[Cytometry] about CD14-negative mature monocytes

Sieghart Sopper sieghart.sopper at i-med.ac.at
Wed Feb 26 12:02:23 EST 2014


One point, I would like to add to the discussion is that there exists a subset of CD56+ monocytes. Thus, using CD56 together with other lineage markers just to gate out all unwanted cells will also remove some monocytes. If used properly in a two dimensional plot with CD14, then CD56 can definitely help (together with MHCII) to differentiate between NK-cells and non classical monocytes. 

In the end, it all comes to multicolor analyses. Nowadays, one cant properly define monocytes with just two markers.

Sieghart

Am 26.02.2014 um 10:39 schrieb mario picozza <mariopicozza at hotmail.com>:

> Ok, let me
> clarify and, I hope, add matter of debate.
> 
> 
> 
> Nuno is right, his combination of Abs is sufficient to identify
> monocyte subsets. 
> 
> 
> 
> Also, I strongly suggest the reading of the consensus paper from the IUIS
> committee (Ziegler-Heitbrock L., et al Nomenclature of monocytes and dendritic
> cells in blood. Blood. 2010 Oct 21;116(16):e74-80) on the cytometric definition
> of the subsets. The commitee states that non classical monocytes are CD14+
> CD16++, where + and ++ denote a ten and a hundred fold expression above the
> isotype respectively. Although this definition is not technically perfect, I
> will discuss here what it roughly means, that there is a metric based on
> isotype control (FMO looks the same) to assign monocytes to one or another
> subset. For most individuals, and whatever clone you use, the degree of CD14 in
> non classical monocytes partly overlap with that of negative cells. This is the
> reason why one subset of myeloid dendritic cells (negative for lineage markers
> including CD14-19-3 and 56) which expresses CD16  was early recognized
> (Mac Donald, Blood, 2002) and why there is a debate about the nature (DC or
> monocytes?) of so called slanDC. 
> 
> 
> 
> I think the solution straightforward, and, instead of making use of isotype controls,
> solves the non classical subset separating it from the background (as recommended by Hulspas
> et al., cytometry part b, 2009). Indeed, CD11c is usually used  to look at
> myeloid dendritic cells. If used in conjunction to HLA-DR it can pre-select
> myeloid mononuclear cells from both neutrophils and CD16+ lymphocytes. After
> HLA-DR, it is a 2nd dimension to define all monocytes and myeloid DC together.
> Now, gated on DR+ CD11c+, lets the CD16 vs CD14 plot speaks itself. 
> 
> 
> 
> As it can be intuited by the literature, there is a large amount of FCS data
> bytes coarsely spread all over the world with a panel like "lineage (CD3 14 19
> and 56), DR, CD11c and CD16", originally designed to select CD16 positive and
> negative mDC (there are also a number of vendors selling so called lineage2).
> As can be easily argued, this material can be re-analyzed by first gating on
> CD11c+ DR+ mononuclear cells, than plotting in Lineage vs CD16 to visualize
> mono-subsets along the CD14 vs CD16 rainbow (practically no lineage
> positive cells other than monocytes express CD11c and DR together) and mDC (see
> figure 1 A and B of my article, Picozza et al., Blood, 2013). 
> 
> 
> 
> P.S. Instead of CD11c one can use CD86 (in resting ex vivo conditions)
> 
> 
> 
> Mario Picozza, Ph.D.
> 
> Laboratory of Applied Dermatology
> 
> Istituto Dermopatico dell'Immacolata, IDI I.R.C.C.S.
> 
> via dei Monti di Creta 104, 00167
> 
> Rome, Italy
> 
> 
> 
> 
> 
> 
> 
> Date: Fri, 21
> Feb 2014 22:06:03 +0000
> 
> From: cravista at gmail.com
> 
> To: TULUC at email.chop.edu; cytometry at lists.purdue.edu
> 
> Subject: Re: [Cytometry] about CD14-negative mature monocytes
> 
> Hi Nuno,
> 
> 
> 
> I saw your message on the Cytometry list. I wanted to ask if you know what
> 
> percentage of mature, circulating monocytes are CD14-negative and how can
> 
> one prove that those CD14-negative cells are indeed monocytes. If you know
> 
> of a paper that describes these cells please let me know.
> 
> 
> 
> Thank you,
> 
> Florin
> 
> 
> 
> 
> 
>>>>>>>>>>> Hi,
> 
> 
> 
> 
> 
> I will answer below.
> 
> 
> 
> As far as I know, the distribution of monocytes in the peripherical blood
> 
> is this:
> 
> - 80 % are "classical" monocytes with CD14+ CD16-;
> 
> - 10 % are "non-classical" monocytes with CD14- CD16+;
> 
> - 10 % are, let´s call "intermediate" monocytes with CD14+ CD16+
>>>> this
> 
> population can be divided in two subpopulations, depending on the
> 
> expression of HLA-DR: one has high expression and the other one not so high;
> 
> 
> 
> All (or almost all) of the monocytes are IREM2 positive (CD300e) and CD64
> 
> positive as well. I think that all of them are CD36 positive as well...
> 
> Dendritic CD16+ cells also express IREM2.
> 
> 
> 
> So, if you want to study PB monocytes and you just have 4 or 5 colours,
> 
> just put CD14, CD16, HLA-DR and CD45.
> 
> 
> 
> I hope it helps.
> 
> 
> 
> 
> 
> Greetings,
> 
> 
> 
> Nuno Oliveira, IPOCFG - Flow Cytometry Division
> 
> 
> 
> 
> 
> 
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PD Dr. Sieghart Sopper
Flow Cytometry Unit
Hematology and Oncology
Medical University Innsbruck
ZVG 7-G5-009A
Anichstr. 35
A-6020 Innsbruck
Tel    	++43 512 504 26332, -82596
Fax    	++43 512 504 25615
E-mail	sieghart.sopper at i-med.ac.at



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