ICSH review of internal quality control policy for blood cell counters.

INTRODUCTION: This paper is a report of an ICSH review of policies and practices for internal quality control (IQC) policy for haematology cell counters among regulatory bodies, cell counter manufacturers and diagnostic laboratories. It includes a discussion of the study findings and links to separate ICSH guidance for such policies and practices. The application of internal quality control (IQC) methods is an essential pre-requisite for all clinical laboratory testing including the blood count (Full Blood Count, FBC, or Complete Blood Count, CBC). METHODS: The ICSH has gathered information regarding the current state of practice through review of published guidance from regulatory bodies, a questionnaire to six major cell counter manufacturers (Abbott Diagnostics, Beckman Coulter, Horiba Medical Diagnostic Instruments & Systems, Mindray Medical International, Siemens Healthcare Diagnostics and Sysmex Corporation) and a survey issued to 191 diagnostic laboratories in four countries (China, Republic of Ireland, Spain and the United Kingdom) on their IQC practice and approach to use of commercial IQC materials. RESULTS: This has revealed diversity both in guidance and in practice around the world. There is diversity in guidance from regulatory organizations in regard to IQC methods each recommends, clinical levels to use and frequency to run commercial controls, and finally recommended sources of commercial controls. The diversity in practice among clinical laboratories spans the areas of IQC methods used, derivation of target values and action limits used with control materials, and frequency of running commercial controls materials. CONCLUSIONS: These findings and their implications for IQC Practice are discussed in this paper. They are used to inform a separate guidance document, which proposes a harmonized approach to address the issues faced by diagnostic laboratories.

ICSH guidance for internal quality control policy for blood cell counters.

This paper is a description of the ICSH guidance for internal quality control (IQC) policy for blood cell counters. It follows from and links to a separate ICSH review for such policies and practices. The ICSH has gathered information regarding the current state of practice through review of published guidance from regulatory bodies, a questionnaire to six major cell counter manufacturers and a survey issued to 191 diagnostic laboratories in four countries (China, the Republic of Ireland, Spain, and the United Kingdom) on their IQC practice and approach to the use of commercial IQC materials. This has revealed diversity both in guidance and in practice around the world. There is diversity in guidance from regulatory organizations in regard to IQC methods each recommends, clinical levels to use and frequency to run commercial controls, and finally recommended sources of commercial control materials. The diversity in practice among clinical laboratories spans the areas of IQC methods used, derivation of target values, and action limits used with commercial control materials, and frequency of running commercial controls materials. These findings and their implications for IQC Practice are addressed in this guidance document, which proposes a harmonized approach to address the issues faced by diagnostic laboratories.

Age and sex specific reference intervals of 13 hematological analytes in Chinese children and adolescents aged from 28 days up to 20 years: the PRINCE study.

OBJECTIVES: Pediatric Reference Intervals in China (PRINCE) is a nationwide initiative that aims to establish and validate harmonized reference intervals (RIs) for Chinese children and adolescents, in which 15,150 healthy volunteers aged up to 20 years were recruited from 11 centers to establish RIs and 7,557 children and adolescents were enrolled from 21 centers to validate RIs. METHODS: The complete blood cell counts (CBC) of venous whole blood were measured by hematology analyzers through Sysmex systems in different centers. Age- and sex-specific RIs were calculated according to the guidelines. RESULTS: Unlike adults with certain levels of analyte concentrations, hematological parameters of children changed through growth and development. Red blood cell counts, hemoglobin, and hematocrit increased with age, and revealed higher concentrations in boys than girls after puberty. White blood cell counts and platelet counts showed significant higher levels than adults before 2 years of age, and then gradually decreased without distinct sex differences. In addition, lymphocyte counts decreased with age while neutrophil counts showed an opposite trend. The lower and upper limits of pediatric RIs of CBC were different from those of adults. CONCLUSIONS: The validation of RIs indicated that the PRINCE study provided a version of RIs suitable for most of regions in China. This first harmonized pediatric RIs of CBC across China provided a robust database to understand the dynamic changes of hematologic parameters from birth to adolescence, and will contribute to clinical diagnosis and prognosis evaluation for pediatric patients as well.

Comparability of sample results and commutability of reference materials among different measurement procedures for protein C activity assays.

BACKGROUND AND AIMS: Several types of measurement procedures (MPs) for protein C activity assays are currently available. Clinical sample (CS) results among different MPs should be comparable. The commutability of reference materials (RMs) is an essential requirement to achieve comparability of CS results. MATERIALS AND METHODS: Considering the total error calculated using reliable biological variation (BV) data and external quality assessment (EQA) criteria, we chose the allowable limits of comparability and criterion of commutability. According to Clinical and Laboratory Standardization Institute EP9 and our previous studies, 92 CSs were used to evaluate the comparability among the three MPs (Sysmex CS-5100, IL ACL TOP 700, and STA-R Evolution). The difference in bias method recommended by International Federation of Clinical Chemistry and Laboratory Medicine was used to assess the commutability of six RMs, including World Health Organization (WHO) IS 02/342. RESULTS: The compliance rates of CSs were 94.6-100% with the corresponding calibration mode. WHO IS, HemosIL calibration plasma, and candidate RMs, PC20201 and PC20202, were commutable between each pair of the three MPs. CONCLUSION: It is feasible to set the allowable limits of comparability and the criterion of commutability based on the BV and EQA criteria.

Study of total error specifications of lymphocyte subsets enumeration using China National EQAS data and Biological Variation Data Critical Appraisal Checklist (BIVAC)-compliant publications.

Objectives: It is important to select proper quality specifications for laboratories and external quality assessment (EQA) providers for their quality control and assessment. The aim of this study is to produce new total error (TE) specifications for lymphocyte subset enumeration by analyzing the allowable TE using EQAS data and comparing them with that based on reliable biological variation (BV). Methods: A total of 54,400 results from 1,716 laboratories were collected from China National EQAS for lymphocyte subset enumeration during the period 2017-2019. The EQA data were grouped according to lower limits of reference intervals for establishing concentration-dependent specifications. The TE value that 80% of laboratories can achieve were considered as TE specifications based on state of the art. The BV studies compliant with Biological Variation Data Critical Appraisal Checklist (BIVAC) were used to calculate the three levels of TE specifications. Then these TE specifications were compared for determining the recommended TE specifications. Results: Four parameters whose quality specifications could achieve the optimum criteria were as follows: the percentages of CD3+, CD3+CD4+ (high concentration) and CD3-CD16/56+ cells, and the absolute count of CD3-CD16/56+ cells. Only the TE specifications of CD3-CD19+ cells could achieve the minimum criteria. The TE specifications of remaining parameters should reach the desirable criteria. Conclusions: New TE specifications were established by combining the EQA data and reliable BV data, which could help laboratories to apply proper criteria for continuous improvement of quality control, and EQA providers to use robust acceptance limits for better evaluation of EQAS results.

Comparison among different measurement systems for fibrinogen using fresh samples and frozen samples.

BACKGROUND: Many laboratories in China have several types of coagulation analyzers. Differences in fibrinogen results among different systems may cause inappropriate medical decisions. Our aim was to set the comparability evaluation criteria and evaluate comparability of different fibrinogen measurement systems using fresh and frozen samples. METHODS: Biological variation (BV) publications on fibrinogen were reviewed. Total error based on reliable BV data and external quality assessment (EQA) criteria were combined to set allowable limit. The compliance rate of samples for the limit should achieve at least 80% if the results obtained from different systems were comparable. Fifty-seven samples before and after freeze-thaw were measured by three measurement systems and the percentage of compliant samples was calculated. RESULTS: The allowable limit was 11.3%. The compliance rates of fresh samples were 78.2-84.2%, and the rates of frozen samples were 54.5-93.0%. The comparability results were different using two kinds of samples. CONCLUSIONS: It is feasible to set allowable limits of comparability based on BV and state of the art; and fresh samples are recommended for evaluating systems comparability. The results of comparability are related to samples' concentrations distribution, which should range over the concentration ranges in routine laboratory tests.

Long-term biological variation estimates of 13 hematological parameters in healthy Chinese subjects.

Background The complete blood count (CBC) is a basic test routinely ordered by physicians as a part of initial diagnostic work-up on their patients. To ensure safe clinical application of the CBC, reliable biological variation (BV) data are needed to establish analytical performance specifications. Our aim was to define the BV of CBC parameters using a rigorous protocol that is compliant with the Biological Variation Data Critical Appraisal Checklist (BIVAC) provided by the European Federation of Clinical Chemistry and Laboratory Medicine. Methods Blood samples drawn from 41 healthy Chinese subjects (22 females and 19 males; 23-59 years of age) once monthly for 6 consecutive months were analyzed using an ABX Pentra 80 instrument. The instrument was precisely calibrated. All samples were analyzed in duplicate for 13 CBC parameters. The data were assessed for outliers, normality, and variance homogeneity prior to nested ANOVA. Gender-stratified within-subject (CVI) and between-subject (CVG) BV estimates were calculated. Results The number of remaining data for each subject was 442-484 after removing outliers. No significant differences existed between female/male CVI estimates. Except for leukocytes, neutrophils, and lymphocytes, the mean values of 10 parameters differed significantly between genders, rendering partitioning of CVG data between genders. No significant differences were detected between most BV estimates and recently published estimates representing a Europid population. Conclusions Most BV estimates in BIVAC-compliant studies are similar. The turnover time of blood cells and age distribution of participants should be considered in a CBC BV study. Our study will contribute to global BV estimates and future studies.

Commutability assessment of reference materials for the enumeration of lymphocyte subsets.

Background Flow cytometric enumeration of lymphocyte subsets in peripheral blood can provide important information about immune status. Commutable reference materials (RM) are crucial for maintaining accurate and comparable measurement results over time and space. Commutability assessment of RMs for lymphocyte subsets enumeration has not been reported elsewhere. Methods Lymphocyte subsets were measured in triplicate on 56 patient samples and eight RMs using two measuring systems commonly used in laboratories (FACS Canto II and Cytomics FC500). The first step was to determine the suitability of RMs and comparability of different systems with patient samples. After the requirements of suitability and comparability were met, the second step was to assess commutability following regression approach and difference in bias approach. Results Two RMs were not measurable on FC500 system for CD3-CD16/56+ and CD3-CD19+ percentages. The results of comparability showed no significant difference in the two systems. Eight RMs for CD3+CD4+ cell count, six RMs for CD3+ and CD3+CD8+ percentages, five RMs for CD3-CD16/56+ percentage, and three RMs for CD3-CD19+ percentage were commutable using the two approaches. For CD3+, CD3+CD8+ and CD3-CD19+ percentages, the results of regression approach showed that one RM was non-commutable for each parameter, while the other approach showed that the RM was commutable. Conclusions The suitability of RM and comparability of different measuring systems are prerequisites for assessing commutability. This study indicated that different approaches led to different results. The difference in bias approach is recommended for criteria relating to medical requirements and performance characteristics of measuring systems in use.

Pediatric reference intervals in China (PRINCE): design and rationale for a large, multicenter collaborative cross-sectional study.

There is a lack of accurate pediatric reference intervals (RIs) in China, with most commonly used RIs established without consideration of the effect of age and gender. The Pediatric Reference Intervals in China (PRINCE) project aims to establish and verify pediatric RIs for 31 common laboratory measurands. The project will be a large, multicenter cross-sectional study: 14,490 healthy children and adolescents aged up to 19 years will be surveyed by 10 children's hospitals and one pediatric department of a university hospital. To evaluate the feasibility and efficiency of the study methods, 602 children were surveyed in the pilot phase of the PRINCE study in April 2017: it found that some measurands were distinctly age dependent and that there were differences between values for males and females. The results of the pilot study affirmed the necessity of the PRINCE project for Chinese pediatrics. The pilot also indicated potential difficulties in the full survey, e.g., difficulties in recruiting children aged under 3 years and insufficient collection of blood samples from infants. The operation of the PRINCE project has been modified based on the findings in the pilot study toward improving the validity of the PRINCE project and promoting its openness and transparency.

[Commutability of reference materials for coagulation factor VIII and factor IX activity on three measurement systems].

OBJECTIVE: To evaluate the comparability of measurement results for coagulation factor VIII (FVIII)and factor IX (FIX) activity and the commutability of reference materials on different measurement systems. METHODS: The study was performed according to CLSI guideline EP30 and China health standard WS/T 356-2011. Clinical samples with different levels of FVIII and FIX which covered over the clinical analytical range, five lots of homemade reference materials (F20140601-F20140605) and a coagulation reference material (SSCLOT4) provided by NIBSC were detected for FVIII and FIX activity on three popular measurement systems in China, which including Stago STA-R Evolution, IL ACL TOP700 and Sysmex CA7000 automatic coagulation analyzers using supplementary reagents. The results between measurement systems were analyzed pairwise. To evaluate the comparability, the linear regression and the biases between the results of clinical samples from two measurement systems were calculated. The comparability was evaluated by the regression coefficient and the biases inside the acceptable range. After eliminated outliers from the results, linear regressions were run again and the 95% confidence intervals were calculated. The commutability of the homemade reference materials and NIBSC reference material were evaluated by comparing the results with the limits of the intervals. RESULTS: The ranges of FVIII and FIX level of clinical samples were 0.5%-218.0% and 1.6%-156.5%, which covered the sample levels in routine work and fit the requirements for commutability evaluation. The square of correlation coefficients (R²) of measurement results of clinical samples for FVIII and FIX activity assays were 0.89-0.94 and 0.81-0.93. The proportions of outliers were all less than 10%. The comparability of measurement results of FVIII and FIX in different measurement systems was acceptable.According to the acceptable criteria for bias, the measurement results of 42, 41 and 45 clinical samples for FVIII and 44, 42 and 41 clinical samples for FIX were used in the commutability evaluation for homemade reference materials and NIBSC reference material after deleting the outliers. The five lots homemade reference materials and NIBSC reference material were all within the 95% prediction intervals from the linear regression of clinical samples' results. CONCLUSIONS: Good correlation and comparability are found for FVIII assay and those for FIX assay are acceptable. All homemade reference materials and NIBSC reference material are commutable for the three measurement systems.

The effect of thalassemia on erythrocyte reference intervals in a representative Han Chinese adult population.

BACKGROUND: The data from apparently healthy individuals with thalassemia has been demonstrated to have an effect on the reference intervals for the erythrocyte indices in areas with a high incidence of thalassemia. METHODS: Six clinical centers screened apparently healthy individuals using a questionnaire and a physical examination. Then, the qualified reference individuals were selected by hematological indices and a genotypic thalassemia diagnosis. Statistical comparisons were conducted for the erythrocyte reference intervals in the Chinese population with and without thalassemia. The constituent ratios and the mean (SD) of erythrocyte indices according to the thalassemia genotype were calculated. The relationship between the MCV values and the thalassemia genotype was also estimated. RESULTS: 4,636 reference individuals were included using hematological indices and genotypic thalassemia screening. The results of the erythrocyte reference intervals for individuals in Guangzhou with thalassemia demonstrated that the RBC, MCV, and MCH values significantly differed by gender compared with other regions (p < 0.01). In contrast, for individuals without thalassemia, the results tended to be similar and clinically acceptable. In addition, the results of the erythrocyte indices revealed significant differences among α-thalassemia patients, ß-thalassemia patients, and the control group. CONCLUSIONS: Apparently healthy individuals with thalassemia in the high prevalence zone of thalassemia could not be excluded by the questionnaire, physical examination or laboratory indices (Fe < 6 µmol/L, Hb < 90 g/L). The screening of genotypic thalassemia based on the MCV or MCH values to exclude unqualified individuals is the most effective way to obtain accurate and reliable reference intervals for the erythrocyte indices.

Complete blood count reference intervals for healthy Han Chinese adults.

BACKGROUND: Complete blood count (CBC) reference intervals are important to diagnose diseases, screen blood donors, and assess overall health. However, current reference intervals established by older instruments and technologies and those from American and European populations are not suitable for Chinese samples due to ethnic, dietary, and lifestyle differences. The aim of this multicenter collaborative study was to establish CBC reference intervals for healthy Han Chinese adults. METHODS: A total of 4,642 healthy individuals (2,136 males and 2,506 females) were recruited from six clinical centers in China (Shenyang, Beijing, Shanghai, Guangzhou, Chengdu, and Xi'an). Blood samples collected in K2EDTA anticoagulant tubes were analyzed. Analysis of variance was performed to determine differences in consensus intervals according to the use of data from the combined sample and selected samples. RESULTS: Median and mean platelet counts from the Chengdu center were significantly lower than those from other centers. Red blood cell count (RBC), hemoglobin (HGB), and hematocrit (HCT) values were higher in males than in females at all ages. Other CBC parameters showed no significant instrument-, region-, age-, or sex-dependent difference. Thalassemia carriers were found to affect the lower or upper limit of different RBC profiles. CONCLUSION: We were able to establish consensus intervals for CBC parameters in healthy Han Chinese adults. RBC, HGB, and HCT intervals were established for each sex. The reference interval for platelets for the Chengdu center should be established independently.

[Establishment of hematological malignancy model in ex vivo cell culture].

The aim of this study was to develop an ex vivo cell culture system for establishing the hematological malignancy model. Mouse bone marrow cells were transfected with GFP-expressed retroviral vectors encoding various leukemia/lymphoma-associated fusion proteins (TEL-PDGFR, Rabaptin5-PDGFR, p210BCR-ABL, AML1-ETO, NPM-ALK). After transfection, the cells were cultured in IMDM containing 10% FCS without growth factors, or with one of the following growth factor combinations: (1) murine c-kit ligand (KL) plus human flt3 ligand (FL); (2) IL-3, thrombopoietin, G-CSF, and hyper-IL-6 (3/T/G/H6); (3) KL/FL plus 3/T/G/H6. The flow cytometry was used to detect the ability of combinations of growth factors to complement the oncogene fusion protein to support self-renewal of the transfected cells. The results showed that the transfected cells could be amplified sustainably in the logarithmic growth way. The indicated combination of c-Kit ligand (KL) with flt-3 ligand (FL) supported the self-renewal of the marrow cells transfected with vectors encoding TEL-PDGFR, Rabaptin5-PDGFR, AML1-ETO and NPM-ALK, in addition to KL/FL, the self-renewal of p210 BCR-ABL transfected-marrow cells also required IL-3. The morphology of cells emerged from culture can be the predictor of the corresponding oncogene-associated malignancy. It is concluded that this study establishes a culture system ex vivo which provides a generalized method for studying hematological malignancies, and may facilitate the screening for therapeutic agents.

[Long-term expansion of T cell progenitors by JAK3 gene transduced primitive hematopoietic cells in vitro].

OBJECTIVE: To explore whether activation of the JAK3-signaling pathway can stimulate long-term expansion of the earliest T cell progenitors from transduced primitive hematopoietic cells and evaluate their potential ability of committed differentiation. METHODS: A retrovirus vector (RV) containing JAK3 gene, two binding sites for chemical inducers of dimerization (AP20187), and green fluorescence protein (GFP), MGI-F(2)JAK3, was constructed. The RV vector MGI-F(2)JAK3 was then transduced into murine bone marrow hematopoietic cells. The transduced murine bone hematopoietic marrow cells were divided equally into four groups blank control group (No drug group), AP20187 group (added with AP20187 only), SCF group (added with stem cell factor only), and AP20187 + SCF group. Cytometry was used to detect the GFP marker and observe the survival of cells. The murine bone hematopoietic marrow cells expanded for 50 days were divided into 2 groups: one group was washed to remove the cytokines to observe their survival, and AP20187 + SCF was added into the culture fluid of other group. Cytometry and CELLQuest v3.1 software analysis were used to analyze the phenotype of the cells of AP20187 + SCF after 50 days' expansion. The C-kit(hi) CD44(+)CD25(-)TN cells after 50 days' expansion were further cultured under the condition of SCF + IL7 + IL3 for 5 days. The differentiation rate of Thy1.2 positive cells was observed by cytometry. Five Ly5.2 mice underwent radiation of the dose of 600 cGy, and then injected with the expanded cells into the thymus. Three weeks later the mice were killed and their thymus glands were taken out to prepare suspension of single cells to undergo cytometry to observe the proportions of GFP positive CD3 and CD4 mature T lymphocytes. RESULTS: One week later the cells of the No Drug group all died, the cells of the AP20187 group and SCF group died 2 - 4 weeks later, however, the cells of the AP20187 + SCF continued to grow and expanded up to 10(12)-fold after 50 days' culture. The cells of the AP20187 + SCF group with the cytokines washed died 2 more weeks later, and those with the cytokines washed and added with AP20187 + SCF continued to grow. The phenotype of the expanded proportion was identified as the earliest T cell progenitors expressing C-kit(hi) CD44(+) CD25(-)TN (triple negative). These progenitors could differentiate into Thy1.2 + T cells in the presence of SCF + IL-7 + IL-3 culture condition. 32% - 96% of the mice thymus cell were GFP positive, 5% +/- 0.8% of the thymus cells were GFP + CD(3) double positive, and 11.0% +/- 2.1% of the T lymphocytes were GFP + CD4(+) double positive. CONCLUSION: AP20187 combined with SCF mediating the activation of JAK3 signaling can dramatically expand the earliest T cell progenitors subpopulation, and acquires the capacity to induce the differentiation into T mature cells. This system may help understand the T cell biology and provide a fundamental basement for gene therapy to immunodeficiency disease in the future.