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3/2015 23-7739-03

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Becton, Dickinson and CompanyBD Biosciences2350 Qume DriveSan Jose, CA 95131 USA

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Becton Dickinson Pty Ltd,4 Research Park Drive,Macquarie University Research Park,North Ryde NSW 2113, Australia

Becton Dickinson Limited,8 Pacific Rise, Mt. Wellington,Auckland, New Zealand

[email protected]

IVD

BD™ Plasma Count Kit

50 Tests per kit—Catalog No. 338331

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CONTENTS

1. INTENDED USE ............................................................................ 5

2. SUMMARY AND EXPLANATION ................................................ 5

3. PRINCIPLES OF THE PROCEDURE................................................ 5

4. REAGENT..................................................................................... 6

Precautions ................................................................................... 7

Storage and Handling ................................................................... 7

5. INSTRUMENT............................................................................... 8

6. SPECIMEN COLLECTION AND PREPARATION............................ 9

Interfering Conditions .................................................................. 9

7. PROCEDURE ................................................................................ 9

Reagents Provided ........................................................................ 9

Reagents and Materials Required but Not Provided ..................... 9

Staining Procedure...................................................................... 10

BD FACSVia Instrument Quality Control................................... 12

BD FACS Brand Instrument Setup .............................................. 12

Acquiring Samples ...................................................................... 13

Data Analysis ............................................................................. 15

8. RESULTS..................................................................................... 18

Calculating Absolute Counts ...................................................... 18

9. LIMITATIONS............................................................................. 19

10. PERFORMANCE CHARACTERISTICS.......................................... 20

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Precision for BD FACSCalibur flow cytometers.......................... 20

Low Range Study ....................................................................... 24

Linearity for BD FACSCalibur flow cytometers.......................... 27

Stability for BD FACSCalibur flow cytometers ........................... 28

Accuracy for BD FACSCalibur flow cytometers ......................... 28

Precision for BD FACSVia flow cytometers ................................ 30

Linearity for BD FACSVia flow cytometers ................................ 31

Accuracy for BD FACSVia flow cytometers................................ 32

WARRANTY..................................................................................... 35

TROUBLESHOOTING ....................................................................... 36

REFERENCES .................................................................................... 36

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BD™ PLASMA COUNT KIT

1. INTENDED USE

The BD™ Plasma Count kit is for in vitro diagnostic use on the BD FACSVia™ system using the BD FACSVia™ clinical software, and on the BD FACSCalibur™ flow cytometer using BD CellQuest™ Pro and BD FACSComp™ software. The kit is intended for in vitro diagnostic use to identify and enumerate residual leucocytes (rWBCs), red blood cells (rRBCs), and platelets (rPLTs) in fresh human plasma, using flow cytometry.

2. SUMMARY AND EXPLANATION

The quality of fresh frozen plasma (FFP) can be determined by counting rWBCs, rRBCs, and rPLTs prior to freezing.1-3 Flow cytometric applications provide a rapid, quantitative, and reproducible method to identify and enumerate residual cell populations.4-9 The BD Plasma Count assay incorporates BD Trucount™ tubes to determine absolute cell counts of rWBCs, rRBCs, and rPLTs using a single tube.

3. PRINCIPLES OF THE PROCEDURE

A single-tube assay is performed by staining a known volume of plasma sample with BD Plasma Count reagents in an individual BD Trucount tube. Fluorochrome-labeled antibodies bind specifically to cell-surface antigens, while the nucleic acid dye stains the DNA and RNA of all nucleated cells. The lyophilized pellet in the BD Trucount tube dissolves, releasing a known number of fluorescent beads.

During analysis, the absolute number (cells/µL) of residual cells in the sample can be determined by dividing the number of residual cellular events by the number of fluorescent bead events, and then multiplying the result by the BD Trucount bead concentration.4

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4. REAGENT

The BD Plasma Count kit contains BD Plasma Count reagent and BD Trucount tubes, sufficient for 50 tests. The BD Plasma Count kit contains the following components. Each reagent is provided in phosphate-buffered saline (PBS) with stabilizer and 0.1% sodium azide.

• Reagent Vial A: Thiazole orange in stabilization buffer. • Reagent Vial B: FITC-labeled CD235a, clone GAR-2(HIR-2) and

PerCP-Cy™5.5*-labeled CD41a, clone HIP8, pre-titrated in stabilization buffer.

• BD Trucount tubes, each containing a lyophilized pellet of 4.2-µm fluorescent-dyed beads.

Thiazole orange is a nucleic acid dye that penetrates cell membranes and binds with RNA and DNA.10,11

Anti-glycophorin A (CD235a) reacts with a major sialoglycoprotein present on the human erythrocyte membrane.12 Anti-glycophorin A is composed of mouse IgG2b heavy chains and kappa light chains.

Glycophorin A is a transmembrane dimeric complex of 31 kilodaltons (kDa) with its carboxyterminal ends extending into the cytoplasm of the red cell.13 Glycophorin A is expressed on human red blood cells, normoblasts, and erythroid precursor cells.14 Mature non-nucleated red blood cells are characteristically glycophorin A positive but CD45 negative.15

* Cy™ is a trademark of GE Healthcare. This product is subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and is made and sold under license from GE Healthcare. This product is licensed for sale only for in vitro diagnostics. It is not licensed for any other use. If you require any additional license to use this product and do not have one, return this material, unopened, to BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, and any money paid for the material will be refunded.


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CD41a recognizes the calcium-dependent gpIIb/IIIa complex, with a molecular weight of 140 kDa.16 This complex is found on platelets and platelet precursors. It acts as a receptor for fibrinogen, von Willebrand factor (vWf), fibronectin, and vitronectin, and it mediates platelet adhesion and aggregation.17-19 The CD41a antibody is composed of mouse IgG1 heavy chains and kappa light chains.

Precautions

• For In Vitro Diagnostic Use.• The addition of a precise volume of sample is critical. Pipets must

be calibrated to deliver exactly 25 µL and 50 µL of sample. If necessary, perform the reverse pipetting technique according to the pipet manufacturer’s instructions.

• Care should be taken to avoid microbial contamination, which could cause aberrant results.

• Do not use the reagents if you observe any change in appearance. Precipitation or discoloration indicates instability or deterioration.

• Before use, inspect the BD Trucount tube to make sure the pellet is intact and below the retainer.

• Bead count varies by lot of BD Trucount tubes. It is critical to use the bead count shown on the lot of BD Trucount tubes that you are currently using when calculating absolute cell counts. Do not mix multiple lots of tubes in the same assay.

WARNING All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection20,21 and dispose of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, and gloves.

Storage and Handling

• Store the BD Plasma Count kit at 2°C–8°C. Do not use after the expiration date shown on the label.

• Avoid unnecessary exposure of the reagent to light.


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• Do not freeze the reagents or expose them to direct light during storage or incubation with cells. Keep the reagent vials dry.

• Store BD Trucount tubes in their original foil pouch at 2°C–25°C. To avoid potential condensation, open the pouch only after it has reached room temperature and carefully reseal the pouch immediately after removing a tube. Examine the desiccant each time you open the pouch. If the desiccant has turned from blue to lavender, discard the remaining tubes. Use tubes within 1 hour after removal from the foil pouch. Once the pouch has been opened, the tubes are stable for 1 month. Do not use beyond the expiration date indicated on the packaging.

5. INSTRUMENT

The BD Plasma Count kit is designed for use on a flow cytometer equipped with appropriate computer hardware and software. The cytometer must have a 488-nm laser capable of detecting forward scatter (FSC) and side scatter (SSC) and three-color fluorescence, and must be able to threshold or discriminate using the FSC channel.

You can use a BD FACS™ brand cytometer, such as the BD FACSCalibur flow cytometer running BD FACSComp software (version 4.0 or later), with the optional BD FACS™ Loader.

We recommend using BD Calibrite™ beads to set photomultiplier tube (PMT) voltages, fluorescence compensation, and to check instrument sensitivity before use.

You can also use the BD FACSVia flow cytometer with the optional BD FACSVia™ Loader. For this cytometer, use BD™ CS&T beads for instrument QC.

Results can be achieved using other platforms. For flow cytometers manufactured by companies other than BD Biosciences, set up the cytometer for three-color acquisition following the manufacturer’s recommendations.


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6. SPECIMEN COLLECTION AND PREPARATION

A minimum of 200 µL of plasma is required for this procedure. For residual cell enumeration, use plasma samples with additive, or fresh plasma samples with no additive within 18 hours of collection. Store plasma samples with no additive at room temperature (20°C– 25°C) under constant agitation until ready for staining.

Interfering Conditions

• Do not use previously fixed and stored plasma.• Samples without additive that are refrigerated before staining

could give aberrant results.• Hemolyzed, lipemic, or clotted samples should be rejected.• RBC agglutination in stained samples increases during storage.

Therefore, acquire samples within 1 hour of staining.

7. PROCEDURE

Reagents Provided

• Reagent Vial A • Reagent Vial B• BD Trucount tubes

Reagents and Materials Required but Not Provided

• Falcon®† disposable 12 x 75-mm polypropylene test tubes or equivalent

• Micropipettor with tips• Vortex mixer and roller mixer• BD FACS™ Clean solution (Catalog No. 340345)

For BD FACS brand cytometers running BD FACSComp software:

• BD Calibrite beads (Catalog No. 349502)• BD Calibrite PerCP-Cy5.5 beads (Catalog No. 345036)

† Falcon is a registered trademark of Corning Incorporated.


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• BD Calibrite™ 3 beads (Catalog No. 340486)• BD FACSFlow™ sheath fluid (Catalog No. 342003) or equivalent

CAUTION Use only BD FACSFlow sheath fluid diluent to dilute BD Calibrite beads.

• Wash buffer of phosphate-buffered saline (PBS) with 0.1% sodium azide

• BD FACS™ Rinse solution (Catalog No. 340346)

For BD FACSVia cytometers:

• BD CS&T beads (Catalog Nos. 656504, 656505)

Staining Procedure

Before beginning the staining procedure, do the following:

• Allow Reagent Vial A to come to room temperature.• Filter the wash buffer (PBS) through a 0.2-µm filter to avoid

increased background signals in the stained plasma sample.

CAUTION Pipette the sample and reagents onto the side of each tube just above the stainless steel retainer. If the sample remains on the side of the tube, it will not be stained with the reagent. Do not touch the bead pellet.

1. For each blood component sample, dispense a minimum of 200 µL of fresh plasma into labeled 12 x 75-mm Falcon tubes.

NOTE Use a new tip for each sample.

2. Remove a BD Trucount tube from the foil pouch, and label the tube.

NOTE Before use, verify that the bead pellet is intact and under the metal retainer at the bottom of each tube. If not, discard the tube and replace it with another.


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3. Using a calibrated pipet, transfer the appropriate amount of blood component sample (25 or 50 µL [Table 1]) into each labeled BD Trucount tube.

NOTE Counting precision (expressed as the coefficient of variation [CV]) is determined by the number of events counted. Counting fewer events results in a higher CV. In rare event analysis, the CV is influenced by sample concentration and sample volume. Thus, analyzing a higher cell concentration and counting more cells results in a lower CV.22 We recommend that you consider the precision required for your application when choosing a sample volume (Table 1).

4. Add 100 µL of Reagent A to each BD Trucount tube.

5. Add 20 µL of Reagent B to each BD Trucount tube.

6. Cap the tubes and vortex gently to mix.

7. Incubate the tubes for 15 minutes in the dark at room temperature.

8. Add 1 mL of filtered wash buffer (PBS) to each tube to stop the staining process.

9. Cap the tubes and vortex gently.

The cells are now ready to be analyzed on the flow cytometer. Protect stained samples from direct light.

Acquire samples within 1 hour after preparation. If stained samples are not acquired immediately after preparation, store samples in the dark at 2°C–8°C for up to 1 hour. Vortex thoroughly just before acquisition.

Table 1 Volume of sample used to enumerate different ranges of rWBCs in plasma

0.5–50 rWBC/µL

>50–500 rWBC/µL

Sample Volume 50 µL 25 µL


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If stained samples are to be run on the BD FACS Loader or BD FACSVia Loader, vortex each tube immediately before placing into the BD FACS Loader carousel or BD FACSVia Loader arm.

NOTE Do not load more tubes on the rack than can be run within 1 hour.

BD FACSVia Instrument Quality Control

Instrument QC checks instrument performance, updates compensation values accordingly, and assesses the background instrument noise.

We recommend running a control sample daily to optimize instrument settings and as a quality control check of the system.23

See the cytometer’s Instructions For Use (IFU) for instructions on how to run instrument QC.

BD FACS Brand Instrument Setup

This section provides guidelines for instrument setup.

Setup is not required for the BD FACSVia flow cytometer.

Preparing for Instrument Setup

Before running stained plasma samples on the flow cytometer, do the following:

• Clean the instrument’s flow cell with BD FACS Clean solution followed by BD FACS Rinse solution. Refer to your cytometer IFU.

• Check the noise level of the BD FACSFlow sheath fluid. The overall noise level should not exceed 30–45 events/second when FSC mode is set to Log and FSC threshold is set between 100–150 channels. If the noise level exceeds this recommendation, use a new cubitainer of sheath fluid. If the problem persists, change the sheath filter.


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Setting Up the Instrument

For BD FACS brand cytometers running BD FACSComp software, we recommend using BD Calibrite beads (FITC, PE, and PerCP-Cy5.5) with BD FACSComp software using the three-color Lyse/No-Wash (LNW) setup. See the BD Calibrite Beads IFU and the BD FACSComp Software User’s Guide for instructions.

For flow cytometers manufactured by companies other than BD, use either BD Calibrite beads and PerCP-Cy5.5 beads or BD Calibrite 3 beads to adjust compensation. Ensure the unlabeled beads are in the lower-left quadrant of the two-dimensional fluorescence plots.

Acquiring Samples

For sample acquisition instructions using the BD FACSVia flow cytometer, see the BD FACSVia System IFU.

For BD FACS brand cytometers running BD FACSComp software, we recommend using BD CellQuest™ Pro software for residual cell enumeration.

NOTE To ensure thorough resuspension of cells and beads, gently vortex samples immediately before acquisition.

1. In BD CellQuest Pro software, create an Experiment document containing five Acquisition-to-Analysis dot plots with the following parameters: FSC vs SSC, FL1 vs FL2, FL2 vs FL3, FSC vs FL3, FL1 vs FL3.

2. After using BD FACSComp software to set up your cytometer, load the CalibFile.LNW file and modify:

• Set FSC (P1) and SSC (P2) to Log mode.• Set FSC threshold to channel 100–150.

3. Vortex the first sample and place it on the cytometer.

4. Set the fluid control to HI and begin acquiring the sample in Setup mode.


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If you are using the BD FACS Loader to acquire samples, see the BD FACS Loader User’s Guide for complete instructions.

5. Adjust instrument settings, if necessary.

6. Stop acquisition, and save the optimized instrument settings.

7. Draw the following regions around the bead population in the corresponding plots.

See Figure 1 for R1, R2, and R3 locations.

8. In the Gate List, define the bead gate as R1 and R2 and R3 and change the label from G4 to Beads.

9. Change the following settings in the Acquisition & Storage window:

• Select Beads from the Acquisition stops when 10,000 events are acquired menu.

• Verify that instrument resolution is set to 1,024 and the P1, P2, P3, P4, and P5 checkboxes are selected.

10. Acquire samples, saving the data.

Data Analysis

This section provides data analysis instructions for BD FACS brand cytometers running BD FACSComp software. For data analysis instructions using the BD FACSVia flow cytometer, see the BD FACSVia Plasma Count Assay Guide.

Plot Region

FL1 vs FL2 R1

FL2 vs FL3 R2

FL1 vs FL3 R3


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Setting rWBC Gates

1. Draw the following regions around the WBC population in the corresponding plots (Figure 1).

2. In the Gate List, change the G9 label to rWBC and combine the following regions:

rWBC = R4 and R5 and R6 and R7

3. Select the Hilite checkbox for rWBC.

Setting rRBC Gates

4. Draw the following regions around the RBC population in the corresponding plots (Figure 1).

5. In the Gate List, change the G12 label to rRBC and combine the following regions:

rRBC = R8 and R9

NOTE All Glycophorin A-FITC positive RBCs appear in R9. Morphologically altered RBCs do not appear in R8, and are therefore excluded from rRBC calculation by the rRBC gate definition.5,7,24

Plot Region

FSC vs SSC R4

FL1 vs FL2 R5

FL2 vs FL3 R6

FL1 vs FL3 R7

Plot Region

FSC vs SSC R8

FL1 vs FL2 R9


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Setting rPLT Gates

6. Draw the following regions around the PLT population in the corresponding plots (Figure 1).

7. In the Gate List, change the G15 label to rPLT and combine the following regions:

rPLT = R10 and R11

Viewing Analysis Plots

8. Arrange the gates in the Gate List in descending order: Beads, rWBC, rRBC, rPLT.

The analysis plots should look similar to those shown in Figure 1.

9. Display Region and Gate statistics for the sample data.

10. Calculate absolute counts. (See Calculating Absolute Counts in the Results section.)

Plot Region

FSC vs FL3 R10

FL1 vs FL3 R11


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Figure 1 Representative plots showing gating strategy for BD Plasma Count assay


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8. RESULTS

Calculating Absolute Counts

NOTE Results are automatically calculated for the BD FACSVia flow cytometer.


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For other instruments, the absolute number of residual cells is determined by dividing the number of residual cell events acquired by the number of fluorescent bead events acquired, and multiplying the result by the bead concentration.

Use the following equation to calculate the absolute cell count of the residual cell population.

(GX/G4) x (BPT/V) = residual cells/µL, where:

• GX = the number of residual cells in the rWBC, rRBC, or rPLT gate

• G4 = the number of beads in the G4 gate• BPT = beads/tube, which is found on the BD Trucount foil pouch

and can vary from lot to lot• V = sample volume

Figure 2 shows the statistics view for residual cell analysis of a plasma sample and rWBC, rRBC, and rPLT calculations.

Figure 2 Statistics view and resulting calculation for rWBC, rRBC, and rPLT analysis of a plasma sample

(140/10,000) x (48,554/25 µL) = 27 rWBC/µL plasma


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(2,086/10,000) x (48,554/25 µL) = 405 rWBC/µL plasma

(129,720/10,000) x (48,554/25 µL) = 25,194 rWBC/µL plasma

9. LIMITATIONS

• Performance has not been established on stream-in-air cytometer systems.

• The addition of a precise volume of blood is critical when BD Trucount tubes are used. The pipet used should be calibrated to deliver 25 µL or 50 µL of sample.

• Gently vortex samples immediately prior to running them on the flow cytometer to ensure thorough resuspension of cells and beads.

• Nucleated red cells contain nucleic acid and could be detected as rWBCs in this assay. However, nucleated red cells are not present in detectable quantities in blood from normal individuals. Their expected frequency in fresh plasma will be even lower and thus less interfering.6

• As for any assay that involves counting statistics, the number of events counted could influence the result.23 We recommend collecting at least 100,000 total events when analyzing leucoreduced plasma, or at least 10,000 BD Trucount beads for leucocyte-depleted plasma.

• Samples containing >38,000 rPLTs have not been validated with this assay.


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10. PERFORMANCE CHARACTERISTICS

Precision for BD FACSCalibur Flow Cytometers

Based on combined dilution series, following ICH and CLSI guidelines, five different concentration levels were tested in triplicate on five different days.25,26 Target values for the five concentration levels were obtained from dilutions of one cell stock solution that was measured on a Sysmex®‡ F-820 automatic cell counter. Purified WBC, RBC, and PLT samples were spiked into cell-free plasma. Cell-free plasma was used as blank. Fresh samples were prepared on each of the five days.

Prepared samples were stained according to the Cell Staining procedure in the Procedure section. See Table 2 for results and Figure 3 for mean values.

‡ Sysmex is a registered trademark of Sysmex Corporation

Table 2 Precision of BD Plasma Count assay for each concentration level (n=15)

Cell Type Level (cells/µL) Mean (cells/µL) SDa (cells/µL) %CVb

WBC 50 51 3.9 7.5

275 291 15.0 5.2

500 508 43.8 8.6

725 758 40.5 5.3

950 967 58.6 6.1

RBC 600 606 55.6 9.2

3,300 2,892 306.8 10.6

6,000 4,737 572.5 12.1


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The following table is a legend for all plots of Figure 3.

PLT 2,000 1,995 215.7 10.8

11,000 10,858 952.8 8.8

20,000 19,566 1,807.7 9.2

29,000 28,446 2,425.6 8.5

38,000 36,546 3,143.6 8.6

a. SD = standard deviationb. CV = coefficient of variation

Item Description

• Mean value (n=15)

— Linear regression of mean value

- - - Expected value

Table 2 Precision of BD Plasma Count assay for each concentration level (n=15)

Cell Type Level (cells/µL) Mean (cells/µL) SDa (cells/µL) %CVb


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Figure 3 Linearity of BD Plasma Count assay (Mean values for each cell type at the five different levels are taken from Table 2.)

Statistic Value

r2 0.999

Slope 1.023

Intercept 3.26


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Statistic Value

r2 0.996

Slope 0.789

Intercept 106.41


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Low Range Study

In a separate study, accuracy of WBC and RBC low range results from the BD Plasma Count assay were compared to estimated cell counts obtained from dilutions of one cell stock solution that was measured on a Sysmex F-820 cell counter. Samples were prepared, diluted, and analyzed using the same methods as in the general comparison study.

Statistic Value

r2 0.999

Slope 0.966

Intercept 146.81


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See Table 3 and Table 4 for results and Figure 4 for plots.Table 3 Precision of BD Plasma Count assay for detection of WBCs and RBCs in the

low cell range (n=15)

Cell Type Level (cells/µL) Mean (cells/µL) SDa (cells/µL)

a. SD = standard deviation

%CVb

b. CV = coefficient of variation

WBC 0.6 0.7 0.28 40.4

1.95 2.4 0.35 14.5

3.30 3.7 0.69 18.8

4.65 5.7 0.93 16.3

6.00 6.0 1.13 18.7

RBC 600 597 48.80 8.2

1,950 1,625 155.14 9.6

3,300 2,707 203.46 7.5

4,650 3,215 226.27 7.0

6,000 4,472 563.16 12.6

Table 4 Accuracy of BD Plasma Count assay vs expected low cell rangea of each cell type (n=90)

a. Concentration levels were obtained from dilutions of one cell stock solution that was measured on a Sysmex F-820 cell counter.

Cell Type Range (cells/µL) Slope Intercept Correlation (r2)

WBC 0–6.0 1.052 0.199 0.979

RBC 0–6,000 0.716 133.47 0.990


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Figure 4 Linearity of BD Plasma Count assay vs expected low cell range (mean values for the five different concentration levels of each cell type are taken from Table 3).

Item Description

• Mean value (n=15)

— Linear regression


Statistic Value

r2 0.979

Slope 1.052

Intercept 0.199


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Linearity for BD FACSCalibur Flow Cytometers

Based on combined dilution series, following ICH and CLSI guidelines, five different concentration levels were tested in triplicate on five different days.25,26 Target values for the five concentration levels were obtained from dilutions of one cell stock solution that was measured on a Sysmex F-820 automatic cell counter. Purified WBC, RBC, and PLT samples were spiked into cell-free plasma. Cell-free plasma was used as blank. Fresh samples were prepared on each of the five days. Prepared samples were stained according to the Cell Staining procedure described in the Procedure section. See Figure 4 for linearity results.

NOTE The rRBC values shown indicate a gradual agglutination effect with increasing concentration, leading to the underestimation of rRBCs at concentrations over 3,000 cells/µL. See statistics in Table 2.

Statistic Value

r2 0.990

Slope 0.716

Intercept 133.47


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Stability for BD FACSCalibur Flow Cytometers

Samples stained with BD Plasma Count reagents for the enumeration of the three cell types in FFP were stable for up to 1 hour after staining when stored at 4°C in the dark.

Accuracy for BD FACSCalibur Flow Cytometers

General Comparison vs Expected Values

In one study, the cell concentration results from the BD Plasma Count assay were compared for accuracy with expected values of red cell, white cell, and platelet counting.5 The samples were obtained from dilutions of one stock solution that was measured on the blood cell counter (Sysmex F-820 model). The stock solution was prepared from spiked RBCs, WBCs, and PLTs into cell-free plasma. The mean value results are shown in Table 5.

Both WBC and PLT results from the flow cytometer compare directly to the expected values. The RBC values shown indicate a gradual agglutination effect with increasing concentration, leading to the underestimation of RBCs at concentrations over 3,300 cells/µL (bias >10%).5

Table 5 Accuracy of BD Plasma Count assay vs expected cell rangea of each cell type (n=90)

a. Concentration levels were obtained from dilutions of one cell stock solution that was measured on a Sysmex F-820 cell counter

Cell Type Range (cells/µL) Slope Intercept Correlation (r2)

WBC 0–950 1.023 3.26 0.999

RBC 0–6,000 0.789 106.41 0.996

PLT 0–38,000 0.966 146.81 0.999


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BD Plasma Count Assay vs Microscopic Reading

The BD Plasma Count assay and the Fuchs-Rosenthal assay were compared for accuracy of residual red blood cell enumeration. A total of 79 samples of healthy donors were stained according to the Cell Staining procedure described in the Procedure section. Absolute rRBC counts were determined by manual counting on the microscope using a Fuchs-Rosenthal counting chamber and by flow cytometry. Study results show excellent comparison between the flow cytometric determination and the microscopy method. Results are shown in Figure 5.


Item Description

+ Value (n=79)

— Linear regression



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Figure 5 Accuracy of BD Plasma Count assay vs Fuchs-Rosenthal manual counting method

Precision for BD FACSVia Flow Cytometers

The system precision study for the enumeration of rWBC, rPLT, and rRBC absolute counts was determined across two BD FACSVia flow cytometers (one manual and one with an automated loader). Two operators acquired two concentration levels of Plasma Count test samples stained in duplicate using the BD Plasma Count Kit and one donor per day over five days.

Precision study samples were created at two concentration levels (high and low) by spiking concentrated RBCs, WBCs, and PLTs from autologous whole blood into cell-free plasma. Concentration levels (WBC/µL, RBC/µL, and PLT/µL) of the test samples were established using the BD FACSCalibur flow cytometer.

Statistic Value

r2 0.960

Slope 0.927

Intercept 41.65


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NOTE The precision measurement includes the following variance components: Instrument, Operator, Between Run, Within Run.

Linearity for BD FACSVia Flow Cytometers

System linearity of the BD FACSVia flow cytometer when using BD Plasma Count Kit was determined using triplicate measurements of 11 equally spaced concentrations of autologous rWBCs, rPLTs, and rRBCs spiked into cell-free plasma. Linearity was assessed with the following ranges:

Table 6 Precision for BD FACSVia flow cytometers

Cell Type Mean SD CV

WBC Low 102.1 7.0 6.9%

WBC High 286.5 17.6 6.2%

RBC Low 1705.5 76.4 4.5%

RBC High 9880.5 816.0 8.3%

PLT Low 10,329.3 550.0 5.3%

PLT High 75,909.5 3323.1 4.4%

Table 7 Linearity for BD FACSVia flow cytometers

Cell Type Range

WBC 4–1,133

RBC 5–8,608

PLT 172–134,507


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Accuracy for BD FACSVia Flow Cytometers

Deming regression analysis was performed for the BD FACSVia manual and BD FACSCalibur manual. Plasma samples were prepared by centrifugation or apheresis method and autologous rWBC, rPLT, and rRBC subsets were added to achieve various concentrations for measurement.

Cell concentrations of test samples were established on the BD FACSCalibur prior to inclusion in the BD FACSVia accuracy study.

Figure 6 Accuracy of the BD Plasma Count kit on BD FACSVia vs BD FACSCalibur for rWBC in plasma

Variable N R2 Slope Intercept

rWBC 86 0.99 0.98 (0.97, 1.0) 1.6 (-1.8, 5.0)

1000

900

800

700

600

500

400

300

200

100

0

BD

FA

CS

Via

rWB

C (c

ells

/µL)

BD FACSCalibur rWBC (cells/µL)

0 100 200 300 400 500 600 700 800 900 1000


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Figure 7 Accuracy of the BD Plasma Count kit on BD FACSVia vs BD FACSCalibur for rRBC in plasma


rRBC 86 0.99 0.98 (0.96, 1.01) -26.3 , 38.3

11,000

10,000

9000

8000

7000

6000

5000

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Figure 8 Accuracy of the BD Plasma Count kit on BD FACSVia vs BD FACSCalibur for rPLT in plasma

WARRANTY

Unless otherwise indicated in any applicable BD general conditions of sale for non-US customers, the following warranty applies to the purchase of these products.

THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND NONINFRINGEMENT. BD’S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF


rRBC 86 1.0 0.97 (0.95, 0.98) 45.8 (-329.9, 421.6)

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BD FACSCalibur rPLT (cells/µL)

0 20,000 40,000 60,000 80,000 100,000 120,000


36

THE PRODUCTS OR REFUND OF THE PURCHASE PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT.

TROUBLESHOOTING

REFERENCES1 Guide to the preparation, use and quality assurance of blood components. (10th ed.)

Council of Europe Publishing; 2004:260.

2 Guidelines for the production of blood and blood components and for the use of blood products (Haemotherapie). Bundesgesundheitsbl-Gesundheitsforsch-Gesundheitsschutz; 2000;43:555-589.

3 Heiden M, Seitz R. Quality of therapeutic plasma-requirements for marketing authorization. Thromb Res. 2002;107 Suppl 1:47-51.

Problem Possible Cause Solution

Poor resolution between debris and residual cells

Inappropriate instrument settings

Follow proper instrument setup procedures; optimize instrument settings as required.

Staining dim or fading, or no residual cells

Reagents out of specification

Store BD Plasma Count reagents at 2°C–8°C and do not use after expiration date; repeat staining using new reagent.

Insufficient reagent Repeat staining with fresh sample; use recommended volumes; properly mix sample with reagents.

Cells not analyzed within 1 hour of staining

Repeat staining with fresh sample; analyze promptly or store samples in the dark at 4°C up to 1 hour.

Improper medium preparation (sodium azide omitted)

Use sodium azide in PBS if used as sample dilution medium.

Few or no BD Trucount beads detected

Bead pellet damaged or not present

Before use, inspect BD Trucount tube; use tubes within 1 hour after removal from foil pouch; do not use beyond expiration date.

Cytometer malfunctioning

Troubleshoot instrument.


37

4 Vowells S, Cadden M, Wagner C, Nuñez R. Detection and Enumeration of Residual White Blood Cells in Leucoreduced Red Blood Cell and Platelet Products Using the LeucoCOUNT Kit. Becton, Dickinson, and Company; 1998.

5 Lambrecht B, Spengler HP, Bauerfeind U, Mohr H. Simultaneous quantitation of contaminating leukocytes, erythrocytes, and platelets in fresh frozen plasma in a single tube assay by flow cytometry. Infusion Therapy and Transfusion Medicine. 2001;28 Suppl 1:52-53.

6 Jilma-Stohlawetz P, Marsik C, Horvath M, Siegmeth H, Hocker P, Jilma B. A new flow cytometric method for simultaneous measurement of residual platelets and RBCs in plasma: validation and application for QC. Transfusion. 2001;41:87-92.

7 Krailadsiri P, Seghatchian J. Residual red cell and platelet content in WBC-reduced plasma measured by a novel flow cytometric method. Transfusion and Apheresis Science. 2001;24:279-286.

8 Frey B, Furrer M, Wettstein M, Minder U. Quantification of residual red blood cells in platelet concentrates and fresh frozen plasma by flow cytometry. Infusion Therapy and Transfusion Medicine. 2001;28 Suppl 1:53.

9 Pichler J, Printz D, Scharner D, Trbojevic D, Siekmann J, Fritsch G. Improved flow cytometric method to enumerate residual cells: minimal linear detection limits for platelets, erythrocytes, and leukocytes. Cytometry. 2002;50:231-237.

10 Lee LG, Chen CH, Chiu LA. Thiazole orange: a new dye for reticulocyte analysis. Cytometry. 1986;7:508-517.

11 Terstappen LW, Loken MR. Five-dimensional flow cytometry as a new approach for blood and bone marrow differentials. Cytometry. 1988;9(6):548-556.

12 Gahmberg CG, Jokinen M, Andersson LC. Expression of the major sialoglycoprotein (glycophorin) on erythroid cells in human bone marrow. Blood. 1978;52:379-387.

13 Wise GE, Oakford LX, Dzandu JK. Ultrastructure of a transmembrane glycoprotein, glycophorin A. Tissue & Cell. 1988;20:219-227.

14 Rogers CE, Bradley MS, Palsson BO, Koller MR. Flow cytometric analysis of human bone marrow perfusion cultures: erythroid development and relationship with burst-forming units-erythroid. Exp Hematol. 1996;24:597-604.

15 de Vries E, De Bruin-Versteeg S, Comans-Bitter WM, et al. Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes. Arch Dis Child Fetal Neonatal Ed. 1999;80:226-229.

16 von dem Borne AE, Modderman PW, Admiraal LG, Nieuwenhuis HK. Platelet antibodies, the overall results. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989:951-977.

17 de Haas M, von dem Borne A. CD41/CD61 Workshop Panel report. In: Kishimoto T, Kikutani H, von dem Borne, et al, eds. Leucocyte Typing VI: White Cell Differentiation Antigens. New York, NY: Garland Publishing, Inc.; 1997:643-645.


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18 Feng R, Shimazaki C, Inaba T, et al. CD34+/CD41a+ cells best predict platelet recovery after autologous peripheral blood stem cell transplantation. Bone Marrow Transplant. 1998;21:1217-1222.

19 Jennings LK, Ashmun RA, Wang WC, Dockter ME. Analysis of human platelet glycoproteins IIb-IIIa and Glanzmann's thrombasthenia in whole blood by flow cytometry. Blood. 1986;68:173-179.

20 Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline —Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2005. CLSI document M29-A3.

21 Centers for Disease Control. Perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR. 1988;37:377-388.

22 Dzik S. Counting low numbers of leukocytes in leukoreduced blood components. Infusion Therapy and Transfusion Medicine. 1999;26:62-65.

23 Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Approved Guideline—Second Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2007. CLSI document H42-A2.

24 Linneweber J, Chow TW, Takano T, et al. Direct detection of red blood cell fragments: a new flow cytometric method to evaluate hemolysis in blood pumps. Asaio J. 2001;47:533-536.

25 Preliminary Evaluation of Quantitative Clinical Laboratory Measurement Procedures; Approved Guideline—Third Edition. Wayne, PA: Clinical Laboratory Standards Institute; 2002. CLSI document EP10-A3 AMD.

26 ICH Q2(R1). Validation of Analytical Procedures: Text and Methodology. 1996.


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