G.G. Agarwal

N. Husain

P.K. Patibandla

R.C. Ahuja

S. Awasthi

V. Venkatesh

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J Vect Borne Dis 44, March 2007, pp. 69–71Effect of EDTA and ACD on blood storage for the detection ofPlasmodium falciparum by PCRP.K. Patibandlaa, V. Venkatesha, G.G. Agarwalb, S. Awasthic, N. Husaind, R.C. Ahujae,S. Swastikaa & S.K. AgarwalaaDepartment of Microbiology, King George’s Medical University, Lucknow; bDepartment of Statistics, University of Lucknow,Lucknow; cDepartment of Paediatrics, dDepartment of Pathology, eDepartment of Medicine and CEU, King George’s MedicalUniversity, Lucknow, IndiaKey words ACD – EDTA – malaria – PCR – Plasmodium falciparumBlood samples are extensively used for PCR-baseddiagnosis of microbial infections and screening forblood banking1. It is not possible to do PCR withfresh blood samples each time. This preliminarystudy is aimed to compare the effect of blood storage(–20oC) on PCR for detecting Plasmodium falciparumin the two most commonly used anticoagulants suchas ethylenediaminetetra acetic acid (EDTA) and acidcitrate dextrose (ACD)2,3.The study was conducted in Department of Micro-biology, King George’s Medical University (KGMU),Lucknow, Uttar Pradesh, India. The protocol wasapproved by the institutional ethical committee,KGMU, Lucknow. Informed consent was obtainedfrom relatives of the patients.Seven microscopy and rapid diagnostic test (RDT)positive blood samples for falciparum malaria, threemicroscopy and RDT negative (healthy persons)blood samples (Haemoglobin  <12%) were collectedin EDTA and ACD anticoagulants and stored at –20oC for a period varying between 2 and 10 months.These samples were collected before start of theantimalarial therapy during the period from August2005 to April 2006. All patients had acute febrile ill-ness and later they had responded to antimalarialtherapy. Giemsa stained thick and thin blood filmswere observed under microscope. RDT for detectingthe HRP-2 antigen of P. falciparum was performedusing a commercially available kit (Paracheck Pf, Or-chid Biomedical Systems, Goa, India) and resultswere interpreted as per manufacturer’s instructions.The protocols according to Patsoula et al4, andWitoon et al5, were followed on EDTA and ACD storedblood. Either of these methods in their exact formatcould not work in our settings. The reason may be thatthese authors had used fresh EDTA and sodiumdodecyl sulphate blood. So we selected primer setsused by these authors but standardised PCR mix andPCR protocol in our laboratory which produced op-timal results.The rapid DNA extraction method according toMichael et al6, and Raj et al7 was selected with a fewmodifications. Briefly, 500 μl of ice-cold 5 mM so-dium phosphate (pH-8) was added to 20 μl of veni-puncture blood and vortexed. After centrifugation for6 min/14500 rpm in a micro centrifuge tube at 4oC,the supernatant was discarded. The pellet was sus-pended in 100 μl PBS containing 0.01% saponin andwashed twice with same buffer by repeating theabove steps of vortexing and centrifugation. Afterwashing, the pellet was suspended in 50 μl of sterile70 J  VECT  BORNE  DIS  44, MARCH 2007Fig. 1: Agarose gel electrophoresis pattern of PCR productsfrom P. falciparum positive long-term stored bloodsamples. Lanes 1, 2 (primer set 1) and lanes 3, 4 (primerset 2) produced 346 bp and 206 bp with EDTA, ACDrespectively, M–100 bp ladderFig. 2: Effect of duration of blood storage in EDTA and ACDanticoagulants on PCR for detecting P. falciparummalariatriple distilled water, vortexed and boiled for 20 minat 100oC. It was again vortexed before use for PCR.PCR was done separately for both sets of primers onall ten samples in the month of June 2006. Twoprimer sets were used as follows:Primer set 1 (Patsoula et al4)PL3 (5’ ATGGCC GTT TTT AGT TCG TG 3’)PL5 (5’ ACG CGT GCA GCC TAG TTT AT 3’)Primer set 2 (Witoon et al5)PF1 (5’ GCT  ACA  TATGCT AGT TGC CAG AC 3’)PF2 (5’ CGT GTACCA TAC ATC CTA CCA AC 3’)For primer set 1, PCR reaction was performed in amix, with a final volume of  50 μl  containing 1 × PCRbuffer, 1.5 mM MgCl2, 0.2 mM dNTP mix, 20 pm ofeach PL3 and PL5 primer  2.5 units Taq-DNA poly-merase (Invitrogen, Brazil). For primer set 2 reactionmix was same as above except that primers used werePF1 and PF2.In each run, an initial 4-min denaturation at 94oC wasfollowed by 35 cycles, each of denaturation at 94oCfor 30 sec, annealing at 52oC for 35 sec, extension at72oC for 30 sec, and then a final extension at 72oC for5 min. PTC100 (MJ Research, Watertown, massmodel, USA) was used in all experiments. The PCRproducts were subjected to electrophoresis in 1.5%agarose gel (Fig. 1).For primer set 1, EDTA stored blood gave 100%(7/7) sensitivity and 100% (3/3) specificity whereasACD stored blood gave only 14.28% (1/7) sensiti-vity and 100% (3/3) specificity.  For primer set 2,EDTA stored blood gave 57.14% (4/7) sensitivity and100% (3/3) specificity, whereas ACD stored bloodgave only 28.57% (2/7) sensitivity and 100% (3/3)specificity.The results from our preliminary study showed thatEDTA with primer set 1 produced positive resultsfrom blood samples stored as old as 10 months,whereas EDTA with primer set 2, produced positiveresults for blood samples old up to eight months only.These results along with the results of ACD (both setsof primers) are shown in Fig. 2.This suggests that long-term EDTA stored blood(–20oC) when used with Patsoula et al4, primer andour standardised PCR mixture and cycles producedresults with 100% sensitivity and 100% specificity,however, there was decrease in  sensitivity with ACDstored blood with both sets of primers. One of thePATIBANDLA et al :  EFFECT OF ANTICOAGULANTS ON PCR 71Corresponding author: Phani Kumar Patibandla, Department of Microbiology, King George’s Medical University,Lucknow–226 003, India.E-mail: phani_pati@rediffmail.comReceived: 27 June 2006 Accepted in revised form: 3 January 2007reasons could be that the dilution effect caused by theliquid formulation of ACD decreased the nucleic acidrecovery8–10. However, the exact reasons are not known.So based on this preliminary study EDTA appearedto be a better anticoagulant than ACD for delayedblood processing in case of PCR analysis for P. fal-ciparum. Our analysis, though, based on a small sam-ple size,  produced optimal results. However, a studyis needed with a large number of stored blood sam-ples to get a stronger evidence of the above results.AcknowledgementThis work was supported by Intramural FacultyGrants 2005 awarded to Dr. V. Venkatesh, KingGeorge’s Medical University, Lucknow, India.References1. 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Effect of EDTA and ACD on blood storage for the detection of Plasmodium falciparum by PCR

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