Enumeration of CD34+ Stem

Cells using Flowcytometry

Agus S Kosasih

Clinical Pathology Laboratory

Dharmais Cancer Hospital
 What is a Stem Cell?
• Stem cell is the cell with the ability to self-
replicate throughout the life of an organism
and can differentiate or give rise to many
cell types upon stimulation and given the
right condition.
• Stem cells have the potential to develop
into functional mature cells characteristic
of different tissue or organs
 Stem cell types
• Totipotent
– Differentiate into all type of cells
• Pluripotent
– Differentiate into 3 germinal layers: ectoderm,
mesoderm and endoderm
– Embryonic stem cells
• Multipotent
– Differentiate into several types of cells
– Hematopoietic stem cells
• Unipotent
– Can only produce 1 type of cell
 Source of stem cells
• Zygote
• Embryonic stem cells
• Cord blood stem cells
– Hematopoietic stem cells
• Adult stem cells
– Bone Marrow:
• Hematopoietic stem cells
• Mesenchymal stem cells
 Source of stem cells
• Peripheral blood :
– Hematopoietic stem cells
• Other Tissue
– Adipocyte
– Muscle
– Nerve
– Bone
Hematopoietic stem cells
 How to Identify Stem cells ?
• Flowcytometry
• CD 34 (Hematopoietic stem cells)
• CD 133 (Epithelial Progenitor Cells)
• Dharmais Hospital
- Facs Calibur 4 colors (Becton Dickinson)
CD 34 : CD45 FITC/CD34 PE/7AAD
CD 133 : CD45 FITC/CD133PE/7AAD
– Single Platform (Trucount tube)
 What Is Flow Cytometry?
Cyto = cell Metry = measurement

• Simultaneous measurements of multiple

characteristics of a single cell
• Measurements are made on a per-cell
basis at routine rates of 500 to 4000
cells per second
Flowcytometry
 Size / Granularity
Whole blood sample
3
RBC lysed

Grans
Size:
2
FS (Size)

Grans > Monos >

Lymphs
1
Monos

Granularity:
Lymphs
Grans > Monos >
Lymphs
SC (Granularity)
 Lysed Whole Blood
largest and most
complex population

1000
800 Neutrophils
Side Scatter

600
400

Monocytes
200

smallest and least Lymphocytes

complex population
0

0 200 400 600 800 1000

Forward Light Scatter

 What is Fluorescent Light?
HO O
λ = 488 nm λ = 530 nm
C
Incident CO2H Emitted
Light Energy Fluorescent Light
Fluorescein Antibody Energy
Molecule

The fluorochrome absorbs energy from the laser

The fluorochrome releases the absorbed energy by:

– Vibration and heat dissipation
– Emission of photons of a longer wavelength
 Fluorescence
Emitted Fluorescence Intensity ∝ Binding Sites

FITC FITC
FITC
FITC
FITC
FITC FITC
FITC
Number of Events

FITC
FITC

Fluorescent Intensity
 Collection Optics–
Optics–
FACSCalibur Cytometer
Detector Filter Color Fluorochrome

FL1 530/30 nm Green FITC

FL2 585/42 nm Yellow/Orange PE

FL3 670nm LP Dark Red PerCP, PerCP-Cy5.5

FL4 661/16 nm Red APC

CD 45 gating
 CD45 Gating
most informative marker
good for all cell types

10 0 10 1 10 2 10 3 10 4
CD45 PerCP ->
M220327.001
Side Scatter

gran

mono

NRBC lymph
Forward Light Scatter
10 0 10 1 10 2 10 3 10 4
CD45 PerCP ->
M220327.001
CD 34
 Stem Cell

CD 45 FITC

Other cell
CD45 FITC

Stem cell

The image cannot

be displayed. Your

CD34 PE
computer may not
have enough
memory to open
the image, or the
image may have
been corrupted.
Dual Parameter Histograms
1 2
-/+ +/+
20% 40%

PE
60%

10% 30%
40%
3 -/ - +/--
+/ 4
FITC

30% 70%
 Standards for Cellular Therapy
Product Services (AABB)
Ref. Std 5.14A – Processing tests for HPC,
apheresis and HPC, marrow products

• Tests to be performed :
– Total nucleated cell count
– CD34 analysis or comparable assay
– Microbial contamination (culture for bacterial
and fungal elements)
– Cell viability
 Dharmais Experience

• Stem Cell Therapy for AMI

• Autologous Peripheral Blood Stem Cell
Transplantation (PBSCT)
• Autologous Bone Marrow Stem Cell
Transplantation (NOGA)
• Stem Cell Count using Flowcytometry
– CD 34
– CD 133
 Dharmais Experience
TEST PANEL for PBSCT
• CBC + diff count
• Lymphocyte subsets + CD14
• Blood viscosity
• CD 34+ Stem Cell Count (FacsCalibur)+ viability
• day 1 (before G-CSF inj)
• day 3
• day 5 (before Harvest)
• day 5 (after Harvest)
• Culture for bacteria (harvest product)
• Culture for fungi (harvest product)
Dharmais Experience CD 34 count
in PBSCT
• Harvesting of Stem cells:
– Day 5 after G-CSF injection
– Using Cobe Spectra Apheresis system
– 1 million stem cells /ml
CD34+ stem cell enumeration

• Single platform flowcytometry using

FACSCalibur
• Analysis: Cellquest software
• CD45 gating
• Viability testing : 7-aad
• Template from ISHAGE protocol
 STEPS

1. Preparation of specimen
2. Acquisition
3. Gating and Data Analysis
 Preparation of specimen

10 µl CD 45 FITC / CD34 PE 50 µl specimen

Trucount tube

Incubate 15‘ dark, room temperature

Add 1 ml Lysing solution

Add 10 ul Cell Viability Solution (7aad), vortex gently

Incubate 15‘ dark, room temperature

ACQUISITION
 Acquisition
Instrument setting
ISHAGE Template
Acquire 75000 cells
Cell Quest software
 Data analysis
CD45 gating
CD45+ absolute cell count
CD34+ percentage
CD34+ absolute cell count
CD34+ viability
 Result of
CD34 count
Dharmais Experience CD 34 count
in PBSCT
• Two phase :
– Phase I : 6 patients – 6 days protocol
– Phase II : 12 patients – 5 days protocol
 CD 34 count First phase

Pat Day 6 pre

# Day 1 Day 3 Day 5 harvest Harvest

1 13,120 35,260 115,720 92,980 1,071,110

2 2,290 7,670 27,370 23,970 830,310

3 11,390 27,900 49,050 47,140 2,084,380

4 3,970 27,030 67,550 255,020 1,940,900

5 650 4,730 19,170 17,290 732,680

6 1,010 7,180 25,490 23,920 1,405,010

 CD34 count in patients for stem cell therapy
00
(phase I)
,0
00
,0
10
CD34 count (LOG 10 cell/ml)

Pat #1
0
00
0,

Pat #2
00
1,

Pat #3
0
00
0,

Pat #4
10

Pat #5
00
,0
10

Pat #5
0
00
1,

Day 1 Day 3 Day 5 Day 6 pre Harvest

0
10

harvest
 CD 34 count 2nd phase
Pat # Day 1 Day 3 Day 5 Harvest
7 1,310 3,740 23,690 820,830
8 870 2,810 11,680 540,920
9 1,380 14,120 124,470 2,127,850
10 2,530 12,550 43,160 994,070
11 1,310 14,510 64,170 1,072,080
12 2,260 7,360 32,600 1,608,280
13 670 5,010 48,230 1,397,020
14 2,370 20,590 80,090 1,715,660
15 900 3,500 14,320 68,110
16 5,030 8,410 15,260 688,000
17 1,050 5,060 76,990 1,539,080
18 600 19,020 29,380 956,570
 CD34+ count in patients undergoing stemcell therapy
(phase 2)

Pt # 7
0 0
CD34+ count (log 10 cells/ ul)

00

Pt # 8
00
10

Pt # 9
Pt # 10
0
00

Pt # 11
00
10

Pt # 12
Pt # 13
0 0
00

Pt # 14
10

Pt # 15
Pt # 16
0
00

Pt # 17
10

Pt # 18
00

Day 1 Day 3 Day 5 Harvest

10
 Harvest Product
• Consists of :
– Stem cells
– Mononuclear cells
– Platelet
– RBC
– Polymorphonuclear cells
 Comparison between cell composition of
baseline and harvest product
Whole blood
base line Harvest product
CD34 (x 103 cell/ml) 2.9 + 3.6 1,133.4 + 523.8
WBC (x 103 cell/ul) 10.04 + 3.46 309.65 + 105.1
MN absolute (x 103 cell/ul) 2.7 + 1.2 124.2 + 34.5
CD4 (%) 41.3 + 8.4 39.2 + 8.7
CD8 (%) 20.9 + 7.9 20.9 + 8.5
CD19 (%) 15.6 + 4.9 14.7 + 8.0
CD14 (%) 7.4 + 3.6 13.2 + 6.2
CD56 (%) 19.4 + 7.8 18.4 + 7.9
RBC (x 106 cell/ul) 4.55 + 0.47 0.25 + 0.19
PLT (x 103 cell/ul) 289.61 + 133.52 1,572.12 + 1024.16
 Dharmais Experience
TEST PANEL for NOGA
• Baseline:
– Marrow cell count + diff count
– CD 34+ Stem Cell Count (FacsCalibur)+
viability
– CD 133+ Stem Cell Count (FacsCalibur)+
viability
 Dharmais Experience
TEST PANEL for NOGA
• Product :
– Cell count + diff count
– Lymphocyte subsets + CD14
– CD 34+ Stem Cell Count (FacsCalibur)+
viability
– CD 133+ Stem Cell Count (FacsCalibur)+
viability
– Culture for bacteria
– Culture for fungi
Result of CD34+ and CD133+ in NOGA
 RESULT OF STEM CELL ENUMERATION
IN NOGA
Pt #1 Pt #2 Pt #3 Pt #4
Post Post Post
Pre ficoll Pre ficoll Pre ficoll Pre Post ficoll
CD45
(x10^3/ul) 10.7 16.12 9.3 12.76 20.33 35.73 17.23 15.07
CD34 (/ml)
74940 483100 67800 449390 164740 525990 117640 554980
CD133
(/ml) 64200 450840 72100 - 87290 22780 92080 207260
WBC
(x10^3/ul) 13.23 20.55 11.15 14 21.68 47 20.3 18.9
MN
(x10^3/ul) 4.35 12.88 3.16 8.37 29.00 55 17.00 42
Viability
(%) 99.93 99.84 99.71 99.94 99.95 99.88 99.89 97.76
 Conclusion
• Accurate stem cells counting is very
important for Stem Cell therapy
• Flowcytometry is the Gold standard
method for stem cell enumeration
THANK YOU FOR YOUR
ATTENTION