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COMPARISON OF NEW MICROSCOPIC SYSTEM

FOR THE MEASUREMENT OF RESIDUAL


LEUCOCYTES IN APHERESIS PLATELETS (APLT)
WITH FLOW CYTOMETRY AND MANUAL
COUNTING

NUNUNG M I UMAR, RAEHANA SAMAD, AGUS ALIM ABDULLAH

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ABSTRACT
Background and Objectives : since 2001, all blood
components in Germany must be leucocyte depleted.
Recently, a new method for quality control of depletion
was introduced. Our study aimed at the validation of the
methode for routine use in apheresis platelet
concentrates.
Material and Methods : we compared the new ADAM-
rWBC device with manual counting in the Nageotte
chamber and flow cytometry, two standard methods, by
measuring residual leucocytes in 40 units of apheresis
platelet concentrates and in six geometrical dilution
series.

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ABSTRACT
Results : Cell counts of residual leucocytes in
the 40 units were below 106 cells per
componenet with all method, although mean
cell counts were approximately 5 and 6 times
higher in flow cytometry and ADAM-rWBC,
respectively, compared to the Nageotte
chamber. No unit with <106 leucocytes was
regarded as contaminated.

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ABSTRACT
The dilution series showed acceptable
accuracy, especially in the range around the
cut-off (aproximately 4-5 cells/µl in
components with a volume of 220 ml) for
regarding a concentrate as contaminated with
leucocytes. No sample spiked with more than
4-5 cell/µl was counted as having less.

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ABSTRACT
Conclusion : In comparison with manual
counting and flowcytometry, the ADAM-rWBC
device performed equally. The method is
suitable for routine screening of leucocyte
contamination of apheresis platelets.

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INTRODUCTION
Blood Transfusion  high amounts of leucocytes
used to induce a lot of side effect
The most important :
- associated graft-versus-host disease or
immunomodulation
- alloimmunization to HLA antigents with
consecutive lack of increment after platelet
transfusion,
- febrile transfusion and infections with leucocyte
dependent viruses like HTLV or CMV

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INTRODUCTION
 Target of leucocyte depleted : < 106
leucocytes per component.
 Following the manufactures descriptions,
established haematologic cell counters
based on flow cytometryc techniques.
 Other method as manual counting is
Nagoette chamber

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INTRODUCTION
New divice Adam-rWBC_microscopic
cell counter measuring on plastic slide with
flourescent staining (propidium iodide).

Flourescent spots on the picture


analysed via image counted as
cell by the software.

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MATERIAL AND METHODS
40 units leucocyte APLT concentrate

Manual counting Flowcytometry Adam-rWBC


(Nageotte Chamber)

• Adam_rWBC uses a green LED as light source


• 203 frames analysed
• Microscopic cell counter measures leucocytes on a plastic
slide after flourescent staining a blood sample
• The pictures of 203 frame taken by CCD camera via
image analysis software counted as cell.
• The measured concentration is shown on displasy of software
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tool.
MATERIAL AND METHODS
Dilution series with 6 APLT samples spiked with
leucocytes from 128 - 0,031 leucocytes per µL

Prior to spiking, APLTs were filtered


to eliminate residual leucocytes

The leucocytes for spiking were taken


from a cell separator and measured with
a cell counter KX-21

Leucocytes and APLT were mixed to first


produce a concetration of 1000 cells/µL , wihich was
confirmed by another measurement cell counter
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MATERIALS AND METHODS
Leucocytes and APLT were mixed to first produce
a concetration of 1000 cells/µL, wihich was
confirmed by another measurement cell counter

Another calculated dilution of this Solution


APLT should produce the Following cell
concentration :128, 64, 32, …,….,…., down to 0,031/µL

This series were also measured


with the three methoeds : manual counting,
flowcytometry, And Adam-rWWBC
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STATISTICAL ANALYSIS
• All statistical test were using SPSS Version
20
• Normal distribution of variables was tested
with Saphiro-Wilks test
• Group comparasion was perfomed with the
Wilcoxon rank-sum
• Correlation between two methodes were
analysed with th Kandall Tau-b test.

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STATISTICAL ANALYSIS
• Linearity was tested by regression analysis
• The coefficient of variation (CV) was
calculated by dividing the standared
deviation (SD) by the mean and multiplying
the result by 100 (CV (%) = (SD/mean) x
100%)

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RESULT
Tabel 1. Mean Leucocytes concetration and absolute cell count, minimum
and maximum leucocytes cell concentration and absolute cell count of 40
APLT units measurered by three different methods and P values

Method Mean cell Min-Max cell P value P value of


concentration concentration compared Adam-rWBC
± SD per µL per µL to Nageotte compared to
Chamber Flowcytometry

Nageotte 0.0725±0.088 0.0-0.3 - -


Flow-
Cytometry 0.410±0.579 0.0-2.81 0.0000318 -
Adam-
r_WBC 0.468±0.417 0.0-2.16 0.0000002 0.031 14
RESULT
Mean absolute cell Min-Max absolute
coun t± SD x 106 cell count x 106

Nageotte 0.015950±0.019287 0.0-0.066

Flowcytometry 0.090200±0.0127345 0.0-0.6182

Adam_rWBC 0.102960±0.091694 0.0-0.4752

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Tabel.2 Mean leucocyte cell count/µL ± SD of six dilution
series as absolute cell count and calculated relation
Mean ± SD; n = 6
Absolute cell count per µ
Target Adam Flow Nageotte
128 141±28 121±15 115±23
64 74±15 61±7 55±12
32 40±7 31±4 25 ±5
16 20 ±4 17 ±4 13 ±2
8 10 ±1 8 ±2 7 ±1
4 6 ±2 5 ±1 3 ±1
2 3 ±1 2 ±0 2 ±0
1 2 ±0.5 1 ±1 1 ±0
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0.500 1 ±0.3 0.5 ±0.2 0.4 ±0.04
Target Adam Flow Nageotte

0.250 0.6 ± 0.3 0.5 ±0.2 0.4 ±0.04


0.125 0.9 ±0.3 0.1 ± 0.1 0.2 ± 0.08
0.063 0.2 ± 0.09 0.1 ± 0.08 0.02 ± 0.04
0.031 0.3 ± 0.2 0.06 ± 0.07 0±0

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Calculated relation of dilution in %
Target Adam Flow Nageotte
100 100 100 100
50 52.3±6.6 50.7±2.9 47.3±4.9
25 28.6±6.0 25.8±3.0 21.4±2.9
12.5 14.0±2.4 14.4±2.2 11.5±1.5
6.25 7.2±0.5 6.9±1.0 6.7±2.3
3.125 4.0±0.8 3.7±0.6 2.8±0.8
1.563 2.0±0.6 1.9±0.2 1.4±0.4
0.781 1.2±0.4 1.2±0.7 0.7±0.1
0.391 0.7±0.2 0.4±0.2 0.3±0.08
0.195 0.4±0.2 0.2±0.1 0.2±0.05
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Target Adam Flow Nageotte

0.098 0.6±0.3 0.1±0.08 0.1 ±0.05


0.049 0.1±0.05 0.1±0.08 0.01±0.03
0.024 0.3±0.2 0.05±0.07 0±0

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Figure 1. (a) Regression analysis of six dilution series20
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(b) Regression analysis of six dilution series
DISCUSSION
• In our study, cell counts differed
significantly.
• The Adam_rWBC highest cell counts,
followed by flowcytometry and Nageotte
chamber
• The Adam_rWBC best acuracy and
precision, especially in the range of
concentrations most important.

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Table 3. Accuracy and precision of ADAM_rWBC, flowcytometry and
Nageotte chamber
ADAM
Accuracy
Target Mean±SD (%) CV (%)

8/µL 8.36±0.33 100 3.96


4/µL 4.30±0.36 100 8.29
2/µL 2.11±0.42 80 19.82
1/µL 1.37±0.23 40 16.78
0.5/µL 0.65±0.08 20 11.62

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FLOWCYTOMETRY
Accuracy
Target Mean±SD (%) CV (%)

8/µL 7.33±0.74 80 10.12


4/µL 3.65±0.39 80 10.56
2/µL 1.58±0.19 20 12.26
1/µL 1.09±0.28 60 25.66
0.5/µL 0.55±0.14 60 25.39

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Nageotte Chamber

Accuracy
Target Mean±SD (%) CV (%)

8/µL 6.44±0.57 60 8.83


4/µL 3.22±0.46 20 14.30
2/µL 1.46±0.19 60 13.35
1/µL 0.90±0.21 80 23.57
0.5/µL 0.24±0.11 20 47.51

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DISCUSSION
• Our data mainly confirm the result of Bae et
al, who presented their data on
measurement of residual leucocytes in red
blood cell concentrates.
• In contrast to Bashir et al, we did not
observe much extra-regional events in
flowcytometry in our series.

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DISCUSSION
A limitation of our study :
• Difficulty of producing diluted samples that
really contain the calculated concentrations,
especially in very low ranges
• While pipetting the dilution series, white
blood cell might not distribute evenly in the
sample, as aggregates of cells migth
develop, or cell might sediment to the
bottom of the tube despite mixing.
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O In concluision, all three methodes are
suitable for measuring residual in aphresis
platelet.

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