Evaluation of a Point-of-Care Testing Analyzer for Measuring Peripheral Blood Leukocytes

Summary

A protocol to measure peripheral blood leukocytes using a POCT card-based leukocyte analyzer is presented here. Same blood samples were tested by two automated hematology analyzers to evaluate the consistency and accuracy of the results. The results showed that the evaluated analyzer had a good correlation with the reference system.

Abstract

White blood cell (WBC) is an important indicator of inflammation in the body, and it can help distinguish between bacterial and viral infections. At present, most primary medical institutions in China have a poor percentage of adoption of blood-testing technology, and a hematology detection system with a high price to performance ratio and easy operation is urgently needed in primary healthcare centers. This paper introduces the principle and operation procedures of a point-of-care testing (POCT) card-based leukocyte analyzer (evaluated system), which was used to detect WBC indexes such as neutrophils, lymphocytes, and intermediate group cells (including eosinophils, basophils, and monocytes) in whole blood. The results from the evaluated system were compared to those from two commercial automatic hematology analyzers (reference system). The correlation and consistency between the evaluated system and the commercial reference systems were analyzed. The results showed that WBC count and number of granulocytes detected by the evaluated and reference systems showed a strong positive correlation (r_s = 0.972 and 0.973, respectively), while the number of lymphocytes showed a relatively low correlation (r_s = 0.851). A Bland-Altman plot showed that the major difference between the values detected by the evaluated system and the reference systems is within 95% limits of agreement (LoA), indicating that the two systems are in good agreement. In conclusion, the evaluated system has an excellent correlation, robust consistency, and a reliable comparison with the results of the widely used automatic hematology analyzers. It is ideal for WBC detection in primary medical institutions where a full-automatic five-category hematology analyzer is unavailable, especially during the COVID-19 normalized prevention and control period.

Introduction

White blood cell (WBC)count or differential is an important indicator to reflect the inflammation of the body, which can distinguish bacterial infection from viral infection. WBC analysis is also helpful to guide the follow-up diagnosis and treatment¹. At present, the five-classification fully automatic hematology analyzer has been widely used in large and medium-sized medical units, because it is automatic, has high efficiency, yields accurate and reliable results, and effectively reduces the work intensity of laboratory technicians. It plays an important role in clinical examination^2,3. However, most primary medical institutions, such as community healthcare centers and private clinics, have a low adoption rate of a hematology analyzer. According to a nationwide multicenter study on clinical laboratory construction in China, the laboratory construction of primary medical institutions is insufficient, as evidenced by the small size of laboratories, the insufficient talents transmission, and the spread of science and technology to the countryside, amongst other factors⁴.

Since December 2019, COVID-19 began to spread all over the world and developed into a global pandemic. In the 'post-epidemic era', a series of national policies have been proposed to implement the normalized prevention and control measures of epidemic situations. The laboratory of primary medical institutions plays an important role in grassroots diagnosis and treatment and disease prevention and control. It is the first line of defense and control in epidemic situations, and it is critical to COVID-19 prevention and control⁵. Some studies have shown that the detection of peripheral blood lymphocytes and neutrophils will contribute to COVID-19 patient screening, diagnosis, and treatment, and that the neutrophil/lymphocyte ratio can also be used as clinical early warning indicators of severe and critical COVID-19^6,7. Moreover, leukocyte detection has the benefit of providing a quick report. Primary medical and health institutions can extensively carry out leukocyte detection to help detect and screen suspected infections in time.

POCT card-based leukocyte analyzer (evaluated system; see Table of Materials) is a three-classification blood cell analyzer based on the gold standard "Coulter principle". The evaluated system provides quantitative analysis results of one WBC histogram and seven blood parameters including WBC count, number of granulocytes (Gran#), percentage of granulocytes (Gran%), number of lymphocytes (Lym#), percentage of lymphocytes (Lym%), number of intermediate cells (Mid#), and percentage of the intermediate cells (Mid%). It adopts the card-based innovative technology and has advantages such as the availability of single-person detection kit, absence of liquid waste, fast detection in 30 s, being free from routine maintenance, and user-friendly operation. Therefore, it is particularly well-suited to primary medical institutions. This study aims to evaluate the clinical detection performance of POCT card-based leukocyte analyzer by comparing against two fully automatic commercial hematology analyzers (reference system 1 and reference system 2; see Table of Materials) from laboratories of two large-scale public hospitals.

Protocol

This study and the use of human blood samples were approved by the Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University (GYYY-2016-73). All participants have given their written consent independently or through their parents (in the case of children).

1. Basic information of the study group

NOTE: Venous blood was collected from patients who visited the First Affiliated Hospital of Guangzhou Medical University (Hospital 1) and the Fifth Affiliated (Zhuhai) Hospital of Zunyi Medical University (Hospital 2). The instrument used for blood routine examination in Hospital 1 is Reference system 1, while Hospital 2 uses Reference system 2.

1. A total of 1066 blood samples were collected from patients who visited Hospital 1 (532) and Hospital 2 (534) and underwent blood routine examinations during January 2021.
   ​NOTE: Patients were randomly selected, came from multiple departments and suffer from various diseases.
2. Exclude patients with incomplete medical records, and those who were not cooperative or refused to give informed consent. Exclude those patients whose blood samples exhibited hemolysis, chyle blood, or cloudiness, or if the blood was inadequate in volume or stored for more than 24 h.

2. Study flow and measurements of interest

NOTE: The evaluated system needs 5 µL of the blood sample for determining WBC and the three classification parameters. After collecting blood, the evaluated system and the reference system were used for blood routine examination.

1. Detect WBC and the three classification parameters of 532 and 534 blood samples using the reference system and the evaluated system, respectively.
   1. Let a highly trained technician randomly renumber the selected blood samples after completing the clinical test with the reference system. Then, hand over the samples to another highly trained technician for detection of the WBC and classification parameters using the evaluated system.
2. Reveal the results of the two systems.
3. Ask a third technician to analyze the five indicators (namely WBC count, Gran#, Gran%, Lym#, Lym%) shared by both evaluated and reference systems only.

3. Procedure for using the evaluated system

NOTE: The evaluated system uses the electrical impedance principle (Coulter principle) to count WBC in the detection element. The testing protocol is divided into six parts: start the analyzer, test preparation, blood collection, reagent mixing, sample analysis, and turn off the analyzer.

1. Start the analyzer
   1. Turn the [O/I] power switch on the back of the analyzer to [I]. Check that the indicator light of the analyzer is on.
   2. Enter the correct username and password in the login dialog box and click on Login. Ensure that the system performs self-check and start-up initialization automatically and then displays the Sample Analysis home page.
2. Test preparation
   NOTE: A complete blood sample test requires four consumables: blood lancet, hemolytic reagent, quantitative pipette with a capillary tube inside, and blood cell detection module (Figure 1).
   1. Click on Next Sample, correctly enter the gender, name, and other clinical information (as required), and select an appropriate reference group. Click on OK to save the information.
      NOTE: Different age groups have their own reference interval, so choosing the right reference group can get a more suitable alarm prompt. Newborn: 1-28 days old; Children: 29 days to 14 years old; Adult Male/Female/General: more than or equal to 15 years old.
   2. Tear the thin film of the blood cell detection module, press the Entry/Exit warehouse button, and place the blood cell detection module into the machine warehouse correctly, with its orifice facing outward.
   3. Puncture the hemolytic reagent sealing film with the tip of a quantitative pipette.
3. Blood collection
   1. Capillary blood collection: Disinfect the left ring finger with a cotton swab dipped in alcohol one way and once. After the alcohol naturally dries out, use a blood lancet to puncture the skin of the left ring finger.
      1. Gently squeeze out the first drop of blood and wipe it with a cotton swab. Squeeze out enough blood to form a full "waterdrop" and collect 5 µL of the blood sample using the capillary tube inside the quantitative pipette.
   2. Venous blood collection: Collect 5 µL of the pre-obtained venous blood sample using the capillary tube inside the quantitative pipette. All tests in this study used venous blood which was collected from each patient (5 mL) using a vacuum vessel containing EDTA-K₂ anticoagulant. Complete all tests within 30 min to 24 h.
4. Reagent mixing
   1. Insert the quantitative pipette into the hemolytic reagent (2.5 mL) and press it tightly to release the blood sample from the capillary tube.
   2. Mix the blood in the capillary tube and hemolytic reagent by turning it upside down 15-20 times at a constant speed, until no obvious red blood remains in the capillary tube. In this study, the blood sample is mixed with hemolytic reagent at a ratio of 1:500.
5. Sample analysis
   1. Open the lid and squeeze the solution into the blood cell detection module.
   2. Press the Entry/Exit warehouse button. After the blood cell detection module enters the warehouse, press the Counting button.
      NOTE: A flashing green indicator light indicates that the analyzer is counting. The blood cell detection module will automatically exit the warehouse after counting, and it should be removed and disposed of properly. Each test takes only 30 s.
   3. On the analyzer interface, click on the OK button twice to confirm that the blood cell detection module has been taken out.
   4. On the analyzer interface, click on the Print button to print the test results.
6. Turn off the analyzer
   1. On the analyzer interface, click on the shutdown button, and select Yes in the dialog box that pops up on the interface. Check that the system starts to execute the shutdown sequence.
   2. Set the [O/I] switch on the back of the mainframe to [O] after the shutdown sequence is completed.

4. Statistical analysis

1. Detect the outliers using the generalized extreme studentized deviate (ESD) method and eliminate these outliers for follow-up statistical analysis according to the requirements of the American Association for Clinical Laboratory Standardization (CLSI) EP9-A3 document⁸.
2. Calculate the descriptive parameters such as means and standard deviations (SDs) for normally distributed continuous data; medians and 25%-75% interquartile ranges for nonnormally distributed data; and frequencies and percentages for categorical data.
3. Use Pearson χ2 test or Fisher's exact test to determine the degree of relationship between categorical variables. Use the Paired-Sample T-test or Mann-Whitney U test to compare numerical data between groups.
4. Show the distribution and linear association of the detected results of the two systems by scatter plots. Apply Spearman's nonparametric correlation test to access the degree of relationship between the quantitative variables. Use Bland-Altman plots and intraclass correlation coefficient (ICC) to verify the agreement between quantitative values detected by the two systems.
5. Analyze the data by statistical software of choice. P-value < 0.05 is considered statistically significant.

Results

Sample data
A total of 1066 patients were enrolled in two research centers, including Hospital 1 (n = 532) and Hospital 2 (n = 534). The patient characteristics are shown in Table 1. The percentage of males is 49.9% and the median age is 52 (32, 66) years. Patients enrolled in the study were comprised of inpatients (51.1%), outpatients (39.0%), and physical examination patients (8.4%). The samples tested were from patients who visited the internal medicine departments (30.6%), surg...

Discussion

With the advancement of modern laboratory medicine, it is now typical to see several detection technologies utilized in the same or different laboratories to identify the same clinical marker. As a result, more emphasis should be placed on the consistency of test results to assist clinics in making accurate interpretations and judgments of test results. According to the investigation, the total value of laboratory equipment in tertiary hospitals and independent laboratories is substantially higher than that in primary ho...

Materials

Name	Company	Catalog Number	Comments
Blood cell detection module	Chuanghuai Medical Technology Co., Ltd.(Shenzhen, China)		consumables for evaluated system
Blood lancet	Chuanghuai Medical Technology Co., Ltd.(Shenzhen, China)		consumables for evaluated system
Hemolytic reagent	Chuanghuai Medical Technology Co., Ltd.(Shenzhen, China)		consumables for evaluated system
IBM SPSS Statistics 25	International Business Machines Corp., Armonk, NY		Software for data analysis
MedCalc 11.4.2.0	2021 MedCalc Software Ltd		Software for data analysis
Microsoft Excel 2019	Microsoft		Software for data analysis
Point-of-care testing (POCT) card-based leukocyte analyzer	Chuanghuai Medical Technology Co., Ltd.(Shenzhen, China)	CX-2000	Evaluated system
Quantitative pipette with capillary tube inside	Chuanghuai Medical Technology Co., Ltd.(Shenzhen, China)		consumables for evaluated system
Siemens fully automatic hematology analyzer and its related reagents and consumables	Siemens Healthcare Diagnostics Inc.	ADVIA 2120i	Reference system 2
UniCel DxH 800 Coulter Cellular Analysis System and its related reagents and consumables	Beckman Coulter, Inc.	DxH 800	Reference system 1