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: tarnok@medizin.uni-leipzig.de

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