Enumeration of CD34+ Stem Cells using Flowcytometry

Clinical Pathology LaboratoryDharmais Cancer Hospital

Agus S Kosasih

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/7AADCD 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

FlowcytometryFlowcytometry

Size / GranularitySize / Granularity

11

FS (

Size

)

SC ((Granularity)

33

22

Lymphs

Monos

Grans

Whole blood sample

RBC lysed

Size:

Grans > Monos > Lymphs

Granularity:

Grans > Monos > Lymphs

Sid

e S

catte

r

Forward Light Scatter

0 200 400 600 800 1000

020

040

060

080

010

00

Lymphocytes

Monocytes

Neutrophils

largest and most complex population

smallest and least complex population

Lysed Whole BloodLysed Whole Blood

TheThe fluorochrome absorbs energy from the laserfluorochrome absorbs energy from the laser

The fluorochrome releasesThe fluorochrome releases the absorbed energy bythe absorbed energy by::–– Vibration and heat dissipationVibration and heat dissipation

–– Emission of photons of a longer wavelengthEmission of photons of a longer wavelength

λ = 488 nm

Antibody

IncidentLight Energy

EmittedFluorescent Light

Energy

λ = 530 nm

FluoresceinMolecule

HO

CO2H

O

C

What is Fluorescent Light?What is Fluorescent Light?

FluorescenceFluorescence

FITC FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

Emitted Fluorescence Intensity ∝ Binding Sites

FITC

Fluorescent Intensity

Num

ber o

f Eve

nts

Detector

FL1

FL2

FL3

FL4

Filter

530/30 nm

585/42 nm

670nm LP

661/16 nm

Fluorochrome

FITC

PE

PerCP, PerCP-Cy5.5

APC

Color

Green

Yellow/Orange

Dark Red

Red

Collection OpticsCollection Optics––FACSCalibur CytometerFACSCalibur Cytometer

CD 45 gatingCD 45 gating

CD45 GatingCD45 Gatingmost informative markermost informative markergood for all cell typesgood for all cell types

10 10 10 10 100 1 2 3 4

M220327.001CD45 PerCP ->

10 10 10 10 100 1 2 3 4

M220327.001CD45 PerCP ->

NRBC

gran

mono

lymph

Sid

e S

catte

r

Forward Light Scatter

CD 34CD 34

Other cellOther cell

Stem cellStem cell

CD 45 FITCCD 45 FITC

CD45 FITCCD45 FITC

The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted.

CD34 PECD34 PE

Stem CellStem Cell

70%70%30%30%

60%60%

40%40%

1 2

3 4FITC

PE

20%20% 40%40%

30%30%10%10%+/+/--

+/++/+--/+/+

--//--

Dual Parameter HistogramsDual Parameter Histograms

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 ExperienceTEST 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 enumerationCD34+ stem cell enumeration

• Single platform flowcytometry using

FACSCalibur

• Analysis: Cellquest software

• CD45 gating

• Viability testing : 7-aad

• Template from ISHAGE protocol

STEPSSTEPS

1.1. Preparation of specimenPreparation of specimen2.2. Acquisition Acquisition 3.3. Gating and Data Analysis Gating and Data Analysis

10 µl CD 45 FITC / CD34 PE

Incubate 15‘ dark, room temperature

Add 1 ml Lysing solution

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

ACQUISITION

Trucount tube

50 µl specimen

Preparation of specimenPreparation of specimen

Incubate 15‘ dark, room temperature

AcquisitionAcquisition

Instrument settingInstrument settingISHAGE Template ISHAGE Template Acquire 75000 cellsAcquire 75000 cellsCell Quest softwareCell Quest software

Data analysisData analysis

CD45 gatingCD45 gatingCD45+ absolute cell countCD45+ absolute cell countCD34+ percentageCD34+ percentageCD34+ absolute cell countCD34+ absolute cell countCD34+ viabilityCD34+ 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 1 Day 3 Day 5

Day 6 pre 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 (phase I)

100

1,000

10,000

100,000

1,000 ,00010,000,000

Day 1 Day 3 Day 5 Day 6 preharvest

Harvest

CD34

cou

nt (L

OG 1

0 ce

ll/m

l) Pat #1Pat #2Pat #3Pat #4Pat #5Pat #5

CD 34 count 2nd phasePat # Day 1 Day 3 Day 5 Harvest

7 1,310 3,740 23,690 820,8308 870 2,810 11,680 540,9209 1,380 14,120 124,470 2,127,850

10 2,530 12,550 43,160 994,07011 1,310 14,510 64,170 1,072,08012 2,260 7,360 32,600 1,608,28013 670 5,010 48,230 1,397,02014 2,370 20,590 80,090 1,715,66015 900 3,500 14,320 68,11016 5,030 8,410 15,260 688,00017 1,050 5,060 76,990 1,539,08018 600 19,020 29,380 956,570

CD34+ count in patients undergoing stemcell therapy (phase 2)

1000

1000

0

1000

00

1000

000

1000

0000

Day 1 Day 3 Day 5 Harvest

CD

34+

coun

t (lo

g 10

cel

ls/ u

l)

Pt # 7Pt # 8

Pt # 9

Pt # 10

Pt # 11

Pt # 12Pt # 13

Pt # 14

Pt # 15

Pt # 16

Pt # 17

Pt # 18

Harvest Product 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 + 523.8WBC (x 103 cell/ul) 10.04 + 3.46 309.65 + 105.1MN absolute (x 103 cell/ul) 2.7 + 1.2 124.2 + 34.5CD4 (%) 41.3 + 8.4 39.2 + 8.7CD8 (%) 20.9 + 7.9 20.9 + 8.5CD19 (%) 15.6 + 4.9 14.7 + 8.0CD14 (%) 7.4 + 3.6 13.2 + 6.2CD56 (%) 19.4 + 7.8 18.4 + 7.9RBC (x 106 cell/ul) 4.55 + 0.47 0.25 + 0.19PLT (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

Pt #1 Pt #2 Pt #3 Pt #4

PrePost ficoll Pre

Post ficoll Pre

Post ficoll Pre Post ficoll

CD45 (x10^3/ul) 10.7 16.12 9.3 12.76 20.33 35.73 17.23 15.07CD34 (/ml)

74940 483100 67800 449390 164740 525990 117640 554980CD133

(/ml) 64200 450840 72100 - 87290 22780 92080 207260WBC

(x10^3/ul) 13.23 20.55 11.15 14 21.68 47 20.3 18.9MN

(x10^3/ul) 4.35 12.88 3.16 8.37 29.00 55 17.00 42Viability

(%) 99.93 99.84 99.71 99.94 99.95 99.88 99.89 97.76

RESULT OF STEM CELL ENUMERATION IN NOGA

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