D e v e l o p m e n t , H i s t o r y, a n d

F u t u re o f A u t o m a t e d C e l l
Counters
a, b
Ralph Green, MD, PhD, FRCPath *, Sebastian Wachsmann-Hogiu, PhD

KEYWORDS
 Automated cell counter  Hematology  Blood  Hemoglobin
 Electrical impedance counting  Flow cytometry  Point of Care  Light scattering

KEY POINTS
 The invention of the microscope allowed the differentiation and counting of blood cells.
 Automated blood cell counters have greatly improved the speed and accuracy of cellular
blood analysis, using optical light scattering or changes in electrical current induced by
blood cells flowing through an electrically charged small opening.
 Novel methods under development include the addition of new parameters to the com-
plete blood count and white cell differential and the development of smaller, portable
instrumentation and of devices that allow in-vivo analysis of blood cells.

“.for the blood is the life”

—Deuteronomy 12:23
INTRODUCTION

Even in antiquity, blood was recognized as a singular bodily fluid that was the essence
of life, possessing mysterious properties that provided sustenance for human survival.
The unraveling of those mysterious properties only became possible once blood could
first be characterized according to the appearance and number of its particulate com-
ponents. Those critical steps comprised, in the first instance, the development of
microscopy, enabling the visualization of component blood cells, and subsequently
advances made possible through the techniques to measure physical properties of
the formed elements of the blood as well as the electronic means of capturing this
information.

a
Department of Pathology and Laboratory Medicine, UC Davis Medical Diagnostics, University
of California, Davis Health System, 4400 V Street, PATH Building, Sacramento, CA 95817, USA;
b
Department of Pathology and Laboratory Medicine, Center for Biophotonics, University of
California, 2700 Stockton Boulevard, Sacramento, CA 95817, USA
* Corresponding author.
E-mail address: ralph.green@ucdmc.ucdavis.edu

Clin Lab Med - (2014) -–-

http://dx.doi.org/10.1016/j.cll.2014.11.003 labmed.theclinics.com
0272-2712/14/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.
2 Green & Wachsmann-Hogiu

Abbreviations

CBC Complete blood count

FOV Field of view
RBC Red blood cell
SRS Stimulated Raman scattering
WBC White blood cell

The development of the optical microscope made it possible to visualize individual

human and plant cells, as well as bacteria. Even the simplest microscope developed
by van Leeuwenhoek more than 300 years ago allowed the observation that blood is
composed of small red globules, and their size was eventually determined. Progres-
sive developments in optical microscopy followed during the next 2 centuries, such
as the addition of an eyepiece to form a compound microscope and improved op-
tics, including objective lenses comprising several lenses to correct for distortions.
The application of dyes by Paul Ehrlich in the late 1870s allowed, for the first time,
differentiation between different white cell types.1,2 By then, it was evident that
the number of blood cells changes in many diseases and it therefore became clear
that it was important to more accurately quantify cell number by doing a blood
count. Laborious manual measurements were introduced in which measured vol-
umes of blood were placed on a calibrated slide chamber and cells were counted
one by one, the total number being calculated from the known volume and geometry
of the chamber. Although still in practice today because of its simplicity, manual
counting of cells is prone to large errors and is both time consuming and labor inten-
sive. Automated methods for counting blood cells became a necessity, and engi-
neers began to work together with hematologists to find solutions to this problem.
Over the ensuing decades, flow-based cytometers using light, impedance measure-
ments, or both, were developed. These devices provided a large number of param-
eters related to enumerating and identifying blood elements, including erythrocytes
(red blood cells [RBCs]), leukocytes (white blood cells [WBCs]), and thrombocytes
(platelets), and differentiating the various leukocyte subtypes. In addition, with the
recognition that qualitative differences in the appearance of red cells relating to their
size, shape, and degree of hemoglobinization connoted certain types of anemia, the
ability to simultaneously and independently measure hemoglobin concentration and
hematocrit (or packed cell volume), together with the red cell count, provided
Maxwell Wintrobe3–5 with the tools to promulgate a set of red cell indices that
included the mean corpuscular volume, mean corpuscular hemoglobin, and mean
corpuscular hemoglobin concentration. This development allowed the morphologic
classification of anemias into subtypes according to whether microcytic, normo-
cytic, or macrocytic; and hypochromic, normochromic, or even hyperchromic ac-
cording to degree of hemoglobinization. Later, it also became possible to quantify
the size distribution of red cell populations, expressed as the red blood cell distribu-
tion width, thus enabling a further distinction into homogeneous and heterogeneous
categories within each morphologic subtype.6

UTILITY OF A BLOOD COUNT

Of all the tissues in the body, the blood in circulation is the easiest and least invasive
to sample. As such, a blood count represents a biopsy obtained through a simple
venipuncture. A complete blood count (CBC) is therefore one of the most commonly
used clinical laboratory tests. It provides a rapid and cost-effective assessment of
various modalities of a patient’s state of health as well as important clues to the
 Automated Cell Counters 3

possible presence of disease. A CBC provides information about the cellular compo-
nents of the blood: RBCs, WBCs, and platelets. CBC data include not only informa-
tion about the numbers of the 3 basic cell types but also provide information about
the size, shape, and degree of hemoglobinization of the RBCs as well as the
morphologically identifiable types of WBCs; the so-called WBC differential. The
CBC provides clinicians with important information relevant to a patient’s state of
health and possible type of underlying disease; these data, along with clinical and
other laboratory data, are often critical in constructing a differential diagnosis for a
patient as well as for monitoring the progression of a disease and its responsiveness
to treatment. At its most rudimentary level, the CBC gives information about low
RBC levels (anemia), high RBC levels (erythrocytosis), low WBC levels (leukopenia),
high WBC levels (leukocytosis), low platelet levels (thrombocytopenia), and high
platelet levels (thrombocytosis). In addition, data on low and high absolute numbers
of the different leukocyte types provide a wealth of information regarding the likely
type and cause of underlying disease, whether infectious, inflammatory, neoplastic,
or other. The blood cell count can therefore be an important first indicator of disease
for many illnesses and is therefore a pivotal starting point in forming a clinical diag-
nosis, screening for changes in patient health, and for monitoring of disease
progression or treatment. For example, in leukocyte disorders, the number of leuko-
cytes reported and leukocyte differential can assist in assessing infections as well as
in evaluating for hematologic malignancies (leukemias). Given this wealth of informa-
tion that comes from what is essentially a single laboratory test, the move toward
automation of the CBC was inevitable once the technical capabilities related to mea-
surement, recording, reporting, and rapidity of throughput became available. More-
over, continued improvements in the various components of these integral steps of
automated blood counting have led to progressive advances in both the quality and
quantity of information that is obtained from a CBC. Modern blood counting instru-
ments are able to closely simulate the qualitative information obtained through con-
ventional microscopy with quantitative measurements of the numbers, dimensions,
and properties of the cellular components of the blood, providing a composite multi-
parameter assessment of the state of the tissue that is the circulating blood.

HISTORY AND CURRENT METHODS

Counting of blood cells was one of the first quantitative methods for blood testing, and
for some time has been the most widely used of tests in clinical settings. Initial mea-
surements were based on careful wet sample preparation on a slide chamber, visual-
ization, and manual counting with the aid of an optical microscope. Later techniques
involved flow methods coupled with either impedance measurements or light scat-
tering/fluorescence techniques to automatically enumerate cells one by one. There
has been a more recent resurgence of microscopy-based techniques as a result of
groundbreaking advances in imaging and image processing tools for automated cell
counting (Table 1).

Manual Counting Methods

The first blood count is credited to Karl Vierordt7,8 at the University of Tübingen, who
published his research on the topic in a series of articles in 1852. His method involved
drawing blood into a capillary tube and spreading a known volume onto a slide, fol-
lowed by microscopic analysis. During the following years, incremental improvements
of this method followed, including better specimen preparation and refinements in the
counting chamber (including the use of elliptically shaped capillary tubes by Malassez9
4 Green & Wachsmann-Hogiu

Table 1
Brief history of early milestones in blood counting methods leading to automation

Discoverer, Year Methodology

Vierordt,7,8 1852 Microscope
Oliver,11 1896 Light scattering and absorption measured by eye
Marcandier et al,12 1928 Light scattering and absorption measured with a photodetector
Coulter,17 1953 Impedance measurement
Fulwyler,13 1965 Impedance measurement and electrostatic cell sorting
Dittrich & Göehde,14 1968 Fluorescence-based flow cytometry
Julius et al,15 1972 Fluorescence-activated cell sorting
George & Groner,16 1973 Light scattering in flow cytometry

Data from Refs.7,8,11–17

as well as the addition of etched perpendicular grids in the chamber for easier enumer-
ation of cells). These improvements resulted in counts becoming more accurate but
still tedious and slow to perform. A description of subsequent modifications was
reviewed and published by Gray.10 Because of its simplicity, this method is still
used in low-resource laboratories around the world.

Automated Counting Methods

Toward the end of the nineteenth century, the prototypes of automated blood coun-
ters were first developed, with rapid advances made throughout the twentieth century.
These instruments performed measurements using either the light scattered and
absorbed by blood cells, or changes in the electrical current induced by blood cells
flowing through a small, electrically charged opening.

Methods based on optical measurements

In 1896 a new method for blood cell counting was proposed by George Oliver,11 based
on the measurement (by eye) of light loss caused by scattering and absorption in a test
tube filled with diluted blood. This method could be considered the forerunner of even-
tually the automated blood count, and it provided an RBC count without the need for
manual counting of individual cells. However, the inability to accurately quantify the
light loss as well as problems related to variations in cell size, shape, or hemoglobin
content prevented this method from becoming widely used. Nevertheless, during
the 1920s, novel developments in photodetectors (made possible by the discovery
of the photoelectric effect and the invention of the photodiode) revived interest in using
light scattering and absorption for blood cell counting. Marcandier and colleagues12
showed in 1928 that, by measuring the light transmitted through a solution of diluted
blood, a blood count could be derived after properly calibrating the photometer. How-
ever, as in the previously described method by Oliver,11 the count was not accurate if
there were variations in cell size, shape, or hemoglobin content. To address these is-
sues, a flow device that isolated cells in small liquid droplets was first developed by
Fulwyler13 in 1965 for the purpose of separating cells based on their size. In 1968, Dit-
trich and Göhde14 coupled a laser beam to this flow device and successfully demon-
strated fluorescence-based cytometry. Because of the high speed of the flow, in
which thousands of cells pass through the laser beam per second, high-throughput
cytometry became a reality. This development may be seen as a watershed in the tran-
sition from manual to automated blood counting. In the early 1970s, Julius and
 Automated Cell Counters 5

colleagues15 demonstrated fluorescence-based cell sorting, which he called

fluorescence-activated cell sorting. The use of fluorescence labels enabled the iden-
tification, in addition to the separation, of many types of cells and therefore added a
new dimension to the blood count. Up to 11 different fluorophores could be used
simultaneously, and, through the use of deconvolution algorithms that allow separa-
tion of overlapping spectra, the number could be even larger. Light scattering was
also implemented in flow cytometers by George and Groner16 in 1973, allowing the
discrimination of different types of WBCs based on their size/scattering properties.
In addition, using light scattering measurements at 2 different angles, they were
able to obtain measurements of red cell size (low-angle light scatter) and hemoglobin
content (higher-angle scatter) after the cells were isovolumetrically sphered.16
Methods based on electrical measurements
During the late 1940s, Wallace Coulter was working on a method to assess particu-
lates in paint. Spurred by his experiences in the Navy and witnessing the effects of
the atomic bombs dropped at the end of the Second World War, he looked for
ways to apply his technique to blood cell counting. In a discovery that came to be
known as the Coulter effect, he started to develop a simplified blood cell analysis
tool that could be used for rapid screening of blood from large numbers of people.
The Coulter effect is based on the phenomena that cells are poor electrical conductors
compared with a saline solution and individual particles passing thorough an orifice at
the same time as an electric current produce a change (decrease) in the current
caused by the particle-induced increase in electrical impedance. Furthermore, the
change in impedance (and therefore in the measured current) is proportional to the
volume of the particle, which is the foundation for size-based counting and separation.
This simple but elegant concept laid the groundwork for subsequent development of
modern automated blood cell counters. After Coulter’s17 first patent on this topic in
1953, many subsequent developments followed, including the popular flow cell
format, in which the cells are directed through a flow channel or chamber rather
than being passed through an aperture. A significant modification to the system
was made in 1965 by Fulwyler,13 who used a Coulter counter to measure cell volume
and then partitioned the cells into droplets of the medium. Because the charge of the
droplets is related to the cell volume, an electrostatic field applied to the droplets can
deflect them into a collection vessel. Cells can thus be sorted and later reused. Like
the fluorescence-based approach pioneered by Julius and colleagues,15 this
approach also played an important part in laying the foundation for automated cell-
sorting techniques.
The addition of RNA-binding dyes such as acridine orange enabled automated
discrimination and enumeration of reticulocytes as a component of the blood count
and substantially improved the accuracy, speed, and precision of reticulocyte count-
ing. Another milestone was the addition of precise size discriminators in electrical and
light scattering instruments, which revolutionized platelet counting by enabling auto-
mation of this previously laborious measurement.

NOVEL METHODS UNDER DEVELOPMENT

Despite the significant improvements in the technologies described earlier, there are
still several limitations to current automated blood counters, such as complex and
time-consuming sample preparation, limited number of parameters that can be
detected, the need for a blood draw, and lack of capability for continuous monitoring.
With rapid technological advancements in optics, electronics, and microfluidics,
further improvements are now being directed toward (1) the development of novel
6 Green & Wachsmann-Hogiu

contrast agents and methods that will allow higher throughput and multiplexing capa-
bilities, as well as adding new parameters to the measurements such as the analysis of
subpopulations of blood cells or counting of smaller particles in blood; (2) the devel-
opment of smaller, portable, automated blood counters; and (3) the development of
in-vivo blood counting techniques.

New Contrast Agents and Methods

Current flow-based blood counters can detect up to approximately 13 parameters
simultaneously (11 color and 2 scattered light [side and forward scatter])18 in a high-
speed laminar flow (up to 20 m/s), leading to the discrimination of phenotypic subpop-
ulations of cells and a throughput of up to 100,000 cells per second. However, the
heterogeneity and complexity of the immune system as reflected in lymphocyte sub-
sets that have clinical diagnostic relevance may require even higher speed and multi-
plexing capabilities.
This improvement can be achieved by adding new contrast agents, such as quan-
tum dots or rare earth metals that have narrower emission bands, or through the use of
intrinsic markers that are easier to multiplex, such as those based on Raman scat-
tering.19 The main disadvantage of the Raman-based technique is the significantly
reduced speed of measurement caused by the low Raman intensity compared with
fluorescence or light scattering. To address this issue, molecular markers that can
be used in combination with surface enhanced Raman spectroscopy and are capable
of multiparameter blood analysis have been developed.20 Other possibilities include
the use of techniques that can provide additional contrast, such as those based on
photothermal and photoacoustic measurements. However, increasing the multidi-
mensionality of the information requires novel computational tools to help analyze
these complex datasets and reveal new information, such as the identification of
diverse populations of cells. Shekhar and colleagues21 give an example of a tool
that enables automated classification of nearly 40 different proteins to recover the
large diversity of CD81 T cells. In addition, with the recent interest in cellular exo-
somes, the ability of flow cytometers to measure particles significantly smaller than
1 mm is currently being explored.
Because thousands of blood counts are performed each day in conventional large
clinical laboratories, the development and improvement of machine-aided flagging al-
gorithms is also a necessity, because they help reduce the number of false-positives
and false-negatives by triggering morphologic review by an expert.

The Need for Portability

Current automated blood counting systems are large devices, the use of which is
generally restricted to centralized laboratories. However, the need for delivery of
improved but less expensive health care has posed newer challenges to scientists
and engineers for the development of automated blood counters that are portable
and can be used, for example, in doctor’s offices, in disaster areas, in low-resource
or remote areas, for monitoring astronauts in space, or even for home testing. The
many developments over the past 20 years in microfluidics, optics, electronics, com-
puters, and integration and miniaturization of devices based on such principles are
bringing that goal ever closer. Sample preparation has been simplified by the use of
microfluidic devices that require much smaller volumes of blood to perform a blood
count. Although miniaturized optical components allow the development of smaller
devices, faster electronic detectors help increase the throughput of such measure-
ments. In addition, the analysis of recorded data is now aided by computers that
 Automated Cell Counters 7

can help improve the accuracy of the counts by using better statistical analysis or
pattern recognition algorithms.

Miniaturization of flow-based cytometers

Several research groups recently reported the development of miniature flow devices.
Such examples include on-chip impedance spectroscopy for a WBC differential count
that has a 95% correlation against a commercial flow-based optical/Coulter
counter,22 or fluorescence-based measurements within a sheathless microflow device
for a 4-part leukocyte differential count.23

The resurgence of image-based blood counters

Before the development of flow-based techniques, blood counting was performed
by hand, with technicians examining cells under a microscope using a hemocytom-
eter. The procedure was extremely laborious and it often required hours to generate
a blood count. Despite this, image-based counting has some advantages, such as
easier sample preparation and handling, and the ability to provide morphologic in-
formation that can be useful for the classification of immature and abnormal cells. In
addition, imaging systems can be significantly smaller, more portable, and more
robust than flow systems. For that reason, there has been a revival of image-
based counters, particularly spurred by the development of high-quality, inexpen-
sive camera sensors and of complex, robust image analysis algorithms. Ceelie
and colleagues24 recently examined the performance of 2 state-of-the-art auto-
mated image-based instruments and concluded that their accuracy in providing
morphologic classification of RBCs and WBCs depends on the type of pathologic
changes in the blood sample. One main drawback of image-based blood counters
is the limited field of view (FOV), which depends on the magnification and is usually
in the range of hundreds of micrometers. For accurate and fast measurements of
WBCs and rare cells, the examination of larger areas is needed. Although scanning
and tiling together multiple images is possible, a significant advance has been
made by the development of a lens-free, in-line holographic imaging technique
that uses partially coherent light to record the shadow of cells on an imaging
sensor. Cell information can be extracted through the recovery of the phase infor-
mation of each cell for very large FOVs, limited only by the size of the imaging chip.
RBC, WBC (with granulocyte, monocyte, and lymphocyte differential), and
hemoglobin concentrations could be measured in this way.25 More recently, an
automated image-based blood counting method that includes RBCs, WBCs with
3-part differential, and platelets has been reported. This method uses a simple
sample preparation (that includes WBC and platelet staining) and automated image
analysis for performing a blood count on very small volumes of blood and can be
adapted to portable devices.26

In-vivo Automated Blood Count

Despite the advantages of automated ex-vivo blood counting, the ability to perform an
automated in-vivo blood count is appealing because of the potential for noninvasive
and near-continuous measurements. Other advantages include that no sample prep-
aration is needed and sampling of larger volumes of blood is possible such that the
detection of rare abnormal cells might become feasible.
The blood cells naturally flow through blood vessels, and therefore an adaptation of
flow devices first comes to mind. However, there are many challenges that prevent a
simple adaptation of the ex-vivo technology to in-vivo measurements. First, the flow is
significantly slower (w0.002 m/s in microvessels and w0.2 m/s in large vessels,
8 Green & Wachsmann-Hogiu

compared with w10 m/s in flow cytometers) in humans, which would make the mea-
surements significantly slower. Second, poor in-vivo optical conditions require the
development of novel contrast mechanisms. Third, the speed of cells is constantly
changing, which makes quantitative measurements difficult. An excellent review of
in-vivo blood measurements has been published by Tuchin and colleagues27 and it
describes recent efforts to apply optical (absorption, fluorescence, elastic scattering,
inelastic scattering [Raman]) as well as ultrasonographic, photothermal, and photoa-
coustic methods for this purpose. Other potential contrast methods are those that
allow deeper penetration of light in the tissue. Examples include optical coherence to-
mography, nonlinear microscopies such as second harmonic generation, multiphoton
fluorescence excitation microscopy, coherent anti-Stokes Raman scattering, and
stimulated Raman scattering (SRS). As an example, SRS has been used for in-vivo
label-free visualization of RBCs flowing through a capillary.28

FUTURE

The difficulty in predicting what the future holds for blood cell counting is best exem-
plified by considering how difficult it would have been to predict the current situation
50 years ago. However, there will be the dichotomy of building better blood counters
for improved diagnostics versus smaller, more portable, or even wearable and
implantable in-vivo devices that can provide certain parameters that may be useful
as early indicators for changes in disease status and more elaborate tests.
Better, more accurate, and more comprehensive instruments will include more pa-
rameters in the blood count, such as the detection of rare-event blood cells, microve-
sicles, and exosomes, or subpopulations of cells with particular phenotypes that may
be of significant clinical value. Measurement of dynamic functional changes in circu-
lating cells is another possibility. Being able to provide multiplexed chemical informa-
tion will also be important additions to future blood counting devices. Higher
throughputs will also likely be the focus of future instruments, and could be achieved
by developing new multimodal methodologies based on existing technologies or
through the application of newer contrast technologies.
Smaller, portable devices that could do a blood count at the point of care and de-
vices designed for in-vivo measurements will benefit from the use of high-resolution
cameras and high-speed transmittance digital microscopy. In this way, cells can be
examined under native conditions within the circulation through the use of photoa-
coustic detection, opening up new possibilities for assessment of cell-cell interac-
tions, detection of circulating tumor cells, in-vivo cell deformability in diseases like
sickle cell anemia and other intrinsic cell membrane disorders, and visualization of
platelets during thrombus formation.27
Ten or more years into the future, automated blood counting instruments are likely
to be significantly different from what they are today. Whether any of the directions
predicted in this article will come into being is unknown at this time. The opportunity
to contemplate this question is one that scientists are free to engage in. It is from such
exercises that past developments stemmed and are likely to arise in the future.

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