International Journal of Laboratory Hematology The Official journal of the International Society for Laboratory Hematology

ORIGINAL ARTICLE

INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY

Comparison of performances of five capillary blood collection tubes Z. PENG*, J. MAO † , W. LI*, G. JIANG*, J. ZHOU*, S. WANG ‡

*Department of Clinical Laboratory, The Children’s Hospital Zhejiang University School of Medicine, Hangzhou, China † Department of Nephrology, The Children’s Hospital Zhejiang University School of Medicine, Hangzhou, China ‡ Department of Oncology, Zhejiang Xiaoshan Hospital, Hangzhou, China Correspondence: Jianhua Mao, No. 57 Zhugan Lane, Hangzhou, Zhejiang, China. Tel.: (+86)0571-88873432; Fax: (+86)0571-88873071; E-mail: [email protected] doi:10.1111/ijlh.12231

Received 10 December 2013; accepted for publication 25 February 2014 Keywords Capillary blood, blood collection tubes, performance comparison

S U M M A RY Introduction: The research demonstrated the way to compare performances of five capillary blood collection tubes for capillary blood collections among patients with pediatrics utilizing the Clinical and Laboratory Standards Institute (CLSI) document EP9-A2. Methods: A questionnaire survey was distributed to evaluate the accessibility and reliability of capillary blood collection tubes from different sources. Visual examination, microscopic examination of blood smears, and instrumental analysis were performed to evaluate the blood sample quality in different tubes. The background test, comparison test, and reliability test were used to analyze the engineering quality and the performance of additives in the tubes, and to determine the reliability of the tubes in routine blood tests. Results: The tubes of brand A were found to be easy to access, with a shorter blood collection time and greater acceptability by users, and the quality of blood samples in those tubes was superior, with no coagulation and less blood cell disruption. Conclusion: Commercially available capillary blood collection tubes were superior to ‘in-house’ tubes. In clinical practice, ‘in-house’ capillary blood collection tubes are not recommended. The guideline of ‘method comparison and bias estimation using patient samples’ from CLSI could also be used in comparing the performances of capillary blood collections.

INTRODUCTION Accurate and effective results rely largely on highquality samples. In complete blood cell count assays for patients with pediatrics, obtaining high-quality blood samples has always been challenging. Venous blood collection is often accompanied by pain and mental stress [1], so many researchers have attempted to improve this unpleasant experi56

ence [2, 3]. Venipuncture for patients with pediatrics incurs even more difficulties and risks from lack of co-operation of young patients who fear pain and blood collection itself [4]. For premature newborns, over collections may result in anemia [5]. Deep veinpuncturing runs the risk of complications, such as infection, cardiac arrest, a tendency toward bleeding and venous thrombosis. Therefore, capillary blood collection is commonly performed in young patients, © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 56–62

Z. PENG ET AL. | COMPARISON OF BLOOD COLLECTION TUBES

and these blood samples are used for complete blood cell count assays. It is worth mentioning that engineering and additives to the tubes for capillary blood collection can directly affect the accuracy of the results of a complete blood cell count. The present study used a questionnaire survey and other tests to compare the performance characteristics, for example, the clinical accessibility and reliability, of four commercially available and one ‘in-house’ capillary blood collection tubes in patients with pediatrics for complete blood cell count assays. The investigation also attempted to set an example to compare the performances of capillary blood collections making use of the guideline of ‘method comparison and bias estimation using patient samples’ from Clinical and Laboratory Standards Institute (CLSI).

M AT E R I A L S A N D M E T H O D S Materials







Commercially available capillary blood collection tubes: The four brands of commercially available tubes were Microtainer of BD, Vaccutee MiniCollect of Greiner Bio One, Impromini of Improve Medical, and GD005 of U-real Medical, marked as tubes A, B, C, and D, respectively. All tubes contain K2EDTA as anticoagulants, and the blood was scooped into tubes without any capillary tubules. ’in-house’ capillary blood collection tubes: The tube was marked as tube E and was made from Safe Lock Tubes of Eppendorf International. Analytical grade of K2EDTA was purchased from Shanghai Sansi Reagent Co. ltd., Shanghai, China. The capillary tube was made according to the requirements of venous blood-to-anticoagulant ratio in CLSI document 6710 : 1995 [6] and SFDA standard YY 0314-2007 [7], using K2EDTA as anticoagulant. The concentration of K2EDTA was 4.55  0.85 lmol/mL blood (1.85  0.35 lmol/mL blood). For each tube, 20 lL of 2% K2EDTA were added for anticoagulation of 200 lL whole blood with final concentration of K2EDTA at 2.0 mg/mL. The tubes were dried at 37 °C and ready to use. Evacuation blood collection tube of the K2EDTA blood collection tubes from BD Inc. (tube F) was the reference tubes. (BD Vacutainer, LOT 2219406).

© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 56–62





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Capillary lancet: Single-use capillary lancet (BD Microtainer Contact-Activated Lancet, LOT S4U82E1) which could control the depth of skin puncture test. Instruments: blood samples were tested on XS-800i automatic blood analyzer from SYSMEX Inc. Kobe, Japan and the reagents, calibrators and quality controls were all from the same manufacturer. The instrument participated in National External Quality Assessment Program of China Clinical Test Center and was in good status. Quality control measures were enforced to maintain instrument stability. The KJMR-II blood mixer was purchased from Jiangsu Kangjian Medical Instrument co., Taizhou, China and the mixing rates were set to 40 swings/ min and 40 rolls/min.

Methods





Blood collection comparison and questionnaire survey: The brand marks of all the tubes were covered, and the tubes were inspected by five experienced clinical laboratory workers, as the participants, for overall appearance and quality. Two hundred and fifty children, aged from 12 to 36 months old, were divided into 25 groups. The double-blinded capillary blood collections to the children were performed by five participants, each person for five groups with five brands tubes, respectively. BD capillary lancets were used for skin puncture, and the collection procedure followed the requirements in CLSI document H04-A6 [8] and the product inserts of the blood collection tubes. Blood flow entered the tube freely along the tube wall. ‘Scratch’ and ‘extrusion’ collections were prohibited. Approximately 400 lL of blood were collected and were mixed with the blood mixer. The time between skin puncture and the end of blood collection was recorded as the collection time. The laboratory workers were asked to complete the questionnaire to provide evaluations for each brand of tubes, based on appearance, accessibility, plain collection, tube wall smoothness, and accessibility to the analyzer. Blood quality examination: All the blood samples were inspected for coagulation. Two blood smears were then made of each sample and were reviewed by two experienced laboratory workers in

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Z. PENG ET AL. | COMPARISON OF BLOOD COLLECTION TUBES

a double-blinded fashion. Microclots, massive cell disruption, and epithelial cells were recorded. Each blood sample was analyzed by an automatic analyzer to record the flag tags and to review any abnormal figures in white blood cell plots, red cells, or platelet charts. Background tests: 400 lL sample diluents of the blood analyzer were added to each tube, and a total of 40 tubes each brand was prepared. The tubes were shaken in a blood mixer and were analyzed in the instrument, following the testing procedure as the blood samples. The results were recorded and analyzed. Comparison of results: The comparison tests were performed according to CLSI document EP9A2 [9].





Sample preparation. The tests were performed for 5 days, and every day, eight fresh blood samples were collected and tested, for a total of 40 samples. All the samples were drawn by syringes. Each sample was separated into two parts immediately, first of 2 mL blood was injected into a BD-evacuated collection tube (purple closure, K2EDTA as anticoagulant), and other blood was added into one of each brand of the capillary blood collection tube up to the scale mark of 400 lL. All the tubes were mixed with the blood mixer and were analyzed by the instrument in both directions after 0.5 h. The results from the capillary blood collection tubes (tubes A–E) and the evacuation tube (tube F) were compared, and clinical compatibility was calculated.



• The main method of comparison tests in CLSI document EP9-A2 contains five sections, such as outlier tests on within-method duplicates, plotting the data and visual checking for linear relationships, visual checking for between-method outliers or performing of calculations, an adequate range of X was tested with the correlation coefficient and the scatter and bias plots were examined for constant scatter. In our tests, we imitated the method comparison and bias estimation as the capillary tube performance evaluation. The detail of statistical procedures could be consulted in the guideline of CLSI document EP9-A2. Stability test: 10 tubes of each brand in method 4.1 were selected and entered into the stability test, with five tubes a day. The samples were analyzed by the instrument two times in plain and inverted sequence on the right day at the time points of 0.5, 1, 2, and 4 h after injected, and the mean values of two tests were recorded as the results. The maximum biases of each tube at different times during the analysis were compared individually. Statistical analysis: The t-test was used to compare the collection time differences between tubes.

R E S U LT S



In the blood collection test, the collection times for the five participants, using brands A, B, C, D, or E, respectively, to collect blood samples for 50 young children, were differed statistically significant (P < 0.05) with the shortest time of brand A. The results of questionnaire survey for five brands of

Table 1. Results of questionnaire survey Subject

Details

Appearance Accessibility

High-quality engineering and materials, etc. Easy to open and close the closure without blood spilling Clear cut on the skin, smooth tube opening without risk to damage patients’ skin Blood flow passing tube opening without delay and smoothly going down along tube wall Easy to mix and to analyze without leakage when tube is closed Sum

Easy to collect Smoothness of tube wall Accessibility to instrument analysis

A

B

C

D

E

5 5

5 3

4 4

4 4

1 1

5

4

4

3

1

5

3

3

4

1

4

5

4

4

1

24

20

19

19

5

Each subject ranked from 1 to 5, and the total score was 25. Higher score means better.

© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 56–62

Z. PENG ET AL. | COMPARISON OF BLOOD COLLECTION TUBES



capillary blood collection tubes from five participants are summarized in Table 1. The visual examination results for the blood samples from the five groups of blood collections are

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listed in Table 2. Epithelial cells or cell disruptions resulted in inaccuracies in the blood cell counts and white cell differential counts, see Figure 1.

Table 2. The results of abnormal samples in visual examination A

Visual clots Microclots Cell disruption Epithelial cells Instrument alarm Abnormal chart plots

B

C

D

E

Cases

%

Cases

%

Cases

%

Cases

%

Cases

%

0 0 2 4 1 5

0 0 4 6 2 10

0 0 2 10 2 11

0 0 4 24 4 22

2 3 5 17 5 18

4 6 10 34 10 36

1 3 4 18 4 18

2 6 8 36 8 36

4 7 7 20 7 24

8 14 14 40 14 48

The examination indicated that some of the capillary blood collection tubes showed microclots and even coagulation in the whole tube. There are two predicted reasons for such a phenomenon in the observed tube plots. First, an uneven spray of anticoagulants can be observed at inner wall of some tubes, and blood coagulation can be triggered by insufficient contact with anticoagulants. The second reason might be the engineering quality. Some blood remained in the tops of tubes in which anticoagulant was limited, forming microclots then mixed into the remaining blood. The abnormal chart plots which sat out of the cell counting range were caused by the epithelial cells or blood cell fragments entering the blood samples during collection process.

(a)

(b)

Impurities

Epithelial cell

Ghost

Figure 1. Impurities in blood sample collected by the capillary blood collection tubes. In the blood collection tests, impurities could be found in some blood samples. This blood samples showed abnormal noise points in the white blood cell plots during the blood analysis, indicating impurities in the samples (a). Some noise points caused by the epithelial cells entering the samples during collection, located in the upper right corner, were caused by epithelial cells entering the samples during collection. It also could be found in the blood smears (b). The small impurities located in the lower positions closer to x-axis (called the Ghost) were usually disrupted blood cells. Sharp edges of the collection tubes might bring force the epithelial cells by skin injury and disrupt the blood cells into fragment during the collection process.

© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 56–62

–0.50

4.00

140.00

120.00

3.00 4.00 5.00 Mean of tube F

0.00

F-A F F-B F F-C F F-D F F-E F

120.0

100.0

A F B F C F D F E F

15.0

10.0

5.0

0.0

600.00

500.00

400.00

A F B F C F D F E F

A F B F C F D F E F

50.00

25.00

0.00

F-A F F-B F F-C F F-D F F-E F

–25.00

–3.00

–15.0

60.0

80.0 100.0 Mean of tube F

120.0

R Sq Linear = 0.988 R Sq Linear = 0.968 R Sq Linear = 0.963 R Sq Linear = 0.978 R Sq Linear = 0.967–50.00

200.00

R Sq Linear = 0.965 R Sq Linear = 0.973 –10.0 R Sq Linear = 0.968 R Sq Linear = 0.975 R Sq Linear = 0.679

60.0

45.00

45.00

300.00 –5.0

30.00 35.00 40.00 Mean of tube F

30.00 35.00 40.00 Mean of tube F

The bias of PLT results

The plots of PLT results A F B F C F D F E F

80.0

25.00

25.00

Mean of tube F

The bias of MCV results

–1.00 –2.00

R Sq Linear = 0.981 R Sq Linear = 0.955 R Sq Linear = 0.956 R Sq Linear = 0.954 R Sq Linear = 0.9

25.00 80.00 90.00 100.00 110.00 120.00 130.00 140.00

Mean of tube F

A F B F C F D F E F

A F B F C F D F E F

35.00

–10.00 80.00 90.00 100.00 110.00 120.00 130.00 140.00

6.00

Mean of tubes A-E

1.00

Bias of tubes F to A-E

2.00

3.00 4.00 5.00 Mean of tube F

The plots of MCV results

The bias of HCT results 3.00

40.00

30.00

80.00 2.00

6.00

10.00

A F B F C F D F E F

R Sq Linear = 0.986 R Sq Linear = 0.979 –5.00 R Sq Linear = 0.981 R Sq Linear = 0.985 R Sq Linear = 0.824

–1.50

2.00

15.00

The plots of HCT results 45.00

F-A F F-B F F-C F F-D F F-E F

0.00

100.00

R Sq Linear = 0.981 R Sq Linear = 0.979 R Sq Linear = 0.972 R Sq Linear = 0.97 R Sq Linear = 0.802

2.00

20.00

5.00

–1.00

3.00

The bias of HGB results A F B F C F D F E F

A F B F C F D F E F

Mean of tubes A-E

0.00

F-A F F-B F F-C F F-D F F-E F

Bias of tubes F to A-E

0.50

A F B F C F D F E F

Bias of tubes F to A-E

A F B F C F D F E F

Bias of tubes F to A-E

Mean of tubes A-E

5.00

The plots of HGB results

The bias of RBC results

The plots of RBC results 6.00

Figure 2. According to the EP9-A2 document, if no linear portion is evident, or if the linear portion is too small, stop the evaluation. Therefore, for a nonlinear relationship in the scatter plot and a more significant bias with the reference tubes of tube E, the testing of the performance of tube E had to be stopped. The reason must have been more serious influencing factors and the lower uniformity of the ‘in-house’ capillary blood collection tubes. Other tubes passed the visual check test and could access the following tests. Check for between-method outliers, the results of four times of the mean absolute difference between brands (4E) of the five brands as the test limit (TLE), and no more than 2.5% of the results were identified as the outlier. An adequate range of X was tested with the correlation coefficient, r. Linear regression for the candidate tubes (A–D) to the reference tube (F). All of the r2 ≥ 0.95, so the X range of the reference tube (F) is adequate. The simple linear regression could be used to estimate the slope and intercept and then could compute average bias through the linear regression procedure. The parameters, such as the estimate of the predicted bias (Bc) at a given Medical Decision Level Xc, the 95% confidence interval for Bc, and

Mean of tubes A-E



According to the requirements of the instrument blank, the background levels were defined as:

Comparison of performances of five capillary blood collection tubes.

The research demonstrated the way to compare performances of five capillary blood collection tubes for capillary blood collections among patients with...
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