It may be effect of heparin protein to granulocytes????

From my experience, there is no advantage to collect blood sample on EDTA-coated tubes for functional analysis with granulocytes because of the chelation of Ca/Mg needed for some granulocyte function tests. Heparin is less stable than EDTA and can be degraded by 24h old travelling, if your samples have to travel to reach your lab than you can use citrate-coated tubes even if you have also the chelation problem more or less (less drastic effect). However some enzymatic activities measured in granulocyte homogenates for lysosomal diseases detection are not influenced by EDTA but for direct functional analysis of granulocytes I do not recommand EDTA -coated tubes.

In my experience citrate tubes were the best choice e.g. in hospitals: they had the least effect on human PMNs, compared to samples acquired in the lab.. (superoxide production: basal, PMA, fMLP and bacterial stimuli; bacteria elimination capacity against S.aureus and E.coli). Heparin tubes had an interesting additional effect in E. coli elimination experients: we have seen rapid NET formation in some cases, in contrast to S.aureus elimination or to normally acquired PMNs.

when working with neutrophils (humans, horses, cattle, camelids) we have always used the yellow top ACD tubes commercially availalble. You can use EDTA with good result, if you remember to put some cations back in after purifying PMN. I have done a little work with heparizined blood, but not enough to comment. I am intrigued about the net formation in Heparinized blood.

Thank you all for your answers, they are very helpful. It looks like EDTA-coated tubes are best for most purposes, unless you want to look specifically at NET formation in response to E. coli and other bacteria. We usually add EDTA during purification of granulocytes, so I couldn't really see an added benefit to using it as an anticoagulant during blood collection. However, we keep heparin-coated tubes in storage for long periods of time (we don't need for the tubes to travel anywhere), so it's possible that the heparin could degrade over time, which would also be an added advantage of using EDTA-coated tubes. We use eosinophils and neutrophils for degranulation assays, immunofluorescence, superoxide release assays, and subcellular fractionation. And we always put the neutrophils back into cation-containing RPMI when we start our experiments to make sure they have plenty of Ca and Mg.

About NET with Na-Heparin: we have seen it only 2 times amoung 7 samples of patient with severe sepsis, only in the presence of opsonized E. coli, but not S. aureus. In contrast, samples from same patients in citrate did not formed NET during the same incubation period (30 min, 37. C, gentle shake).

It depends on which type of blood test you are after. I believe for standard coagulation studies stick to cit. sodium also, for TEG studies but for other studies such as D.Dimer and fibrinogen I believe that there will be no big difference

Excess of EDTA or improper mixing of blood affects both red cells and leucocytes, causing shrinkage and degenerative changes.EDTA has also been shown to cause leucoagglutination affecting both neutrophils and lymphocytes.Lithium or sodium salt of heparin at a concentration of 10-20 iu per ml of blood is a commonly used anticoagulant for chemistry, gas analysis, and emergency tests. It does not alter the size of the red cells, and it is recommended when it is important to reduce to a minimum the chance of lysis occurring after blood has been withdrawn. Heparin is not suitable for blood counts because it often induces platelet and leucocyte clumping.

I isolated human granulocytes into the tubes coated with heparin from different donors many times without any problems and with high yields of neutrophils or eosinophils. I have not any experience with tubes coated with EDTA. To achieve high yields of native eosinophils and neutrophils it is very important to start their isolation from the blood immediately after its taking away from a donor. Chromatin of neutrophils is much less stable comparing the other blood cells.

Best regards Emilie

EDTA preserves the cell structure other anticoagulants distort or destry the cell structure for microscopic observation. EDTA is the coagulant of choice.

To Csaba Timar: I have found NET formation in neutrophils to be highly inconsistent between preparations. An added complexity is that it is not very easy to carry out live imaging of NET formation as exposure to intense light actually induces neutrophil apoptosis and necrosis.

Heparin activate the PMN cells. EDTA is better if you want to avoid priming reaction to inföammatory cells. The priming happens anyway but slowlier with EDTA.

To Paige Lacy: I do agree with You, it seems to be incosistent, due to - as I wrote - it happened only 2 times from a lot others, but we have seen it only the presence of NaHeparin and opsonized E.coli. However, in the reported experiments we did not focused on NET production, it was only a side finding, when we wanted optimize the sample acquiring methods from severly ill patients. So this 2 sporadic events forced us to not using Na-Heparin tubes in this relation.

About identification NET: we just wanted to know wether it is NET or clot, so we did not use sophisticated and fine experiments, just acquired sample from the "coagulum-like" jelly, and threated with DN-ase, and observed in fluorescent microscopy histones and extracell. DNA.

Thanks for the update, Csaba. It's useful to have in case we need to study NETs!

The EDTA coated tubes prevent the clotting of patient blood because we need to determine the granulocytes, if we use the heparin-coated tubes this lead to clotting RBC's, WBC's (granulocytes and others) and platelets together and we could not able to determine and purify the granulocytes.

It depends what you want to do with neutrophils and eosinophils. If you want to measure receptors or activity then use EDTA. Removing calciun and magnesium reduce activation and priming processes but heparin does not.

Thanks, Pertti! That appears to be true. Heparin is only able to prevent clotting but cannot affect the activation status of neutrophils and eosinophils as well as EDTA.

I'm thinking it's because EDTA chelation of calcium and magnesium prevents the activation of adhesion molecules (beta intregrins), which promote clumping in neutrophils and eosinophils. Would this be the most accurate reason for the effects of EDTA?

Yes. The CR3 receptor complex (CD11b/CD18) is dependent on calcium. It may be central in receptor capping. In flow cytometry we use Ca2+ and Mg2+ -free buffer with some EDTA just to prevent cell clumping. If cells are measured soon (1-3 hours) after blood sample taking we normally avoid unnecessary cooling and warming up. There will be less clumping and cell activation if cells are just at room temperature. After isolation we use 0,1% gelatin (from swine skin) in buffer to reduce cell adhesions.

EDTA is preferred

EDTA is better than Heparin for this purpose, one of the advantages of EDTA is chelation of Ca++ result in inhibition of Neutrophil clumping caused by activation of adhesion molecule on Neutrophil