Abstract Background: We have empirically noted that many physicians routinely request anti-phospholipid antibodies (aPL) without a correct clinical indication. The aim of this study was to evaluate retrospectively whether aPL testing at our Thrombosis Centre was justified. Methods: Medical records from 520 subjects for aPL screening tests for various clinical conditions were reviewed. The aPL screening tests were: lupus anticoagulant (LA), anti-cardiolipin antibodies (aCL) and anti-β(2) glycoptotein I (aβ(2) GPI). Requests for aPL screening were divided into justified, potentially justified or not adequately justified. Results: aPL testing requests were considered justified in 358 (69%) patients, potentially justified in 66 (12.6%) and not adequately justified in 96 (18.4%). LA was positive in 65 (18%) of justified requests and in only one (1%) of the 96 potentially justified requests. None of the 66 not adequately justified for aPL testing was positive for LA. aβ(2) ..

Barcellona D

Mameli A

Marongiu F

Vannini ML

English

BARCELLONA, DORIS

MARONGIU, FRANCESCO

Archivio istituzionale della ricerca - Università di Cagliari

Clin Chem Lab Med 2011;49(4):695–698  2011 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2011.1112010/386Article in press - uncorrected proofHigh frequency of inadequate test requests forantiphospholipid antibodies in daily clinical practiceAntonella Mameli, Doris Barcellona, Maria LuigiaVannini and Francesco Marongiu*Dipartimento di Scienze Mediche Internistiche‘‘M. Aresu’’ and Azienda Mista Ospedaliero-Universitaria,University of Cagliari, Cagliari, ItalyAbstractBackground: We have empirically noted that many physi-cians routinely request anti-phospholipid antibodies (aPL)without a correct clinical indication. The aim of this studywas to evaluate retrospectively whether aPL testing at ourThrombosis Centre was justified.Methods: Medical records from 520 subjects for aPL screen-ing tests for various clinical conditions were reviewed. TheaPL screening tests were: lupus anticoagulant (LA), anti-car-diolipin antibodies (aCL) and anti-b2 glycoptotein I (ab2GPI). Requests for aPL screening were divided into justified,potentially justified or not adequately justified.Results: aPL testing requests were considered justified in358 (69%) patients, potentially justified in 66 (12.6%) andnot adequately justified in 96 (18.4%). LA was positive in65 (18%) of justified requests and in only one (1%) of the96 potentially justified requests. None of the 66 not ade-quately justified for aPL testing was positive for LA. ab2GPI was positive in 63 (17.6%) of the 358 justified, in four(6%) of the 66 potentially justified and in five (5.2%) of the96 not adequately justified requests; aCL IgG were positivein 59 (16.4%) of the 358 justified and in five (7.5%) and six(6.2%) of the potentially justified and not adequately justi-fied requests, respectively. The presence of the triple aPLpositivity was found exclusively in the justified requests.Conclusions: This study suggests that requests for aPL testsshould be addressed more adequately. This work could be anexample of how to focus attention on requests for laboratorytests especially on the basis of valid clinical criteria beforethe analyte is measured.Keywords: anti-cardiolipin antibodies; anti-b2 glycoproteinI antibodies; Dilute Russell’s viper venom time (dRVVT);inadequate test request; silica clotting time (SCT).*Corresponding author: Prof. Francesco Marongiu, Dipartimentodi Scienze Mediche Internistiche ‘‘M. Aresu’’, University ofCagliari, SS 554, Monserrato 09042 (Cagliari), ItalyPhone: q39-706754188, Fax: q39-7051096201,E-mail: marongiu@medicina.unica.itReceived July 1, 2010; accepted September 25, 2010;previously published online February 9, 2011IntroductionLaboratory investigation in the diagnosis of antiphospholipidsyndrome (APS) is based on the presence of lupus antico-agulant (LA) in plasma and/or in combination with anti-car-diolipin (aCL) and anti-b2 glycoprotein antibodies (ab2 GPI)(1, 2). Taken together, they represent the laboratory patternof anti-phospholipid antibodies (aPL). Suggested indicationsfor aPL testing are: prolongation of activated partial throm-boplastin time (APTT) mixing test with normal plasma,venous or arterial thrombosis, especially if occurring beforethe age of 50 years, thrombosis at unusual sites or associatedwith autoimmune diseases, or complications of pregnancy(2). However, a ‘‘grey zone’’ of potentially clinical utilitymay exist in the case of autoimmune diseases. In particular,there is good evidence that patients with systemic lupus ery-thematosus (SLE) may acquire features of APS and vice-versa (3). Requests for aPL testing in other conditions are,in general, discouraged to avoid false-positive results (4–6).The aims of this study were to evaluate retrospectivelywhether aPL testing at our Thrombosis Centre (TC) betweenJuly 2008 and August 2009 was justified and followed theabove criteria, and to analyse the physicians approach to aPLtesting which may be just one example of how laboratorytests are excessively requested in daily clinical practise. Ingeneral, the phenomenon of ordering superfluous tests is wellrecognized, but it is difficult to contrast. Physicians often donot know the meaning of the tests they order. This may bethe case with aPL (7).Materials and methodsPatientsThe records of 720 consecutive unselected subjects, referred to ourTC from July 2008 to August 2009 for aPL screening tests, wereconsidered. Two hundred records were excluded as the reasons forthe request were unknown. A total of 520 requests were considered.The reasons for performing the tests were retrospectively identifiedby consulting the medical records. Requests were divided into threegroups: justified (clinical evidence of thrombotic events and mis-carriage), potentially justified (autoimmune disease without clinicalevidence of thrombotic events and miscarriage) or not adequatelyjustified (all other conditions).Coagulation testsSilica clotting time (SCT) The test consists of paired APTTs per-formed on test plasmas with micronized silica (HaemosilTM SilicaClotting Time, Instrumentation Laboratory, Milan, Italy) as activatorand two phospholipid concentrations (low SCT1 and high SCT2)as platelet substitutes. The test was performed using an automatedUnauthenticated | 94.39.64.132Download Date | 11/15/12 9:22 AM696 Mameli et al.: Antiphospholipids and inadequate test requestsArticle in press - uncorrected proofcoagulometer (ACL Advance, International Laboratory, Milan, Ita-ly) and is based on the ratio between the SCT screen and SCTconfirm. A SCT screen ratio was first calculated by dividing theclotting time obtained in the patients using low SCT1 by the meanclotting time obtained in a group of normal subjects. A SCT confirmratio was calculated by dividing the clotting time obtained in thepatients using high SCT2 by the mean clotting time obtained in agroup of normal subjects. A final ratio was obtained by dividingthe results of SCT screen by that of SCT confirm. If the cut-offvalue of 1.21 was exceeded, the test was repeated after mixing 1:1the plasma sample with normal plasma. It was considered positivewhen a 1:1 mixture of test plasma and normal plasma yielded aratio more than 1.21. This cut-off value was calculated using the99th percentile of coagulation times obtained in frozen plasmasamples from 50 healthy subjects.Dilute Russell’s viper venom time (dRVVT) The test consistsof paired clotting times (HemosilTM dRVVT LAC screen and con-firm, Instrumentation Laboratory, Milan, Italy) performed using anautomated coagulometer (ACL Advance, Instrumentation Labora-tory, Milan, Italy). It is based on the ratio between the dRVVTscreen (low phospholipids concentration) and dRVVT confirm (highphospholipids concentration). For the generation of the final ratiowe followed the same procedure described for SCT. It was consid-ered positive when a 1:1 mixture of test plasma and normal plasmayielded a ratio more than 1.34. This cut-off value was calculatedusing the 99th percentile of coagulation times obtained in frozenplasma samples from 50 healthy subjects.ab2 GPIIgG and IgM anti-b2 GPI antibodies were measured using ELISAkits (EUROIMMUN Medizinische Labordiagnostika AG, Lu¨beck,Germany). Concentrations of ab2 GPI antibodies were expressed inunits/mL and values of more than 8 and 12.5 were considered pos-itive for IgM and IgG isotypes, respectively. The cut-off values havebeen derived locally from 50 healthy subjects using the 99thpercentile.aCLThe presence of IgG and IgM aCL were detected using quantitativeELISA kits (Anticariolipine Bouty, Italy) according to the manufac-turer’s instructions. Measured values of aCL were expressed inunits/mL, and values of more than 12 U/mL for either IgG or IgMwere considered positive. The cut-off values have been locallyderived from 95 healthy subjects using the 99th percentile.Only abnormal tests (LA, aCL and ab2 GPI), confirmed after3 months, were considered positive.Statistical analysisStatistical analysis was performed using MEDCALC software (ver-sion 10.0.1.0). Data were expressed as median and range ormean"SD where appropriate. Frequency data were compared usingFisher’s exact test. p-Values -0.05 were considered statisticallysignificant.ResultsA total of 720 tests for an aPL screen performed in our TCfrom July 2008 to August 2009 were examined. Requests for520 of these requests were correctly detected by examiningthe medical record. A total of 358 out of 520 requests foraPL screening were considered as justified. Justified, poten-tially justified and non-adequately justified requests are listedin Table 1.A total of 168 patients (32%) tested positive on two occa-sions for at least one of the considered tests (dRVVT, SCT,ab2 GPI, aCL). To determine whether the presence of aPLwas more prevalent in individuals with justified requests, thedistribution of test positivity in all groups (Table 2) was eval-uated. Sixty-five (18%) of justified requests were positivefor SCT and/or dRVVT, only one (1%) of the 96 potentiallyjustified requests was positive for SCT, none of the 66 notadequately justified were positive for SCT and/or dRVVT;ab2 GPI was positive in 63 (17.6%) of the 358 justified, infour (6%) of the 66 potentially justified and in five (5.2%)of the 96 not adequately justified requests; aCL IgG waspositive in 59 (16.4%) of the 358 justified and in five (7.5%)and six (6.2%) of the potentially justified and non-adequatelyjustified requests, respectively. When the combination of aPLtest was considered, the presence of triple aPL positivity, i.e.,LA, aCL and a ab2 GPI was found exclusively in the ade-quate request patients only. The other possible combinationsshowed a higher frequency of aPL positivity in the group ofpatients with an adequate request, but the differences werenot significant because the subgroups were too small.DiscussionWe have empirically noticed that many physicians routinelyrequest aPL screening for patients in whom these requestsdo not seem to be adequate. We therefore questioned theadequacy of aPL screening at our TC.There are several reasons why physicians request aPLscreening for their patients in daily practice: the occurrence(accidentally identified) of prolongation of the APTT withoutknown aetiology, to provide an explanation for arterial orvenous thrombosis, to assess the risk of miscarriage (preg-nancy loss) recurrence, thrombosis in unusual sites or asso-ciated with autoimmune diseases (2). These requests havebeen classified in this study as justified. Even in autoimmunediseases, requests for aPL may be ordered since a relation-ship between some of these analytes and antiphospholipidsyndrome does exist (3). In this case, we classified therequests as potentially justified.Generalised searches on asymptomatic individuals otherthan the above should be discouraged to avoid the risk offalse-positive results, of wasting time and resources, and ofobtaining a positive result which could be misleading in thediagnosis and management of the single patient leading tothe use of inappropriate anti-thrombotic drugs. Theserequests were classified as not justified.Results of this retrospective study show that an importantpercentage of potentially justified and not justified requestswere processed in our laboratory. In particular, only onepatient showed LA positivity in the group of patients withpotentially justified request, while none showed LA positi-Unauthenticated | 94.39.64.132Download Date | 11/15/12 9:22 AMMameli et al.: Antiphospholipids and inadequate test requests 697Article in press - uncorrected proofTable 1 Justified, potentially justified and not adequately justified requests for aPL screening tests.Diagnosis n %Justified (ns358)Deep venous thrombosis 98 18.9Recurrent pregnancy losses 96 18.5Antiphospholipid syndrome 47 9Ischaemic stroke 33 6.3Transient ischemic attack 31 5.9Superficial venous thrombosis 16 3Pulmonary embolism 16 3Myocardial infarction 16 3Avascular bone necrosis 3 0.5Ischaemic neuritis 1 0.1Prolonged APTT 1 0.1Potentially justified (ns66)Autoimmune diseasesa 66 12.6Not adequately justified (ns96)Artrhalgias or arthritis 27 5.1Positivity for autoantibodies 13 2.5Raynaud phenomenon 8 1.5Familial history of thrombosis 7 1.3Other conditionsb 18 3.4aAutoimmune diseases: systemic lupus erythematosus (ns24, 36.3%), systemic sclerosis (ns27, 41%), rheumatoid arthritis (ns10, 15%),Sjo¨gren’s syndrome (ns5, 7.5%), without clinical evidence of thrombotic events or pregnancy losses. bOther conditions: headache, infectivediseases, hepatitis, urticaria, venous insufficiency, dermatitis, ecchimosis, fever, neoplasm, erythema nodosum, pericarditis, aftosis.Table 2 Distribution of aPL positivity in adequate and non-adequate requests.Justified Potentially Not adequatelyns358 justified ns66 justified ns96LA positivitya 65 (18.1%) 1 (1.5%) 0LAqab2 GPIqaCL 46 0 0LAqab2 GPI 3 0 1 (1%)LAqaCL 3 0 0ab2 GPI-IgGb 63 (17.6%) 4 (6%) 5 (5.2%)ab2 GPI-IgM 59 (16.5%) 4 (6%) 7 (7.2%)aCL IgG 59 (16.5%) 5 (7.5%) 6 (6.2%)aCL IgM 47 (13.1%) 6 (9%) 10 (10.4%)Values are given in numbers (percentage). LA, lupus anticoagulant; aCL, anticardiolipin antibody; ab2 GPI, anti-b2 glycoprotein antibody.ap-0.0001 vs. potentially and not justified requests. bp-0.004 vs. potentially and not justified requests.vity in the group of patients with unjustified requests. More-over, it is worth noting that triple positivity (LA, aCL andab2 GPI) was absent in both the potentially justified and notjustified group, but present in the justified request group.Ab2 GPI and aCL were present in the potentially justifiedand not justified groups, but we observed that in differentcombinations, LA was never present. Even though the groupof patients with a potentially justified request was small, ourresults show that the percentage of positive aCL and ab2 GPItests was as low as that of the patients with unjustifiedrequests. It should be remembered that only LA was signif-icantly associated with thromboembolism. Both aCL and ab2GPI were found to not be so closely related with thrombo-embolism (8, 9).Requests for aCL and ab2 GPI appear less useful in themanagement of the single patient without clinical and envi-ronmental evidence of either a thrombotic event or foetalloss. An educational program could be planned in order tolimit inappropriate aPL requests (10, 11). This approach hasbeen successfully adopted to improve, for instance, theappropriateness of antiepileptic drug monitoring (12). Acomputer-based intervention could be built, forcing physi-cians to order aPL tests only if the clinical criteria of appro-priateness are satisfied. A proposal for an algorithm ispresented in Figure 1. Requests for aPL testing should beconsidered if there is clinical suspicion of APS. In particular,LA testing should be the first test performed. If the requestis not justified, physicians should be discouraged from order-ing aPL tests. In fact, asymptomatic cases meeting only thelaboratory criteria for APS are not to be treated with oralanticoagulants. A particular condition may be that related topatients with potentially justified requests, that is, those withautoimmune disease. In these patients, ordering aPL testingmay be useful only if anti-thrombotic prophylaxis is required.Unauthenticated | 94.39.64.132Download Date | 11/15/12 9:22 AM698 Mameli et al.: Antiphospholipids and inadequate test requestsArticle in press - uncorrected proofFigure 1 Proposed algorithm for aPL screening.This paper has some limitations. It is a retrospective study,but we wanted to investigate the daily clinical and laboratorypractice of aPL testing at our TC with the aim of limitinginappropriate requests in the future. We hope that this workis an example of how to focus attention on the requests forlaboratory tests, especially on the basis of valid clinical cri-teria to determine test appropriateness before it is measured.Conflict of interest statementAuthors’ conflict of interest disclosure: None declared.Research funding: None declared.Employment or leadership: None declared.Honorarium: None declared.References1. Arnout J. Antiphospholipid syndrome: diagnostic aspects oflupus anticoagulants. J Thromb Haemost 2001;86:83–91.2. Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL,Cervera R, et al. International consensus statement on an updateof the classification criteria for definite antiphospholipid syn-drome (APS). J Thromb Haemost 2006;4:295–306.3. Tincani A, Andreoli L, Chighizola C, Meroni PL. The interplaybetween the antiphospholipid syndrome and systemic lupuserythematosus. Autoimmunity 2009;42:257–9.4. Tripodi A. Testing for lupus anticoagulants: all that a clinicianshould know. Lupus 2009;18:291–8.5. Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, etal. Update of the guidelines for lupus anticoagulant detection.J Thromb Haemost 2009;7:1737–40.6. Favaloro EJ, Wong RC. Laboratory testing and identificationof antiphospholipid antibodies and the antiphospholipid syn-drome: a potpourri of problems, a compilation of possible solu-tions. Semin Thromb Hemost 2008;34:389–410.7. Sood R, Sood A, Ghosh AK. Non-evidence-based variablesaffecting physicians’ test – ordering tendencies: a systematicreview. Neth J Med 2007;65:167–77.8. Galli M, Luciani D, Bertolini G, Barbui T. Anti-beta 2-glyco-protein I, antiprothrombin antibodies, and the risk of thrombosisin the antiphospholipid syndrome. Blood 2003;102:2717–23.9. Galli M, Luciani D, Bertolini G, Barbui T. Lupus anticoagu-lants are stronger risk factors for thrombosis than anticardioli-pin antibodies in the antiphospholipid syndrome: a systematicreview of the literature. Blood 2003;101:1827–32.10. Walraven C, Naylor CD. Do we know what inappropriate lab-oratory utilization is? J Am Med Assoc 1998;280:550–8.11. Hindmarsh JT, Lyon AW. Strategies to promote rational clinicalchemistry test utilization. Clin Biochem 1996;29:291–9.12. Chen P, Tanasijevic MJ, Schoenenberger RA, Fiskio J, Kuper-man GJ, Bates DW. A computer-based intervention for improv-ing the appropriateness of antiepileptic drug level monitoring.Am J Clin Pathol 2003;119:432–8.Unauthenticated | 94.39.64.132Download Date | 11/15/12 9:22 AM

High frequency of inadequate test requests for antiphospholipid antibodies in daily clinical practice

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High frequency of inadequate test requests for antiphospholipid antibodies in daily clinical practice

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