The need for counting residual leukocyte in blood products
Allogeneic leukocytes in red blood cell units and platelet concentrates have
been involved in a variety of transfusion reactions, including HLA
alloimmunization, the transmission of cell-associated viruses and prions, and
immunosuppressive effects.
In the US, sanitary regulations require that residual leukocytes are lower than
5 x 106 (~ 20 WBC/μL) per product in RBC and apheresis platelets and lower
than 0.86 x 106 per product in whole blood platelet preparations (~ 2 WBC/μL).
Because of this, simple and accurate routine methods for the enumeration of
residual leukocytes was needed.
Nageotte chamber and standard hematology analyzers have low sensitivity
when considering the expected low leukocyte contamination.
Flow Cytometry in residual leukocyte counting
Flow cytometry (FCM) has become a choice method to evaluate leukoreduced
blood products, and such application is a typical example of rare cell
detection.
FCM is used currently for accurate determination of residual leukocytes in
blood components by DNA staining with more reproducibility and
throughput than conventional counting methods.
Current residual leukocyte counting by FCM based on DNA :
• Is a time-consuming, “rare-event” issue.
• Requires fixation/permeabilization.
• Is performed on a monoparametric basis.
higher
Flow Cytometry in residual leukocyte counting
Thus, FCM studies on leukoreduction might benefit from:
• Faster acquisition rate:
• To allow acquiring statistically significant numbers of leukocytes in
substantially shorter periods of time.
• Multiparametric /polychromatic analysis:
• To allow simultaneous immunophenotypic analysis.
• Use of permeable DNA-binding dyes:
• To allow functional analysis.
• Volume-based count calculation:
• To allow determination of cell concentration without adding beads.
Attune™ Flow Cytometer: Advantages for Rare Cell Counting
Direct Counting
Acoustic Focusing
Violet Laser
Multiple Discriminators
Sample rate control
Attune™ Flow Cytometer: Advantages of Violet Laser
The violet laser (405 nm) has become increasingly popular in flow cytometry due to its
ability to increase the multiplexing capabilities of the flow cytometer.
Reagents for the violet laser include cell cycle analysis, cell viability and vitality, cell
proliferation, and apoptosis.
Acoustic Focusing and Violet Laser for Leukocyte Counting
We have developed a no-wash no-lyse FCM method for counting residual
leukocyte in leuko-reduced blood using the Attune™ Acoustic Focusing Flow
Cytometer and Vybrant® DyeCycle™ Violet, a cell membrane-permeant dye
for DNA analysis in living cells excited by violet laser.
No-wash no-lyse Method for Residual Leukocyte Counting
The method involves incubation of leuko-reduced samples with 50 M
Vybrant DyeCycle Violet for 30 min at 37ºC without any further manipulation.
1. 100 μL of sample + 1 μL of 5 mM Vybrant® DyeCycle™ Violet
2. Incubate for 20 minutes in the dark.
3. Make up to 3 mL with RPMI medium.
4. Analyze in an Attune flow cytometer using violet and blue lasers
Leukocyte Identification with Vybrant DyeCycle Violet
VL1 -H (450 nm)
VL1 (450 nm):
DNA content by Vybrant
DyeCycle Violet
BL2 (610 nm):
Autofluorescence
BL2 -H (610 nm)
Cell concentration is
directly obtained in the
Attune flow cytometer
Leukocyte Identification with existing PI-Based Methods
Leucocount /BD
Leucocount /FC500
Leukosure /EPICS XL
A
B
Cell concentration must be calculated from a formula:
WBC/L = WBC events/Bead events x Stated Beads per tube/Sample volume
No-wash no-lyse Method for Residual Leukocyte Counting
Assay performance has been validated by assessing:
- Internal consistency of the Attune cytometer by running serial dilutions of
fluorescent microspheres (FlowCount, Beckman-Coulter) and of whole
blood in RPMI medium.
- Correlation between leukocyte counts obtained with the Attune cytometer
and with Neubaer or Nageotte chambers.
- Correlation of leukocyte counts obtained with the Attune cytometer and
with a standard hydrodynamic-focusing cytometer (Cytomics FC500
MCL, Beckman-Coulter) using a commercial kit based on DNA staining
with Propidium Iodide plus concentration-calibrated beads (Leucocount
Kit, Becton Dickinson).
Counting Serial Dilutions of Beads: Effect of Flow Rate
120
Attune
100
80
25 µL/min
60
100 µL/min
200 µL/min
40
500 µL/min
1000 µL/min
20
0
0
20
40
60
80
100
120
Manual
Flow rate
25 µL/min
100 µL/min 200 µL/min 500 µL/min 1000 µL/min
Equation
y = 0,913x y = 0,970x y = 1,017x y = 0,913x y = 0,967x
Regression
R² = 0,999 R² = 0,999 R² = 0,999 R² = 0,999 R² = 0,999
Residual Leukocyte Counting in Blood Preparations
• Human whole blood and serial dilutions in RPMI medium.
• Concentration-calibrated cellular preparations for residual leukocyte
counting (RBC LeukoTrol cells Beckman-Coulter):
• Low control: 2 cells/μL
• High control: 20 cells/ μL
• Leuko-reduced RBC bags for transfusion.
Counting Serial Blood Dilutions: Effect of Flow Rate
25
ATTUNE
20
15
100 µL/min
200 µL/min
10
500 µL/min
5
1000 µL/min
0
0
5
10
15
20
25
MANUAL
Flow rate
100 µL/min
200 µL/min
500 µL/min
1000 µL/min
Equation
y = 0,990x
y = 0,991x
y = 0,930x
y = 0,895x
Regression
R² = 0,999
R² = 0,998
R² = 0,996
R² = 0,996
Counting of Leuko-Trol RBC High: Correlations
y = 1.1116x
R² = 0.9996
Attune counting
25
20
15
10
5
0
0
10
15
Manual counting
20
y = 0.9314x
R² = 0.9972
25
Attune counting
5
20
15
10
5
0
0
10
20
Leukocount Kit counting
30
Leukocyte Identification with Vybrant DyeCycle Violet
18.0
WBC/ μL
18.9
WBC/ μL
200 μL/min; 200 μL
500 μL/min; 500 μL
Leuko-Trol RBC HIGH:
18.2
WBC/ μL
0.66 events/μL x 30 (dilution) = 19.8 WBC/ μL
Expected count: 20 WBC / μL.
Verified with Nageotte chamber.
1000 μL/min; 1000 μL
Leukocyte Identification with Vybrant DyeCycle Violet
1.5/ μL
200 μL/min; 200 μL
1.8/ μL
500 μL/min; 500 μL
Leuko-Trol RBC LOW:
0.06 events/μL x 30 (dilution) = 1.8 WBC/ μL
2.1/ μL
Expected count: 2 WBC / μL.
Verified with Nageotte chamber.
1000 μL/min; 1000 μL
Leukocyte Identification with Vybrant DyeCycle Violet
LEUKOREDUCED SAMPLE (RBC bag):
0.01 events/ μL x 30 (dilution)= 0.3 WBC/μL
Comparison of the Different Counting Methods
Sample
Theoretical counting
(WBC/μL)
Nageotte
(WBC/μL)
Leukocount Kit
(WBC/μL)
Attune
(WBC/μL)
RBC LOW
2.0 ± 1.5
1.90 ± 0.23
1.80 ± 0.25
2.33 ± 0.78
RBC HIGH
21.5 ± 5.5
18.48 ± 1.18
22.09 ± 0.91
20.52 ± 2.6
RL
<2
0.13 ± 0.23
0.026 ± 0.059
0.2 ± 0.13
Conclusions
Results show an excellent correlation of this method with
other methods for residual leukocyte enumeration, as well as
high internal consistency in the analysis of serial dilutions of
whole blood, red blood cell bags and commercial controls with
low- and high leukocyte count for quality control procedures.
This is a simple method requiring less degree of sample
manipulation and does not require the use of concentrationcalibrated beads.