CELL SEPARATIONPRODUCTS
For HLA Analysis

Scientists Helping Scientists™ | WWW.STEMCELL.COM

TABLE OF CONTENTS
Cell Separation Solutions for HLA Laboratories
4

Cell Separation Solutions for Crossmatch Analysis
Case Study: Development and Validation of a Rapid Optimized
Flow Cytometric Crossmatch (FCXM) Assay

8

Cell Separation Solutions for HLA Serological Typing

10

Cell Separation Solutions for Chimerism Analysis
Case Study: Fully Automated Sequential Cell Isolation of Four Different
Cell Types from a Single Sample with RoboSep™-S
The Importance of Purity Assessment in Chimerism Analysis

14

EasySep™: Rapid Immunomagnetic Cell Isolation

15

EasySep™ Direct: Cell Isolation Directly from Whole Blood

16

RoboSep™: Fully Automated Immunomagnetic Cell Isolation

18

SepMate™: Hassle-Free PBMC Isolation

19

RosetteSep™: Unique Immunodensity Cell Isolation

20

High-Quality and Reliable Antibodies

21

Selected Publications

Product Listing
22

HLA Applications

23

Cell Separation Magnets & Accessories

Scientists Helping Scientists™
STEMCELL Technologies is a leader in the development of specialty cell culture media, cell separation products,
and accessory reagents for life science research. Driven by science, we deliver over 1500 products to morethan 70 countries worldwide. To learn more about how STEMCELL Technologies helps to make research work,
visit www.stemcell.com.

Cell Separation Solutions for HLA Laboratories
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 has reinforced the importance of HLA matching for graft survival
and transplantation efficiency.
The specific and varied distribution of HLA antigens requires precise, accurate, and consistent HLA testing. In a busy HLA lab, finding powerfultools that facilitate reliable HLA testing while processing a high number of samples is of great importance.
At STEMCELL Technologies, we provide highly optimized cell separation solutions for a variety of HLA applications to facilitate high-volume
sample processing and clear, reliable test results. Our products have been formulated for the isolation of highly purified cells
to be used in:•

Crossmatch Analysis (see pages 4-5)•

HLA Serological Typing (see pages 8-9)•

Chimerism Analysis (see pages 10-11)

Cell Isolation Systems Available for HLA Tests:

EasySep

TM

Fast and Easy Immunomagnetic Cell Isolation
EasySep™ isolates cells quickly and easily without the use of columns (see pages 14 - 15).
Most EasySep™ kits can be completely automated using RoboSep™, the fully automatedcell separator (see pages 16 - 17).

RosetteSep

TM

Unique Immunodensity Cell Isolation
RosetteSep™ isolates highly purified cells directly from human whole blood during density
gradient centrifugation (see page 19). RosetteSep™ is easily combined with SepMate™,
the specialized density gradient tube, for fast, easy, and consistent cell isolation(see page 18).

Our cell isolation systems are particularly suitable for HLA labs, as all procedures are:
GENTLE. Obtain viable, functional cells without the need for columns.
FAST AND EASY. Isolate cells with easy-to-follow protocols.
CONVENIENT. Perform cell isolations at room temperature.

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3

CELL SEPARATION PRODUCTS FOR HLA ANALYSIS

Cell Separation Solutions for Crossmatch Analysis
To facilitate solid organ transplant, several screening tests, such as
the complement-dependent cytoxicity (CDC) assay and the flow
cytometric crossmatch (FCXM) assay, are performed to assess
whether the organ recipient has donor specific antibodies (DSA)
that may contribute to transplant rejection. When performing
any type of crossmatch assay to test for the presence of DSA inrecipient 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 alterantigen expression, possibly causing erroneous results and nonspecific, high background staining.
EasySep™, RoboSep™, and RosetteSep™ have been optimized foruse in downstream HLA tests, including the CDC and FCXM assays,
and can be performed at room temperature for easiersample processing.

EasySepTM•

EasySep™ is a column-free immunomagnetic cell isolation technology to isolate cells from a variety of sample sources(see pages 14 - 15).

•

EasySep™ Direct products isolate untouched cells directly from whole blood, spleen, or lymph node samples without density
gradient centrifugation or red blood cell lysis.

•

EasySep™ is ideal for obtaining highly purified lymphocytes that can immediately be used in the CDC and FCXM assays.

RoboSepTM•

RoboSep™ instruments offer true walk-away automation of cell separation using EasySep™ reagents (see pages 16 - 17).

•

Cells are isolated from up to 16 samples simultaneously.

•

Separation protocols are easy to run and can be standardized for high throughput labs.

•

Laboratory personnel save time by automating sample processing.

RosetteSep

TM•

RosetteSep™ is a fast and easy immunodensity procedure for the isolation of untouched cells directly from whole blood(see page 19).

•

Desired T and/or B cells are purified during the standard density gradient centrifugation step.

•

Easily combined with SepMate™ (see page 18) to maximize reproducibility and minimize the need for user training.

4

STEMCELL Technologies Inc.

StartCD3 FITC

CD3 PerCP-Cy5.5

CD3 FITC

Isolated

CD19 APCCounts

CD19 APC

Isolated

CountsStart

CD3 PerCP-Cy5.5

Figure 1. EasySep™ Direct HLA Crossmatch T Cell Isolation Kit(Catalog #19671)

Figure 2. EasySep™ Direct Human Total Lymphocyte Isolation Kit(Catalog #19655)

Starting with human whole blood from normal healthy donors, the typicalT cell (CD3+) content of the non-lysed final isolated fraction is 97 ± 3% (gated onCD45). In the above example, the T cell (CD3+) content of the lysed whole blood
start sample and non-lysed final isolated fraction is 38.0% and 93.2% (gated onCD45), respectively.

Starting with human whole blood from normal healthy donors, the typical total
lymphocyte (CD3−CD19+ and CD3+CD19−) content of the non-lysed final isolated
fraction is 96.7 ± 1.5% (gated on CD45) or 95.8 ± 2.2% (not gated on CD45). In
the above example, the total lymphocyte (CD3−CD19+ and CD3+) content of thelysed whole blood start sample and non-lysed final isolated fraction is 30.5% and93.2% (gated on CD45), respectively.

Cell Separation Products for Crossmatch Analysis
EasySep™ / RoboSep™ (Catalog #)Cell Type

Selection

1

Whole Blood , Spleen

1.

Lymph Node and MNC2

rosetteSep™ (Catalog #)Whole Blood1

T Cells

Negative

19671, 896714

15061, 15061HLA3

B Cells

Negative

19684, 896844

15064, 15064HLA3

Total Lymphocytes

Negative

19655

19961HLA

15263, 15263HLA3

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

2. Kit works on mononuclear cells (MNCs) isolated from peripheral blood or bone marrow. MNC: Mononuclear Cell
3. This kit has been CE-marked as a Class I In Vitro Diagnostic (IVD) product in the European Union according to the 98/79/EC In Vitro Diagnostic Device Directive and complies with its requirements.
4. This kit carries the CE mark and is available as a Class I IVD device in Australia, Canada, and the European Union. To learn more about the regulatory status of this productin your specific region, contact info@stemcell.com.

In Vitro Diagnostic (IVD) Reagents
Several of our EasySep™ Direct and RosetteSep™ reagents have been CE-marked as in vitro diagnostic devices in specific regions.
We strive to offer reagents you can trust and demonstrate our commitment to the highest standard of quality control.

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5

CELL SEPARATION PRODUCTS FOR HLA ANALYSIS

Case Study
Development and Validation of a Rapid Optimized Flow Cytometric
Crossmatch (FCXM) Assay
HLA Laboratory, Department of Pathology, Dalhousie University, Halifax, Canada

BackgroundTo facilitate solid organ transplantation, the flow cytometric crossmatch (FCXM) assay is usedas part of a pre-transplant risk assessment to determine whether the organ recipient has donor
specific antibodies (DSA).1 Performing the FCXM assay is time consuming and the various steps
in the protocol, including the donor lymphocyte enrichment step, are not performed consistently
across laboratories. This lack of standardization may lead to inconsistent results.
In order to optimize the standard FCXM assay, Dr. Robert Liwski from Dalhousie University in
Halifax, developed, optimized and validated two FCXM procedures, the Halifax, and Halifaster
FCXM protocols.2,3 The different parameters that were optimized, included the cell isolation
method, assay platform, cell number, serum volume, and incubation times. In this case study wefocus on the different cell isolation methods followed by these protocols.

“It is about time.”
Dr. Robert Liwski, MD, PhD,
from the Deparment of Pathology at
Dalhouse University commenting
on the need for faster and standardized
protocols for the FCXM assay.

Methods
The FCXM assay can be divided in two parts. The first part consists of the donor lymphocyte isolation and pronase and DNAse treatment.
The second part consists of the crossmatch assay. The FCXM assays were performed following the standard, Halifax, and Halifaster protocols
as previously described.2,3 In the standard and Halifax protocols, cells were isolated from whole blood by density gradient centrifugation
using the density gradient medium Lympholyte® (~45 minutes/sample). The lymphocyte purity obtained using density gradient centrifugation
varies between 15-90% depending on the donor and sample quality.4 In the Halifaster protocol, cells were isolated directly from whole blood
with the EasySep™ Direct Human Total Lymphocyte Isolation Kit Catalog #19655 (~25 minutes/sample) following instructions on the productinformation sheet (see schematic on page 15). This lymphocyte isolation method is fast, and the typical total lymphocyte purity is 95.8 ± 2.2%
(not gated on CD45). The results of the FCXM assays following the different protocols were compared.
In addition, the impact of lymphocyte purity on FCXM results was assessed.5 Briefly, lymphocytes (Ly), neutrophils (Nu), and monocytes (Mo)were isolated from 5 volunteer donors using the corresponding EasySep™ Direct kits (Catalog #19655, #19666, and #19669, respectively).
Whole leukocyte (WL) preparations were obtained by adding Ly, Nu, and Mo cells in equal proportions (1/3 of each). The results of the FCXM
assays using WL (low lymphocyte purity) and Ly (high lymphocyte purity) preparations were compared.

Results
B
250

200

200

150
100
50

150
100
50

50

100

150

200

250

FSC-H

6

Lympholyte
30

Number of cells x 10 6

250

SSC-A

SSC-A

A

C

STEMCELL Technologies Inc.

50

100

150

FSC-H

200

250

®

EasySep™ Direct

25
20
15
10
5
0

Total Cells Lymphocytes

Figure 3. Using EasySep™ Direct Human Total
Lymphocyte Isolation Kit (Catalog #19655)
Increases Lymphocyte Purity While Maintaining
Cell Yield
Cells were isolated from the same deceased donor whole
blood sample using (A) the density gradient separation®
medium Lympholyte (~45 minutes/sample) or (B) EasySep™
Direct (~25 minutes/sample) and analyzed by flow cytometry.
Cleaner samples were obtained with EasySep™ Direct.
(C) Samples isolated using EasySep™ Direct contained fewer
contaminating cells and yet maintained overall number of T
(CD3+) and B (CD19+) cells compared to samples isolated using®
Lympholyte . Each column with error bar represents the mean
± SD from 24 mL whole blood (n = 20 donors). Data kindly
provided by Dr. Robert Liwski.

B Cell FCXM

A
WL

400
300
200
100

B

B Cell XM
600

Standard FCXM
MCSF from Cutoff

MCSF shift from NC

Ly

-200

400
300
200

y = 0.9799x + 1.6227
R 2 = 0.9753

100
0

0

200

400

-100

0
1/50

1/100

1/200

1/400

1/50

1/100

1/200

-200

1/400

Dilution

B Cell XM
600

500

600

Halifaster FCXM
MCS from cutoff

T Cell FCXM
500

-200

500
400
300
200

y = 1.1521x + 17.776
R 2 = 0.9789

100
0

0

200

400

-100

Halifax FCXM
MCSF from Cutoff

-200

600

Halifax FCXM
MCSF from Cutoff

Figure 4. Use of Highly Enriched Lymphocytes Isolated with
EasySep™ Direct Improves DSA Detection Compared to Whole
Leukocyte Cell Preparations

Figure 5. Isolation of Lymphocytes from Whole Blood Using
EasySep™ Direct Does Not Compromise Sensitivity of the
FCXM Assay

Lymphocytes (Ly), neutrophils (Nu), and monocytes (Mo) were isolated fromvolunteer donors (n=5) using EasySep™ Direct Catalog #19655, #19666, and#19669, respectively. Whole leukocyte (WL) preparations were obtained by adding
Ly, Nu, and Mo cells in equal propotions. WL (low lymphocyte purity) and Ly (highlymphocyte purity) preparations were treated with pronase and then used toperform the FCXM assay against negative control (NC) sera or several dilutionsof positive control sera. The median channel fluorescence shifts (MCFS) weregenerated by using the negative control sera samples as a baseline. The MCFS
between WL and Ly were then compared. Each column with error bars represents
the mean ± SEM (n = 5 donors). Data kindly provided by Dr. Robert Liwski.

B cell FCXM assays were performed in parallel using cells isolated with EasySep™
Direct (Halifaster Protocol) or with the density gradient separation medium®
Lympholyte (standard and Halifax Protocol). FCXM results were compared
between (A) standard and Halifax and (B) Halifaster and Halifax FCXM protocols.
Linear regression analysis of median channel fluorescence shifts (MCFS) showed
an excellent correlation for the B cell and T cell (not shown here) FCXM assays
between the two different isolation protocols. Data are expressed as MCSF fromthe cutoff level defined as the mean + three standard deviations. Data kindly
provided by Dr. Robert Liwski.

Summary•

The Halifaster protocol incorporates EasySep™ Direct for theisolation of lymphocytes. This resulted in fewer contaminating
cells compared to using the density gradient centrifugation
method with Lympholyte®.

•

Performing FCXM assays with highly enriched lymphocytes
isolated with EasySep™ Direct improved detection of DSA andreduced the variability of FCXM results.

•

The Halifaster FCXM protocol reduced the overall time tocomplete the FCXM assay to less than 2 hours withoutcompromising quality or sensitivity. This was in part by reducing
the lymphocyte isolation step to less than 30 minutes.

200

Time Required (Minutes)

Cell Isolation
Pronase & DNAse
Treatment

150

Crossmatch Assay

100

50

0
Standard

Halifax

HalifasterProtocol Followed

Figure 6. Following the Halifaster FCXM Protocol Reduces
the Overall Time to Complete the FCXM assay to Less Than
2 Hours
The cell preparation part for the FCXM assay consists of the donor lymphocyte
isolation step followed by treatment with pronase and DNAse. The Halifaster
FCXM protocol reduced the time required for the cell preparation by almost 40%(from 90 to 55 minutes). This was achieved by using EasySep™ Direct technology
for the lymphocyte isolation step. This technology is significantly faster than thedensity gradient centrifugation method used by the standard and Halifax FCXM
protocols. The approximate total times it takes to complete the FCXM assay(including cell preparation) when following the standard, Halifax, and Halifaster
FCXM protocols is 175 minutes, 125 minutes, and 85 minutes, respectively.3 Data
kindly provided by Dr. Robert Liwski.

Cited References
1. Rebibou JM et al. (2004) T-cell flow-cytometry crossmatch and longterm renal graft survival. Clin Transplant 18(5): 558–563.
2. Liwski RS et al. (2015) Development and Validation of a Rapid
Optimized Flow Cytometry Crossmatch (FCXM) Assay, the Halifax
and Halifaster FCXM Protocols. ASHI Quarterly (Third Quarter 2015):
19–25.
3. Liwski RS et al. (2018) Rapid optimized flow cytometric crossmatch(FCXM) assays: The Halifax and Halifaster protocols. Hum Immunol
79(1): 28–38.
4. Hamrick C & Lebeck L. (2000) Flow cytometric T and B cellcrossmatching. ASHI Lab Man 4th Ed.(Philadelphia: American Societyfor Histocompatibility and Immunogenetics): 41–45.
5. Liwski R et al. (2016) P099 The impact of lymphocyte purity on flow
cytometry crossmatch (FCXM) assay. It’s not purely theoretical. Hum
Immunol 77, Supple: 110–111.

Data kindly provided by Dr. Robert Liwski, HLA Laboratory, Department of
Pathology, Dalhousie University, Halifax, Canada. Robert.Liwski@nshealth.ca

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CELL SEPARATION PRODUCTS FOR HLA ANALYSIS

Cell Separation Solutions for HLA Serological Typing

For clear and reliable HLA serological typing, RosetteSep™ kits are the ideal solution for isolating clean cell preparations that are compatible
with HLA typing reagents.
RosetteSep™ is a fast, easy, and cost-effective immunodensity procedure for the isolation of untouched cells directly from whole blood. Cells
are isolated during the density gradient centrifugation step, which reduces sample handling time and maximizes convenience.

RosetteSep

TM•

RosetteSep™ is a fast, easy, and cost-effective cell isolation technology for obtaining untouched cells directly fromwhole blood (see page 19).

•

Desired T and/or B cells are purified during the standard density gradient centrifugation step without columns or magnets.

•

Cells are viable, untouched, and immediately ready for downstream testing.

•

Cell purities of up to 95% allow for accurate serological HLA testing.

•

Can be used with SepMate™ for consistent and fast cell separations.

8

STEMCELL Technologies Inc.

Figure 7. Results of a Complement-Dependent Cytotoxicity Assay
Cells were separated using STEMCELL Technologies' HLA reagents and analysed in a complement-dependent cytotoxicity (CDC) assay.

Typical Performance Data for Kits Used for Serological HLA Testing
Isolated

CD3 FITC

Start

Isolated

CD3 FITCCountsCountsCountsCountsStart

CD19 PE

CD19 PE

Figure 8. RosetteSep™ HLA T Cell Enrichment Cocktail(Catalog #15061HLA)

Figure 9. RosetteSep™ HLA B Cell Enrichment Coktail(Catalog #15064HLA)

Starting with human fresh whole blood the CD3+ cell content of the non-lysed
final isolated fraction typically ranges from 90% - 97% (gated on CD45).
In the above example, the T cell (CD3+) content of the lysed whole blood startsample and non-lysed final isolated fraction is 20% and 96% (gated on CD45),
respectively.

Starting with human fresh whole blood, the typical B cell (CD19+) content of the
non-lysed final isolated fraction typicall ranges from 59.3% - 97.2% (gated onCD45). In the above example, the B cell (CD19+) content of the lysed whole blood
start sample and non-lysed final isolated fraction is 3.8% and 94.6% (gated onCD45), respectively.

Cell Separation Products for Serological HLA Typing
RosetteSep™ (Catalog #)Cell Type

Selection
1

Whole Blood
T Cells

Negative

15061, 15061HLA2

B Cells

Negative

15064, 15064HLA2

1. Kit also works on other red blood cell containing samples (i.e. cord blood, buffy coat).
2. This kit has been CE-marked as a Class I IVD product in the European Union according to the 98/79/EC In Vitro Diagnostic Device Directive and complies with its requirements.

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CELL SEPARATION PRODUCTS FOR HLA ANALYSIS

Cell Separation Solutions for Chimerism Analysis
Chimerism analysis is important for detecting and monitoring theestablishment of donor leukocytes in a recipient following bonemarrow transplantation.1,2 Investigating chimerism within a specificcell population, also referred to as lineage-specific chimerism,
can increase sensitivity over analyzing the entire leukocyte
population.3,4 However, lineage-specific chimerism requires cellisolation techniques that result in high cell purity.
In addition, cross-contamination between samples is always
a concern since the presence of even a few non-target cells or cells
from another sample can alter the integrity of downstream genetic
analyses. For reliable lineage-specific chimerism analysis, EasySep™
provides a fast and easy method to obtain highly purified cells
and flexible protocols to isolate multiple cell types from the same
sample. To save time and increase laboratory throughput, EasySep™
can be automated with RoboSep™, an instrument that offers fullautomation of immunomagnetic cell separation.

EasySepTM•

EasySep™ positive selection kits provide highly purified cells ready for downstream lineage-specific chimerism analysis(see pages 14 - 15).

•

Flexible protocols allow for sequential isolations to maximize cell recovery of multiple cell types from a single sample.

•

Cells can be isolated directly from a variety of sources including buffy coat or whole blood.

RoboSepTM•

RoboSep™ instruments offer true walk-away automation of immunomagnetic cell separation using EasySep™ reagents(see pages 16 - 17).

•

Disposable tips and a column-free system eliminate the risk of sample cross-contamination.

•

Protocols for simultaneous and sequential cell isolations allow for automated sample processing of up to 16 samples.

RosetteSep

TM•

RosetteSep™ allows for efficient cell isolations directly from whole blood (see page 19).

•

Desired cells are isolated during density gradient centrifugation and are immediately ready for downstream applications.

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STEMCELL Technologies Inc.

Typical Performance Data for Kits Used by Chimerism Laboratories
Start

CountsCountsCD3 FITC

Isolated

CountsIsolated

CountsStart

CD14/CD66b FITC

CD2 PE

CD14/CD66b FITC

Figure 11. EasySep™ HLA Whole Blood CD3+ Positive Selection Kit(Catalog #17871)

Figure 12. EasySep™ HLA Chimerism Whole Blood Myeloid Cell
Positive Selection Kit (Catalog #17884)

Starting with human whole blood, the CD3+ cell content of the isolated fraction(as assessed by staining the start and isolated fractions with anti-CD3 or anti-CD2
antibodies, respectively) typically ranges from 96 - 99%. In the above example,
the T cell content of the lysed whole blood start sample and the non-lysed final
isolated fraction is 18.2% and 98.1% (gated on CD45), respectively.

Starting with human whole blood, the CD14 /CD66b cell content of the isolated
fraction is typically 94.5 ± 4.1%. In the above example, the myeloid cell++
(CD14 /CD66b ) content of the lysed whole blood start sample and the non-lysed
final isolated fraction is 66.6% and 98.5% (gated on CD45), respectively.

++

Cell Separation Products for Chimerism Analysis

Cell Type

EasySep™ / RoboSep™ (Catalog #)RosetteSep™ (Catalog #)Positive Selection

Negative Selection

Selection Marker

Whole Blood1

MNC2

Whole Blood1

CD3

17871

17851

15271, 15271HLA3

CD19

17874

17854

−

CD19/ CD20

17886

−

−

CD15

17881

18651

CD33

17885

17876

CD33/66b

17884−

Granulocytes

CD66b

17882

−

−

Monocytes

CD14

17878

17858

−

NK Cells

CD56

17875

17855

−

Hematopoietic Progenitor Cells

CD34

17879

17856

−

T Cells
B Cells

Myeloid Cells

15272, 15272HLA3

For automated cell isolation EasySep™ kits are available as RoboSep™ Reagent kits (RF).
1. Kit also works on other red blood cell containing samples (i.e. cord blood, buffy coat).
2. Kit works on mononuclear cells (MNCs) isolated from peripheral blood or bone marrow. MNC: Mononuclear Cell
3. This kit has been CE-marked as a Class I IVD product in the European Union according to the 98/79/EC In Vitro Diagnostic Device Directive and complies with its requirements.

Cited References
1.

Bader P et al. (2005) How and when should we monitor chimerism after allogeneic stem cell transplantation? Bone Marrow Transplant 35(2): 107–19.

2. Levrat E et al. (2015) Very long term stability of mixed chimerism after allogeneic hematopoietic stem cell transplantation in patients with hematologic
malignancies. Bone Marrow Res 2015: 176526.
3. Breuer S et al. (2012) Early recipient chimerism testing in the T- and NK-cell lineages for risk assessment of graft rejection in pediatric patients undergoing
allogeneic stem cell transplantation. Leukemia 26(3): 509–19.
4. Rupa-Matysek J et al. (2011) Correlation between the kinetics of CD3+ chimerism and the incidence of graft-versus-host disease in patients undergoing
allogeneic hematopoietic stem cell transplantation. Transplant Proc 43(5): 1915–23.

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CELL SEPARATION PRODUCTS FOR HLA ANALYSIS

Case Study
Fully Automated Sequential Cell Isolation of Four Different Cell Typesfrom a Single Sample with RoboSep™ -S
Florida Hospital Tissue Typing Laboratory, Orlando, Florida

Background
Many analyses, such as chimerism testing, are often performed on small blood samples (e.g. pediatric samples). As a result, analysis of purified
cell subsets requires techniques that can isolate more than one cell type from an undivided starting sample. Here we describe
a method used by the Florida Tissue Typing Laboratory to sequentially isolate B cells, T cells, myeloid cells, and NK cells starting froma single sample of HetaSep™-treated blood for their chimerism analysis.

Methods
Anti-CD19
cocktail

Nucleatedcells

Anti-CD3
cocktail

Anti-CD33/66b
cocktail

NK Cellenrichment

Supernatant
CD19-depleted

SupernatantCD19, CD3-depleted

SupernatantCD19, CD3, andmyeloid-depleted

B Cells

T Cells

Myeloid

NK Cells(in quadrant 1)(in quadrant 2)(in quadrant 3)(in quadrant 4)

Fully Automated Sequential Separation with RoboSep™ Instruments
Figure 13. Automated Sequential Separation of B Cells, T Cells, Myeloid Cells, and NK Cells from a Single Sample of Hetasep™Treated Blood

Results

Why Use RoboSep™ for Sequential
Isolation of Immune Cells?

100% Purity90

FLEXIBLE. Perform sequential cell isolations of 4 cell
types from a single sample using RoboSep™-S or fromfour samples using RoboSep™-16.

80

FULLY AUTOMATED. Perform all isolations during
a single machine cycle with minimal “hands-on” time.

70

EFFICIENT. Isolate cells from small volumes (0.5 - 4.5mL) of blood to run DNA analysis or other down stream
applications.

60

50

B Cells

T Cells

Myeloid Cells

NK Cells

Figure 14. Purity of Four Different Cell Types Isolated from 18
Different Samples Using RoboSep™-S Sequential Separation
Data kindly provided by Max Marschner, Supervisor, Florida Hospital Tissue
Typing Lab.

12STEMCELL Technologies Inc.

VERSATILE. Customize protocols for any cell type andsample source.
INCREASED THROUGHPUT. Streamline cell isolations

and increase laboratory throughput.

The Importance of Purity Assessment in Chimerism Analysis
Lineage-specific chimerism analysis is an important tool formonitoring the outcome of allogeneic hematopoietic celltransplantations (allo-HCT). Chimerism status in transplant
recipients can indicate the potential dynamics of disease relapse,
graft-versus-host disease (GVHD), graft versus tumor (GVT) effects,
and other outcomes.1-5
Therefore, investigating chimerism within specific cell subsets,
particularly in recipients of non-myeloablative allo-HCT6,7,
is an increasingly common practice that offers the following
advantages over analyzing the entire leukocyte population:••

Increased assay sensitivity. If a patient has mixed chimerism
in only one or a few cell lineages, the overall proportion of hostcells may be too low for detection by whole blood assays.8
The significance of mixed chimerism differs between cellsubsets. For instance, one recent study found that the degreeof mixed chimerism within T and NK cells, but not myeloid cells,
allowed patients to be classified into different risk groups forgraft rejection.9 In another study, full donor T cell chimerism
consistently preceded GVT effects.13 Recent literature suggeststhat this type of lineage-specific analysis is highly informative
for chimerism labs.14

The benefits of lineage-specific chimerism analysis depend upon
the purity of isolated cell subsets, as contamination by nontarget cells decreases the reliability of the results. Assessing thepurity of isolated cell populations is therefore an essential
quality control step. The official European Federation for
Immunogenetics (EFI) and American Society for Histocompatibility
and Immunogenetics (ASHI) guidelines stipulate that the purityof sorted cell populations must be documented and taken into
account when results are analyzed.

When HCE (Haemopoietic Chimaerism and Engraftment)
testing is performed on cellular subsets isolated by cellsorting, the purity of the sorted population must be
documented and taken into account in the analysis of theresults. If this is not possible it must be clearly stated in
the report.
EFI STANDARDS VERSION 6.3 (2015), SECTION I6.9

Document the purity obtained if processing involves
isolation of cell subsets. If purity is not assessed,
this must be documented on the test report.
ASHI STANDARDS 2016, SECTION D.5.3.4.1.8

To read the complete review on the importance of lineage-specificchimerism analysis and guidelines for assessing purity using
flow cytometry visit www.stemcell.com/chimerism-purityassessment.

Cited References
1. McCann SR et al. (2005) Hemopoietic chimerism following stem cell transplantation.
Transfus Apher Sci 32(1): 55–61.
2. Khan F et al. (2004) Significance of chimerism in hematopoietic stem celltransplantation: new variations on an old theme. Bone Marrow Transplant 34(1):
1–12.
3. Park M et al. (2011) Clinical implications of chimerism after allogeneic hematopoietic
stem cell transplantation in children with non-malignant diseases. Korean J Hematol
46(4): 258–64.
4. Rettinger E et al. (2011) Preemptive immunotherapy in childhood acute myeloid
leukemia for patients showing evidence of mixed chimerism after allogeneic stem celltransplantation. Blood 118(20): 5681–8.
5. Bader P et al. (2008) Monitoring of post-transplant remission of childhood
malignancies: is there a standard? Bone Marrow Transplant 42 Suppl 2: S31-4.
6. Childs R et al. (1999) Engraftment kinetics after nonmyeloablative allogeneic
peripheral blood stem cell transplantation: full donor T-cell chimerism precedes
alloimmune responses. Blood 94(9): 3234–41.
7. Urbano-Ispizua A et al. (2001) The number of donor CD3(+) cells is the mostimportant factor for graft failure after allogeneic transplantation of CD34(+) selected
cells from peripheral blood from HLA-identical siblings. Blood 97(2): 383–7.
8. Antin JH et al. (2001) Establishment of complete and mixed donor chimerism after
allogeneic lymphohematopoietic transplantation: recommendations from a workshop
at the 2001 Tandem Meetings of the International Bone Marrow Transplant Registry
and the American Society of Blood an. Biol Blood Marrow Transplant 7(9): 473–85.
9. Breuer S et al. (2012) Early recipient chimerism testing in the T- and NK-cell lineages
for risk assessment of graft rejection in pediatric patients undergoing allogeneic stem
cell transplantation. Leukemia 26(3): 509–19.
10. Jens G et al. (2011) Mixed Chimerism after Allogeneic Stem Cell Transplantation Focus on Double Cord Blood Transplantation. Emma W (Ed.). J Blood Disord Transfus
2(S1).
11. Moratto D et al. (2011) Long-term outcome and lineage-specific chimerism in 194
patients with Wiskott-Aldrich syndrome treated by hematopoietic cell transplantationin the period 1980-2009: an international collaborative study. Blood 118(6):1675–84.
12. Goh R-Y et al. (2011) Lineage-specific chimerism analysis in nucleated cells,
T cells and natural killer cells after myeloablative allogeneic hematopoietic stem celltransplantation. Korean J Hematol 46(1): 18–23.
13. Bornhäuser M et al. (2009) Monitoring of donor chimerism in sorted CD34+
peripheral blood cells allows the sensitive detection of imminent relapse after
allogeneic stem cell transplantation. Haematologica 94(11): 1613–7.
14. Buño I et al. (2015) Mixed Chimerism in T Cells after Allogeneic SCT Allows Early
Diagnosis of Graft Rejection and Successful Treatment with Immunosuppression
Withdrawal and/or Donor Leukocyte Infusion. Blood 106(11): 5289 LP-5289.

WWW.STEMCELL.COM

13

CELL SEPARATION PRODUCTS FOR HLA ANALYSIS

EasySep™
Rapid Immunomagnetic Cell Isolation
EasySep™ is a powerful immunomagnetic cell selection procedurethat combines the specificity of monoclonal antibodies with thesimplicity of a column-free magnetic system for the effortless
isolation of highly purified cells. Cells are targeted for either
depletion (negative selection) or selection (positive selection) using
antibody complexes directed to specific cell surface antigens.
The antibody complexes link targeted cells to magnetic particles.
Labeled cells are pulled to the sides of the tube when the sample isplaced in an EasySep™ magnet.

Manual or Automated Cell Isolation
Automate cell isolation to save hands-on time andincrease laboratory throughput with RoboSep™
instruments (see pages 16 - 17).
For manual cell separation choose from a wide range of
EasySep™ magnets that provide flexibility in the sample
volume and throughput of cells(see page 23).

EasySep™ kits are available for the isolation of human T cells,
B cells, granulocytes, lymphoid, or myeloid cells from a variety ofsample sources, including whole blood, spleen, lymph nodes, andmononuclear cells (MNCs) from whole blood or bone marrow.

Typical EasySep™ Human Positive Selection Protocol

1

2

4

Place tube in
EasySep™ Magnet
and incubate for3 minutes*

Ea

syS

ep

TM

Pour off desired fraction
into a new tube

TM

Add EasySep™ magnetic particles
to cell suspension

EasySep

EasySepTM

Add EasySep™ Isolation Cocktail
to single cell suspension

33

Incubate
3 minutes*

Incubate
3 minutes*

4

Place tubes in EasyEights™ EasySep™
Magnet andincubate for 10 minutes*

Pipette off desired
fractions into new tubes

Negative Selection:
Desired cells are poured/
pipetted into new tube.

*Times shown are typical for positive selection kits using the purple
EasySep™ Magnet. Time for each kit will vary depending on the exact
isolation protocol and magnet used.

Start

EasyEights™EasySep™

Positive Selection:
Desired cells remain
in magnet. EasySep™
magnetic particles
do not interfere withdownstream applications
and are flow cytometrycompatible.

Isolated

CountsCountsFigure 15. EasySep™ Human CD3 Positive Selection Kit II(Catalog #17851)

CD3 PE

14

STEMCELL Technologies Inc.

Starting with a single cell suspension of human peripheral blood mononuclear cells,
the CD3+ cell content of the isolated fraction is typically 99.2 ± 0.2% (mean ± SD
using the purple EasySep™ Magnet). In this example, the total CD3+ cell content of
the start sample and isolated fraction is 55.3% and 99.0%, respectively.
CD3 PEEasySep™ Direct
Cell Isolation Directly from Whole Blood
EasySep™ Direct isolates untouched and highly purified cells
directly from whole blood, spleen, or lymph nodes without density
gradient centrifugation, RBC lysis, or other pre-processing steps.
Cells are immediately ready for downstream applications, including
flow cytometry crossmatch and gene expression analysis. Individual
samples of 0.5 – 30 mL can be processed in
as little as 20 minutes.

Why Use EasySep™ Direct?
FAST. Isolate highly purified total lymphocytes, T cells, or

B cells directly from whole blood, spleen, or lymph nodes
without lysis or centrifugation.
HIGH PURITY. Obtain high cell purity with no RBC

contamination, even on older blood samples.
CONVENIENT. Automate cell isolations and minimize

sample handling with RoboSep™ instruments.

Typical EasySep™ Direct Protocol for Whole Blood and Buffy Coat

1

2

Add EasySep™ Direct Isolation
Cocktail and EasySep™ Direct
RapidSpheres™ to whole blood

3

Place tube in EasySep™
Magnet and incubate
for 5 minutes*

Incubate
5 minutes*

Ea

sy

Se

p

TM

4

5

Place tube in EasySep™
Magnet and incubate
for 5 minutes*

Pour off desired fraction into
a new tube and add EasySep™
Direct RapidSpheres™

Pour off desired
fraction into
a new tube

Incubate
5 minutes*
Ea

sy

Se

p

TM

*Times for each kit will vary depending on sample source and isolation protocol. Please refer to the product information sheet of each product for sample compatibility
and specific isolation protocol.

VIDEO
Learn how EasySep™ Direct works
www.EasySepDirect.com

WWW.STEMCELL.COM

15

CELL SEPARATION PRODUCTS FOR HLA ANALYSIS

RoboSep™
Fully Automated Immunomagnetic Cell Isolation
RoboSep™ instruments offer true walk-away automation of immunomagnetic cell separation. Using EasySep™ reagents, RoboSep™-S andRoboSep™-16 perform all cell labeling and magnetic isolation steps for up to four and sixteen samples, respectively. Sample handling is
minimized and the use of disposable tips in these column-free systems ensures that isolated cells of interest are immediately available for any
downstream application.

RoboSep -S

RoboSep -16

The compact design of RoboSep™-S
brings the convenience of automated cellisolation to any busy laboratory.

The enhanced liquid-handling capabilities of
RoboSep™-16 allow high-volume users to efficiently
isolate desired cells with speed and confidence.

TM

TM

Why Use RoboSep™?
FULLY AUTOMATED. Just load reagents and samples and walk away.
SIMULTANEOUS OR SEQUENTIAL CELL ISOLATION. Perform simultaneous cell isolations for up to 4 samples using RoboSep™-S
and 16 samples using RoboSep™-16 or sequentially isolate different cell types from the same sample.
NO CROSS-CONTAMINATION. Eliminate the risk of cross-contamination with a column-free system and single-use disposable tips.
VERSATILE. Use EasySep™ technology to isolate virtually any cell type from any species or sample, including whole blood. Programs

are cell type-specific and customizable.

16

STEMCELL Technologies Inc.

How RoboSep™ Works
RoboSep™-S and RoboSep™-16 fit easily into the workflow of any lab that needs the multi-sample processing capacity, speed, reliability,
and convenience of automated cell isolation. Start your cell isolation protocol with as little as 5 minutes of “hands-on” time with
RoboSep™-S and RoboSep™-16. Both pre-programmed routine protocols and customizable protocols are available to meet your uniquecell isolation needs.

Typical RoboSep™-S Protocol

1

Select protocol. Load sample,
EasySep™ reagents, buffer, and tips
in carousel.

2

Press “Run”.

3

2

Press “Run”.

3

Return in 25 to 60 minutes tocollect your separated cells.

Typical RoboSep™-16 Protocol

1

Select protocol. Load sample,
EasySep™ reagents, buffer, and tips.

“We like the reliability of the RoboSep™, theminimization/elimination of specimen handling by thetech during subset separation, and the low maintenanceof the instrument. These factors are important to us withsuch a high throughput of samples processed.”

Return in 25 to 60 minutes tocollect your separated cells.

PRODUCT TOURSee RoboSep™ Instruments in Action
www.RoboSep.com

Wendy Leong, Senior Clinical Laboratory Scientist
PATHOLOGY/BLOOD CENTER LABORATORY

WWW.STEMCELL.COM

17

CELL SEPARATION PRODUCTS FOR HLA ANALYSIS

SepMate™
Hassle-Free PBMC Isolation
SepMate™ is a specialized tube for fast and easy PBMC isolationin just 15 minutes. The SepMate™ tube contains a unique insert
that prevents the density gradient medium1 (e.g., Ficoll-Paque®
or Lymphoprep™) and blood sample from mixing. The density
gradient medium is pipetted through a central hole in the insert
and the sample is poured or rapidly pipetted on top of the insert.
This eliminates the need to carefully layer the sample directly
onto the density gradient medium, an otherwise time-consuming
and highly laborious step. Only 10 minutes of centrifugation are
required, and this step can be performed with the brake on,
further reducing the total time necessary for separation. Aftercentrifugation, plasma and PBMCs are simply poured into a
new tube.

Why Use SepMate™?
EASY. Avoid the need for slow and laborious sample

layering over the density gradient medium.
FAST. Centrifuge for just 10 minutes with the brake on

and simply pour off PBMCs into a new tube.
CONSISTENT. Eliminate errors and minimize variability
between users.
VERSATILE. Combine with RosetteSep™ to isolate

purified cell subsets from whole blood in 25 minutes.
REGISTERED. Use with whole blood or bone marrow
samples for In Vitro Diagnostic (IVD) applications.2

SepMate™ is registered as an In Vitro Diagnostic (IVD)2 device
in select regions.

Typical SepMate™ Protocol

1

Products

Add density gradientmedium throughSepMate™ insert

Blood Volume
Processed (mL)

Product

Catalog #Unit Size

SepMate™-15 (IVD2)

85415

SepMate™-15 (RUO3)

86415

SepMate™-50 (IVD2)

85450

SepMate™-50 (RUO3)

86450

Product

Catalog #Density

Unit Size

Lymphoprep™

07801
07851
07811
07861

1.077 g/mL1

250 mL
500 mL
4 x 250 mL
6 x 500 mL

0.5 - 5
SepMate™ insert

2

100 tubes

Density gradient medium1(e.g. Ficoll-Paque® or
Lymphoprep™)

4 - 17

Pipette sample
onto insert

SepMate™ insert

1.
2.

3

Centrifuge for10 min (brake on)
3.

PlasmaPBMCs
Density gradient medium
RBCs andgranulocytes

18

STEMCELL Technologies Inc.

Lymphoprep™ has the same density as Ficoll-Paque ® and can be substituted for
Ficoll-Paque ® without any need to change your existing protocols.
SepMate™ 85415/85450 is available in Australia, Canada, Europe, and the United Statesof America, where it is registered as an In Vitro Diagnostic (IVD) device for the isolationof mononuclear cells from human whole blood, cord blood, and bone marrow by density
gradient centrifugation. This product is also available in China where it is considered
a non-medical device by the China Food and Drug Administration (CFDA) and should
therefore be used as general laboratory equipment.
SepMate™ 86415/86450 is intended for Research Use Only (RUO) and is available inregions where SepMate™ 85415/85450 is not available.

4
Pour

VIDEO
PBMCs

How to Use SepMate™
www.stemcell.com/SepMateVideo

RosetteSep™
Unique Immunodensity Cell Isolation
RosetteSep™ is a fast and easy immunodensity procedure for the isolation of untouched cells directly from whole blood. By crosslinking
unwanted cells to red blood cells (RBCs) present in the sample, RosetteSep™ eliminates the need for a separate magnetic separation step
because cells are purified during standard density gradient centrifugation. This approach significantly reduces sample handling time andmaximizes convenience. Select kits have been CE-marked as Class I IVD products in the European Union. For a complete listof RosetteSep™ products visit www.RosetteSep.com.

Typical RosetteSep™ Protocol

1

2Collect cells

TM

TM

4

p

p

Centrifuge for 20 minutes*

se

tte

Se

TM

p

Se

Se

tte

3

Layer blood over density
gradient medium

Ro

tte

se

se

Ro

Ro

Incubate
20 minutes
Ro

TM

TM

se
Ro tteS
e
se
tte p
Se
p

TM

p
Se
tte
se

tte
se
Ro

Ro

Se

p

TM

Add RosetteSep™
Antibody Cocktail

PlasmaEnriched cells
Whole
blood

Density gradient medium

Density gradientmediumHighly purified cells
are left untouched

Red blood cells andunwanted cells (rosetted)*Use SepMate™ to reduce centrifugation time to 10 minutes with brake on.

Enriched

Start
Rosetted(unwanted)
cellCountsCountsCell to enrich

Red Blood
Cells
Magnification 400X

Figure 16. Picture of a Blood Sample After Addition of
the RosetteSep™ Cocktail and Prior to Density GradientCentrifugation

CD3 FITC

CD3 FITC

Figure 17. RosetteSep™ HLA T Cell Enrichment Cocktail(Catalog #15061HLA)
Starting with fresh whole blood the CD3+ cell content of the enriched fraction
typically ranges from 90% - 97%. In the example above, the T cell (CD3+) content
of the lysed whole blood start sample and the non-lysed final isolated fraction is
20% and 96%, respectively.

RosetteSep™ and SepMate™
Simplified and Standardized Cell Isolation
RosetteSep™ is easily combined with SepMate™ to rapidly and reproducibly isolate PBMC subsets from whole blood. By using the unique
SepMate™ tube, sample throughput is increased and errors associated with improper sample layering are eliminated. This allows even users
with minimal training to consistently perform cell isolation by density gradient centrifugation in a busy laboratory environment.

WWW.STEMCELL.COM

19

CELL SEPARATION PRODUCTS FOR HLA ANALYSIS

Verified and Reliable Antibodies
Take the Guesswork Out of Finding the Right Antibody
Using compatible antibodies is an essential component foryour research. Antibodies from different vendors can provide
variable and potentially sub-optimal staining profiles. STEMCELLTechnologies offers a line of high-quality primary and secondary
antibodies that have been verified to work with our cell isolationand cell culture reagents in specific applications, ensuring thatyour downstream cell analysis, including phenotyping and purity
assessments, works consistently.

Why Use STEMCELL’s Antibodies?
LARGE PORTFOLIO. Large selection of primaryand secondary antibodies and isotype controls.
VERSATILE. Proven performance for multiple applications.
RELIABLE. Trusted by our dedicated teamof research scientists.

Recommended Antibodies for Purity Assessment
Cell Type
Leukocytes

Recommended Labeling Antibodies
MarkerAntigen, Clone

Catalog #CD45

Anti-Human CD45, HI30

60018

CD2

Anti-Human CD2, RPA-2.10

60007

Anti-Human CD3, UCHT1

60011

CD3
T Cells

B Cells

NK Cells
Myeloid Cells

Monocytes

Anti-Human CD3, SK7

60127

CD4

Anti-Human CD4, OKT4

60016

CD5

Anti-Human CD5, UCHT2

60082

CD8

Anti-Human CD8a, RPA-T8

60022

CD19

Anti-Human CD19, HIB19

60005

CD20

Anti-Human CD20, 2H7

60008

CD22

Anti-Human CD22, HIB22

60083

CD43

Anti-Human CD43, CD43-10G7

60085

CD56

Anti-Human CD56 (NCAM), HCD56

60021

CD15

Anti-Human SSEA-1 (CD15), MC-480

60060

CD33

Anti-Human CD33, HIM3-4

60096

CD66b

Anti-Human CD66b, G10F5

60086

CD14

Anti-Human CD14, M5E2
Anti-Human CD14, MoP9

60004
60124

CD36

Anti-Human CD36, FA6-152

60084

Granulocytes

CD66bAnti-Human CD66b, G10F5

60086

Hematopoietic Progenitor Cells

CD34

Anti-Human CD34, 581

60013

Antigen Presenting Cells

HLA-DR

Anti-Human HLA-DR, LN3

60164

Secondary Antibodies
Target Antigen

Host Species

Type

Conjugation

Catalog #Mouse IgG (H + L)

Goat

Polyclonal

FITC

60138

New antibodies are added to our growing inventory on a continuing basis. To find labeling antibodies compatible with a specificcell isolation kit, please refer to the corresponding product information sheet. For a complete list of our available antibodies visit
www.stemcell.com/antibodies.

20

STEMCELL Technologies Inc.

Selected Publications
Selected RoboSep™ Publications
1. Decot V et al. (2008) Chimerism analysis following
nonmyeloablative stem cell transplantation using a new cellsubset separation method: Robosep. Biomed Mater Eng 18(1 Suppl): S19–26.
2. Eggimann L et al. (2015) Kinetics of peripheral blood chimerism
for surveillance of patients with leukemia and chronic myeloid
malignancies after reduced-intensity conditioning allogeneic
hematopoietic SCT. Bone Marrow Transplant 50(5): 743–5.

Selected EasySep™ and RosetteSep™
Publications
1. Alheim M et al. (2013) Evaluation of a new flow cytometry
crossmatch procedure for simultaneous detection of cytotoxicity
and antibody binding. Tissue Antigens 82(2): 125–30.
2. Alheim M et al. (2015) Improved flow cytometry based
cytotoxicity and binding assay for clinical antibody HLA
crossmatching. Hum Immunol 76(11): 849–57.

3. Fernandez-Bango C et al. (2017) P230 Automation for flow
cytometry crossmatch (FCXM) lymphocyte isolation usingrobosep. Hum Immunol 78: 224.

3. Aljurf M et al. (2016) Chimerism Analysis of Cell-Free DNA in
Patients Treated with Hematopoietic Stem Cell Transplantation
May Predict Early Relapse in Patients with Hematologic
Malignancies. Biotechnol Res Int 2016: 8589270.

4. Hanson V et al. (2013) Assessment of the purity of isolated cell
populations for lineage-specific chimerism monitoring post
haematopoietic stem cell transplantation. Tissue Antigens 82(4):269–275.

4. Crespo E et al. (2015) Pre-Transplant Donor-Specific T-Cell
Alloreactivity Is Strongly Associated with Early Acute Cellular
Rejection in Kidney Transplant Recipients Not Receiving T-Cell
Depleting Induction Therapy. PLoS One 10(2): e0117618.

5. Lee HC et al. (2015) Mixed T Lymphocyte Chimerism after
Allogeneic Hematopoietic Transplantation Is Predictive for
Relapse of Acute Myeloid Leukemia and Myelodysplastic
Syndromes. Biol Blood Marrow Transplant 21(11): 1948–54.

5. Falbo DK et al. (2015) Flow cytometric crossmatch results using
lymphocytes isolated from donor peripheral blood and spleen
tissue on five consecutive days. Hum Immunol 76(Supplement):
62.

6. McQueen KL et al. (2013) 115-P: Automated High Throughput
Isolation of Lymphoid and Myeloid Cells Directly from Whole
Blood using RoboSep-HT. Hum Immunol 74(Supplement): 129.

6. Fidler SJ. (2012) Crossmatching by complement-dependent
lymphocytotoxicity. Methods Mol Biol 882: 359–77.

7. Rennert H et al. (2011) Bone Marrow Engraftment Analysis
BT - Diagnostic Molecular Pathology in Practice: A Case-BasedApproach. In I. Schrijver, ed. Diagnostic Molecular Pathology
in Practice. Berlin, Heidelberg: Springer Berlin Heidelberg, pp.
147–157.
8. Szewczyk K et al. (2016) Flow cytometry crossmatchreactivity with pronase-treated T cells induced by non-HLA
autoantibodies in human immunodeficiency virus-infectedpatients. Hum Immunol 77(6): 449–55.
9. van Besien K et al. (2017) Cord blood chimerism and relapse
after haplo-cord transplantation. Leuk Lymphoma 58(2):288–297.

7. Lemp NA et al. (2014) 1016-LBP: RosetteSep HLA –An exceptional method for isolation of lymphocytes fromperipheral blood. Hum Immunol 75(6): 487.
8. Liwski RS et al. (2012) 30-OR: Canada-Wide Evaluationof Rapid Optimized Flow Crossmatch (ROFCXM) Protocol. Hum
Immunol 73: 26.
9. Liwski RS et al. (2018) Rapid optimized flow cytometriccrossmatch (FCXM) assays: The Halifax and Halifaster protocols.
Hum Immunol 79(1): 28–38.
10. Park MH. (2014) P022: Flow Cytometric Crossmatch for
Deceased Donor Transplant Candidates Using Small Numberof Cells and Serum Volume in Microplate. Hum Immunol
75(Supplement): 64.

WWW.STEMCELL.COM

21

CELL SEPARATION PRODUCTS FOR HLA ANALYSIS

Product Listing
Cell Isolation Products
Catalog Numbers of Kits Commonly Used for HLA Applications
Crossmatch
Cell Type

Total
Lymphocytes

Selection

SerologicalTypingChimerism

Whole Blood1

Whole Blood1,
MNC 2, LN3
SpleenWhole
Blood1Whole
Blood1

MNC 2, LN3

15263,
15263HLA4

19655

19961HLA

−

−

−

−

−

−

−

17871

17851

15061
15061HLA4

19671
89671519671
89671515061
15061HLA4−−

Lymphoid(CD3+)

−

−

−

−

15271
15271HLA4

−

CD19

−

−

−

−

17874

17854

CD19/CD20

−

−

−

−

17886

−

15064
15064HLA4

19684
89684519684
89684515064
15064HLA4

−

−

CD15

−

−

−

−

17881

18651

CD33

−

−

−

−

17885

17876

CD33/66b

−

−

−

−

17884

−

CD66b

−

−

−

−

17882

−

Negative

Myeloid

−

−

−

−

15272
15272HLA4−

Positive

CD14

−

−

−

−

17878

17858

Positive

CD56

−

−

−

−

17875

17855

−

−

−

−

−

17955

−

−

−

−

17879

17856

Negative
Positive

CD3

T Cells
Negative

Positive
B Cells
Negative

Myeloid Cells/
Granulocytes

Monocytes

Positive

NK Cells
Negative
Hematopoietic
Progenitors

Positive

EasySep™/RoboSep™ kits
1.
2.
3.
4.
5.

CD34

RosetteSep™ kits

Kit also works on other red blood cell containing samples (i.e. cord blood, buffy coat).
Kit works on mononuclear cells (MNCs) isolated from peripheral blood or bone marrow. MNC: Mononuclear Cell
LN: Lymph NodeThis kit has been CE-marked as a Class I In Vitro Diagnostic (IVD) product in the European Union according to the 98/79/EC In Vitro Diagnostic Device Directive and complies with its requirements.
This kit carries the CE mark and is available as a Class I IVD device in Australia, Canada and the European Union. To learn more about the regulatory status of this product in your specific region,
contact info@stemcell.com.

Our commitment to your research does not end when you purchase our products. STEMCELL Technologies' Technical Support experts
and comprehensive RoboSep™ service packages offer top quality support and maintenance, freeing you to focus on your science.
Please visit www.RoboSep.com to learn more.

22

STEMCELL Technologies Inc.

Cell Separation Magnets and Accessories
EasySep™ Magnets for Manual Cell Isolation
The Iconic ColumnFree Cell Isolation
Platform

For Larger ColumnFree Cell Isolations

EasySep™ Magnet

For Very Large
Volume Cell
Isolations"The Big Easy"
EasySep™ Magnet

For Simultaneous
Processing ofMultiple Samples

Easy 50
EasySep™ Magnet

For Simultaneous
Small Volume Cell
Isolations

EasyEights™
EasySep™ Magnet

EasyPlate™
EasySep™ Magnet

Catalog #

18000

18001

18002

18103

18102

Number of Samples

1

1

1

8 on each side = 16 total

96

0.25 - 2.0 per 5 mL tube
0.5 - 8.0 per 14 mL tube

0.05 - 0.2 mL

Working Volume1

0.1 - 2.5 mL

0.2 - 10 mL

0.5 - 40 mL

Collection Method

Pour off

Pour off

Pipette off

Pipette off

Pipette off

Polystyrene 50 mL Tube
(Catalog #38010)

Polystyrene 5 mL Tube
(Catalog #38007)
Polystyrene 14 mL Tube
(Catalog #38008)

96-Well Round-Bottom
Microplate(Catalog #38018)

Recommended Plasticware

Polystyrene 5 mL Tube
(Catalog #38007)

Polystyrene 14 mL Tube
(Catalog #38008)

RoboSep™ Instruments & Accessories

SepMate™ Products

Product

Catalog #RoboSep™-S

21000

RoboSep™-16

23000
2

RoboSep™ Buffer (250 mL)

20104

RoboSep™ Buffer 5X Concentrate (250 mL)

20124

RoboSep™ Filter Tip Racks2 (1 box of 8 racks)for RoboSep™-S

20125

Sterile Filtered Conductive Tips for RoboSep™-16

23101

Non-Sterile Filtered Conductive Tips
for RoboSep™-16

23102

Density Media
Product

Catalog #Unit Size

Lymphoprep™

07801
07851
07811
07861

250 mL
500 mL
4 x 250 mL
6 x 500 mL

HetaSep™

07806
07906

20 mL
100 mL

RosetteSep™ DM-L

15705

100 mL

RosetteSep™ DM-M

15725

100 mLSpinSep™ Density Medium

17531

100 mL

Product

Catalog #SepMate™-15 (IVD3)

85415

4

SepMate™-15 (RUO )86415

SepMate™-50 (IVD3)

85450

SepMate™-50 (RUO4)

86450

Blood Volume
Processed (mL)

Unit Sizte

0.5 - 5100 tubes
4 - 17

1.		
Please refer to the product information sheet of each product for the corresponding
volume range and cell concentration that can be processed with each EasySep™ magnet.
2. 		 RoboSep™ Buffer and 1 - 2 boxes of RoboSep™ Filter Tip Racks are included with every
purchase of a RoboSep™ Reagent Kit.
3.		 SepMate™ 85415/85450 is available in Australia, Canada, Europe and the UnitedStates of America, select regions where it is registered as an In Vitro Diagnostic (IVD)
device for the isolation of mononuclear cells from human whole blood, cord blood andbone marrow by density gradient centrifugation. This product is also available in China
where it is considered a non-medical device by the China Food and Drug Administration(CFDA), and should therefore be used as general laboratory equipment.
4.		 SepMate™ 86415/86450 is intended for Research Use Only (RUO) and is available
in regions where SepMate™ 85415/85450 is not available.

Copyright © 2019 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping Scientists, EasySep,
RoboSep, RosetteSep, SepMate, EasyStand, EasyEights, EasyPlate, RapidSpheres, HetaSep, and SpinSep are trademarks of STEMCELL Technologies Canada Inc. Lymphoprep is a trademark of Alere
Technologies. Ficoll-Paque is a registered trademark of GE HealthCare Ltd. Lympholyte is a registered trademark of Cedarlane Laboratories Ltd. All other trademarks are the property of their respective
holders. While STEMCELL has made all reasonable efforts to ensure that the information provided by STEMCELL and its suppliers is correct, it makes no warranties or representations as to the accuracy orcompleteness of such information.
STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC
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DOCUMENT #29045

VERSION 8.0.0

APRIL 2019