An Automated Cell Prepration Method For FlowCARE™ Pan-Leukogated (PLG) CD4

Assay in a 96-Well Plate Offers Increased Throughput and Ease of Use
Jin Zhang, Valentin Quesada, Juan De Castro, Jennifer Zawislak, Jorge Acevedo, Ed Jachimowicz, and Liliana Tejidor

Beckman Coulter, Inc., Cellular Analysis Business Group, Miami, FL
Abstract

Background. The measurement of CD4+ cells in HIV-infected patients
has evolved into a routine and high volume test in flow cytometry. CD4+
cell enumeration is measured with differing combinations of monoclonal
antibodies, balancing performance with cost. A good balance is the PLG
CD4 assay, using two monoclonal antibodies: anti-CD45 for resolving
leukocytes and anti-CD4 for enumerating CD4+ cells. The current PLG
assay is semi-automated and allows for a maximum of 32 blood samples
in tubes to be processed in a batch.
Methods. We have modified the FlowCare™ PLG CD4 assay so that the
addition of blood, antibodies, lysing, quenching, fixation reagents, and
Flow-Count™ Fluorospheres is performed in 96-well plates by an
automated sample and liquid handling system (BioMek® NXP). The plate
is then transferred to a flow cytometer equipped with a multi-plate reader
for enumeration of CD4+ cells. For identification purposes, each individual
sample is tracked throughout by bar code. There is an approximately 30%
reduction in time between the semi-automated and automated systems.
Results. Performance testing of 24 non-clinical and 70 HIV samples
demonstrated a significant correlation between the FlowCare™ PLG CD4
assay prepared in 96-well plates on the BioMek® NXP and the semiautomated tube method (r2 = 0.99). Aging studies with 9 non-clinical and
15 HIV specimens (up to 5 days post blood draw) showed no significant
change in the absolute CD4+ counts and percent of CD4+ cells (p =
0.41/0.99). This application is undergoing clinical evaluation.
Conclusions. The results indicate that the PLG assay using automated
sample preparation in a 96-well plate system gives comparable
performance to the semi-automated tube method. The ability to process
larger batches of clinical samples in a shorter period of time allows for
reduced costs, higher throughput, and more efficient use of laboratory
staff.
Materials and Methods

Phosphate Buffered Saline Solution (PBS)

Sodium dihydrogenphosphate
Disodium hydrogenphosphate 7H2O
Sodium azide
Sodium chloride
QS to 1 liter with reagent grade water
Osmolarity
0.2g
2.0g
0.1g
9.4g
pH approximately 7.4
315 to 345 H2O
Analog Processing Medium 1

isodium hydrogenphosphate 7H2O
D
Sodium dihydrogenphosphate
Sodium chloride
Glutaraldehyde (25%)
QS to 1 liter with reagent grade water
Osmolarity
2.0 g
0.2 g
24.5 g
40 ml
pH approximately 7.4
900 mOsm/kg H2O
Analog Processing Medium 2
odium chloride
S
Glutaraldehyde (25%)
QS to 1 liter with reagent grade water
Osmolarity
31.7 g
40 ml
pH approximately 6.5
1000 mOsm/kg H2O
Analog Processing Medium 3
Disodium hydrogenphosphate 7H2O

Sodium dihydrogenphosphate
Sodium chloride
Glutaraldehyde (25%)
2.0 g
0.2 g
16.5 g
40 ml
Suspension Medium 1

Xanthine compound
2.0-7.0 g
Adenosine monophosphate
0.2-0.8 g
Inosine
0.2-0.8 g
pH adjusting agents sufficient
to obtain
pH 6.0-6.5
Osmolarity adjusters sufficient
to obtain
250-350 mOsm/kg H2O
Preservative
2.0-6.0 g
QS to 1 liter with reagent grade water
Suspension Medium 2
Propyl paraben
Methyl paraben
Procaine hydrochloride
Deoxycholic acid
Lactose
Actidione
Trisodium citrate dehydrate
Citric acid monohydrate
Sodium dihydrogenphosphate
monohydrate
Phenergan hydrochloride
Colistimethate, sodium
Penicillin G., sodium
Kanamycin sulfate
Neomycin sulfate
5’-AMP
Adenine
Inosine
Dihydrostreptomycin sulfate
Tetracycline hydrochloride
30% Bovine albumin
QS to 1 liter with reagent grade water
0.3 to 1.0 g
0.5 to 1.0 g
0.1 to 0.5 g
0.1 to 0.9 g
0.0 to 50.0 g
0.1 to 0.6 g
3.0 to 8.0 g
0.3 to 0.9 g
0.8 to 2.5 mg
0.1 to 1.0 g
0.2 to 0.9 g
1.75 x 106 units
0.2 to 0.8 g
0.2 to 1.0 g
0.4 to 1.0 g
0.2 to 0.8 g
0.4 to 1.0 g
0.2 to 1.0 g
0.2 to 1.0 g
100 to 350 ml
Preparation of immature granulocyte analog and
integration into a Reference Control
WASHING STEPS
• Use 50 ml of non human whole blood from selected species
collected in citrate anticoagulant.
• Centrifuge whole blood and aspirate the top layer including white
blood cells, platelets and plasma.
• Wash packed red blood cells three times with PBS. The cell
washing steps were a series of centrifugations (1000 rpm/5
minutes), followed by removal of the supernatant and resuspension of the packed cells with PBS.
• Re-suspend washed cells in the residual PBS.
I
NTEGRATED CONTROL COMPOSITION
• Provide a predetermined volume of Suspension Medium 1
containing stabilized human Red Blood Cells and a Platelet
component.
• Add predetermined amounts of lymphoid and myeloid analogs.
• Add a predetermined amount of immature granulocyte analog.
• The analogs are added in proportions to resemble white blood
cell subpopulations and immature granulocytes in Normal and
Abnormal human whole blood.
• The following integrated reference control compositions were
formulated:
Composition A
30% Lymphoid analog made of human RBC
70% Myeloid analog made of processed goose RBC
Composition B
30% Lymphoid analog made of Human RBC
49% Myeloid analog made of processed Goose RBC
21% Immature granulocyte made of processed Emu RBC
Composition C
25% Lymphoid analog made of Human RBC
58% Myeloid analog made of processed Emu RBC
21% Immature granulocyte made of processed Alligator RBC
USE OF THE HEMATOLOGY REFERENCE CONTROL ON A

HEMATOLOGY ANALYZER FOR MEASUREMENT OF
IMMATURE GRANULOCYTES USING A DC IMPEDANCE
MEASUREMENT
The reference control compositions were analyzed on an
experimental hematology analyzer. In the analysis, an aliquot of 28
µl of the reference control or a blood sample was diluted with 6 ml
of Coulter® LH Series Diluent (Beckman Coulter, Inc., Miami,
Florida) in a WBC bath, mixed with 1 ml of a lytic reagent to lyse
red blood cells. The lytic reagent contained 25.0 g/L of
Tetradecyltrimethylammonium bromide (from Dishman), 15.0 g/L of
Igepal SS-837 (an ethoxylated phenol from RhÔne-Poulenc),
4.0 g/L of Plurofac A38 prill surfactant (from BASF Corp.), and had
a pH of 6.2.
The experimental hematology analyzer was a modified LH750
(product of Beckman Coulter, Inc., Miami, Florida), equipped
with non-focused apertures of a length of 100 µm and a width
of 80 µm for measuring the prepared sample mixtures as described
above. The sample mixture was drawn through a
set of three apertures by a constant vacuum. The white blood cells
were enumerated by DC impedance measurement, and
a WBC histogram (size vs cell count) produced after pulse
editing.
2
illustrates a DC histogram of a clinical sample containing
immature granulocytes. The manual reference reported about 12% of
immature granulocytes (IG), including metamyelocytes, myelocytes and
promyelocytes, which showed on the right side of the myeloid
subpopulation. This sample had only 7% lymphocytes, and the majority
of the white blood cells were myeloid population.
Figure

2A 
illustrates 
DC histogram of another clinical sample containing
about 6% of immature granulocytes including metamyelocytes and
myelocytes, which are indicated by the large cells extending into the
right-most region of the histogram. The manual reference also reported 5
NRBC per 100 WBC in this sample. As shown, NRBCs are located on the
left side of the lymphoid population, which was differentiated from the
white blood cells.
Figure

Figure

7 illustrates the DC histogram of reference control composition C
analyzed on the experimental hematology analyzer with the same
dynamic range shown in Fig. 5, which resembles the cell distribution of
the clinical sample containing immature granulocytes.
Results
ANALOG PROCESSING STEPS
• Add 1 ml of packed washed red blood cells to 49 ml of the Analog
Processing Medium 1 and mix by inversion to form a
cell processing suspension.
• Roll mix at a slow speed overnight at room temperature.
• Wash processed cells three times with PBS, as described in
Washing Step.
• Re-suspend processed cells in Suspension Medium 1 or 2.
• Store resulting Immature Granulocyte Analog at room
temperature
3 and 4 
illustrates 
DC histograms of reference control
compositions A and B. As shown, the histogram of the reference control
composition A resembles the cell distribution of the normal blood sample,
and the histogram of the reference control composition B resembles the
cell distribution of the clinical sample containing immature granulocytes.
Figure

Figure

1 illustrates a DC histogram of a normal whole blood sample
analyzed on the experimental hematology analyzer. The white blood cells
in the normal sample exhibit a bi-modal distribution, with the lymphoid
subpopulation on the left and the myeloid subpopulation on the right. No
cell population located on the right side of the myeloid subpopulation.
5 and 6 
illustrates 
DC histograms of a normal whole blood
sample and a clinical sample containing immature granulocytes analyzed
on the same experimental hematology analyzer with a larger dynamic
range of the measurement. With the larger dynamic range, normal white
blood cells distributed in approximately only half of the histogram. Some
of the immature granulocytes in this example were very large, and
extended to the extreme right of the histogram.
Figure

onclusions
C
• The control composition offers an automated cost effective way of
monitoring immature granulocytes enumeration on low cost
analyzers.
• The flexibility of the process allows the manufacture of a wide range
of analogs that encompass multiple detection systems. The analog
can be measured by radio frequency, impedance and optical
methodologies that reflect the size of cells.
• Analogs can be combined to simulate the broad range distribution
of immature granulocytes seen in clinical samples.
• The immature granulocyte analog can be used as a stand alone
product or integrated into a reference control containing stabilized
red blood cells, mature white blood cells, platelets, reticulocytes and
NRBC components enabling quality control of hematology systems
with a single product
Acknowledgements
We are grateful to Jing Li, Staff Scientist, Beckman Coulter Inc,
Miami, Florida for her collaboration in the evaluation of the Immature
Granulocyte Reference Control.