Escolar Documentos
Profissional Documentos
Cultura Documentos
Agus S Kosasih
Grans
Size:
2
FS (Size)
Granularity:
Lymphs
Grans > Monos >
Lymphs
SC (Granularity)
Lysed Whole Blood
largest and most
complex population
1000
800 Neutrophils
Side Scatter
600
400
Monocytes
200
FITC FITC
FITC
FITC
FITC
FITC FITC
FITC
Number of Events
FITC
FITC
Fluorescent Intensity
Collection Optics–
Optics–
FACSCalibur Cytometer
Detector Filter Color Fluorochrome
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
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
1. Preparation of specimen
2. Acquisition
3. Gating and Data Analysis
Preparation of specimen
Trucount tube
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 #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,
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