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bosis and Haemostasis. Thromb Haemost 1994; 71: 520–5.

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4 Baronciani L, Federici AB, Cozzi G, Canciani MT, Mannucci PM.

von Willebrand factor collagen binding assay in von Willebrand dis-

ease type 2A, 2B, and 2M. J Thromb Haemost 2006; 4: 2088–90.

5 Baronciani L, Federici AB, Beretta M, Cozzi G, Canciani MT,

Mannucci PM. Expression studies on a novel type 2B variant of the

von Willebrand factor gene (R1308L) characterized by defective col-

lagen binding. J Thromb Haemost 2005; 3: 2689–94.

Standardization of circulating endothelial cell enumeration bythe use of human umbilical vein endothelial cells

P . K . Y . GOON, T . WATSON, E . SHAN TS I LA , C . J . BOOS and G. Y . H . L I PHaemostasis, Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham, UK

To cite this article: Goon PKY, Watson T, Shantsila E, Boos CJ, Lip GYH. Standardization of circulating endothelial cell enumeration by the use of

human umbilical vein endothelial cells. J Thromb Haemost 2007; 5: 870–2.

Since Jaffe et al. [1] successfully cultured viable human

umbilical vein endothelial cells (HUVECs), HUVECs have

been essential to modern vascular research. They are consid-

ered the archetypal example of mature endothelial cells (ECs),

with a distinct and demonstrable endothelial phenotype. With

the recent advent of mature circulating endothelial cell (CEC)

analysis, the need for a reliable �positive control� cell populationfor CECs is mandatory, with HUVECs being an obvious

choice.

In recent published work, HUVEC use has been central to

the validation of CEC techniques such as immunomagnetic

bead (IB)methodology and flow cytometry (FC) [2,3]. The lack

of consensus on CEC methodology aptly illustrates the innate

difficulty facing researchers. Based on our own CEC validation

work [4], we have observed that CEC/HUVEC capture is

crucially influenced by a number of cellular characteristics of

the isolation technique used (FC vs. IB). To investigate this

further, we studied three distinct patient groups: breast cancer,

acute myocardial infarction, and following traumatic vene-

puncture. Cultured HUVECs were spiked into known quan-

tities of phosphate buffer solution and venous blood, and

analyzed using IB and FC.

Based on a well-established and validated technique [3,4], IB

capture of HUVECs was effected by incubating the sample

with CD146-conjugated IBs. CD146+ cells were then magnet-

ically separated and incubated with fluorescein isothiocyanate

(FITC)-labeled Ulex europaeus lectin. The samples were

washed and viewed under fluorescence microscopy. CECs

were defined as cells 10–50 lm in diameter, staining for

U. europaeus lectin, and bound by ‡ 4 CD146 beads [3,4]. In

the phosphate buffer solution samples, recovery of lectin-

stained HUVECs was in excess of 80%, and when spiked into

blood samples, HUVECs were easily identifiable as being

rounder in shape, significantly larger, and readily sequestrated

by large numbers of beads, compared with CECs found in

unspiked blood samples (Fig. 1).

Using the same samples, the analysis was repeated using FC.

As previously described [4], the whole blood sample was lyzed

(10 min or less), and blocked (using serum and specific Fc

receptor immunoglobulin), before final staining with conju-

gated antibodies (FITC–CD45, phycoerythryn [PE]-CD146,

PE Cy5–CD34). Crucially, red cell lysis (FACS Lysing

Solution, Becton Dickinson, Oxford, UK) before staining did

not significantly alter the relevant (CD45, CD34, CD146)

antigenic sites [confirmed during validation, using both IB and

FC methods, and with non-fixative lysing solution, e.g. High-

Yield Lyse (Caltag Medsystems, Buckingham, UK); data not

shown], whereas with 2% paraformaldehyde fixation, there

were significant differences (e.g. reduced CD146 antigen–

antibody binding). Using sequential gating with the appropri-

ate forward scatter (FSC) and side scatter (SSC) profiles

(representing size and granularity, respectively) to exclude

irrelevant events, including dead cells and anucleated fragments

(supplementary Fig. S1), CECs were defined as CD45)/146+/

34+events, a definition in keeping with previous reports [2,5–8].

Ideally, the progenitor marker CD133 would be needed to

exclude with certainty cells representing the endothelial

progenitor cell (EPC) population. However, others have shown

Correspondence: Gregory Y. H. Lip, Haemostasis, Thrombosis and

Vascular Biology Unit, University Department of Medicine, City

Hospital, Birmingham, B18 7QH, UK.

Tel.: +44 121 5075080; fax: +121 554 4083; e-mail:

g.y.h.lip@bham.ac.uk

Received 26 October 2006, accepted 5 January 2007

870 Letters to the Editor

� 2007 International Society on Thrombosis and Haemostasis

that the vast majority of CD133+ EPCs (including CD133+/

146+ EPCs) are invariably CD45dim or CD45+ cells [6],

whereas others have reported that CD146+ EPCs represent

only 4% of the total CD146+ CEC population [9]. CECs

enumerated by our method were exclusively CD45), thereby

excluding the majority of EPCs.

Using FC, the capture rate was low for HUVECs (5–10%).

This was a result of significant cellular differences between

HUVECs and peripheral blood CECs. Firstly, the majority of

HUVECs were significantly larger than lymphocytes, mono-

cytes, granulocytes and blood CECs, as evident from the high

FSC (supplementary Fig. S1G). With microscopy, a charac-

teristic HUVEC was seen to be approximately 1.5–2.0-fold

larger than CECs (Fig. 1). Secondly, HUVECs had greater

granularity than peripheral blood mononuclear cells (PBMCs),

as seen by the high SSC (supplementary Fig. S1). Thirdly,

unstained HUVECs showed a high degree of autofluorescence

(supplementary Fig. S1), as evidenced by positive fluorescence

in all three-color channels that had been standardized using

PBMCs. As a direct consequence, most HUVECs were gated

out into the �dump� channel (i.e. CD45+ cells), being wrongly

perceived by the cytometer to be �positive� for the CD45

marker. Inevitably, this corresponds to erroneously �low�HUVEC capture. Conversely, whenHUVEC autofluorescence

was adjusted for, only similar-sized CECs were counted (i.e.

very large CECs), excluding the majority of CECs, which were

comparable in size to lymphocytes and monocytes [confirmed

with microscopy and IB, using traumatic and atraumatic

venepuncture blood specimens (Fig. 1; supplementary

Fig. S1)], and which would then appear understained for

CD146 and CD34. Similarly, some CD45+ mononuclear cells

would erroneously appear as �understained� for CD45, thereby

contaminating G2 and contributing wrongly to the final CEC

analysis. Compared to HUVECs, CECs are more heterogene-

ous in morphology (Fig. 1), with the majority being also

smaller in size, and thus more comparable to the general

lymphocyte/monocyte population.

Overall, these findings suggest that use of HUVECs as a

positive-control population for CEC analysis using FC is

inadvisable, as HUVECs may only represent rarer, larger

CECs in peripheral blood. Depending on how the flow

cytometer is standardized, the assay could potentially lead to

spuriously low or high CEC counts when running blood

samples, making any conclusions unreliable. With the IB

technique, this is much less of an issue, as the capture of ECs

depends on CD146 antigenicity, and method-specific problems

of autofluorescence and gating are not encountered. This is

reflected by the good recovery rate of spiked HUVECs in the

tested samples.

Several obvious limitations, however, need to be mentioned.

Firstly, we only included three patient groups for our definition

of the �typical CEC�, and this may not necessarily equate to

CECs in other diseases (e.g. vasculitis, sepsis, diabetes mellitus,

and transplantation). Secondly, we did not evaluate other

available examples of cultured ECs (e.g. human umbilical

artery ECs, human dermal microvascular ECs, human brain

microvascular ECs), which could conceivably be more repre-

sentative of the general CEC phenotype. Thirdly, HUVEC

culture (including culture mediums, passaging, and trypsiniza-

tion) might crucially alter cell characteristics. A fourth consid-

eration regards the comparison of FC parameters – as the

numbers of CECs found in blood tend to be much smaller than

the numbers of spiked HUVECs, the discrepancy in numbers

makes a direct comparison of (for example) SSC and autoflu-

orescence limited. Finally, we recognize the importance of

using the CD133 progenitor marker in FC assays in order to

confidently exclude all EPCs from the CEC population. We

stress that the observations and conclusions are based on our

particular FC strategy and, as such,may not be generalizable to

other FC protocols currently in use.

CECs are increasingly recognized as sensitive markers of

endothelial damage/dysfunction. Consequently, it is essential

that CEC assays are rigorously validated, with emphasis on the

use of positive controls. Our observations suggest several

potential limitations of using HUVEC validation for CEC

work. This is particularly applicable to FC (vs. IB), and is

largely explained by morphologic and phenotypic differences.

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

Fig. 1. Circulating endothelial cells (CECs) captured by immunomagnetic

bead technique. (A, B, C) Representative examples of typical mature

CECs captured by the immunomagnetic bead (IB) method and viewed

with fluorescence microscopy, representing three different patient groups

[traumatic venepuncture sample in a healthy individual, a breast cancer

patient and an acute myocardial infarction (AMI) patient respectively],

demonstrating Ulex lectin uptake (green–yellow fluorescence); Note that

in (C), the CEC is viewed using both white light and fluorescing light. (D)

This represents a typical human umbilical vein endothelial cell captured by

the IB method. Note that the cell appears rounder, is much larger, and has

a higher concentration of CD146 IBs rosetting it, differing significantly

from CECs [which, in the two disease states (B, C), typically have a more

attenuated, irregular appearance] (individual beads are approximately

4.5 lm in diameter; all cells viewed with the same magnification).

Letters to the Editor 871

� 2007 International Society on Thrombosis and Haemostasis

Supplementary Material

The following supplementary material can be found at http://

www.blackwell-synergy.com/loi/jth:

Fig. S1. Flow cytometric analysis of peripheral blood for

circulating endothelial cell (CEC) capture. (A, B, C). Typical

forward scatter (FSC)/side scatter (SSC) profile of blood

leukocytes [in this case, a patient with acute myocardial

infarction (AMI)], demonstrating sequential gating strategy

for mature CEC enumeration. G2 represents a gate to

exclude all non-relevant CD45+ cells, and cells of high SSC,

R1 represents CD45)/146+/34+ CECs, and R2 represents

CD34+ progenitor cells. (D, E, F) Typical FSC/SSC plots

from three different groups of subjects, demonstrating FSC/

SSC characteristics of CECs (in bold for clarity). Note that

the majority of CECs are comparable to normal peripheral

blood mononuclear cells (PBMCs). (G) A typical scatter-plot

of an unstained blood sample (from an AMI patient) spiked

with cultured HUVECs (highlighted in bold for clarity),

demonstrating their considerably different FSC/SSC charac-

teristics compared to PBMCs (A). (H) In the same unstained

sample, unstained HUVECs (boxed area) also possess a

greater degree of autofluorescence compared with other

blood leukocytes (fluorescence in FL1); compare this with a

similarly unstained sample without HUVECs (I, J). (K) A

representative plot of a pure unstained HUVEC population,

further demonstrating autofluorescence in FL2 and FL3

channels, and (L) when stained specifically for CD146 and

CD34.

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Influence of the Thr325Ile polymorphism onprocarboxypeptidase U (thrombin-activable fibrinolysisinhibitor) activity-based assays

J . L . WILLEMSE ,* V . MATUS ,� E . HEY LEN ,* D . MEZZANO� and D . F . HEN DR IKS**Laboratory of Medical Biochemistry, University of Antwerp, Antwerp, Belgium; and �Laboratory of Thrombosis and Haemostasis, P. Catholic

University of Chile, Santiago, Chile

To cite this article: Willemse JL, Matus V, Heylen E, Mezzano D, Hendriks DF. Influence of the Thr325Ile polymorphism on procarboxypeptidase U

(thrombin-activable fibrinolysis inhibitor) activity-based assays. J Thromb Haemost 2007; 5: 872–5.

Carboxypeptidase U is a potent attenuator of the fibrinolytic

rate present in the circulation as its zymogen procarboxypept-

idase U [proCPU, thrombin-activable fibrinolysis inhibitor

(TAFI)].

During the last few years, a large number of studies have

investigated the role of proCPU as a possible risk factor for

thrombotic disease. The outcome, however, is confusing. High

proCPU levels have been reported to be a risk factor for venous

Correspondence: Dirk F. Hendriks, Laboratory of Medical

Biochemistry, University of Antwerp, Antwerp, Belgium.

Tel.: +32 3820 27 27; fax: +32 3 820 27 45; e-mail:

dirk.hendriks@ua.ac.be

Received 30 November 2006, accepted 3 January 2007

872 Letters to the Editor

� 2007 International Society on Thrombosis and Haemostasis