Analysis of variability in determining intact parathyroid hormone (iPTH) according to the method used to process the sample
ANÁLISIS DE LA VARIABILIDAD EN LA DETERMINACIÓN DE LA PTH SEGÚN EL MÉTODO EMPLEADO PARA PROCESAR LA MUESTRA
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"paginaFinal" => "335" ] ] "autores" => array:1 [ 0 => array:3 [ "autoresLista" => "Ana Isabel Morales García, José Luis Górriz Teruel, Mª Carmen Plancha Mansanet, Verónica Escudero Quesada, Luis Manuel Pallardó Mateu" "autores" => array:5 [ 0 => array:4 [ "nombre" => "Ana Isabel" "apellidos" => "Morales García" "email" => array:1 [ 0 => "amoralesg@senefro.org" ] "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "affa" ] ] ] 1 => array:3 [ "nombre" => "José Luis" "apellidos" => "Górriz Teruel" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "affa" ] ] ] 2 => array:3 [ "nombre" => "Mª Carmen" "apellidos" => "Plancha Mansanet" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "affa" ] ] ] 3 => array:3 [ "nombre" => "Verónica" "apellidos" => "Escudero Quesada" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "affa" ] ] ] 4 => array:3 [ "nombre" => "Luis Manuel" "apellidos" => "Pallardó Mateu" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "affa" ] ] ] ] "afiliaciones" => array:1 [ 0 => array:3 [ "entidad" => "Servicio de Nefrología y Servicio de Medicina Nuclear Hospital Universitario Dr. Peset Valencia, Valencia, España, " "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "affa" ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "ANÁLISIS DE LA VARIABILIDAD EN LA DETERMINACIÓN DE LA PTH SEGÚN EL MÉTODO EMPLEADO PARA PROCESAR LA MUESTRA" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:8 [ "identificador" => "fig1" "etiqueta" => "Tab. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "copyright" => "Elsevier España" "figura" => array:1 [ 0 => array:4 [ "imagen" => "11818078_t1_333.jpg" "Alto" => 231 "Ancho" => 400 "Tamanyo" => 27381 ] ] "descripcion" => array:1 [ "en" => "Descriptive features of the study population" ] ] ] "textoCompleto" => "<p class="elsevierStylePara"><span class="elsevierStyleBold">INTRODUCTION</span></p><p class="elsevierStylePara">Alterations in the calcium-phosphorus metabolism are a prevalent problem and of great importance to patients with CKD (chronic kidney disease)<span class="elsevierStyleSup">.1-8</span> Appropriate and early evaluation, as well as treatment is vital for optimum control of our patients.</p><p class="elsevierStylePara">The ideal diagnostic method for the grade and type of renal osteodystrophy is still iliac crest biopsy following tetracycline double labelling,<span class="elsevierStyleSup">9-25</span> but, due to its invasive and toilsome nature, it is of difficult application in daily clinical practice.<span class="elsevierStyleSup">26</span> At present, diagnosis and treatment of these alterations are based on the determination of plasmatic levels of intact parathyroid hormone (iPTH).<span class="elsevierStyleSup">10,17,27-31</span> However, the determination of this hormone is not simple and is subjected to great variability.<span class="elsevierStyleSup">32-34</span> In addition to this, methods used to process the iPTH samples are not standardised, a fact which could be an important source of pre-test variability.<span class="elsevierStyleBold"></span></p><p class="elsevierStylePara"><span class="elsevierStyleBold">AIM OF THE STUDY</span></p><p class="elsevierStylePara">To analyse variability in iPTH determination results comparing different methods of processing the same sample of plasma treated with ethylenediaminetetraacetic acid (EDTA) in patients with CKD at different stages of development.</p><p class="elsevierStylePara"><span class="elsevierStyleBold">MATERIAL AND METHOD</span></p><p class="elsevierStylePara">In our study, we analysed 294 blood samples from 49 patients with chronic kidney disease (CKD) at different stages of development, 18 were from transplanted patients at the Renal Transplant Unit (36.7%) and 31 from the Chronic Haemodialysis Unit at our hospital (63.3%).</p><p class="elsevierStylePara">All the blood samples were collected in the morning (between 8 and 9 am) after an eight hour fast and predialysis for the patients undergoing substitutive kidney treatment. The blood was drawn at the Haemodialysis Service of our hospital. Three tubes of EDTA were extracted from each patient: two 5ml tubes and one 10ml tube. Immediately after the extraction, they were sent to the refrigerator at the Nuclear Medicine Department, where each was processed differently, as will be described herewith.</p><p class="elsevierStylePara"><span class="elsevierStyleBold">Reference group or group 1 (EDTA 5ml tube):</span></p><p class="elsevierStylePara">- Blood sample collected (and quickly sent refrigerated to the Nuclear Medicine Department).</p><p class="elsevierStylePara">- Refrigerated centrifugation.</p><p class="elsevierStylePara">- Plasma immediately frozen.<span class="elsevierStyleBold"></span></p><p class="elsevierStylePara"><span class="elsevierStyleBold">Group 2 (EDTA 10ml tube):</span></p><p class="elsevierStylePara">- Blood sample collected.</p><p class="elsevierStylePara">- Maintained one hour at room temperature.</p><p class="elsevierStylePara">- Normal centrifugation.</p><p class="elsevierStylePara">- Separated into 3 tubes:</p><p class="elsevierStylePara">- One of the tubes is immediately frozen. Group 2A</p><p class="elsevierStylePara">- One tube is refrigerated for eight hours and then frozen. Group 2B</p><p class="elsevierStylePara">- Another tube is refrigerated for > 24 hours and frozen. Group 2C<span class="elsevierStyleBold"></span></p><p class="elsevierStylePara"><span class="elsevierStyleBold">Group 3 (EDTA 5ml tube):</span></p><p class="elsevierStylePara">- Blood sample collected.</p><p class="elsevierStylePara">- Maintained > 3 hours at room temperature.</p><p class="elsevierStylePara">- Normal centrifugation.</p><p class="elsevierStylePara">- Separated into two tubes:</p><p class="elsevierStylePara">- Immediately frozen. Group 3A</p><p class="elsevierStylePara">- One tube is refrigerated for > 8 hours and frozen. Group 3B</p><p class="elsevierStylePara">The processing methods used for the blood samples in our study were those usually applied in this domain. The iPTH was measured using the immunoradiometric assay (IRMA Total Intact Scantibodies assay, 2nd generation). The withinrun variability obtained was less than 2.5%. Group 1 was considered the gold standard, since these are the generally recommended conditions for this type of samples, independently of the method or preservative used (EDTA or citrate).</p><p class="elsevierStylePara">A two by two comparison was performed between the iPTH mean values in the reference group and the others. Therefore, the distribution normality was checked first by applying tests of homogeneity variance and normality. Subsequently, we performed a comparison by pairs with the t-test including the Bonferroni correction. The level of significance was established at p < 0.05.</p><p class="elsevierStylePara"><span class="elsevierStyleBold">RESULTS</span></p><p class="elsevierStylePara">The average age of the population studied was 60.88 ± 17.65 and the age range between 26 and 90 years. Of the 49 patients included in the study, 25 were female and 24 male, which corresponds to 51 and 49%, respectively. The main cause of chronic kidney disease is non-affiliated, followed by vascular and glomerular origin. The remaining descriptive features of our study population are displayed in table 1.</p><p class="elsevierStylePara">The mean value in the reference group was 202.25 ± 199.72pg/ml. The mean values of intact iPTH in the other groups analysed, displayed in table 2, were 196 ± 203.23pg/ml, 202.8 ± 200.2pg/ml, 200.06 ± 194.87pg/ml, 204.08 ± 204.073pg/ml and 197.94 ± 182.31pg/ml. As can be seen in figure 1, the results were practically identical for each group, with no important differences with respect to the reference group (p = 0.87, p = 0.99, p = 0.95, p = 0.96, p = 0.90), when comparing with groups 2a, 2b, 2c, 3a and 3b, respectively).<span class="elsevierStyleBold"></span></p><p class="elsevierStylePara"><span class="elsevierStyleBold">DISCUSSION</span></p><p class="elsevierStylePara">Parathormone serum determination is difficult to quantify since it is quite unstable both in serum and in the blood<span class="elsevierStyleSup">43-45</span> (av. 5 minutes). The reference method is based on refrigerated spinning and freezing the sample immediately after extraction. However, this procedure is very difficult in daily clinical practice, since nephrology activity is not only limited to the hospital domain. In general, samples are drawn at the clinics and then sent to the referral hospitals for analysis. On occasion, these clinics are at quite a distance from the hospitals. Most of the samples are not spun or separated in the place of extraction because they do not have the means required to do so. In addition to this, the samples are spun at room temperature. We can derive from this the importance of knowing and reducing the variability to a maximum in the handling of samples.</p><p class="elsevierStylePara">At present, there is no standardised method of processing them. There is some controversy on the stability of parathormone in serum or plasma treated with EDTA. Several studies state that the determination of iPTH in serum is less stable than that obtained from plasma and blood sample conserved in tubes treated with EDTA.<span class="elsevierStyleSup">35-42</span> However, Omar et al.38 in 2001 observed that the levels of iPTH in EDTA decreased 14.8% after remaining at room temperature for 48 hours. In addition to this, recently, Cavalier et al.<span class="elsevierStyleSup">42</span> proved that samples processed with EDTA are not always more stable than in serum. They argue that the stability of the iPTH after 24 hours at -20ºC is greater in serum than for plasma treated with EDTA.</p><p class="elsevierStylePara">In almost all the studies performed to date, serum and EDTA are compared.<span class="elsevierStyleSup">35-42</span> However, one of the limitations which our study presents is that a control group with serum (citrate) was not established. Therefore, we cannot evaluate the determinations of the iPTH in serum and plasma (EDTA) once frozen at -20ºC, nor compare this with the findings of Cavalier et al. In addition to this, we have not analysed what occurs after 48 hours at room temperature in order to compare with the Omar et al. results.</p><p class="elsevierStylePara">The findings of this study show no significant differences between the reference group and the other groups analysed. It is confirmed that the plasma samples treated with EDTA are stable and its use is recommended with refrigerated centrifugation and immediate freezing is difficult to achieve.<span class="elsevierStyleBold"></span></p><p class="elsevierStylePara"><span class="elsevierStyleBold">CONCLUSION</span></p><p class="elsevierStylePara">The use of EDTA as a preservative in the processing of analytical samples for the blood determination of iPTH allows for greater processing time without the requirement of immediate freezing, displaying a minimal variability in the results obtained according to different methods of processing. These results can help to establish logistical strategies for the processing of blood samples in CKD patients.</p><p class="elsevierStylePara"><a href="grande/11818078_t1_333.jpg" class="elsevierStyleCrossRefs"><img src="11818078_t1_333.jpg" alt="Descriptive features of the study population"></img></a></p><p class="elsevierStylePara">Table 1. Descriptive features of the study population</p><p class="elsevierStylePara"><a href="grande/11818078_t2_333.jpg" class="elsevierStyleCrossRefs"><img src="11818078_t2_333.jpg" alt="Mean iPTH value (pg/ml) for each group studied"></img></a></p><p class="elsevierStylePara">Table 2. Mean iPTH value (pg/ml) for each group studied</p><p class="elsevierStylePara"><a href="grande/11818078_f1_334.jpg" class="elsevierStyleCrossRefs"><img src="11818078_f1_334.jpg"></img></a></p><p class="elsevierStylePara">Figure 1. </p>" "pdfFichero" => "P-E-S-A118-EN.pdf" "tienePdf" => true "PalabrasClave" => array:2 [ "es" => array:4 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec439881" "palabras" => array:1 [ 0 => "Procesamiento muestras" ] ] 1 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec439883" "palabras" => array:1 [ 0 => "PTH" ] ] 2 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec439885" "palabras" => array:1 [ 0 => "Estabilidad" ] ] 3 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec439887" "palabras" => array:1 [ 0 => "Variabilidad" ] ] ] "en" => array:4 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec439882" "palabras" => array:1 [ 0 => "processing conditions samples" ] ] 1 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec439884" "palabras" => array:1 [ 0 => "PTH" ] ] 2 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec439886" "palabras" => array:1 [ 0 => "stability" ] ] 3 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec439888" "palabras" => array:1 [ 0 => "Variability" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "es" => array:1 [ "resumen" => "<p class="elsevierStylePara">Introducción: Las alteraciones del metabolismo óseo-mineral presentan una alta prevalencia en los pacientes con enfermedad renal crónica (ERC), siendo mayor conforme avanza el estadio de enfermedad. El diagnóstico de dichas alteraciones se basa fundamentalmente en la determinación de niveles de hormona paratiroide (PTH-i). Sin embargo, la determinación de esta hormona no es sencilla y está sometida a gran variabilidad. Los métodos para procesar las muestras de PTH-i no están estandarizados, hecho que podría ser una fuente importante de variabilidad preanalítica. Objetivo: Analizar la variabilidad en los resultados de la determinación de la PTH-i comparando distintas formas de procesar la misma muestra de plasma tratado conácido etilendiaminotetraacético (EDTA) en pacientes con ERC. Material y métodos: Se han analizado 294 muestras, correspondientes a 49 pacientes con ERC, 18 procedentes de la Consulta de Trasplante Renal (36,7%) y 31 del programa de Hemodiálisis Crónica de nuestro Centro (63,3%). Se ha procesado la misma muestra de cada uno de nuestros pacientes de seis maneras distintas, comparando las medias entre el grupo de referencia o gold standard y los otros grupos a estudio. Las muestras se procesaron con diferentes condiciones de temperatura y tiempo antes de ser congeladas, constituyendo seis grupos: centrifugación y congelación inmediata (grupo 1, de referencia); muestra a temperatura ambiente una hora, centrifugación y mantenimiento en nevera (2-8 ºC) durante 0, 8 o 24 horas (grupos 2 A, 2B y 2C, respectivamente); mantenimiento de sangre a temperatura ambiente 3 horas, mantenimiento en nevera (2-8 ºC) durante 0 y 8 horas (grupos 3A y 3B). La PTH-i se ha determinado mediante Inmunoradiometria (IRMA Total Intact Scantibodies assay). Se ha realizado el test de homogeneidad de varianzas y normalidad, y depués comparaciones por pares con el t-test con la corrección de Bonferroni. Resultados: La PTH-intacta media en el grupo de referencia fue 202,5 ± 199,72 pg/ml. Las medias de PTH-intacta en distintos grupos analizados fueron 196 ± 203,23 pg/ml, 202,8 ± 200,2 pg/ml, 200,06 ± 194,87 pg/ml, 204,08 ± 204,073 pg/ml, 197,94 ± 182,31 pg/ml. Los resultados fueron prácticamente superponibles, no encontrando diferencias significativas respecto al grupo de referencia (p = 0,87, p = 0,99, p = 0,95, p = 0,96, p = 0,90 al comparar con grupos 2A, 2B, 2C, 3A y 3B, respectivamente). Conclusiones: La utilización de EDTA como conservante en el procesamiento de las muestras analíticas para la determinación sanguínea de PTH-i permite un mayor tiempo de procesamiento de la misma, sin la exigencia de su congelación inmediata, mostrando una mínima variabilidad en los resultados obtenidos según diferentes formas de procesamiento. Estos resultados pueden ayudar a establecer estrategias logísticas para el procesamiento de muestras sanguíneas en los pacientes con ERC.</p>" ] "en" => array:1 [ "resumen" => "<p class="elsevierStylePara">Background: The measurement of i-PTH circulating is not easy due to its analytical variablity. Variability that appears in the process that goes from the sample collection to the final result determination. There are several important aspects that can influence within the pre-test variability: type of sample (serum o plasma), temperature, time elapses from blood extraction to freezing and from freezing to i-PTH quantification. Blood coming from centres far from our laboratory do not always meet the required processing conditions. Our aim was to study the stability of i-PTH with varying conditions of temperature and time until freezing in patients with chronic kidney disease (CKD). Method: We have analyzed 294 blood samples of 49 patients with chronic kidney disease (18 transplantated patients (36.7%) and 31 patients in haemodyalisis (63.3%)). The blood samples were collected using tubes treated with ethylenediaminotetraacetic acid (EDTA); these samples were subjected to different conditions of temperature and time before they were frozen, constituting 6 groups: blood centrifuged and plasma immediately frozen (group A or reference group); blood maintained 1 hour at room temperature and plasma stored at 2-8 ºC during 0, 8 and 24 hours (groups B,C,D); blood maintained 3 hours at room temperature and plasma stored at 2-8 ºC during 0 and 8 hours (groups E,F). The intact PTH (i-PTH) was measured using the immunoradiometric assay (IRMA Total Intact Scantibodies assay). We have analyzed the differences between the PTH-i mean values in the referenced groud and the others. We have applied the tests of homogeneity variance and normality and we have perform a comparation by pairs with the t-test including the Bonferroni correction. Results: The mean value of intact- PTH in the referente Group was 202.5±199.72 pg/ml. The means values of intact-PTH in the other groups were 196 ± 203.23 pg/ml, 202.8 ± 200.2 pg/ml, 200.06 ± 194.87 pg/ml, 204.08 ± 204.073 pg/ml, 197.94 ± 182.31 pg/ml. The results were practically identical for each group. We did not find important differences with respect to the reference group (p = 0.87, p = 0,99, p = 0,95, p = 0,96, p = 0,90 when comparing with groups 2a, 2b, 2c, 3a y 3b). Conclusions: The use of EDTA maintain the PTH stability during a longer period without the necessity of freezing the samples immediately. These results can help to state strategies to management the samples in patients with ERC.</p>" ] ] "multimedia" => array:3 [ 0 => array:8 [ "identificador" => "fig1" "etiqueta" => "Tab. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "copyright" => "Elsevier España" "figura" => array:1 [ 0 => array:4 [ "imagen" => "11818078_t1_333.jpg" "Alto" => 231 "Ancho" => 400 "Tamanyo" => 27381 ] ] "descripcion" => array:1 [ "en" => "Descriptive features of the study population" ] ] 1 => array:8 [ "identificador" => "fig2" "etiqueta" => "Tab. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "copyright" => "Elsevier España" "figura" => array:1 [ 0 => array:4 [ "imagen" => "11818078_t2_333.jpg" "Alto" => 97 "Ancho" => 826 "Tamanyo" => 17417 ] ] "descripcion" => array:1 [ "en" => "Mean iPTH value (pg/ml) for each group studied" ] ] 2 => array:7 [ "identificador" => "fig3" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "copyright" => "Elsevier España" "figura" => array:1 [ 0 => array:4 [ "imagen" => "11818078_f1_334.jpg" "Alto" => 352 "Ancho" => 397 "Tamanyo" => 16577 ] ] ] ] "bibliografia" => array:2 [ "titulo" => "Bibliography" "seccion" => array:1 [ 0 => array:1 [ "bibliografiaReferencia" => array:45 [ 0 => array:3 [ "identificador" => "bib1" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:3 [ "referenciaCompleta" => "1.- Mann JFE, Gerstein HC, Pogue J, et al. 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| Year/Month | Html | Total | |
|---|---|---|---|
| 2024 November | 11 | 8 | 19 |
| 2024 October | 85 | 56 | 141 |
| 2024 September | 89 | 45 | 134 |
| 2024 August | 73 | 69 | 142 |
| 2024 July | 52 | 37 | 89 |
| 2024 June | 66 | 49 | 115 |
| 2024 May | 67 | 40 | 107 |
| 2024 April | 166 | 39 | 205 |
| 2024 March | 76 | 29 | 105 |
| 2024 February | 38 | 41 | 79 |
| 2024 January | 39 | 33 | 72 |
| 2023 December | 41 | 29 | 70 |
| 2023 November | 32 | 31 | 63 |
| 2023 October | 62 | 36 | 98 |
| 2023 September | 60 | 33 | 93 |
| 2023 August | 44 | 25 | 69 |
| 2023 July | 67 | 24 | 91 |
| 2023 June | 58 | 17 | 75 |
| 2023 May | 95 | 31 | 126 |
| 2023 April | 44 | 23 | 67 |
| 2023 March | 69 | 24 | 93 |
| 2023 February | 46 | 26 | 72 |
| 2023 January | 44 | 43 | 87 |
| 2022 December | 43 | 44 | 87 |
| 2022 November | 48 | 44 | 92 |
| 2022 October | 48 | 51 | 99 |
| 2022 September | 58 | 28 | 86 |
| 2022 August | 49 | 42 | 91 |
| 2022 July | 44 | 51 | 95 |
| 2022 June | 46 | 37 | 83 |
| 2022 May | 82 | 18 | 100 |
| 2022 April | 57 | 35 | 92 |
| 2022 March | 48 | 40 | 88 |
| 2022 February | 65 | 36 | 101 |
| 2022 January | 63 | 27 | 90 |
| 2021 December | 48 | 39 | 87 |
| 2021 November | 41 | 36 | 77 |
| 2021 October | 46 | 29 | 75 |
| 2021 September | 31 | 38 | 69 |
| 2021 August | 36 | 22 | 58 |
| 2021 July | 50 | 35 | 85 |
| 2021 June | 42 | 13 | 55 |
| 2021 May | 46 | 32 | 78 |
| 2021 April | 104 | 14 | 118 |
| 2021 March | 100 | 37 | 137 |
| 2021 February | 91 | 24 | 115 |
| 2021 January | 71 | 12 | 83 |
| 2020 December | 45 | 9 | 54 |
| 2020 November | 42 | 13 | 55 |
| 2020 October | 39 | 6 | 45 |
| 2020 September | 35 | 11 | 46 |
| 2020 August | 54 | 4 | 58 |
| 2020 July | 50 | 8 | 58 |
| 2020 June | 51 | 7 | 58 |
| 2020 May | 65 | 15 | 80 |
| 2020 April | 62 | 8 | 70 |
| 2020 March | 95 | 8 | 103 |
| 2020 February | 76 | 17 | 93 |
| 2020 January | 111 | 33 | 144 |
| 2019 December | 104 | 16 | 120 |
| 2019 November | 92 | 19 | 111 |
| 2019 October | 74 | 7 | 81 |
| 2019 September | 83 | 23 | 106 |
| 2019 August | 54 | 7 | 61 |
| 2019 July | 90 | 21 | 111 |
| 2019 June | 84 | 13 | 97 |
| 2019 May | 79 | 9 | 88 |
| 2019 April | 140 | 50 | 190 |
| 2019 March | 68 | 24 | 92 |
| 2019 February | 27 | 15 | 42 |
| 2019 January | 46 | 18 | 64 |
| 2018 December | 66 | 25 | 91 |
| 2018 November | 72 | 15 | 87 |
| 2018 October | 122 | 14 | 136 |
| 2018 September | 65 | 14 | 79 |
| 2018 August | 32 | 14 | 46 |
| 2018 July | 63 | 12 | 75 |
| 2018 June | 41 | 10 | 51 |
| 2018 May | 50 | 18 | 68 |
| 2018 April | 34 | 7 | 41 |
| 2018 March | 42 | 6 | 48 |
| 2018 February | 32 | 8 | 40 |
| 2018 January | 52 | 7 | 59 |
| 2017 December | 46 | 11 | 57 |
| 2017 November | 50 | 8 | 58 |
| 2017 October | 46 | 13 | 59 |
| 2017 September | 51 | 7 | 58 |
| 2017 August | 52 | 14 | 66 |
| 2017 July | 46 | 5 | 51 |
| 2017 June | 69 | 15 | 84 |
| 2017 May | 61 | 22 | 83 |
| 2017 April | 67 | 12 | 79 |
| 2017 March | 41 | 7 | 48 |
| 2017 February | 73 | 17 | 90 |
| 2017 January | 64 | 13 | 77 |
| 2016 December | 53 | 4 | 57 |
| 2016 November | 63 | 10 | 73 |
| 2016 October | 142 | 7 | 149 |
| 2016 September | 161 | 16 | 177 |
| 2016 August | 217 | 9 | 226 |
| 2016 July | 140 | 6 | 146 |
| 2016 June | 142 | 0 | 142 |
| 2016 May | 138 | 0 | 138 |
| 2016 April | 91 | 0 | 91 |
| 2016 March | 87 | 0 | 87 |
| 2016 February | 128 | 0 | 128 |
| 2016 January | 116 | 0 | 116 |
| 2015 December | 121 | 0 | 121 |
| 2015 November | 97 | 0 | 97 |
| 2015 October | 95 | 0 | 95 |
| 2015 September | 87 | 0 | 87 |
| 2015 August | 86 | 0 | 86 |
| 2015 July | 66 | 0 | 66 |
| 2015 June | 42 | 0 | 42 |
| 2015 May | 54 | 0 | 54 |
| 2015 April | 5 | 0 | 5 |
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