsare quick, efficient, and cost-effective for serological HLA testing and crossmatching and allow for simpler scoring with less ambiguity

and greater productivity and confidence (see Table 2).

SEROLOGY

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RosetteSep Enrichment.Cells are clean and untouched.

EasySep Positive Selection.The tiny magnetic particles used in the EasySepprocedure are invisible under the microscope.

Other Magnetic Isolation Method.The large brown magnetic particles cover the cellsand interfere with observation.

T cells were isolated from blood samples using RosetteSep (A), EasySep Positive Selection (B), and another commerciallyavailable magnetic positive selection procedure often used in HLA labs (C). After isolation, the cell suspensions obtained wereanalyzed by microscopy.

TABLE 2: Cell Separation Kits for Serological HLA Testing and crossmatch analysis

CELL TYPE SELECTION

ROSETTESEP EASYSEP ROBOSEP

BUFFY COAT/

WHOLE BLOOD1PBMC, BM,

SPLEEN, LN2BUFFY COAT/



WHOLE BLOOD1

T CELLSPositive

CD2 - 18657HLA 18687HLA 18657HLAR 18687HLAR

CD3 - 18051HLA3 18081HLA3 18051HLAR3 18081HLAR3

Negative 15061HLA 19051HLA 19951HLA 19051HLAR 19951HLAR

B CELLSPositive

CD19CD20

--

18054-

1808418685HLA4

18054R-

18084R18685HLAR4


1. Kit also works on other red blood cell containing samples

(i.e. cord blood, buffy coat, bone marrow).

2. PBMC = Peripheral Blood Mononuclear Cell; BM = Bone Marrow; LN = Lymph Node

3. Please contact STEMCELL Technologies for compatibility with other products.

4. Coming Soon!

FIGURE 2: Comparison of 3 Cell Isolation Methods.

1

2

3

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ROSETTESEP

Ideal for quick and efficient isolation of clean, untouched cells directly from whole blood (see page 10).

Unwanted cells are targeted for depletion by monoclonal antibodies that link them to red blood cells

and pellet during a quick Ficoll density-centrifugation.

Desired T or B cells are immediately available for flow cytometry for cleaner FCXM analysis.

All RosetteSep HLA kits contain HLA-specific protocols that use less reagents per sample and

are available to process 250 mL or 1000 mL of blood per kit for increased savings.

EASYSEP

EasySep negative selection kits isolate untouched cells with purities up to 99% (see page 11).

EasySep positive selection kits label cells with small magnetic particles that are flow cytometry

compatible (see Figure 3).

Positively isolated cells can be directly stained and analyzed by flow cytometry without removing

the magnetic particles (see Figure 4).

Isolates cells with minimal background fluorescence when performing FCXM analysis (see Figure 4).

ROBOSEP

To process numerous samples and save a significant amount of hands-on time, cell isolations can be

fully automated with RoboSep (see pages 12-13).

Uses EasySep reagents to label and separate cells.

Standardizes cell separation protocols with automation for clinical labs.

Eliminates cross-contamination by using unique sets of disposable tips.

Saves technicians time for faster sample processing.

When using FCXM assays to test for the presence of donor-reactive antibodies in a patients serum, it is important that

cell preparations are clean and maintain cell viability. Any damage to or activation of the cells during separation may

compromise the cell membrane or alter antigen expression, possibly causing erroneous results and non-specific, high background

staining.

RosetteSep, EasySep, and RoboSep are ideal products for FCXM analysis as all kits are flow cytometry compatible and all

procedures can be performed at room temperature for easier sample processing (see Table 3).

FLOW CYTOMETRY CROSSMATCH (FCXM)

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TABLE 3: Products for FCXm

CELL TYPE SELECTIONROSETTESEP

EASYSEP

ROBOSEP

BUFFY COAT/





WHOLE BLOOD1

T CELLSPositive




B CELLS Negative 15064HLA 19054HLA 19954HLA 19054HLAR 19954HLAR

TOTALLYMPHOCYTES

Negative 15263HLA - 19961HLA - 19961HLAR



FIGURE 4: Flow Cytometric Crossmatch Analysis Following EasySepHLA Whole Blood CD3 Positive Selection

%

ofMax

AHG FITC

Positively selected cells were incubated withoutserum (blue), with negative serum (red) and with

positive serum (green), and stained with FITC-conjugated goat anti-human globulin (AHG).

FIGURE 3: Scanning Electron Micrograph of B Cell IsolatedUsing EasySep Positive Selection

(10,000X magnification, 200 nm pore size)

The EasySep magnetic particles are small anddo not interfere with flow cytometry analysis.

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ROBOSEP

Avoids cross-contamination between samples since each individual sample is handled with unique tips -

instead of multi-use columns and tubings.

The only machine that can fully automate all cell separation procedures for large, routine labs (see pages 12-13).

Capacity to isolate 1 to 4 samples from a variety of sources including buffy coat or whole blood, and

store standardized and customized protocols in its 30GB hard-drive.

EASYSEP

EasySep positive selection kits provide highly purified cell populations for chimerism analysis (see Table 4).

Cells can be isolated directly from buffy coat or whole blood, which saves time.

EasySep kits are available for isolating myeloid and lymphoid cells, or individual cell populations

such as CD3+, CD14+, CD15+, CD19+, CD33+, CD34+, CD56+ and more (see Table 4).

Sequential isolation protocols are available to maximize cell recovery of multiple cell types

from a single sample.

ROSETTESEP

Ideal for quick and efficient isolation of cells directly from whole blood (see page 10).

Unwanted cells are targeted for depletion by monoclonal antibodies that link them to red blood cells and pellet during a quick Ficoll density-centrifugation.

Desired cells are isolated with high purity.

All RosetteSep HLA kits contain HLA-specific protocols that use less reagents per sample and are available

to process 250 mL or 1000 mL of blood per kit for increased savings.

Chimerism analysis is important for monitoring post-transplant outcome and for determining appropriate therapy after transplantation.

Since it is the basis for providing early treatment decisions, chimerism analysis requires sensitive and reliable techniques to detect

the presence of donor leukocytes in the hosts peripheral blood or bone marrow. In addition, cross-contamination between samples

is always a concern since the presence of even a few cells from another patient can alter the results of the chimerism analysis.

STEMCELL Technologies has developed RoboSep, the only instrument to offer true walk-away automation of immunomagnetic cell

separation using EasySep technology. With proven results and solid references (see page 13), RoboSep is the ideal cell separator

for chimerism labs to process high-volume samples and isolate highly pure cells.

CHIMERISM LABS

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FACS HISTOGRAM RESULTS WITH EASYSEP HLA BUFFYCOAT/WHOLE BLOOD CD3+ CELL SELECTION KIT

TABLE 4: Products for Chimerism Analysis

CELL TYPE SELECTION

ROSETTESEP EASYSEP ROBOSEP

BUFFY COAT/

WHOLE BLOOD1BUFFY COAT/




SPLEEN, LN2

TOTALLYMPHOCYTES

Positive - 18684HLA - 18684HLAR -

Negative 15263HLA - - - -

T CELLSPositive CD3 - 18081HLA 18051HLA 18081HLAR 18051HLAR


B CELLS PositiveCD19

CD19/CD20-

1808418184HLA3

1805418454HLA3

18084R18184HLAR3

18054R18454HLAR3

MYELOID CELLSPositive

CD15CD33

CD33/66b

---

18681HLA18287HLA18683HLA

-18257

-

18681HLAR18287HLAR18683HLAR

-18257R

-


GRANULOCYTESPositive CD66b - 18682 - 18682R -

Negative 15121 - - - -

MONOCYTES Positive CD14 - 18088 18058 18088R 18058R

NK CELLS Positive CD56 - 18085HLA 18055 18085HLAR 18055R

HEMATOPOIETIC

PROGENITORCELLS

Positive CD34 - 18086 18056 18086R 18056R



3. Coming Soon!

TYPICAL PERFORMANCE DATA FOR KITS USED BY CHIMERISM LABORATORIES:

EasySepHLA Buffy Coat/Whole Blood CD3+

Positive Selection Kit - for 60 mL buffy coat/blood(Catalog #18081HLA)

Starting with fresh whole blood, the CD3+ cell content of theenriched fraction typically ranges from 93 - 99%.*Red blood cells were removed by lysis prior to flow cytometry.

EasySep HLA Buffy Coat/Whole Blood Myeloid Cell

Positive Selection Kit for 60 mL buffy coat/blood(Catalog #18683HLA)

FACS HISTOGRAM RESULTS WITH EASYSEP HLA BUFFYCOAT/WHOLE BLOOD MYELOID SELECTION KIT

Starting with fresh whole blood, the CD14+/CD66b+ cell content ofthe enriched fraction typically ranges from 94 - 99%.*Red blood cells were removed by lysis prior to flow cytometry.

CD14 / CD66b FITC

Counts

CD14 / CD66b FITC

Counts

Start*: 52% CD14+/CD66b+ Cells Selected: 98% CD14+/CD66b+ Cells

CD3 FITC CD3 FITC

Counts

Start*: 16% CD3+ Cells Selected: 99% CD3+ Cells

Counts

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Simple immunodensity-centrifugation procedure (see Figure 5). Quickly isolates untouched cells directly from any red blood cell (RBC)-containing samples.

Unwanted cells are linked to RBCs by specific antibodies forming dense immunorosettes which

pellet during centrifugation over the density gradient.

Desired cells are immediately ready for HLA analysis.

FIGURE 5: RosetteSep Procedure

Layer over densitymedium (e.g. Ficoll)

Add RosetteSepcocktail to whole blood

1

2

3

Enriched cells are leftuntouched (unrosetted)

Collect

Unwanted cells arecrosslinked to red blood

cells (rosetted) withtetrameric antibody

complexes

Plasma

Spin

Label

Ficoll is a trademark of GE Healthcare Ltd.

ROSETTESEP

Cell to enrich

Rosetted(unwanted)

cell

Red BloodCells

Unwanted CellTargeted for

Depletion

Tetrameric Antibody ComplexesAnti-

Cell

Anti-

Glycophorin A

Anti-

Glycophorin A

Anti-

Glycophorin A

Incubate 20 minutesat room temperature

Red blood cellsand unwanted cells

(rosetted)

Enriched cells

Ficoll

Picture of a Blood Sample After Additionof the RosetteSep Cocktail, and Prior toCentrifugation Over Ficoll.Magnification 400X

Illustration Showing Rosette of UnwantedCell and RBCs Formed by RosetteSepTetrameric Antibody Complexes

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FIGURE 6: EasySep Procedure

Add EasySep selectioncocktail to single cell

suspension Incubate 15 minutes

Labeled cell suspension

Pour off and collect cellsaccordingly.Negative Selection:

desired cells are decantedinto new tube.Positive Selection:

desired cells remain in tube.

Incubate 10 minutes

Place tube in magnetfor 5 minutes

Add EasySep

magnetic particles

EASYSEP

Immunomagnetic positive or negative selection of T cells, B cells, granulocytes, lymphoid, or myeloid cellswith purities of up to 99% (see Figure 6).

Any sample source including buffy coat, whole blood, peripheral blood mononuclear cell (PBMC),

bone marrow, lymph node, and spleen.

EasySep positive selection kits label desired cells with small magnetic par ticles that are

flow cytometry compatible.

EasySep negative selection kits isolate untouched cells by labeling unwanted cells for removal.

Isolated cells are immediately available for HLA analysis.

EASYSEP MAGNET CATALOG #18000

THE BIG EASY EASYSEP MAGNET CATALOG #18001

1

2

3

EASYSEP MULTISTAND CATALOG #18010

The EasySep Magnet is designedto hold one 12 x 75 mm (5 mL)polystyrene tube to isolate up to2.5 x 108 cells (or up to 5 x 108 cellsfor rare cell [e.g. CD34+ selection])per separation.

The Big Easy EasySep Magnetis designed to hold one 17 x 100mm (14 mL) polystyrene tube toisolate up to 109 cells (or up to 2 x109 cells for rare [e.g. CD34+] cellselection) per separation.

Separate up to 4 samples at onceusing the EasySep MultiStand.

The multistand is designed tohold up to 4 EasySep Magnetsor 4 The Big Easy EasySepMagnets.

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The ultimate system to fully automate cell isolation (Figure 7).

Uses EasySep reagents to process up to 4 independent samples with only 5 minutes of hands-on time,

saving technician time, and decreasing staff fatigue.

Avoids cross-contamination as each sample is handled with a unique set of disposable tips.

Every cell separation run has a user-accessible log file that can detail reagent lot numbers for validation.

ROBOSEP

Select protocol. Load samples,EasySep Selection Cocktail, magneticparticles, buffer and tips in carousel.

Press Run.

RoboSep processes samples(approx. 25 - 60 min/run).

Return to collect your separated cells.

FIGURE 7: RoboSep Procedure

1

2

3

4

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We like the reliability of the RoboSep,the minimization/elimination of specimen handling by the tech during subset separation, andthe low maintenance of the instrument. These factors are important to us with such a high

throughput of samples processed.

Wendy Leong,LAB MANAGERPATHOLOGY/BLOOD CENTER LABORATORY

We get up to eight samples a day that we sort for CD138 + cells. These cells are for differentstudies at the clinic and have to be processed the same day. Using our old system it wouldtake up to 30 minutes per sample because of the volume and wed have to be there all the timeto load, unload and clean the machine. Now we can run four samples at once and have ourenriched cells in an hour. RoboSep uses dedicated pipette tips for each sample so theres no

issue with cross-contamination. Because the process is all automated we can get other thingsdone in the meantime. It makes our job a lot easier when we dont have to check the timer every

ten minutes.

Were a busy lab and RoboSep really saves us a lot of

time.Kim Henderson,RESEARCHER

CLINICAL LAB IN THE MIDWESTERN UNITED STATES

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CELL TYPE SELECTION METHODSEROLOGY FLOW CROSSMATCH CHIMERISM

PBMC1 BLOOD2 PBMC1 BLOOD2 PBMC1 BLOOD2

TOTAL

LYMPHOCYTES

Positive CD3/CD19

- - - - - 18684HLA

- - - - - 18684HLAR

Negative

- - - 15263HLA - 15263HLA

- - - 19961HLA - -

- - - 19961HLAR - -

T CELLS

Positive

CD218657HLA 18687HLA 18657HLA 18687HLA - -

18657HLAR 18687HLAR 18657HLA 18687HLAR - -

CD318051HLA4 18081HLA 18051HLA 18081HLA 18051HLA 18081HLA

18051HLAR4 18081HLAR 18051HLAR 18081HLAR 18051HLAR 18081HLAR

Negative

- 15061HLA - 15061HLA - -

19051HLA 19951HLA 19051HLA 19951HLA - -

19051HLAR 19951HLAR 19051HLAR 19951HLAR - -Lymphoid(CD3+)

- - - - - 15271HLA

B CELLS

Positive

CD19CD20

18054 18084 - - 18054 18084

18054R 18084R - - 18054R 18084R

- 18685HLA5 - - - -

- 18685HLAR5 -

CD19/20- - - - 18454HLA5 18184HLA5

- - - - 18454HLAR5 18184HLAR5

Negative

- 15064HLA - 15064HLA - -

19054HLA 19954HLA 19054HLA 19954HLA - -

19054HLAR 19954HLAR 19054HLAR 19954HLAR - -

MYELOID CELLS/

GRANULOCYTEPositive

CD33/66b

- - - - - 18683HLA

- - - - - 18683HLAR

CD15 - - - - - 18681HLA

- - - - - 18681HLAR

CD33- - - - 18257 18287HLA

- - - - 18257R 18287HLAR

CD66b- - - - - 18682

- - - - - 18682R

MONOCYTES Positive CD14- - - - 18058 18088

- - - - 18058R 18088R

NK CELLS Positive CD56- - - - 18055 18085HLA

- - - - 18055R 18085HLAR

HEMATOPOIETICPROGENITORCELLS

Positive CD34

- - - - 18056 18086

- - - - 18056R 18086R

1. PBMC = Peripheral Blood Mononuclear Cell; kit will also work on single cell suspension of bone marrow, spleen, and lymph node


3. Red = RosetteSep, Purple = EasySep, Black = RoboSep (RoboSep includes EasySep kits plus 250 mL RoboSep Buffer and 1-2 boxes of 8 RoboSep Tip Racks)

4. Please contact STEMCELL Technologies for compatibility with other products.

5. Coming Soon!

Product Listing

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SUPPORT PRODUCTS CATALOG #

RosetteSep HLA Granulocyte Depletion Cocktail 15664HLA

EasySep Glycophorin A Depletion Kit 18352

RoboSep Glycophorin A Depletion Kit 18352R

HetaSep(An erythrocyte aggregation agent to quickly separate nucleated cells from erythrocytes inwhole blood)

07906

Ficoll-Paque PLUS (100 mL)1 07907

Ficoll-Paque PLUS (500 mL)1 07957

Ficoll-Paque PREMIUM (100 mL)1,2 07908

Ficoll-Paque PREMIUM (500 mL)1,2 07958

EasySep RBC Lysis Buffer, 10x Concentrate 20120

Ammonium Chloride Solution (100 mL) 07800

Ammonium Chloride Solution (500 mL) 07850

EasySep Magnet 18000

The Big Easy EasySep Magnet 18001

RoboSep The Fully Automated Cell Separator 20000

RoboSep Buffer (included in kits) 20104

RoboSep Tip Racks (included in kits) 20105

RoboSep Filter Tip Racks 20125

1. Ficoll and Ficoll-Paque PLUS are registered trademarks of GE Healthcare LTD.

2. Ficoll-Paque PREMIUM is manufactured according to GMP and ISO standards, and in accordance with USP recommendations for ancillary materia ls. Ficoll-Paque PREMIUM

is a sterile, ready-to-use reagent with low levels of endotoxin (

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FOR RESEAR CH USE ONLY. NOT FOR THERAP EUTIC OR DIAGNOSTIC USE. CATALOG #290 45 VERSION 4.0.0 MAY 2010

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BR010HLA400Web