I am sure that blood in EDTA vaccutainer will give better count compared with heparin , I can tell u that please dont keep the samples again in refrigerator, try to proceed the isolation immediately after receiving samples in hand , I have done hundreds of samples both heparin and EDTA in parallel I always found that EDTA is best.,please gimme an idea about the protocol that u followed ?

1.DOUBLE DILUTE BLOOD WITH 1X PBS(50 ML CORNING TUBE FOR 5- 15ML BLOOD) 2. BOTTOM LOAD USING FICOL (~1 ML FICOL /1 ML BLOOD OR LESS) 3.SPIN AT 1300 RPM AT RT FOR 20 MINS(acceleration and deceleration should be 1) 4.COLLECT THE PBMC LAYER ALONG WITH PLASMA COMPLETELY LEAVING FICOL ONLY FOR MAX HARVESTING . 5. MAKE UP THE COLLECTED FLUID WITH 1X PBS (5o ml) 6. SPIN AT 1300 RPM (RT) FOR 10 MINS .acceleration and deceleration should be 1) 7. DISCARD THE SUPERNATANT . 8. RESUSPEND THE PELLET IN 10 ML PLAIN RPMI . 9. COUNT THE LYMPHOCTES . 10. ADD 500 UL OF FCS AND 500 UL OF FRREZING MEDAI for 1 million of lymphocytes (20% DMSO CARRIED IN ICE)) WHICH MAKES FINAL CONCENTRATION OF 10 % DMSO . 11. ALQUOT 3 milion cells /cryo vial and keep in iso propanal frosty and transfer immediately to - 80 12. after two days transfer vials to liquid nitrogen. while thawing keep the vial in 37 c water bath till the cell pellet comes down to pea sized and give a wash immediately with 5 ml plain rpmi and continue for further cultures.

We always used heparinized blood up to 24h after collection for whole blood analysis and it worked well. For PBMC you can use even older blood. But you should avoid refrigerating of the blood. Better try to keep it at room temperature during transport. For cell culture and stimulation experiments it is better to use heparinized blood because cells will respond better.