International Journal of Clinical Biochemistry and Research

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Vijayan, Krishna K G, Danniel, Shaji, Mathew, John, Sukumaran, Varkey, Thomas, Thomas, Paul, Ouseph, Paul, and Vasudevan: Mispa count X; The first indigenous indian hematology 3-part analyzer


Introduction

The invention of impedance technology and its application in cell counting by Charles Wallace Coulter heralded the beginning of new era in hematology.1 Sysmex Corporation which used conductance technology in hematology counters shifted to impedance technology in their analyzers.2 Today, automation in Hematology is accepted worldwide, which improves accuracy and speed of cell counting, reduces the manual workload and enables the blood cell counting of other body fluids than blood.3 Adapting to this advanced technology helps to omit errors caused due to the limitations in the traditional microscopic counting; The most common errors in the traditional technique can be classified into three vide: the statistical error (due to lower number of cells counted), distributional error (due to unequal distribution of cells in smear) and the error due to mistakes in subjective interpretation of the technicians.4, 5

Blood cell count is one of the mostly prescribed blood tests, as these directly indicate the overall physiological health status of an individual. Complete blood count and differential blood count are widely used in diagnosis and prognosis of diseases. So the authentication of results produced by any hematology analyzer must be proved.6, 7 A few clinical cases are listed below which support the relation of blood cell counting with disease and treatment progress Dengue fever is characterized by low leukocyte count in the initial stage and platelet count lowers as the disease progresses.8 The mortality rate in case of peritoneal dialysis patients can be envisaged by testing red cell distribution width.9 The health progress in allogeneic peripheral blood stem cell transplantation can be monitored using platelet count.10 Quantification of lamellar bodies in amniotic fluids is a useful method to determine fetal lung maturity.11

Mispa count X, being the first indigenous 3-part hematology analyzer in India can reduce the import of expensive hemtology analyzers to a great extent. With this domestic invention that matches international quality, it is now affordable for common rural population. All evaluation protocols used for verification of Mispa Count X viz., linearity, carry over, precision and comparability are in reference to the CLSI standards and ICSH guidelines.

Materials and Methods

Analyzer

Mispa Count X is a tabletop, compact 3-part analyzer with 275 mm width, 436 mm height and 461 mm length. The analyzer has a 10.4” SVGA resistive touch Screen Display having a resolution of 800x600 with 24-bit colour depth. The equipment works efficiently at an ambient temperature of 15 - 35°C and at a relative humidity of 80% at 32°C. The analyzer can with stand temperature up to 50°C and altitude change up to 3000m without affecting the result.

Mispa Count X is a quantitative and automated 3-part haematology analyzer designed for In vitro Diagnostic use in clinical laboratory. The system is having a throughput of 60 samples /hour. Mispa Count X provides 18 clinical parameters & 3 Histograms (WBC, RBC & PLT), including a 3-part WBC differential. Analyzer has automatic flagging of results that are out of normal range and has a storage capacity of 35000 sample results, including parameter, histogram, and patient information. It has a thermal printer and is also provided with an option of connecting to an external printer.

The verification study of Mispa Count X, a 3-part Hematology analyzer designed and manufactured by Agappe Diagnostics Ltd was performed against Beckman coulter DxH 800 and Sysmex XN1000.

Specimens

The study procedures including blood collection technique, sample tubes, sample number etc., are performed as per the respective CLSI guidelines. Peripheral venous blood was collected in K2-EDTA anti-coagulated vacutainers from 180 patients.12 The specimens were kept at room temperature and tested within 6 hours of sample collection.13

Data analysis

The statistical analyses were conducted by using Microsoft Excel (2013).

Linearity

The linearity test was done following CLSI guideline EP06-A. The linearity of leukocyte count, erythrocyte count, platelet count, hematocrit and hemoglobin concentration were evaluated using the linearity kits of R&D Biotechnie (CBC line 139). Each level of linearity control is run 4 consecutive times and the percentage deviation from the expected measurement is calculated.14

Carry Over

The carry over study evaluate whether the analyzer shows any effect of the high count/ blank measurement on low level count measurement. The carry-over of leukocyte count, erythrocyte count, platelet count and hemoglobin measurement were assessed following CLSI guideline H26-A2. To perform the study three consecutive run of high sample without any interruption were followed by the three consecutive low sample run. The same procedure was repeated with 3 blank run followed by 3 low control run to study the effect of blank run on expressing low count measurement.15, 16 The carry-over is calculated by using the following equation.

Figure 0
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Imprecision

Both long term and short term imprecision were performed following CLSI guideline EP05-A2. The repeatability was tested by running 5 different blood samples of normal measurable range, 31 times each. The coefficient of variation (CV %) was calculated. The reproducibility was established by running 3 levels of whole blood controls (normal, low and high) (Lot: 31030557) twice a day with 4 to 6 hours gap for 20 days. The CV% was calculated and assessed the within laboratory precision.17

Comparability (Correlation)

The comparison of Mispa Count X with Coulter DxH 800 and Sysmex XN 1000 were performed by testing 180 patient samples. The correlation coefficient was calculated for each parameter. The study protocol follows CLSI guideline EP09-A2.18

Figure 1

Comparison of hematology parameters of Mispa Count X (Agappe Diagnositcs) with Coulter DxH 800 (Beckman Coulter). Abbreviations: MCV, mean corpuscular volume; MPV, mean platelet volume; RDW, red cell distribution width; Lym #, lymphocyte count; Mid #, mid cell count; Gran #, granulocyte count.

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/cc442a8d-4ee2-425b-87ca-359c2377cac6/image/b3eb739c-4d96-4f23-8d33-4ea714006c06-uimage.png

Figure 2

Comparison of hematology parameters of Mispa Count X (Agappe Diagnositcs) with Sysmex XN 1000 (Sysmex Corporation)

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/cc442a8d-4ee2-425b-87ca-359c2377cac6/image/9b095b92-738c-4b46-bdaf-6711079e9df3-uimage.png

Figure 3

Sample stability. Normal blood samples incubated at room temperature and tested at 0th, 1st, 2nd, 4th, 8th, 24th and 48th hour.

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/cc442a8d-4ee2-425b-87ca-359c2377cac6/image/7230a4de-e92d-4812-ba58-86d1b0e2023d-uimage.png

Table 1

Linearity. a) Full range linearity controls, b) Low level linearity controls of R&D Biotechnie Linearity kit (L# CBC-Line 130)

WBC

RBC

Hb

PLT

Levels of Control *

Expected value

Obtained value

% Deviation

Expected value

Obtained value

% Deviation

Expected value

Obtained value

% Deviation

Expected value

Obtained value

% Deviation

a) Full range linearity

1

8.65

8.65

0

1.09

1.15

-5.41

3.43

3.425

-0.15

23.3

24

-2.89

2

17.3

16.45

4.91

2.17

2.28

-4.72

6.86

6.9

0.58

233.25

233.25

0

3

38.9

36.83

5.38

2.9

2.96

-1.96

9.15

9.225

0.81

466.5

464

0.535

4

73.5

77.6

-5.54

4.35

4.35

0

13.73

13.725

-0.04

1282.9

1224.3

4.57

5

90.8

95.12

-4.73

6.88

6.59

4.19

21.73

20.4

-6.52

1516.1

1423.8

6.09

6

121.1

123.7

-2.13

7.24

6.91

4.67

22.88

21.35

-7.17

2332.5

2241.5

3.9

b) low range linearity

1

0.415

0.425

-2.41

0.212

0.225

-6.07

0.6

0

0

8.29

6.34

-23.5

2

0.83

0.875

-5.42

0.424

0.44

-3.71

1.2

1.175

-2.13

16.6

17.2

-3.8

3

1.66

1.675

-0.904

0.849

0.878

-3.42

2.4

2.4

0

33.2

35

-5.58

4

4.15

4.225

-1.8

2.12

2.19

-3.005

5.99

6.15

2.60

82.9

87

-4.98

5

5.81

5.925

-1.98

2.97

3.005

-1.18

8.38

8.5

1.41

116

120

-3.5

6

8.3

8.3

0

4.24

4.24

0

11.98

11.975

-0.04

165.8

165.8

0

[i] *Each levels of controls tested consecutively from 1 – 6

Table 2

Carry over. a) On low samples by high value sample run. b) On low samples by blank run

WBC

RBC

Hb

PLT

a)

H1

16.5

5.91

11.6

501

H2

16.2

5.84

11.5

541

H3

16.5

5.81

11.5

489

L1

1.4

3.24

10.8

163

L2

1.5

3.32

10.9

181

L3

1.4

3.28

10.8

165

Carry Over (%)

0 %

-1.52%

0%

-0.595%

b)

B1

0

0

0

0

B2

0

0

0

0

B3

0

0

0

0

L1

1.4

3.28

10.9

164

L2

1.4

3.4

11

158

L3

1.4

3.18

10.9

162

Carry Over (%)

0%

-3.14%

0%

-1.23%

Manufacturer Specification

<1.5%

<1.5%

<1.5%

<2.5%

Table 3

Imprecision

Parameters

Value

Unit

Range

CV % Specification

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

WBC

Mean

103/µL

> 6.0

6.73

5.25

5.63

6.14

6.17

CV%

%

< 4.0%

3.35

3.33

3.44

2.55

1.75

RBC

Mean

106/µL

> 4.0

5.58

4.95

4.65

4.09

4.31

CV%

%

< 4.0%

1.77

1.52

1.5

2.06

1.74

HGB

Mean

g/dL

> 12.0

15.65

14.65

13.04

11.71

13.38

CV%

%

< 2.5%

1.7

1.24

2.26

1.72

1.24

HCT

Mean

%

> 35.0

46.25

41.65

40.67

32.94

40.99

CV%

%

< 2.5%

1.68

1.45

1.63

1.9

1.48

MCV

Mean

fL

>85

82.86

84.12

87.39

80.4

95.08

CV%

%

<2.5%

1.28

0.89

1.75

1.1

0.71

RDW CV

Mean

%

> 12.0

13.31

13.08

13.51

13.08

12.62

CV%

%

< 5%

4.39

2.85

3.21

3.78

3.02

RDW SD

Mean

fL

> 25

41.04

42.21

39.05

40.75

41.23

CV%

%

< 5%

1.09

1.27

1.79

1.69

1.51

PLT

Mean

103/µL

> 200

295.42

353.28

319.14

251.35

215.38

CV%

%

< 10%

6.95

4.57

3.04

3.96

3.35

MPV

Mean

fL

> 7.0

9.44

8.89

8.52

9.58

9.72

CV%

%

< 5%

1.7

1.45

2.54

1.4

1.64

LYMPH %

Mean

%

> 15

30.78

34.68

46.46

40.79

38.15

CV%

%

< 5%

4.13

2.5

4.99

2.47

2.68

MID %

Mean

%

> 5

5.98

5.84

5.61

5.91

6.74

CV%

%

< 10%

5.89

5.69

7.19

6.19

7.27

GRAN %

Mean

%

> 50

63.24

59.48

47.91

53.3

55.12

CV%

%

< 5%

2.37

1.59

4.84

1.76

1.81

[i] Within run Imprecision (Using clinical samples of the specified range)

Table 4

Parameters

Value

Unit

Range

CV % Specification

Normal control

(Lot#)

Low control

(Lot#)

High control

(Lot#)

WBC

Mean

103/µL

> 6.0

6.98

2.26

15

CV%

%

< 4%

2.7

4.8

2.5

RBC

Mean

106/µL

> 4.0

4.34

2.23

5.4

CV%

%

< 4%

3.7

3.5

1.8

HGB

Mean

g/dL

> 12.0

11.91

6

16.2

CV%

%

< 2.5%

2.2

2.3

1.7

HCT

Mean

%

> 35.0

33.12

14.88

42

CV%

%

< 5%

4.3

2.7

4.9

MCV

Mean

fL

>85

77.11

66.66

77.8

CV%

%

<5%

2.4

2.3

4.8

RDW CV

Mean

%

> 12.0

17.11

18.21

17

CV%

%

< 7%

4.2

5.4

6.1

RDW SD

Mean

fL

> 25

49.14

47.98

50.9

CV%

%

< 7%

6.3

6.5

5.8

PLT

Mean

103/µL

> 200

264.86

97.34

554

CV%

%

< 10%

3.8

7.7

7.9

MPV

Mean

fL

> 7.0

12.15

11.22

12.8

CV%

%

< 5%

3.6

2.7

4.4

LYMPH %

Mean

%

> 15

34.15

48.9

19.2

CV%

%

< 5%

3.3

3.8

4.8

MID %

Mean

%

> 5

7.2

7.91

7

CV%

%

< 10%

8.9

8.8

9.7

GRAN %

Mean

%

> 50

59.32

43.18

73.9

CV%

%

< 5%

2.4

3.4

4.8

[i] Long term Imprecision (Using tri-level control)

Table 5

Correlation of Mispa Count X with comparator analyzers (Beckman Coulter DxH 800 & Sysmex XN 1000)

Parameters

Measured range

Comparison with Beckman Coulter DxH 800

Comparison with Sysmex XN 1000

r2 Value

Computed equation

Bias

r2-Value

Computed equation

Bias

Manufacturer Specification

WBC (103/µL)

2.5 - 28

0.9765

y = 0.9499x + 0.7787

± 0.233

0.9726

y = 0.9478x + 1.136

± 0.72

>0.95

RBC (106/µL)

2.25 - 9.0

0.991

y = 1.0276x - 0.1336

± 0.014

0.9514

y = 0.9737x + 0.1534

± 0.038

>0.95

Hb (g/dL)

6.5 - 20

0.9875

y = 0.9873x + 0.1399

± 0.012

0.9579

y = 0.9515x + 0.5684

± 0.027

>0.95

MCV (fL)

60 - 110

0.9593

y = 0.9498x + 4.3585

± 0.129

0.9450

y = 0.9556x + 3.8101

± 0.059

>0.9

Plt (103/µL)

25 - 750

0.9708

y = 1.0143x - 7.7128

± 4.36

0.9550

y = 1.007x - 2.9527

± 1.22

>0.95

MPV

6 - 12

0.8848

y = 0.798x + 1.5664

± 0.0341

0.8330

y = 1.2643x - 1.7127

± 0.258

≥ 0.90

RDW-CV

12 - 30

0.7985

y = 1.011x + 0.0094

± 0.425

0.8234

y = 0.8322x + 2.3449

± 0.0036

≥ 0.75

RDW-SD

35 - 80

0.7533

y = 1.0346x - 1.295

± 0.1625

0.8159

y = 0.9152x + 4.0698

± 0.078

≥ 0.75

Lymph # (103/µL)

0.3 - 7.5

0.926

y = 1.0802x + 0.1256

± 0.300

0.961

y = 1.0016x + 0.2664

± 0.270

≥ 0.90

Mid # (103/µL)

0.1 - 2.0

0.9082

y = 0.9402x + 0.0027

± 0.048

0.8729

y = 0.8132x + 0.0814

± 0.0047

≥ 0.9

Gran # (103/µL)

0.4 - 22

0.9531

y = 0.9658x + 0.56

± 0.376

0.9672

y = 0.9883x + 0.4848

± 0.4273

≥ 0.90

Sample stability

Blood samples collected from 5 healthy volunteers were used for this study. The sample stability was tested at 1st, 4th, 8th and 24th hour after blood collection. The samples are stored at room temperature (24 – 30 °C) and low temperature (4 – 8 °C). The deviation of the test results of the various aged samples and temperatures were calculated and compared.19

Calibration and quality control

The calibration of the test analyzer was performed by running the calibrator 10 times and the average is taken. It has been calibrated against the assigned values of the calibrator. The quality control was performed throughout the study period to ensure the performance stability of the calibration.

Results

Mispa Count X exhibited an average background of 0.1 (103/µL), 0.02 (106/µL) and 4 (103/µL) for total WBC, RBC and platelets respectively. The equipment showed a good linearity range of 0.5 to 120, 0.25 to 7.0, 1.2 – 20 and 20 to 2000 for WBC (103/µL), RBC (106/µL), hemoglobin (mg/dL) and PLT (103/µL) respectively (Table 1). Mispa count X exhibited a negligible carry-over of <1.5% for WBC, RBC and platelets counts as well as haemoglobin measurement from previously run high sample and blank run (Table 2). The CV% for both intra-run precision (a) and inter-run precision (b) of Mispa Count X were < 10% for all parameters which meets the equipment specifications (Table 3). Mispa Count X produced an acceptable r2 values (> 0.90) on comparison with Sysmex XN-1000 and Beckman Coulter DxH800 except for mid cell counts and MPV measurements which was 0.9082 and 0.8729 for mid cell counts and 0.8834 and 0.8330 for MPV. The results of comparability and bias estimation are illustrated in Table 5 and Figure 2, Figure 3. The sample stability shows no significant deviation within 24 hrs of storage at room temperature for any parameters except for Mid%. The average CV% shown were 2.52%, 1.16%, 1.30%, 1.45%, 3.31%, 2.49%, 3.48%, 3.77% and 14.62% respectively for WBC, RBC, Hb, HCT, PLT, MPV, Lym%, Gran% and Mid% (Figure 4).

Discussion

Mispa Count X is the first completely indigenous Hematology analyzer which works on the principle of impedance technology. The technical validation of this newly developed analyzer is performed by following CLSI guidelines and proven for commercialization.

The overall performance of Mispa Count X was satisfying. It delivers the results of all parameters with accuracy and precision. The analyzer exhibited no significant carry over from a previous high specimen run and blank run. The CV% obtained were within the claimed limit (<1.5%) and thus is acceptable according to EP5-A2.17 The linearity range of Mispa Count X (0.5 – 120 x 103/µL for WBC, 0.25 – 7.0 x 106/µL for RBC, 1.2 – 20 mg/dL for Hb and 20 - 2000 x 103/µL for platelet) are appropriate for a 3-part hematology analyzer.14 The analyzer has a good performance correlation with Beckman Coulter DxH800 and Sysmex XN1000 for all parameters except for mid cell count and MPV which produced a lower r2 value < 0.90. The comparator analyzers were much advanced versions than Mispa Count X which could produce more reliable differential counts. Considering the fact that the 3-part analyzer aids only the primary screening of samples and the golden standard of microscopic examination of smear is recommended for any abnormality confirmation, the deviation from specification in monocyte count is acceptable.18 Similar monocyte measurement variation is already been reported.13 Mispa Count X produces flags for all high and low measurements of each parameters which ease the task of the technician by limiting the number of samples for microscopic examination.

According to Imeri, the technology of the analyzer and its performance capability affects the sample stability.19 Mispa Count X produced stable results for 24 hours at room temperature. The analyzer functions with a cyanide free and azide free reagent system which causes no harm to the environment.

The wide linearity ranges, negligible carry over, good precision CV% and acceptable bias and r2 values on comparison with both Coulter DxH800 and Sysmex XN1000 proved that the newly developed analyzer could produce reliable and comparable results and it performs equally well as any other already proven equipment.

Conclusion

Mispa Count X opens up the possibility of replacement of the imported, expensive analyzer with a new, Indian made, relatively less expensive analyser with high level of performance, affordable for the rural population.

Declaration

This manuscript has not been published elsewhere and is not under consideration by another journal.

Conflicts of Interest

Authors declare no conflict of interest.

Informed Consent

Whole blood samples were collected after informed consent and used for this study. No patient details and medical history were used for the manuscript.

Ethics Approval

The institutional ethical committee approval has been taken before conducting the evaluation and the whole study was conducted following the guidelines.

Acknowledgments

The authors acknowledge Agappe Diagnostics, Pattimattom Cochin, Kerala, India for providing the facilities to carry out this work and also for granting permission to publish the data acquired from the study. The authors would like to express their sincere gratitude to Geevarghese Joseph, Eldho Soyi and Emosh P S, R&D Equipment division who supported to make this effort fruitful.

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F Imeri R Herklotz L Risch Stability of hematological analytes depends on the hematology analyzer used: a stability study with Bayer Advia 120, Beckman Coulter LH 750 and Sysmex XE 2100Clin Chim Acta20083971-2687110.1016/j.cca.2008.07.018



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© This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Article type

Original Article


Article page

265-273


Authors Details

Deepa K Vijayan*, Jiby Krishna K G, Dinimol Danniel, Ashily Shaji, Jojo Mathew, Arun John, Ajaikumar Sukumaran, Peeter K Varkey, Thushara Thomas, Riji Thomas, Eldho Paul, Varghese N Ouseph, Jofy K Paul, D M Vasudevan


Article History

Received : 07-09-2021

Accepted : 21-10-2021

Available online : 05-01-2022


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