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Validation of human cell counting methods
Daniella Paiva Bonfim1, Bruno Cosme1, Jailton Carreteiro Damasceno2, José
1 1*
Mauro Granjeiro and Fernanda Leve
1Division of Metrology Applied to Life Sciences (Dimav), National Institute of
Metrology, Quality and Technology (INMETRO), Brazil; Materials Metrology
Division (Dimat), National Institute of Metrology, Quality and Technology
(INMETRO), Brazil
*Corresponding author: fleve@inmetro.gov.br
Abstract. The quantification of biological processes has gained importance in metrology, as its
application in diagnosis and personalized therapy has been increasing the demand of the
biotechnology sector. Considering that the cell is the microscopic and functional unit of living
beings, cell count is critical for the reproducibility of the in vitro study. This work aims to
determine the measurement uncertainty and the commutability of the methods of manual cell
counting in a Neubauer chamber and automatic cell counting, both image-based and widely used.
For this, we used different amounts (1x105-6x105) of HCT-116 cells, a well-established line
whose authenticity was confirmed by STR analysis. Initially, we checked if 20 up/down
movements, performed with the micropipette, are enough to maintain the homogeneous
distribution of cells in the four most outer quadrants of the Neubauer chamber. Then, different
5 5
amounts of cells (1x10 -6x10 ) were prepared using a balance or calibrated micropipettes,
stained with trypan blue (1:1) and counted in an automatic counter or a Neubauer chamber, in
quadruplicate. Data analysis was performed using the GraphPad Prism 6 program, and the
significance analysis was performed using the one-way ANOVA test, Bonferroni post-test, and
simple linear regression. The measurement uncertainty was estimated using the calculations
recommended in the ISO GUM. The results show that the homogenization provided an equitable
distribution (p>0.05) of cells in the four most outer quadrants of the Neubauer chamber,
regardless of the cell concentration used. Manual and automatic counting showed the same linear
correlation between the expected cell number and the observed cell number, showing the
compatibility of the methods. As a function of the cell concentration analyzed, the measurement
uncertainty ranged between 6-18% for automatic counting and between 11-24% for manual
counting. However, we observed that the commutativity of the manual and automatic counting
methods depends on the dilution preparation method and that only the gravimetric dilution
presented commutativity (p>0.05). The results show that performing 20 up/down movements are
sufficient to homogenize the samples for counting in quadruplicate in the Neubauer chamber and
the automatic counter, that counting in 4 and 9 quadrants are equivalent, that the manual and
automatic methods are switchable when the dilutions are prepared by gravimetry and have
similar measurement uncertainty, in the range between 6 and 24%. We intend to use these results
to validate manual counting methodology and automatic counting of human cells, allowing a
possible comparison with other automated methodologies, including flow cytometry and real-
time electrical impedance analysis.
Keywords: Validation; Cell count; Neubauer chamber; Automatic count.
1. Introduction
Cells are the basic units of living organisms and can have different complexities. Cell analysis includes
measurements of intra- and extracellular components and the cells themselves, which may be related to
size, number of cells in a given population, biological function, and quantification of cellular
components (DNA, protein, lipids, etc.). In this context, cell quantification is a fundamental measure
widely used in clinical diagnosis and research, for example, in cytotoxicity assays (SARKAR et al.,
CMB – 11° Congresso Brasileiro de Metrologia
2017). However, with the development and growth of the global biotechnology industry, cell
quantification has become of fundamental importance also in the manufacture of products, including
biological products derived from cells and also the cells themselves, manipulated for therapeutic use,
called Therapy Products Advanced (SILVA JUNIOR et al., 2018).
Thus, together with the growing demand from the biotechnology sector, the need arose to harmonize
and validate classical cell counting methods and carry out more accurate and faster cell measurements
since errors in the counting process can generate unreliable results non-reproducible. Several factors can
interfere in the counting process, including the dynamic properties of cells, such as size and morphology
diversity; the inadequate maintenance of cell lines; the presence of aggregates; the use of non-
homogeneous samples; the lack of operator/analyst training; errors in data acquisition analysis; the use
of inadequate or uncalibrated measuring instruments; the lack of harmonized protocols; in addition to
the difficulty of performing many repetitions in measurements due to the high cost (SIMON et al., 2016).
Together, these factors represent potential sources of variability and can influence the reproducibility of
the data obtained (HIRSCH & SCHILDKNECHT, 2019). Also, there is a difficulty in developing
reference materials based on eukaryotic cells, mainly due to the inherent variability of biological
materials (FARUQUI et al., 2020).
In this sense, some initiatives have been taken to establish reproducible and traceable cell counting
methods. In 2014, a working group on cell analysis called the Cell Analysis Working Group (CAWG)
was created, which is part of the Consultative Committee for Amount of Substance (CCQM), an
advisory committee for chemical and biological metrology, of the Bureau International des Poides et
Measurements (BIPM). The CAWG’s mission is to identify, establish and sustain the global
comparability of cell measurement capabilities, through metrological reference measurement systems,
with traceability to the International Measurement System (SI) or other agreed units (BIPM, 2020).
Another relevant initiative in this sector was the publication of the ISO 20391 standard, a general
guide to cell counting methods. This standard is divided into two parts, where the first published in 2018
(ISO 20391-1) defines the terms and provides general guidance on different methods of counting
adhered or suspended cells; and the second part (ISO 20391-2), published in 2019, guides the procedures
for statistical analysis of data and assessment of the quality of a cell count measurement.
Among the classical methods of cell counting, manual counting through the Neubauer chamber,
followed by obtaining images by optical microscopy, and automatic counting, in a counter, also based
on images, stand out. Furthermore, it is of great importance to estimate cell viability to designate the
number of living cells in a given cell population, a relevant parameter in various applications ranging
from testing the toxicity analysis of a compound and evaluating the success of cryopreservation
techniques, to the infusion of modified cells in a patient (STODDART, 2011). Among the various
protocols developed to determine cell viability, trypan blue exclusion is the primary, simple and
accessible method (CHEN et al., 2017). Briefly, trypan blue staining is a permeability assay based on
cell membrane integrity: while living cells have intact cell membranes, which exclude dyes like trypan
blue, dead cells do not. Thus, dead cells are visualized with blue stained cytoplasm (STROBER, 2001).
It is essential to mention that, although the manual counting method is more routinely used due to its
low cost, it is subject to variation among users and depends on training and time (CADENA-HERRERA
et al., 2015). On the other hand, the automatic method is more costly. However, it quantifies cells in less
time and with less effort, and the literature has pointed to greater yield, precision, reproducibility, and
providing additional parameters, such as percentage of cell viability and diameter (LOUIS & SIEGEL,
2011). Still, it is worth noting that few users perform the correct validation of both methodologies and
are unaware of factors, as mentioned above, that directly influence cell count.
According to ABNT NBR ISO/IEC 17025:2017, validation is confirmation by examination and
provision of objective evidence that the specified requirements for a particular intended use are met. In
other words, validating a method demonstrates that, under the conditions in which it is practiced, the
characteristics necessary to obtain valid and consistent results are obtained.
Considering the role of the National Institute of Metrology in providing metrological traceability to
other laboratories in the country, we propose to evaluate the effectiveness of cell suspension
homogenization for manual and automatic counting, the primary sources of uncertainty for the different
CMB – 11° Congresso Brasileiro de Metrologia
methods of counting human cells, and to calculate the uncertainty associated to the cell counting using
an automatic counter and a Neubauer chamber.
2. Materials and methods
2.1 Cell culture
A well-established human colon carcinoma cell line of epithelial origin, HCT-116 (ATCC, #CCL-247),
was used. Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with
10% fetal bovine serum (FBS) at 37°C in 5% CO2. Cell authenticity tests were performed, including
STR profile analysis, to verify identity, and purity tests, including microbiological and mycoplasma
control tests, by bioluminescence.
2.2 Homogeneity test
5 5 5
Different suspensions containing 1x10 , 3x10 and 6x10 cells were homogenized with 20 up/down
movements with a micropipette and counted in the Neubauer chamber in quadruplicate. Counting made
in each of the four outer quadrants was compared as well, as a comparison was made between the count
in all nine quadrants of the chamber with the count using only the four most outer quadrants.
2.3 Cell quantification assay using trypan blue staining
Different cell suspension dilutions were prepared by gravimetry (calibrated balance) and volumetry
(calibrated micropipettes). Dilutions from 1x105 to 6x105 cells were prepared in quadruplicate,
established considering the ideal number of cells for counting in a Neubauer chamber, according to
LONZA (2009). To ensure homogeneity between the samples, 20 up/down movements were performed
with the micropipette at each preparation stage of the dilutions.
Samples, including live cells and dead cells, were quantified from a counting solution containing the
cell suspension and trypan blue dye, in a 1:1 ratio, by manual counting in a Neubauer and counting
®
chamber automatic, using the Countess (Invitrogen) system.
2.4 Analysis of cell suspension density and obtaining volumes
5 5 5
Dilutions containing 1x10 , 3x10 and 6x10 HCT-116 cells, in triplicate, were prepared from known
volumes, and each replicate was weighed, and density was calculated using the equation:
= , [Eq. 1]
being “mass” equal to the average values obtained after weighing each replicate of the different amounts
of cells; and “volume” equal to the value used to prepare the dilutions and corrected by the pipette
calibration certificate.
After, an average was made between the density values of each dilution to obtain a single density
value for further analyses.
To obtain the volume of HCT-116 cell samples, prepared using the calibrated balance, the equation
was used:
= , [Eq. 2]
5
being “mass” equal to the value obtained after weighing each replicate of the different dilutions (1x10
5
to 6x10 ); and “density” equal to the value obtained after analyzing the cell suspension density.
2.5 Automatic cell count
5 5
Dilutions containing 1x10 to 6x10 HCT-116 cells diluted in trypan blue (1:1) were counted in a
®
Countess Automated Cell Counter (Invitrogen), in quadruplicate. The chosen protocol for counting
was determined according to the characteristics of the cell line, so that cells with a size between 5 and
CMB – 11° Congresso Brasileiro de Metrologia
20 µm were considered and the program provided data on the total cells/mL, including live cells/mL
and dead cells /mL, and the percentage of viability.
To calculate the number of cells observed, the equation was used:
( ∗ )
= , [Eq. 3]
1000
being “initially observed value” equal to the number of cells/mL, provided by the program; “pipetted
volume” equal to the pipetted volume of each sample (in the case of the experiment using a calibrated
balance for the preparation of dilutions, this volume was calculated from the density of the cell
suspension, as per item 2.3); and the value of “1000” corresponding to 1 mL (1000 µL).
2.6 Manual cell count
Quantification of different dilutions (1x105 to 6x105) of HCT-116 cells was performed using the four
most outer quadrants of the calibrated Neubauer chamber.
Considering the information from the calibration certificate, the height, width and depth
measurements of the Neubauer chamber were corrected, and the new measurements were used to
calculate the volume of each quadrant. Thus, the volume of each quadrant was calculated using the
equation:
= ℎℎ ∗ ℎ ∗ ℎ. [Eq. 4]
To calculate the total number of cells, we perform four steps:
➢ Step 1: calculation of the number of cells in each quadrant, using the equation:
= ( ∗ ), [Eq. 5]
in which “number of cells” means the number of cells counted in the quadrant; the “dilution factor”
equal to 2 (since the counting solution contained cell suspension and trypan blue in the proportion of
1:1); and “volume”, previously calculated from the area of the Neubauer chamber and which varies
according to the number of the quadrant;
⮚ Step 2: average of total cells from all quadrants, using the equation:
(1+2+3+4)
= , [Eq. 6]
“Q” = quadrant; 4
⮚ Step 3: obtaining the number of cells/mL, by multiplying the “averages obtained by the
calculations in step 2” by “1000 (1 mL or 1000 µL)”:
( )
/ = ∗1000; [Eq. 7]
⮚ Step 4: and finally, we calculate the observed number of cells using the equation:
( ∗ )
= , [Eq. 8]
1000
“initial observed value” is the number of cells per mL, calculated in step 3; “pipetted volume” equal to
the pipetted volume of each sample to prepare dilutions (in the case of the gravimetric method, the
volume was calculated from the density, as per item 2.3); and the value of “1000”, which corresponds
to 1000 µL.
CMB – 11° Congresso Brasileiro de Metrologia
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