Home Medicine Analytical performance of an automated volumetric flow cytometer for quantitation of T, B and natural killer lymphocytes
Article
Licensed
Unlicensed Requires Authentication

Analytical performance of an automated volumetric flow cytometer for quantitation of T, B and natural killer lymphocytes

  • Simon Degandt , Bart Peeters , Stijn Jughmans , Nancy Boeckx and Xavier Bossuyt EMAIL logo
Published/Copyright: February 21, 2018
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 56 Issue 8

Abstract

Background:

Quantitation of lymphocyte subsets (B cells, T cells, CD4 and CD8 T cells and NK cells) classically relies on quantitation of lymphocytes and immunophenotyping by flow cytometry. AQUIOS CL (Beckman Coulter) is a fully automated system that performs an onboard volumetric cell count, automatically processes the sample (staining, lysing and fixation) and analyzes the results. We compared AQUIOS CL to a dual-platform analysis and evaluated analytical performance.

Methods:

We evaluated precision, sample stability, inter-sample carryover, linearity and interpanel consistency. AQUIOS CL was compared to a dual-platform method (Sysmex XE-5000 and BD FACSCanto-II). A total of 113 patient samples were included: 45 from posttransplant patients, 44 from children and 24 random routine samples. The degree of automation was scored through the need of manual revisions triggered by AQUIOS CL run notifications and run flags.

Results:

Intrarun and interrun variability was <9.1% with dedicated control material and <32.1% with patient samples. Relative values of lymphocyte subsets could be determined up to 48 h after venipuncture when the sample was kept at room temperature. There was no carryover and good linearity. Interpanel consistency was 3.3% for relative values and 9.4% for absolute values. Method comparison showed good analytical correlation between AQUIOS CL and a dual-platform method. Thirty-five percent of the samples triggered a run notification. In 74% of these samples, the results could be accepted without intervention, so in 26% of all samples, an unnecessary notification was generated.

Conclusions:

AQUIOS CL allows for reliable fully automated immunophenotyping of lymphocyte subset quantitation. Gating algorithms could be further improved.

Keywords: AQUIOS; flow cytometry; lymphocyte subsets
Corresponding author: Prof. Dr. Xavier Bossuyt, PhD, MD, Department of Laboratory Medicine, UZ Leuven, Herestraat 49, 3000 Leuven, Belgium
aSimon Degandt and Bart Peeters share first authorship.bNancy Boeckx and Xavier Bossuyt share senior authorship.

Acknowledgments

This study was supported by Beckman Coulter through donations of laboratory equipment and supplies. This private company had no role in study design and the collection of data. They did help in interpreting gating results with run notifications. Beckman Coulter had no role in the preparation of the manuscript or the decision to submit it for publication. We thank Professor David Barnett and Liam Whitby (UK National External Quality Assessment Scheme [UK NEQAS], Sheffield) for providing information and data regarding the UK NEQAS immune monitoring scheme.

  1. Author contributions: BP, NB, XB: study design. SD: drafted the manuscript. BP, SJ: patient recruitment. SD, BP, SJ: data acquisition. SD, BP: statistical analysis. NB,XB checked and improved content and English grammar and style of the manuscript. NB, XB commented on and edited the manuscript, which was read and approved by all authors. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None declared.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References

1. Picard C, Al-Herz W, Bousfiha A, Casanova JL, Chatila T, Conley ME, et al. Primary immunodeficiency diseases: an update on the classification from the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015. J Clin Immunol 2015;35:696–726.10.1007/s10875-015-0201-1Search in Google Scholar

2. Fleisher TA, Madkaikar M, Rosenzweig SD. Application of flow cytometry in the evaluation of primary immunodeficiencies. Indian J Pediatr 2016;83:444–9.10.1007/s12098-015-2011-0Search in Google Scholar

3. Maguire O, Tario JD, Jr, Shanahan TC, Wallace PK, Minderman H. Flow cytometry and solid organ transplantation: a perfect match. Immunol Invest 2014;43:756–74.10.3109/08820139.2014.910022Search in Google Scholar

4. WHO. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection: recommendations for a public health approach, second edition. 2016. Available at: http://www.who.int/hiv/pub/arv/arv-2016/en/. Accessed: 18 Jul 2017.Search in Google Scholar

5. Brando B, Barnett D, Janossy G, Mandy F, Autran B, Rothe G, et al. Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis. Cytometry 2000;42:327–46.10.1002/1097-0320(20001215)42:6<327::AID-CYTO1000>3.0.CO;2-FSearch in Google Scholar

6. Barnett D, Granger V, Whitby L, Storie I, Reilly JT. Absolute CD4+ T-lymphocyte and CD34+ stem cell counts by single-platform flow cytometry: the way forward. Br J Haematol 1999;106:1059–62.10.1046/j.1365-2141.1999.01632.xSearch in Google Scholar

7. Gossez M, Malcus C, Demaret J, Frater J, Poitevin-Later F, Monneret G. Evaluation of a novel automated volumetric flow cytometer for absolute CD4+ T lymphocyte quantitation. Cytometry B Clin Cytom 2017;92:456–64.10.1002/cyto.b.21360Search in Google Scholar

8. Grossi V, Infantino M, Meacci F, Bellio E, Bellio V, Ciotta G, et al. Comparison of methods and TAT assessment: volumetric AQUIOS CL and bead-based FACS CANTO II cytometers. Cytometry B Clin Cytom 2017. doi: 10.1002/cyto.b.21513 [Epub ahead of print].10.1002/cyto.b.21513Search in Google Scholar

9. CLSI. User verification of precision and estimation of bias; approved guideline – third edition. CLSI document EP15-A3. Carey, RN: Clinical and Laboratory Standards Institute; 2014.Search in Google Scholar

10. Centers for Disease Control and Prevention (CDC). Guidelines for performing single-platform absolute CD4+ T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. Morb Mortal Wkly Rep MMWR 2003;52:1–13.Search in Google Scholar

11. Comans-Bitter WM, de Groot R, van den Beemd R, Neijens HJ, Hop WC, Groeneveld K, et al. Immunophenotyping of blood lymphocytes in childhood. Reference values for lymphocyte subpopulations. J Pediatr 1997;130:388–93.10.1016/S0022-3476(97)70200-2Search in Google Scholar

12. Ekong T, Hill AM, Gompels M, Brown A, Pinching AJ. The effect of the temperature and duration of sample storage on the measurement of lymphocyte subpopulations from HIV-1-positive and control subjects. J Immunol Methods 1992;151:217–25.10.1016/0022-1759(92)90120-ISearch in Google Scholar

13. Jalla S, Sazawal S, Deb S, Black RE, Das SN, Sarkar A, et al. Enumeration of lymphocyte subsets using flow cytometry: effect of storage before and after staining in a developing country setting. Indian J Clin Biochem 2004;19:95–9.10.1007/BF02894264Search in Google Scholar

14. Zini G. International council for standardization in haematology (ICSH). Stability of complete blood count parameters with storage: toward defined specifications for different diagnostic applications. Int J Lab Hematol 2014;36:111–3.10.1111/ijlh.12181Search in Google Scholar PubMed

15. Nascimbeni M, Shin EC, Chiriboga L, Kleiner DE, Rehermann B. Peripheral CD4(+)CD8(+) T cells are differentiated effector memory cells with antiviral functions. Blood 2004;104:478–86.10.1182/blood-2003-12-4395Search in Google Scholar PubMed

Supplementary Material:

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2017-0638).

Received: 2017-07-20
Accepted: 2018-01-19
Published Online: 2018-02-21
Published in Print: 2018-07-26

©2018 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Editorials
  3. Procalcitonin for diagnosing and monitoring bacterial infections: for or against?
  4. Is it time to abandon the Nobel Prize?
  5. In Memoriam
  6. Norbert Tietz, 13th November 1926–23rd May 2018
  7. Reviews
  8. Procalcitonin guidance in patients with lower respiratory tract infections: a systematic review and meta-analysis
  9. Telomere biology and age-related diseases
  10. Opinion Paper
  11. Procalcitonin-guided antibiotic therapy: an expert consensus
  12. Genetics and Molecular Diagnostics
  13. mRNA expression profile in peripheral blood mononuclear cells based on ADRB1 Ser49Gly and Arg389Gly polymorphisms in essential hypertension — a case-control pilot investigation in South Indian population
  14. General Clinical Chemistry and Laboratory Medicine
  15. External quality assessment schemes for glucose measurements in Germany: factors for successful participation, analytical performance and medical impact
  16. Long-term stability of glucose: glycolysis inhibitor vs. gel barrier tubes
  17. Observational studies on macroprolactin in a routine clinical laboratory
  18. Anti-streptavidin IgG antibody interference in anti-cyclic citrullinated peptide (CCP) IgG antibody assays is a rare but important cause of false-positive anti-CCP results
  19. Point-of-care creatinine testing for kidney function measurement prior to contrast-enhanced diagnostic imaging: evaluation of the performance of three systems for clinical utility
  20. Hematology and Coagulation
  21. Analytical performance of an automated volumetric flow cytometer for quantitation of T, B and natural killer lymphocytes
  22. Lupus anticoagulant testing using two parallel methods detects additional cases and predicts persistent positivity
  23. Reference Values and Biological Variations
  24. Within-subject biological variation of activated partial thromboplastin time, prothrombin time, fibrinogen, factor VIII and von Willebrand factor in pregnant women
  25. Within-subject and between-subject biological variation estimates of 21 hematological parameters in 30 healthy subjects
  26. Immunoglobulin G (IgG) and IgG subclass reference intervals in children, using Optilite® reagents
  27. Cancer Diagnostics
  28. Serum carbohydrate sulfotransferase 7 in lung cancer and non-malignant pulmonary inflammations
  29. Cardiovascular Diseases
  30. Clinical performance of a new point-of-care cardiac troponin I test
  31. Diabetes
  32. Testing for HbA1c, in addition to the oral glucose tolerance test, in screening for abnormal glucose regulation helps to reveal patients with early β-cell function impairment
  33. Effects of common hemoglobin variants on HbA1c measurements in China: results for α- and β-globin variants measured by six methods
  34. Infectious Diseases
  35. Serum procalcitonin concentration within 2 days postoperatively accurately predicts outcome after liver resection
  36. Predictive value of serum gelsolin and Gc globulin in sepsis – a pilot study
  37. Cerebrospinal fluid free light chains as diagnostic biomarker in neuroborreliosis
  38. Letter to the Editor
  39. Glucose and total protein: unacceptable interference on Jaffe creatinine assays in patients
  40. Interference of glucose and total protein on Jaffe-based creatinine methods: mind the covolume
  41. Interference of glucose and total protein on Jaffe based creatinine methods: mind the covolume – reply
  42. Measuring procalcitonin to overcome heterophilic-antibody-induced spurious hypercalcitoninemia
  43. Paraprotein interference in the diagnosis of hepatitis C infection
  44. Timeo apis mellifera and dona ferens: bee sting-induced Kounis syndrome
  45. Bacterial contamination of reusable venipuncture tourniquets in tertiary-care hospital
  46. Detection of EDTA-induced pseudo-leukopenia using automated hematology analyzer with VCS technology
  47. Early adjustment of antimicrobial therapy after PCR/electrospray ionization mass spectrometry-based pathogen detection in critically ill patients with suspected sepsis
  48. Thromboelastometry reveals similar hemostatic properties of purified fibrinogen and a mixture of purified cryoprecipitate protein components
  49. Bicarbonate interference on cobas 6000 c501 chloride ion-selective electrodes
https://doi.org/10.1515/cclm-2017-0638
Keywords for this article
AQUIOS; flow cytometry; lymphocyte subsets

Articles in the same Issue

  1. Frontmatter
  2. Editorials
  3. Procalcitonin for diagnosing and monitoring bacterial infections: for or against?
  4. Is it time to abandon the Nobel Prize?
  5. In Memoriam
  6. Norbert Tietz, 13th November 1926–23rd May 2018
  7. Reviews
  8. Procalcitonin guidance in patients with lower respiratory tract infections: a systematic review and meta-analysis
  9. Telomere biology and age-related diseases
  10. Opinion Paper
  11. Procalcitonin-guided antibiotic therapy: an expert consensus
  12. Genetics and Molecular Diagnostics
  13. mRNA expression profile in peripheral blood mononuclear cells based on ADRB1 Ser49Gly and Arg389Gly polymorphisms in essential hypertension — a case-control pilot investigation in South Indian population
  14. General Clinical Chemistry and Laboratory Medicine
  15. External quality assessment schemes for glucose measurements in Germany: factors for successful participation, analytical performance and medical impact
  16. Long-term stability of glucose: glycolysis inhibitor vs. gel barrier tubes
  17. Observational studies on macroprolactin in a routine clinical laboratory
  18. Anti-streptavidin IgG antibody interference in anti-cyclic citrullinated peptide (CCP) IgG antibody assays is a rare but important cause of false-positive anti-CCP results
  19. Point-of-care creatinine testing for kidney function measurement prior to contrast-enhanced diagnostic imaging: evaluation of the performance of three systems for clinical utility
  20. Hematology and Coagulation
  21. Analytical performance of an automated volumetric flow cytometer for quantitation of T, B and natural killer lymphocytes
  22. Lupus anticoagulant testing using two parallel methods detects additional cases and predicts persistent positivity
  23. Reference Values and Biological Variations
  24. Within-subject biological variation of activated partial thromboplastin time, prothrombin time, fibrinogen, factor VIII and von Willebrand factor in pregnant women
  25. Within-subject and between-subject biological variation estimates of 21 hematological parameters in 30 healthy subjects
  26. Immunoglobulin G (IgG) and IgG subclass reference intervals in children, using Optilite® reagents
  27. Cancer Diagnostics
  28. Serum carbohydrate sulfotransferase 7 in lung cancer and non-malignant pulmonary inflammations
  29. Cardiovascular Diseases
  30. Clinical performance of a new point-of-care cardiac troponin I test
  31. Diabetes
  32. Testing for HbA1c, in addition to the oral glucose tolerance test, in screening for abnormal glucose regulation helps to reveal patients with early β-cell function impairment
  33. Effects of common hemoglobin variants on HbA1c measurements in China: results for α- and β-globin variants measured by six methods
  34. Infectious Diseases
  35. Serum procalcitonin concentration within 2 days postoperatively accurately predicts outcome after liver resection
  36. Predictive value of serum gelsolin and Gc globulin in sepsis – a pilot study
  37. Cerebrospinal fluid free light chains as diagnostic biomarker in neuroborreliosis
  38. Letter to the Editor
  39. Glucose and total protein: unacceptable interference on Jaffe creatinine assays in patients
  40. Interference of glucose and total protein on Jaffe-based creatinine methods: mind the covolume
  41. Interference of glucose and total protein on Jaffe based creatinine methods: mind the covolume – reply
  42. Measuring procalcitonin to overcome heterophilic-antibody-induced spurious hypercalcitoninemia
  43. Paraprotein interference in the diagnosis of hepatitis C infection
  44. Timeo apis mellifera and dona ferens: bee sting-induced Kounis syndrome
  45. Bacterial contamination of reusable venipuncture tourniquets in tertiary-care hospital
  46. Detection of EDTA-induced pseudo-leukopenia using automated hematology analyzer with VCS technology
  47. Early adjustment of antimicrobial therapy after PCR/electrospray ionization mass spectrometry-based pathogen detection in critically ill patients with suspected sepsis
  48. Thromboelastometry reveals similar hemostatic properties of purified fibrinogen and a mixture of purified cryoprecipitate protein components
  49. Bicarbonate interference on cobas 6000 c501 chloride ion-selective electrodes
Sign up now to receive a 20% welcome discount
Institutional Access
How does access work?
Have an idea on how to improve our website?
Please write us.
© 2026 De Gruyter Brill
Downloaded on 24.1.2026 from https://www.degruyterbrill.com/document/doi/10.1515/cclm-2017-0638/html
Scroll to top button