Evaluation of the TEST 1 erythrocyte sedimentation rate system and intra- and inter-laboratory quality control using new latex control materials

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1 Clin Chem Lb Med 2010;48(7): by Wlter de Gruyter Berlin New York. DOI /CCLM Evlution of the TEST 1 erythrocyte sedimenttion rte system nd intr- nd inter-lbortory qulity control using new ltex control mterils Choong-Hwn Ch 2,, Young Joo Ch 1, *, Chn- Jeoung Prk 2, *, Hyun Kyung Kim 3, Eun-Jong Ch 4, Duck Hee Kim 2, Honghoon 2, Je-Seol Jung 3, Mi-Jung Kim 2, Seongsoo Jng 2, Hyun-Sook Chi 2 nd Dong Soon Lee 3 1 Deprtment of Lbortory Medicine, Chung-Ang University College of Medicine, Seoul, Kore 2 Deprtment of Lbortory Medicine, University of Ulsn College of Medicine nd Asn Medicl Center, Seoul, Kore 3 Deprtment of Lbortory Medicine, Seoul Ntionl University College of Medicine, Seoul, Kore 4 Deprtment of Biomedicl Engineering, School of Medicine, Chungbuk Ntionl University, Cheongju, Kore wcoefficients of vrition (CV) 6.6% 21.7%x with ptient smples nd inter-ssy precision (CV 0.0% 6.8%) with ltex control mterils were stisfctory. The reference rnges of 2 10 mm/h for mles nd 2 19 mm/h for femles were estblished. Inter-lbortory QC dt with ltex control mterils in three hospitls demonstrted good ccurcy nd stisfctory precision (CV 0.0% 14.4%). Conclusions: Our results demonstrte tht the TEST 1 QC is relible nd the ltex control mterils re vluble for inter-lbortory proficiency testing. Clin Chem Lb Med 2010;48: Keywords: erythrocyte sedimenttion rte; ltex control; qulity control; TEST 1. Abstrct Bckground: The erythrocyte sedimenttion rte (ESR) test hs been considered to be simple procedure, not requiring qulity control (QC). However, QC is essentil for ccurcy nd precision. We evluted the TEST 1 ESR system nd performed QC procedures using newly developed ltex control mterils in three hospitls. Methods: Using tripotssium ethylenediminetetrcetic cid blood smples (ns184), we compred TEST 1 ESR vlues with Westergren ESR dt nd evluted intr-ssy precision. Three levels of ltex control mterils were used to ssess inter-ssy precision. Reference rnge ssessment ws done using smples from 220 helthy individuls. Interlbortory QC with ltex control mterils in three hospitls ws performed. Results: Correltion between TEST 1 ESR nd Westergren ESR results ws good (p-0.001). Intr-ssy precision Current ddress: Deprtment of Lbortory Medicine, University of Ulsn College of Medicine nd Gngneung Asn Hospitl, Gngneung, Kore *Corresponding uthors: Young Joo Ch, MD, Deprtment of Lbortory Medicine, Chung-Ang University Hospitl, Heukseok-1dong, Dongjk-gu, Seoul , Kore Phone: q , Fx: q , E-mil: chyoung@cu.c.kr Chn-Jeoung Prk, MD, Deprtment of Lbortory Medicine, Asn Medicl Center, Pungnp-2dong, Songp-gu, Seoul , Kore Phone: q , Fx: q , E-mil: cjprk@mc.seoul.kr Received October 12, 2009; ccepted Jnury 21, 2010; previously published online My 12, 2010 Introduction The erythrocyte sedimenttion rte (ESR) is widely used in clinicl prctice s n indictor of inflmmtion, infection, trum, or mlignnt disese (1). The current reference method for mesurement of ESR is tht proposed by Westergren; the sedimenttion of red blood cells is mesured by observing the level to which cells fll in given time intervl, usully 1 h, in well-described, specific pipette (2). An utomted mesurement procedure, the TEST 1 (Alifx, Pdov, Itly) hs been mrketed. This system llows utomtic mesurement of ESR in specimens collected in both sodium citrte nd EDTA blood smple tubes using microgglutintion method (3). ESR performed using mnul nd semi-utomted methods hs long been considered s simple procedure tht could be performed without the need for qulity control (QC) (4). In clinicl lbortories, relible methods for clibrtion nd the use of pproprite control mterils re required for monitoring the ccurcy nd precision of ny routine method. In the lst few yers, number of commercil QC mterils hve ppered on the mrket, but most re bsed on stbilized whole blood nd re not suitble for use with techniques involving utonlyzers, such s the TEST 1 tht evlutes the kinetics of the ggregtion rection (1, 5). Recently, new ltex control mteril hs been developed to increse instrument relibility nd to enble the performnce of sttisticl QC (6). The gol of this study ws to evlute ESR mesurement using the TEST 1 system nd to investigte QC procedures 2010/543

2 1044 Ch et l.: Evlution nd QC of the TEST 1 ESR system with ltex control bsed on the use of ltex control mterils for the dily intrnd inter-lbortory QC of ESR in the clinicl lbortory. Mterils nd methods Mterils All blood for the study ws collected in 3-mL tripotssium ethylenediminetetrcetic cid (K 3 EDTA) tubes (Becton Dickinson, Frnklin Lkes, NJ, USA). Blood smples were obtined from hospitlized nd mbultory ptients t three tertiry cre hospitls (Hospitl A: Seoul Ntionl University Hospitl, Seoul, Kore; Hospitl B: Chung-Ang University Hospitl, Seoul, Kore; Hospitl C: University of Ulsn College of Medicine nd Asn Medicl Center, Seoul, Kore), nd processed for nlysis. All smples were obtined under stndrdized conditions, in the morning fter night of fsting, nd tested within 4 h of venipuncture, ccording to the recommendtions of the Interntionl Council for Stndrdiztion in Hemtology (ICSH) (7). Westergren method The Westergren method ws performed ccording to ICSH specifictions on undiluted blood nticogulted with EDTA using glss pipettes (Greiner Bio-One GmbH, Kremsmuenster, Austri) (7). During sedimenttion, pipettes were mounted verticlly on pproprite supporting rcks nd kept t room temperture (21 228C). TEST 1 method TEST 1 is closed utomted nlyzer tht determines ESR t 378C by ssessment of red cell ggregtion. A 150 ml blood smple is delivered into cpillry tube. The electricl signl, collected by photodiode detector, is directly correlted to red cell ggregtion in the cpillry. A mthemticl lgorithm converts the rw dt obtined from opticl density signls into ESR results, which re trnsformed into comprble Westergren vlues. The first result is obtined in bout 4 min nd subsequent results re obtined every 20 s (1, 3, 6, 8). The most recent version of TEST 1, the TEST 1 THL, includes new version of the softwre tht llows the nlyzer to run the new ltex controls. The TEST 1 THL nlyzers were set up in the lbortories of the three hospitls. Ltex clibrtors nd control mterils The clibrtors nd control mterils for the TEST 1 re redy-mde turbidimetric stndrds of synthetic ltex solutions. Ech set is composed of three tubes ech contining 3 ml of ltex solution t three different concentrtions (6). Ech QC level hs known turbidity vlue, on which the instrument performs trnsmittnce mesurements correlted to the ESR vlue. The TEST 1 nlyzers were clibrted using the newly developed clibrtors. Accurcy We selected 184 blood smples from the ptients of hospitl A for comprison studies. All hd TEST 1 ESR vlues rnging from 5 to 105 mm/h nd grouped into one of 10 clsses on the bsis of the TEST 1 ESR vlue. The Westergren ESR vlues were obtined t hospitl A. Ech residul smple ws trnsported, under cold storge, to hospitl B. In hospitl B, the smples were returned to room temperture for 45 min nd remixed before the mesurement of ESR by TEST 1. Ech residul smple ws trnsported, gin in the cold, to hospitl C, where the sme procedures s those in hospitl B were performed. In smples with hemtocrit (Hct) F35%, ESR vlues were corrected to Hct of 40% using Fbry s formul: corrected Westergren ESR vlues(westergren ESR=15%)/(55% Hct%) (9). Precision A reproducibility study ws performed t hospitl B using blood smples nd three levels of ltex controls. To test intr-ssy precision, 20 blood smples, stored t room temperture nd with TEST 1 ESR vlues of more thn 5 mm/h, were used. Eight replicte mesurements of 10 blood smples with different ESR rnges were performed under continuous loding, nd nine replicte mesurements of nother 10 blood smples were mde t 30 min intervls. Tble 1 Correltion nd differences between TEST 1 nd Westergren ESR vlues. Method R 2 Difference Limit of greement 95% CI Overll, ns184 Westergren TEST 1 A to to 24.6 Westergren TEST 1 B to to 19.9 Westergren TEST 1 C to to 33.9 Corrected Westergren TEST 1 A to to 7.4 Corrected Westergren TEST 1 B 0.67 y to to 3.0 Corrected Westergren TEST 1 C to to 16.4 Hct F35%, ns95 Westergren TEST 1 A to to 33.7 Westergren TEST 1 B to to 29.1 Westergren TEST 1 C to to 44.3 Corrected Westergren TEST 1 A 0.66 y to to 0.4 Corrected Westergren TEST 1 B 0.72 y to to 3.7 Corrected Westergren TEST 1 C to to 10.5 Hct )35%, ns89 Westergren TEST 1 A to to 16.7 Westergren TEST 1 B to to 11.9 Westergren TEST 1 C to to 24.2 TEST 1 ESR vlues mesured t hospitl A, B, or C. CI, confidence intervl.

3 Ch et l.: Evlution nd QC of the TEST 1 ESR system with ltex control 1045 The inter-ssy precision of three levels of control mteril ws evluted over 5 dys, with five determintions per dy. Stbility test A stbility test ws performed t hospitl C. The stbility of ESR smples over time ws evluted by storing the 184 blood specimens used in the ccurcy study for 24 h t 48C. The smples were returned to room temperture for 45 min, remixed nd then remesured. Reference rnge The reference rnge ws estblished t hospitl C using blood smples collected from 220 helthy individuls (110 mles nd 110 femles) visiting the helth promotion center t hospitl C. To clculte the reference rnge, nd considering the increse in ESR in reltion to ge, ESR vlues were evluted seprtely for ech decde of dult life in men nd women. Precision Intr-ssy reproducibility using blood smples over wide rnge of ESR vlues is shown in Tble 2A. Continuous loding reveled good precision except in the lowest nd highest rnges, but loding t intervls of 30 min (with totl durtion of mesurement of more thn 4 h nd 30 min) showed higher CV compred with continuous loding. Inter-ssy reproducibility using ltex control mterils is shown in Tble 2B. The level 1 control encompsses ESR vlues in the norml popultion nd levels 2 nd 3 encompss the cliniclly significnt ESR rnges. The precision of the three levels of control mterils ws very good. Stbility test Storge of blood smples t 48C for up to 24 h cused significnt decrese in ESR vlues mesured with the TEST 1 Inter-lbortory QC with ltex control mterils in three hospitls ESR mesurements of three levels of ltex control mterils were performed over dys, with one determintion per dy, in ech of three hospitls. Sttisticl nlysis Sttisticl nlysis ws crried out using MS Excel 2003 softwre (Microsoft, Settle, WA, USA), SPSS version 13.0 softwre (SPSS Inc., Chicgo, IL, USA), nd MedClc version (MedClc, Mrikerke, Belgium). The dt from the method comprison studies were nlyzed using Pssing-Bblok regression, non-liner regression nd Blnd-Altmn nlysis. To estimte precision, we computed mens, SDs, nd coefficients of vrition (CVs). Differences between mens for stbility were evluted by the pired Student s t-test. Estimtes for the reference rnge were bsed on non-prmetric sttisticl method using percentiles. Comprison of mens between genders in ny given ge group ws performed using the Mnn-Whitney test. Comprison of mens between gerelted groups in the mle or femle reference popultion ws chieved by the Kruskl-Wllis test. Sttisticl significnce ws considered if p Results Accurcy There ws sttisticlly significnt correltion between the TEST 1 nd Westergren dt (p-0.001), but notble differences were noted lso (Tble 1 nd Figure 1). Pssing- Bblok regression showed non-liner reltionship between both methods. The TEST 1 ESR vlues were lower thn the Westergren vlues. The differences between TEST 1 nd Westergren vlues in 89 blood smples with Hct )35% were smller thn those in 95 blood smples with Hct F35%. In ddition, the correltion coefficients between both methods in smples with Hct )35% were higher thn those in smples with Hct F35%. The differences between TEST 1 nd corrected Westergren vlues were smller thn differences between TEST 1 nd non-corrected Westergren vlues. Figure 1 Comprison of TEST 1 nd Westergren methods for ESR mesurements using dt obtined from hospitl B. (A) Scttergrm of correltions between the ESR mesurement methods (ns184; ys x 2 q0.5823xq1.9242; R 2 s0.72; p-0.001). The solid line indictes regression line nd the dshed lines indicte 95% confidence intervl for the regression line nd the dotted lines indicte 95% prediction intervl for the regression line. (B) Plot of differences between the ESR mesurement methods. Horizontl dshed lines denote the limits of greement nd oblique dotted line denotes regression line of differences vs. verges.

4 1046 Ch et l.: Evlution nd QC of the TEST 1 ESR system with ltex control Tble 2 Intr-ssy (A) nd inter-ssy (B) reproducibility of TEST 1 for mesurement of ESR. (A) Intr-ssy, blood smples (B) Inter-ssy, qulity control mterils, ns25 ESR, Continuous loding 30-min intervl ESR Men"SD, CV, % Climed Bis, % mm/h ns8 ns9 mm/h vlues Men"SD, CV, % Men"SD, CV, % mm/h mm/h " " Level 1 4.9" (4 6) " " Level " (20 26) " " Level " (54 72) " " " " " "5.3 b " " "6.0 b " " " ) "13.5 b " Five different dys, five determintions per dy. b Seven replictes. (Figure 2). The men difference ws 6.05 mm/h (95% confidence intervl , p ), reflecting decrese of 18.7%. The difference incresed in proportion to ESR vlues. Reference rnge The reference rnge for mles ws 2 10 mm/h, nd tht for femles 2 19 mm/h (Tble 3). There were significnt differences between the genders in groups ged 60 yers or less (ps0.028, 0.002, 0.002, nd for groups ged yers, yers, yers, nd yers, respectively). There ws no significnt difference between the genders in the group ged over 60 yers (ps0.144). No significnt differences were seen mong ge-relted groups in both the mle nd femle reference popultion (ps0.562, ps0.652). Figure 2 Stbility of ESR smples fter storge for 24 h t 48C (ns184). The men difference ws 6.05 mm/h (p ). Inter-lbortory QC using ltex control mterils in three hospitls Accurcy nd reproducibility of the ltex control mterils used for QC re shown in Tble 4. All three hospitls demonstrted good ccurcy nd stisfctory precision, even with level 1 QC. Discussion We evluted the TEST 1 system nd investigted QC procedures using newly developed ltex control mterils. The TEST 1 system showed good correltion with the Westergren method nd stisfctory precision. The ltex control mterils showed good ccurcy nd suitble precision for the dily intr- nd inter-lbortory QC of ESR. The proposed newly developed turbidimetric stndrds re vilble, esy to use, commutble with humn whole blood smples, nd the costs re comprble to those of other commercilly vilble control mterils (6). We found tht Westergren ESR vlues were higher thn those obtined using TEST 1, especilly in smples with Hct F35%. It hs been suggested tht the Westergren method overestimtes ESR in smples of low Hct (8). Also, in previous study (10), we suggested tht ESR mesurements by TEST 1 reflect inflmmtion better thn those obtined by the Westergren method in ptients with mlignncy, utoimmune disese, or infection. TEST 1 vlues obtined t hospitl C were lower thn those mesured with the corrected Westergren method. Lower TEST 1 vlues t hospitl C might be cused by the dely in mesurement resulting from trnsporttion of smples from hospitl A, to hospitl B, nd finlly, to hospitl C. All ESR nlyses should be performed within 4 h from the time of collection ccording to ICSH nd Clinicl nd Lbortory Stndrd Institute (CLSI) recommendtion (7, 11). Storge of smples in EDTA tubes t 48C for up to 24 h cused significnt decrese (18.7%) in ESR vlues. This

5 Ch et l.: Evlution nd QC of the TEST 1 ESR system with ltex control 1047 Tble 3 Reference rnges for ESR determined using the TEST 1 for men nd women ccording to ge group. Age, yers Men Women n Men, Lower limit Upper limit n Men, Lower limit Upper limit mm/h (2.5%), mm/h (97.5%), mm/h mm/h (2.5%), mm/h (97.5%), mm/h ) Totl Tble 4 Qulity control results with ltex controls, using three different TEST 1 nlyzers in three hospitls. Ltex control n Expected men Men"SD, CV, % Bis, % (rnge), mm/h mm/h Level 1 Hospitl A 16 5 (4 6) 4.8" y4.0 Hospitl B 16 5 (4 6) 5.1" Hospitl C 11 5 (4 6) 5.0" Level 2 Hospitl A (20 26) 24.1" Hospitl B (20 26) 25.0" Hospitl C (20 26) 25.0" Level 3 Hospitl A (54 72) 66.1" Hospitl B (54 72) 66.5" Hospitl C (54 72) 66.8" Mesured once dy. decrese is greter thn tht ( decrese of 4.1%) reported by Pdro-Miquel nd Fuentes-Arderiu (12), but is in greement with the drop of 20.9% reported by Plebni nd coworkers (3). CLSI recommendtions re tht the test should be run within 4 h if blood smples re left t room temperture or within 12 h if they re stored t 48C (11). The reference rnge dt were nlyzed using non-prmetric sttistics becuse ESR vlues did not show true Gussin distribution. In the present study, there ws no increse in ESR with respect to ge. The reference vlues of the present study were similr to those obtined in study of Korens (2 19 mm/h in men, 2 26 mm/h in women) (13), nd lower thn those in Itlins (2 28 mm/h in men nd 2 37 mm/h in women, ged yers) (14). Differences in the reference popultion, such s rce or region might be the cuse of difference in the reference vlues (15). TEST 1 vlues obtined from three hospitls in the ccurcy study showed significnt vrition with respect to trnsport time. Blood smples showing significnt decrese in ESR vlues with trnsport time re not suitble for interlbortory proficiency testing. Ltex control mterils could be used for inter-lbortory proficiency testing becuse of stisfctory inter-ssy precision. In conclusion, TEST 1 ESR results showed good precision nd good correltion with Westergren ESR dt. Our results, obtined with ltex control mterils demonstrte tht the TEST 1 QC for the mesurement of ESR is relible, nd the ltex control mterils re vluble for inter-lbortory proficiency testing. Acknowledgements This reserch ws supported by Bsic Science Reserch Progrm through the Ntionl Reserch Foundtion of Kore (NRF) funded by the Ministry of Eduction, Science nd Technology ( ). We wish to thnk members of the Jinsung Meditech (Seoul, Kore) for their technicl support. Conflict of interest sttement Authors conflict of interest disclosure: The uthors stted tht there re no conflicts of interest regrding the publiction of this rticle. Reserch funding: None declred. Employment or ledership: None declred. Honorrium: None declred. References 1. Plebni M, Piv E. Erythrocyte sedimenttion rte: use of fresh blood for qulity control. Am J Clin Pthol 2002;117: Westergren A. The technique of the red cell sedimenttion rection. Am Rev Tuberc 1926;14:

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