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    Clinical SS - 2018 Nguyen Khoa - Ann Biol Clin

    Uploaded by

    JoseAngelBarreraAlvarez

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    © All Rights Reserved
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    ee IOLOGIE Original article INIQUE sn Bi Cin 208: 704: 416-20 Sickle SCAN™ (BioMedomics) fulfills analytical conditions for neonatal screening of sickle cell disease Intérét du Sickle SCAN™ (BioMedomics) pour le dépistage néonatal de la drépanocytose Thao Nguyen-Khoa’ Abstract. Sickle SCAN™ is a rapid, qualitative, point-of-care lateral flow Louis Minet immunoassay for the identification of AS, AC, SS/SBYthal, SC and CCICB°thal Bichr Ala? phenotype. We evaluated this testunder the conditions encountered the French newborn screening (NBS) program for sickle cell disease: a total of 104 dried : 6 Sepa ie blood spots (DBSs) were tested with an HPLC reference method and then with Christelle Remus" the Sickle SCAN™ device. Sickle SCAN™ identified the hemoglobin (Hb) Senses chee phenotype comtectly on 96% of cases. In the four non-concordant cases, the Valérie Gauthereaus antibody anti-HbS cross-reacted with HE (n=2), HbD (n=1) o HDX (1t=1) Sarah Enouz+ ‘There were no false negative, In order to test Sickle SCAN™!s sensitivity to | magtebnes (HS) HbA, HDS ot HC pour Tienicalon des phenotypes cinipah nytt at os hapoghooe- AS, AC, SS/SB°thal, SC et CC/CB"thal. Nous avons évalué ce TDR dans les pater eres ace conditions du programe fans de istage ntl de a repanocyose hhormonologie, Hépital Robert Debré, 104 papiers buvards & partir desquels les taches de sang séché ont été testées AP-HP, Paris, France ; avec la méthode chromatographique de référence du programme de dépistage, \Départomont do biothérapie, Hépital OLAS CONTI Ears programme de dépistage jc TDR Sickle SCAN™, Dans 96 % des cas, es phénotypes de Hb universitairo Neckor-Entants malades, a ‘AP-HP, Paris, France ‘ont été correctement idemtifiés, Pour les quatre eas discordant, anticorps anti- 4 Centre investigation cinique, HDS a 1éagi de manigre croisée avec PHbE (n= 2), lHbD (n = 1) et avee une opiaux universtaros Parc-Oucet, HDX (n= 1). Il n'y a pas eu de faux négatif. D’autre part, 21 autres papiers AP-HR,Inserm, Paris, Franco buvards ont servi A tester la sensibilité du Sickle SCAN™ avec des faibles § Fédération parsienne pout lo taux d’HbA ct HbS (0,6 2 4,2 % d’HDA et 2,0 4 69 % eHDS). Les HDA et opistago eta prevention des ; HBS ont toujours 66 deectées lonquelles sient présontes a pls de 1% Tatescapedetrfant, HOptl Unverstare Necker tents malades, et 2 Sb, respectivement, Le TDR Sickle SCAN™ est sensible et spécifique tin Par france pour "identification des nouveat-ns présenant un phénotype drépanocytie Reprints: T Nouyen-Khoa Toc hisaaick: Nguyen Khon T, Mine LAUT, Ribel 1A, Remus C, Simin A, Gaserau 416 | 2 Gio Caracam tt Sec SCAN (desis soa contin nl ena of eel An ed Ch SM ATO See cll as. Sole 1: TG: 1620 abiccatcontse © BioMedomics Ine., Durham, North Carolina, USA. 7 EurocordiMonacors, Hopital Saint-Louis, AP-HP, Pais ol Centre scientifique de Monaco, Monaco, Franco ® Département de génétique, Insitute Imagine, Pris, France Mots clés: gnostic rapide Article received May 04, 2018, accepted May 26, 2018 Sickle SCAN™ is 2 new point-of-care test (POCT) commercialized by BioMedomics, Inc (www.biomedomics.com, USA) for the diagnosis of sickle cell disease (SCD) via the specific identification of Patients with AS or AC trait, SS/SB°thal, SC or CCICBtal phenotype. Hereafter, the term “HDX” is used to refer to ‘unknown Hb, In newborns, fetal Hb (HF) stays at a high level for several months after bitth, and then falls to the adult level by 12 months. The newborn's HDF phenotype is expressed as FA for normal Hb and FAS, FAC, FAD, FAE, FS, FC, and so on for newborns with sickle cell trait Mb, As a POCT, the device must be practical and rapid to tse, and capable of identifying the SCD phenotype with high levels of sensitivity and specificity. Kanter et al. (1] validated the Sickle SCAN™ device using 71 capillary blood samples from 21 children (mean age of 13.5 years) and 50 adults. Furthermore, MeGann ef al. [2] tested 139 venous and capillary blood samples from pediatric and adult patients and also 7 dried blot spot (DBS) samples (by transferring venous blood! onto newborn screening (NBS) card), ‘The Necker pediatric hospital Paris, France) specializes in the treatment of SCD. In 2016, the lle-de-France regional NBS service (Fédération parisienne pour le dépistage et la prévention des handicaps de l'enfant, Pacis, France) tested 132115 DBS cards for SCD (representing 40% of the national cohort); the regional incidence of the disease was I in S41 [3]. ‘The objective ofthe present study was to evaluate the qual- ity of the Sickle SCAN™ procedure with reference to the high-performance procedures used in our laboratories. Under the conditions used in the regional NBS program, we tested 104 DBS cards with an unknown phenotype and 21 DBS for which the phenotype was known (with ‘ high level of HBF and low levels - betwreen I and 10% - of either HbA and sickle cell Hb (HS), as observed in newborns). ‘Ar Bll Cn, 76, 4, jlo 2018 I répond au critéres de qualité des TDR pour Vorientatio ddrépanocytose et sera particuligrement utile dans les pays d'endémie ob "acces aux équipements de laboratoire est trés limite Sickle SCAN™ for neonatal screening of SCD liagnostique de ta irépanoeytose, hémoglobine S, dépistage néonatal, test de dia- Materials and methods Samples ‘The NBS laboratory supplied ourstudy with 104 DBS cards with sufficient dried blood to allow retesting for the Hb trait with the Sickle SCANT device. The Sickle SCAN™ ‘operators were blinded to the phenotype determined with the reference technique. To test the Sickle SCAN™” sen- sitivity, a separate set of 21 DBS cards with a known, low proportion of HbA and HbS were selected. A 3 mm dise ‘was punched out of the DBS card and tested with the Sickle SCAN™ device. High-performance liquid chromatography (HPLC) ‘The regional NBS laboratory analyzes Ho from DBS cards, with an HPLC-based VARIANT nbsTM system (BioRad Laboratories, Matnes-La-Cogueite, France); this served as the reference method in our study. The Sickle SCAN™ procedure Sickle SCAN™ js a chromatographic sandwich immunoassay for the qualitative detection of human HbA, HbS and HBC in a blood sample [1, 2}. The kit includes tubes prefilled with 1.0 mL of hemolysis buffer and a testing cartridge. The dise punched out of a DBS is introduced into a prefilled tube. After 5 minutes, 5 drops of hemolysate are placed in the cartridge inlet. The latter contains mouse monoclonal antibody (Ab) conjugated to blue-colored nanoparticles and directed against the C-terminus of the human Hb a-chain, The conjugated Ab thus forms a complex with any Hb present in the sample. Next, the complex diffuses through an absorbent strip containing three test lines (containing rabbit monoclonal capture Abs against the N-terminal amino acid sequence ‘of human sickle cell Hb (HbS or HbC) and normal Hb (HbA), respectively) and a control line (containing a goat 47 Original article anti-mouse IgG Ab). Each specific Hb-Ab complex is captured at the corresponding test line, where it produces a blue-colored band. Excess conjugate flows past the test lines and is captured on the control line. ‘The test result is, read after 5 minutes of migration, Phenotyping DBS cards were tested first with the HPLC method and then with the Sickle SCAN™ device, All the Sickle SCAN™. tests were performed by the same operator, The presence or absence of Sickle SCAN™ bands was observed by two dif ferent readers, on a double-blind basis. The Sickle SCAN™ phenotype was classified as a function of the Hb band intensity Results Diagnostic accuracy in SCD ‘The Sickle SCAN™ results were the same for the two read= ers, who found the Hb bands easy (0 read (table 1). The ‘operator found the procedure easy to perform, with few handling stops. Sickle SCAN™ jdentified the Hb phenotype correctly on 100 of the 104 (969) DBS cards (lable 1). In the four non-concordant cases, Sickle SCAN™ detected two FAE phenotypesas FAS, one FAD phenotypeas FAS, and a FAX phenotype as FAS (table 1) Interestingly, Sickle SCAN™ detected a sickle cell trait hemoglobin in all four cases; the cexact phenotypes could be determined later using the HPLC reference method Sensitivity for the detection of low levels of HbA and HbS in the presence of high HOF levels We selected 21 samples with low levels of HbA and HbS, 4s determined by HPLC (ranges of 0.6-4.2% and 2.0-6.9%, respectively; fable 2). When the percentage of HbA was lower than that of HbS, the phenotype was supposedly FSBtthal, When the percentage of HbA was higher than that of HbS, the phenotype was supposedly FAS, When the percentages of HbA and HbS were respectively higher than 1% and 2%, these Hb were always detected by the Sickle SCAN™ test - even in the presence of high HbF levels (table 2) Sickle Sean™ thus met the sensitivity criteria for SCD NBS programs. Discussion Sickle SCAN™ is a rapid, qualitative, pointof-eare lat- eral flow immunoassay for the identification of AS, AC, By ‘88/8 °thal, SC and CC/CB%Ahal phenotype. We evaluated this test under the conditions encountered in the French NBS program for SCD: a total of 104 DBSs were tested with an HPLC reference method and then with the Sickle SCANT device. Sickle SCAN™ identified the Hb phe notype correctly in the presence of high HbF levels, on 96% of the DBS cards. In the four non-concordant cases, the antibody anti-HbS cross-reacted with HbE (PAE phe- notype, n=2), HbD (FAD phenotype, n=1) or HBX (FAX phenotype, n=l). However, this would not have had an impact on the newborn's medical care; when an HbX result is obtained, the exact Hb phenotype is determined later using the HPLC reference method. There were no false negatives. Hence, Sickle SCAN™ appears to be an accurate point-of-care method for the identification of AS, AG, SS/SBMhal, SC and CCICMthal phenotypes on DBS, cans. ‘The Sickle SCAN™ device’s analytical qualities have been extensively documented by Kanter ¢7 af [1] and MeGann ¢7 cal, [2] with capillary and/or venous blood samples present- ing & broad panel of abnormal Hl phenotypes. Sensi and specificity were above 98% fore di SC [1, 2}. Out results attest to Sickle SCAN™’s analyti reliability - even in newborns with high levels of HBF, and for HDA and HS percentages as low as 1% and 2.3%, respectively. Ourpresent results demonstrate that the Sickle SCAN™ testis able to discriminate between normal and ‘SCD phenotypes under neonatal sereening conditions. In developed countries, Hb phenotype analysis in NBS programs is based on HPLC, capillary electrophoresis, isoelectric focusing, tandem mass spectrometry (MS/MS) 4}, Other new methods are also currently emerging, e.g MALDI-TOF (matrix assisted laser desorption ionisation - time of flight)-MS, DNA-based methods, However, these ‘methods are costly and require a specialist operator -concl- tions that are not always met in SCD-endemic countries. This is why POCTS might be of great value for NBS pro- ssrams in these countries. A number of POCTs for SCD have been developed, on the basis ofthe particular characteristics of red blood cells (RBCS) in SCD: ~ density measurements [5, 6]. The polymerization of HbS increases the RBCs’ density. However, this approach does not discriminate between AS and AA phenotypes, and cen- (vfugation is required for a rapid result (12 min, versus hours with gravity flow in the absence of centrifugation); — measurements of the dynamic deformability and adhe- sion of sickled RBCs under flow conditions mimicking the post-capillary venule (7]. This approach is limited by inter ference from high levels of HDF, and the complexity of the microfluidic technology: ~ a paper-based sickle cell anemia test, based on the fact that HbS is less soluble than HbA, HbE, and HbC {8-10}. ‘This approach has two disadvantages (i) AS and SC pheno- ‘re Bel Cin, 7614, lle act 2018 Sickle SCAN™ for neonatal screening of SCD. Table 1. Comparison of hemoglobin phenctyping results using tho HPLC Variant nbs"™ reference method (BioRad) andthe Sickle SCANT™ chromatographic immunoassay (BioMedomes Inc). Hemoglobin phenotype PLC Variant nibs™ Sete Scan"™ Number rosults, FA 6 8 ° FAS % —~o =a FAC 7 ° FS (S556) 19 ° Fs0 5 ° FAK 8 TRS FAE 5 i BAS FAD 6 = 1 As. Fo (6000p) 3 0 FABarrs 2 ° FAO-Aab 1 0 Talal 108 4 ‘Table2. Sensitivty ofhomogiabia (Hb) phonclype dotocion using the Sickle SCANT chromatographic immunoassay witniow porcontagos (of HA or HbS determined by HPLC Variant nbs™ referanco mathod. Hb phenotype HPLC HAs HPLC HOS% Sickle Sean™ test FS 00 ar s FSpr 06 s FSpr 08 s FSpe 10 ‘SiAlow Fspe 10 ‘S+Alow FSpe 10) SiAlow Fspe 13 ‘S#A low Fspe 14 ‘SAlow Fspe i 1a SiAlow Fspe — 15. SAlow FSpe 15) if S+Aow FSpe 18. SrAlow FSpe 18. S+Alow FSi 18. SrAlow FSpe 17 SeAlow FSpe 4 ‘SeAlow FAs: 26 aS. FAS. - aS. FAS aS. FAS aS. FAS aS types give the same result, and (i) low levels of HbS cannot de detected in the presence of high levels of HUF; = lateral-low immunochromatographic assays [11] such as HemoTypeSC™ (Silver Lake Research Corporation, CA, USA) [12] and Sickle SCAN™ [1, 2, 13]. The ‘ovo tests are based on the same principle, both using ‘monoclonal Abs. The Sickle SCAN™ test is easier 10 ‘nn 810 Cin, v.78, 4, let 0012088 ‘operate than the HemoTypeSC™ which requires more handling. steps and more reagents. Furthermore, Sickle SCAN™'s optical detection procedure is enhanced by the complex formation with blue colored beads. This means that Sickle SCAN™ has a relative high level of sensitivity when the sample contains low levels of SCD Hb, 419 Original article ‘The Sickle SCAN™ test may be of value in SCD-endlemic countries, where laboratories are not always equipped with HPLC systems. Sickle SCAN™ does not require a labora tory environment or the use of electronic.equipment. It was very easy fo use, and the detection of SCD Hb (enhanced by bbluc dye) was easily read by an operator with no training in laboratory techniques. A study in Nigeria has demonstrated the practicality of the Sickle SCAN™ device when used by non-expert operators [12] Sickle SCAN™ might also be useful in emergency situa- tions, when the laboratory is too faraway for rapid, adequate diagnostic processing, Conclusion ‘When tested on a series of DBS cards from newboms, the Sickle SCAN™ test appears to be an accurate method for the identification of patients with AS or AC wait, SS/SBPthal, SC or CCICBYthal phenotype - even in the presence of high level of HbF and low levels of HA and HbS (> 1% and >2.3%, respectively). Although false posi tive tests were observed for some samples containing HbE ‘or HbD, these would not have had an impact on medical cate, More importantly, no false negatives were found with regard to the identification of HbS and HbC. The Sickle SCAN™ test meets the analytical criteria for NBS for ‘SCD in endemic countries with poor access to laboratory equipment. Conflict of interest: J. S. Kim: employee of BioMe-

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