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Performance Evaluation of Body Fluids on the UniCel DxH 800 Coulter Cellular Analysis System

Introduction

Materials and Methods

T-13199A

The counting of cells in body fluids is a tedious and labor intensive task when performed manually. This task is important in that it discriminates between reactive, infectious and malignant diseases. Synovial, pleural and cerebrospinal fluids are the most common fluids analyzed in the laboratory and with few exceptions the presence of cells in these body fluids confirms a significant medical finding. More and more, there are fewer technical experts and with workload increasing, this has forced the

reliance of the analysis of body fluids to the automated hematology system. Today's more competent analyzers have an automated cycle for this analysis.

This study was initiated to assess the performance of the body fluid analysis on the DxH 800 System and included:

1 . Accuracy of the RBC and total nucleated cell (TNC) counts to manual methods

2. System linearity for RBC and TNC

3. Mode to mode carryover.

The evaluation of system repeatability and carryover using body fluid samples was established and published in "Evaluation of Specifications on the UniCel DxH 800 Coulter Cellular Analysis System."

Specimens

All specimens tested were spent samples covering a wide clinical range collected with EDTA, heparin or without anticoagulant depending on the sample type. All samples were analyzed within eight hours of collection. As per laboratory protocol, all specimens were closely examined for clots. All synovial fluids were pretreated with hyaluronidase

prior to analysis on the UniCel DxH 800 system. Only visibly bloody CSF samples were used for the CSF RBC analysis on the DxH 800. Table 1 includes a breakdown of the body fluid samples collected.

Results

Table 1. Body Fluid Samples

Specimen Type Total Collected

CSF 28

Serous 105

Synovial 14

Grand Total 147

UniCel DxH 800

All studies were performed using the body fluids cycle (BFC) on the DxH 800. The body

fluid cycle on the DxH 800 includes a rinse cycle prior to analysis that ensures the TNC and RBC background results are within acceptable limits, and then a portion of body fluid specimen is diluted for RBC and TNC counting. The TNC dilution includes the addition of a lytic reagent that removes any non-nucleated cells. The RBC and TNC are counted and sized simultaneously with all data captured in digital format and analyzed by the DxH 800 algorithms.(1) The digital processing allows the DxH 800 algorithms to analyze all the cellular

data at a higher resolution and with greater accuracy.

The DxH 800 System was calibrated prior to the start of this evaluation. Daily checks were

performed each day the system was in use and quality control performed using all three levels of COULTER Body Fluid Control.

Reference Method

The Neubauer Chamber method for cell counting was used as the reference method for Accuracy. 100 III of body fluid sample was diluted with 10 III of 0.1 % toluidine blue diluting fluid and incubated. Enumeration of the RBC and TNC was based using a maximum of 11 1-mm squares and included correction for dilution, area counted and depth.(2, 3)

Statistical Analysis

Statistical analysis included N, minimum, maximum, correlation and mean difference.

Accuracy - Body Fluid - TNC and RBC

A total of 147 body fluid samples were collected and from this data set 100 samples used for TNC and 73 were used for RBC analysis. Samples flagged with partial aspiration, R flags and those outside the measuring range were excluded from the analysis. Table 2 includes a

breakdown of the sample types and the summary of the accuracy results. Accuracy scatter plots for the TNC and RBC are illustrated in Figures 1 a and 1 b. System

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Table 2. Accuracy of TNC and RBC Body Fluid Counts

Body Fluids Accuracy Specifications

Parameter Units Measuring Range

TNC Cells/mm3 20 - 89,000

RBC Cells/mm3 10,000 - 6,200,000

Body Fluids Accuracy

Parameter N Method Mean Data Range

Min Max

Manual 5,294 16 87 ,620 TNC 100

DxH 800 5,037 26 82,294

Manual 197,592 720 4,451 ,000 RBC 73

DxH 800 198,994 1,005 4,454,747

TNC

100,000 ,------------------------,

90,000

80,000

70,000

o 60,000 o ro I 50,000 x

o 40 ,000

30,000

20,000

10,000

•

•

•

Bias

±5 or 10%

±500 or 5%

R

0.9924

0.9981

• TNC

- Equality

Bias

-4.85%

0.71 %

20,000 40,000 60,000 80,000 100,000

Manual Chamber Count

Figure 1 a. Body Fluid TNC Accuracy

5 ,000,000

4,500,000

4 ,000,000

3 ,500,000

g 3 ,000,000 ro I 2,500,000 x

o 2,000,000

1,500,000

1,000,000

500,000

RBC

•

•

•

• RBC

- Equality

1,000,000 2,000,000 3 ,000,000 4 ,000,000 5 ,000,000

Manual Chamber Count

Figure 1 b. Body Fluid RBC Accuracy

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, I

Linearity

TNC and RBC linearity was evaluated using prepared dilutions from spent body fluids specimens. Two different linearity sets were prepared to test the linear response at mid and low range concentrations for TNC and RBC as specimens with cell concentrations to test

the upper linearity specification were not available. The correlation coefficients (R2) for the mid range TNC, 0.9989 and the RBC, 0.9998 and the correlation coefficients (R2) for the low range TNC, 0.9895 and the RBC, 0.9995 demonstrate excellent correlation with the R2

>0.95. The linearity regression results for the low range TNC and RBC are illustrated in Figures 2a and 2b and the mid range TNC and RBC are illustrated in Figures 2c and 2d.

Linearity - TNC Body Fluids Low Concentration

250,----------------------------------------------------

"0 (j)

Q) 150 > 0 () (j)

0:::

50

• 0 0 50 100 150

Expected

Figure 2a. Linearity - TNC Body Fluids - Low Concentration

30,000

25,000

"0 20,000 (j)

Q) > 0 15,000 () (j)

0:::

10,000

5 ,000

0 0 5,000

Linearity - RBC Body Fluids Low Concentration

10,000 15,000

Expected

20,000

Figure 2b. Linearity - RBC Body Fluids - Low Concentration

4

200 250

25,000 30,000

!

l

\J Q)

(jj >

Linearity - TNC Body Fluids Mid Range Concentration

7,000 ,--------------------------,

6,000

8 4,500 Q)

n:::

3,000

1,500 ---- -- --- -

O ~~-,_--,_--,_--,_--,_--,_--~

o 1,000 2,000 3,000 4,000 5,000 6,000 7,000

Expected

Figure 2c. Linearity TNC Body Fluids - Mid Range Concentration

\J

Linearity - RBC Body Fluids Mid Range Concentration

2,500,000 ,---------------------,

2,000,000 - - - --- - - - -- - - - - - - -- - - - - --- - -- --- - - - - - - - - - - - - - - - - - - - - --

~ 1,500,000 ------ -------- --------------- --- ------- ---- - - --------

> o () Q)

n::: 1,000,000 - ---- ------- -- - ---- -- ----- -------- ------- - -- ------ -- --

500,000 - - --- ----

O ~---,_---,_--_,---_,---~

o 500,000 1,000,000 1,500,000 2,000,000 2,500,000

Expected

Figure 2d. Linearity RBC Body Fluids - Mid Range Concentration

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Discussion

Acknowledgements

Body Fluids Mode to Mode Carryover

The assessment of Body Fluids Carryover was performed on two different instances during this study. Body Fluid carryover consisted of using a normal whole blood analyzed in triplicate in the CBC mode followed by a diluent blank in the Body Fluids mode. The results indicate

that the carryover analyses met specifications and that the diluent samples results were below the background limits for the Body Fluids cycle. The results for both analyses are included in Table 3.

Table 3. Body Fluids Mode to Mode Carryover

Body Fluids Mode to Mode Carryover

CBC Cycle WBC RBC HGB PLT

Average whole 7.706 4.353 14.25 257.9

blood results

BFC Cycle TNC RBC PASS

Diluent 1 results 7 184

CBC Cycle WBC RBC HGB PLT

Average whole 7.571 3.782 11 .59 331.5

blood results

BFC Cycle TNC RBC PASS

Diluent 1 results 8 339

Limits ~20 ~1000

cells/mm3 cells/mm3

The UniCel DxH 800 Cellular Analysis system includes a dedicated analysis cycle for body fluids. This cycle ensures accurate and reliable enumeration of TNC and RBC on cerebrospinal, serous, and synovial fluids. This useful information is provided without the need for any special system, hardware or aspiration requirements. This automated process also alleviates the technologist from the error prone and labor intensive manual method of body fluid analysis.

In this study, the accuracy of the TNC and RBC counts as compared to the reference Neubauer Chamber count demonstrates very good agreement throughout the measuring

range when compared to specifications.(4) The assessment of the linearity for the RBC and TNC results demonstrate excellent correlation with the results of both exceeding 0.95. The critical nature of cell counts on body fluids at low levels was assessed by carryover studies. The carryover analysis not only ensures that there is no carryover from sample to sample; it also demonstrates that the RBC and TNC background counts are well below background specifications.

The results of this evaluation demonstrates that the DxH 800 system is able to meet or exceed published specifications for body fluids analysis by providing accurate results without carryover throughout the measuring range.

The technical assistance of the laboratory staff at London Health Sciences Center and

Barnes Jewish Hospital are greatly appreciated.

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References 1. UniCel DxH 800 Coulter® Cellular Analysis System Instructions for Use. Beckman Coulter,

Inc. PN 629743, rev. AC. (December 2008)

2. Laboratory evaluation of body fluids. Stiene-Martin EA, Lotspeich-Steininger CA, Koepke JA, eds. Clinical Hematology: Principles, Procedures, Correlations. 2nd ed. Philadelphia,

PA: Lippincott; 1998:400-414.

3. Kjeldsberg C, Knight J. Body Fluids: Laboratory Examination of Cerebrospinal, Seminal, Serous and Synovial Fluids. 3rd ed. Chicago, III: ASCP Press: 1993.

4. Schonenenberg H, Fassl H. Comparison of the coulter-counter-method and the counting­chamber to determine the cell count of the cerebrospinal fluid (in German). Artzl Forsch. 1970;24:118-124.

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Beckman Coulter, the stylized logo, UniCel, and COULTER are registered trademarks of Beckman Coulter, Inc. DxH is a trademark of Beckman Coulter, Inc.

This paper is published as an educational service by Beckman Coulter, Inc. © 2009 Beckman Coulter, Inc. B2009-1 0504 Printed in U.S.A.

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