WhatCellsCanDoWhenTheyDie (andHowWeCanDye It)
Attila T�arnok*
MICROPARTICLES are derived from stressed or activated
cells of diverse origin and can be found in various body
fluids. They are indicators of trauma, stress, radiation
damage among others. Due to their small size of around 30–
100nm in diameter (depending on their cellular source) they
are difficult to quantitate by flow cytometry without addi-
tional specific staining. Typically this labeling includes mem-
brane permeant nucleic acid dyes such as those from the
SYTO family (1). Orozco and Lewis (this issue, page 502)
focused their review on different aspects of microparticle iso-
lation and measurement. They report that critical factors for
enrichment are duration and speed of centrifugation and dif-
ferent setups need to be chosen for different types of micro-
particles. Staining with nucleotide specific fluorochromes
helps to distinguish necrotic from apoptotic microparticles
(MPs). Specific staining of surface antigens then aids to dis-
tinguish MPs from different cellular origin.
Malaria (Plasmodium sp.) infection is a very serious dis-
ease in many countries world-wide, but particularly in tropical
areas. Malaria is often a co-infection to HIV and leads to
aggravation of both diseases. Earlier studies aimed to identify
malaria-infected erythrocytes and to distinguish different mat-
uration stages by the use of nuclear dyes and flow cytometry
or imaging (2). Now, Pattanapanyasat and coworkers (this
issue, page 515) report on the additional detection of external
phosphatidylserine with annexin V as a measure for parasite
maturation. As commented upon by Shapiro and Ulrich (this
issue, page 500), flow cytometry is a helpful support for diag-
nosis of the state of malaria infection. As flow cytometry
became a leading technology in HIV and AIDS diagnosis in
resource poor countries, thanks to the support of many wel-
fare organizations such as the WHO, Gates or Clinton founda-
tion, among others, the instrumental tool is at hand to diag-
nose both malaria and AIDS in the same laboratory setting.
Furthermore, stages of malaria maturation and erythrocyte se-
nescence can be monitored.
The Ki67 nuclear protein is a commonly used immuno-
cytochemical marker of proliferation, and used to detect and
quantify proliferating cells. It is induced when quiescent cells
enter the G1–S phase transition and is expressed throughout
the S, G2 and Mitosis phases, as well. Its expression is elevated
in several human tumor tissues and it is a diagnostic marker
that is inversely correlated with survival rates in a variety of
cancers. It was hypothesized that the proximal promoter of
the Ki67 protein could be used to drive reporter gene expres-
sion to distinguish between subpopulations of cells that are
arrested in the cell cycle from those that are actively transition-
ing through it (Zambon; this issue, page 564). Such a reporter
would provide a valuable tool with a variety of applications.
Expression of GFP using this promoter resulted in the cellular
fluorescence in cells that express endogenous Ki67. The high
fluorescence intensity of the cells allowed visual observation
and their flow cytometric detection. This reporter was used to
detect proliferating cells in living complex three-dimensional
cellular aggregates (embryoid bodies), thereby demonstrating
its potential utility for in vivo studies.
Neuronal degeneration responsible for ataxia telangiecta-
sia is ultimately due to the accumulation of DNA damage due
to loss of the DNA checkpoint function of ataxia telangiectasia
mutated (ATM) protein. ATM protein plays a central role in
the DNA damage checkpoint response. Following DNA
damage, it activates signaling cascades able to promote DNA
repair, block cell cycle progression while repair continues, or
induce apoptosis (3). In many cases neuronal degeneration
has been linked to inappropriate cell cycle entry, including in
AT patients. The expression of cyclin D1 can play a critical
role in this process, since forced expression of this protein in
otherwise normal neurons is toxic. It is believed that a variety
Department of Pediatric Cardiology, Heart Centre Leipzig, Universityof Leipzig, Leipzig, Germany
Received 9 April 2010; Accepted 19 April 2010
*Correspondence to: Prof. Attila T�arnok, Department of PediatricCardiology, Heart Centre Leipzig, University Leipzig, Str€umpellstr.39, 04289 Leipzig, Germany.
E-mail: [email protected]
Published online in Wiley InterScience(www.interscience.wiley.com)
DOI: 10.1002/cyto.a.20917
© 2010 International Society for Advancement of Cytometry
Editorial
Cytometry Part A • 77A: 495�496, 2010
of stimuli, including the production of reactive oxygen species,
induce these fully differentiated cells to re-enter the cell cycle,
to express cyclin D1 and other markers of cell cycle progres-
sion, and to ultimately initiate DNA synthesis, potentially
leading to cell death. Digital fluorescent imaging of increased
Cyclin D1 expression, induced by hydrogen-peroxide, and cell
proliferation in differentiated neural cell cultures shows that
ATM functions to maintain low levels of cyclin D1 expression
in differentiated neurons; and may provide important clues in
understanding neural degeneration in general (Hitomi and
Stacey; this issue, page 524).
Among peripheral blood mononuclear cells (PBMC), cy-
totoxic T-cells and natural killer cells (NK) are mostly respon-
sible for cell killing and cytolysis. For the estimation of this
cell activity, a new cytometric test was developed for Guava
flow cytometers, and actually that assay was adapted for usual
flow cytometers (Cao et al.; this issue, page 534). After incuba-
tion of the effector and target cells in mixed cell cultures by
the vitality test (7AAD exclusion), defined dead cell ratio was
compared to the all cell count. The test functions with frozen
and thawed PBMC cell samples and also with specific CD31CD81 cell lines as effectors and different tumor cell lines as
targets.
Standardization of the presentation of scientific informa-
tion is the key to good scientific practice. In the last few years
it has become common practice that Minimal Information
(MI) has to be provided to allow reproduction of specific ex-
perimental setups. The first such requirements were defined
for gene arrays and for the past two years, the consensus MI
requirements for flow cytometry data are available (4). Now,
Blimkie and colleagues (this issue, page 546) publish the first
manuscript that has been prepared in full accordance to the
MIFlowCyt guidelines. This work deals with the identification
of B-lymphocytes and serves as the first example for MIFlow-
Cyt correctly applied. Since January 2010, manuscripts con-
taining FCM data should follow these guidelines and it will be
mandatory for all papers in the near future.
Hematological analyzers for the determination of whole
blood differential blood pictures render some defined charac-
teristic differences between instruments and technologies
applied (5). A major drawback is that only mature polymor-
phonuclear neutrophilic granulocytes can be quantified and
the counting of immature (band) neutrophils still relies on
manual microscopic analysis of blood smears. Roussel and col-
leagues (this issue, page 552) developed an antibody panel and
an analysis protocol that enables for the full blood differential
counting via flow-cytometry. The panel includes blasts of dif-
ferent cell types as well as several lymphocyte sub-sets. Most
importantly the authors provide for the first time, reference
values for children (0–5yrs), grown-ups (18–70 yrs) and aged
(> 70 yrs) people based on over 100 blood samples from
healthy subjects.
ACKNOWLEDGMENT
Dr. Jozsef Bocsi, Heart Center Leipzig, is acknowledged
for his help with this editorial.
LITERATURE CITED
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3. Zhao H, Albino AP, Jorgensen E, Traganos F, Darzynkiewicz Z. DNA damageresponse induced by tobacco smoke in normal human bronchial epithelial and A549pulmonary adenocarcinoma cells assessed by laser scanning cytometry. Cytometry A2009;75A:840–847.
4. Lee JA, Spidlen J, Boyce K, Cai J, Crosbie N, Dalphin M, Furlong J, Gasparetto M,Goldberg M, Goralczyk EM, Hyun B, Jansen K, Kollmann T, Kong M, Leif R,McWeeney S, Moloshok TD, Moore W, Nolan G, Nolan J, Nikolich-Zugich J, ParrishD, Purcell B, Qian Y, Selvaraj B, Smith C, Tchuvatkina O, Wertheimer A, WilkinsonP, Wilson C, Wood J, Zigon R. International Society for Advancement of CytometryData Standards Task Force, Scheuermann RH, Brinkman RR. MIFlowCyt: the mini-mum information about a Flow Cytometry Experiment. Cytometry A 2008;73A:926–930.
5. Kleine TO, Nebe CT, L€ower C, Lehmitz R, Kruse R, Geilenkeuser WJ, Dorn-BeinekeA. Modifications of haematology analyzers to improve cell counting and leukocytedifferentiating in cerebrospinal fluid controls of the Joint German Society for ClinicalChemistry and Laboratory Medicine. Cytometry A 2009;75A:688–691.
EDITORIAL
496 What Cells Can Do When They Die