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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CURSO DE GRADUAÇÃO EM FARMÁCIA

DILUIÇÕES DE AMOSTRAS: SOLUÇÃO PARA REALIZAÇÃO DE EXAMES LABORATORIAIS EM NEONATOS?

Júlia Guedes de Araújo Duavy

Natal - RN 2020

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Júlia Guedes de Araújo Duavy

DILUIÇÕES DE AMOSTRAS: SOLUÇÃO PARA REALIZAÇÃO DE EXAMES LABORATORIAIS EM NEONATOS?

Natal - RN 2020

Trabalho de Conclusão de Curso apresentado ao Curso de Graduação em Farmácia do Centro de Ciências da Saúde da Universidade Federal do Rio Grande do Norte como requisito parcial para a obtenção do grau de bacharel em Farmácia.

Orientadora: Profa. Dra. Marcela Abbott Galvão Ururahy

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Universidade Federal do Rio Grande do Norte – UFRN Sistema de Bibliotecas – SISBI

Catalogação de Publicação na Fonte. UFRN – Biblioteca Setorial do Centro de Ciências da Saúde – CCS

Duavy, Julia Guedes de Araújo.

Diluições de amostras: solução para realização de exames

laboratoriais em neonatos? / Julia Guedes de Araújo Duavy. - 2020. 43f.: il.

Trabalho de Conclusão de Curso - TCC (Graduação em Farmácia) - Universidade Federal do Rio Grande do Norte, Centro de Ciências da Saúde, Departamento de Análises Clínicas e Toxicológicas. Natal, RN, 2020.

Orientadora: Marcela Abbott Galvão Ururahy.

1. Diluição- TCC. 2. Biochemical tests - TCC. 3. Serum - TCC. 4. Plasma - TCC. 5. Neonates - TCC. 6. Phlebotomy - TCC. I. Ururahy, Marcela Abbott Galvão. II. Título.

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Júlia Guedes de Araújo Duavy

DILUIÇÕES DE AMOSTRAS: SOLUÇÃO PARA REALIZAÇÃO DE EXAMES LABORATORIAIS EM NEONATOS?

__________________________________________________________________ Presidente: Profa. Marcela Abbott Galvão Ururahy, Dra. – Orientadora, DACT/UFRN

__________________________________________________________________ Membro: Prof. Rand Randall Martins, Dr. – DFAR/UFRN

__________________________________________________________________ Membro: Karla Simone Costa de Souza, Dra. – PPgCF/UFRN

Natal, 20 de julho de 2020

Trabalho de Conclusão de Curso apresentado ao Curso de Graduação em Farmácia do Centro de Ciências da Saúde da Universidade Federal do Rio Grande do Norte como requisito parcial para a obtenção do grau de bacharel em Farmácia.

Orientadora: Profa. Dra. Marcela Abbott Galvão Ururahy

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Dedico todo o empenho deste trabalho para a minha família, que sempre incentivou e apoiou meus sonhos, contribuindo diariamente para que eu me torne constantemente uma pessoa melhor.

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AGRADECIMENTOS

A gratidão é um sentimento forte capaz de mudar a perspectiva das situações. Ser grato é se encher de bons sentimentos e ter a certeza de que tudo na vida, de uma forma ou de outra, te soma. Sabendo disso, meu sentimento nesse momento não poderia ser outro que não a gratidão por toda a trajetória que me trouxe até aqui e me ajudou a olhar as coisas de forma mais gentil.

Sou grata, primeiramente, a Deus, por me conhecer profundamente e me apresentar a essa profissão linda, que é ser farmacêutico. Sou grata a Ele e a Nossa Senhora por me sustentar em todas as dificuldades e por me fazer persistir em meio as adversidades para que, hoje, eu possa estar concluindo mais um ciclo.

Agradeço a minha família por sempre batalhar intensamente para possibilitar minha formação, não só educativa, mas como pessoa, principalmente. Aqui, abro espaço para agradecer diretamente aos meus pais, Ana Paula e Paulo, e aos meus irmãos, Sofia e Arthur, essa conquista também é de vocês. Agradeço aos meus avós Paula, Lena, Armando e Roberto e a minha tia-avó Marta, alicerces de quem eu sou, que sempre zelaram pela minha vida e por minha jornada.

Aos meus professores, que sempre prezaram por nos ensinar com excelência, sendo instrumentos e exemplos fundamentais na formação dos futuros profissionais que seremos. A eles, meu reconhecimento.

Agradeço, em especial, à minha orientadora, pela oportunidade oferecida, pela confiança cedida e pela amizade construída. Com quem aprendi para além de conhecimentos técnicos, valores. Meus sinceros agradecimentos e admiração a você, Marcela Ururahy.

Agradeço aos meus colegas de turma, em especial aos meus amigos Amanda Cirne, Arlene da Câmara, Ester Oliveira, Daniele Cavalcante, Gabriela Suassuna, Lucas Andriel e Melissa Farache, com quem dividi os obstáculos, as superações e os bons momentos, que serão sempre marcados em meu coração.

Aos meus colegas do Grupo de Pesquisa em Doenças Metabólicas (GPDM) da Universidade Federal do Rio Grande do Norte, por todo conhecimento compartilhado. Em especial, a Antonnyo Palmielly, com quem pude expandir meus conhecimentos e ao meu amigo Jardson Oliveira, que dividiu comigo de perto esta última etapa do curso.

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Ao meu namorado, Thiago Gadelha, por sempre estar disposto a me ouvir e ajudar. Tendo sido por todos esses anos um grande incentivador e apoiador dos meus sonhos e planos.

As minhas queridas amigas Gabriela Rocha, Nathália Marques e Vanessa Gomes por serem minha base, dividirem a vida, as batalhas e as conquistas, sempre disponíveis para escutar, ser amparo e fortaleza.

A todos que cruzaram meu caminho, deixaram um pouco e levaram outro tanto. Meu sincero, muito obrigado!

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“A melhor forma de prever o futuro é criá-lo” - Peter Drucker.

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SUMÁRIO

1. INTRODUCTION... 03

2. MATERIALS AND METHODS... 05

2.1 SUBJECTS... 05

2.2 SAMPLE COLLECTION, PROCESSING AND DILUTION... 05

2.3 BIOCHEMICAL EVALUATION... 05 2.4 STATISTICAL ANALYSIS... 06 3. RESULTS... 07 4. DISCUSSION... 11 5. CONCLUSION... 14 DECLARATION OF INTEREST... FUNDING SOURCES... ACKNOWLEDGMENT... 14 14 14 REFERENCES... SUPPLEMENTARY TABLE 1... ANEXO 1 - NORMAS DA REVISTA...

15 19 20

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1

DILUIÇÃO DE AMOSTRAS: SOLUÇÃO PARA A REALIZAÇÃO DE EXAMES LABORATORIAIS EM NEONATOS?

SAMPLES DILUTION: SOLUTION FOR CARRYING OUT LABORATORY TESTS IN NEONATES?

Júlia Guedes de Araújo Duavya Antonnyo Palmielly Diógenes Limaa Stephany Ann Costa do Rosarioa Renata Kelly de Freitas Manoa Marcela Abbott Galvão Ururahya,*

aDepartment of Clinical and Toxicological Analyses, Federal University of Rio Grande

do Norte, Natal - RN, Brazil. R. Gen. Gustavo Cordeiro de Farias, S/N - Petrópolis, Natal - RN, 59012-570.

*Author’s contact information: Marcela Abbott Galvão Ururahy

Address: Rua General Gustavo Cordeiro de Farias, S/N. Centro de Ciências da Saúde, Faculdade de Farmácia, Laboratório de Bioquímica Clínica. Bairro: Petrópolis, Natal/RN, Brazil

Phone number: +55 84 3342-9807 Fax number: +55 84 3342-9833

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2

ABSTRACT

Blood collection in children is a challenging reality compared to adults. The low volume of whole blood of a newborn can suffer a strong impact when laboratory tests are required. Laboratory blood spoliation is related to the intensification of physiological anemia of the newborn, which may predispose to the need for blood transfusion. Recognizing the importance of laboratory tests for screening and monitoring of pathologies in neonates, the objective of this study was to investigate serum/plasma dilution as a possible solution to reduce the volume collected in newborns. Blood samples were obtained from 20 adult volunteers. The blood was collected in a tube containing a clot activator and serum gel separator and, also, in a tube containing the ethylenediaminetetraacetic acid (EDTA) anticoagulant. The serum and plasma were diluted in the proportions of 1:2, 1:3, 1:4 and 1:5, and subsequently, analyzing 19 biochemical parameters. For analysis of the effect of dilution on the values in the samples, linear regression of random effects was used (p<0.05). Uric acid, albumin, aspartate aminostransferase, total bilirubin, globulin, total protein and lactate dehydrogenase were stable in both serum and plasma dilutions. These results may indicate that the pre-dilution of serum and plasma samples for these parameters may bring benefits to clinical practice, especially neonatal. However, further studies involving the dilution of serum/plasma and biochemical analyses should be performed to validate this methodology and confirm the results by this study.

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1. INTRODUCTION

The neonatal period may be marked by the presence of some relatively frequent health complications regarding neonatal units, such as Respiratory Distress Syndrome, Peri-Intraventricular Hemorrhage, epileptic seizures, sepsis, jaundice, and glucose, calcium and magnesium metabolism disorders may also occur [1,2,3]. Some factors determine the periodicity of laboratory tests, such as gestational age and the acuity of the disease. In general, premature children who develop an acute condition tend to require more frequent examinations, especially when they evolve with severe cardiorespiratory and infectious diseases, increasing the blood loss by phlebotomy [4,5].

The practice of blood collection in pediatrics is a challenging reality when compared to adults, especially when considering the neonates [4]. For the neonatal population, some pre-analytical variables are extremely important, such as age, the volume to be collected and the proportion of anticoagulant [6]. In small volume samples, the amount of liquid anticoagulant present in the collection tube may dilutes the sample, impairing the expected results, as well as hemolysis, harmful effect of glucose metabolism and evaporation can also happen [4].

The amount of volume required to perform laboratory tests is dependent on some factors, such as the material available and used for collection, the instruments and laboratory equipment, how many tests this equipment performs at the same time and whether the analysis of the reference laboratories need specific requirements regarding the samples volume [7].

It is known that neonates may present a physiological anemia in the postnatal period due to the replacement of fetal hemoglobin by adult, the shorter lifespan of Red Blood Cells (RBC), the increased sensitivity to oxidative lesions and the decrease in erythropoietin [8,9,5]. In addition, the need for laboratory tests for the follow-up of neonates with serious illnesses and iatrogenic blood loss, especially in the first weeks of life, represents an indispensable factor for the event of neonatal anemia [10,11,12,5]. This phenomenon can also be called "Laboratory Hemorrhage", and premature newborns may develop more pronounced anemias (prematurity anemia), which becomes even more significant when expressed in low birth weight and very low birth weight infants [13,9,10,5].

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4 In a pediatric Intensive Care Unit (ICU), the average volume of blood loss due to phlebotomy reached 2.7 ± 2.3 mL by collection and there was an average of three blood collections per day [14]. It is known that neonates correspond to a population with a reduced blood volume, therefore more susceptible to blood spoliation related to laboratory tests [14]. Aladangady et al. [15] and Leung et al. [16] demonstrated total blood volumes of 68.9 mL/Kg and 70.8 mL/Kg, respectively. For the Brazilian Society of Clinical Pathology/Laboratory Medicine [17], the volume of blood circulating in newborns is approximately 75 to 110 mL/Kg and it is considered safe to collect blood up to 3 mL/Kg at each puncture. In cases where there is a need for multiple collections, it is recommended that only 5-10% of the total volume be collected in a period a month [17]. However, in practice, it is known that depending on the clinical situation of the patient, there is a need to perform phlebotomy frequently, leading to iatrogenic anemia, an event that may predispose in this population the need for blood transfusion [14,10,11,17].

Thus, anemia and RBC transfusion are issues present and worrying in pediatric and neonatal ICU [14,9], where neonates with a birth weight of less than 1500 g are likely to receive at least one blood transfusion [18] during hospitalization, and the volume of transfusion is inversely proportional to gestational age and birth weight [13].

The best way to reduce the need of transfusion in neonates is to invest in prevention, which includes reducing blood spoliation to perform laboratory tests and/or the volume collected by phlebotomy [12,19,20,5,10].

So, considering the importance of laboratory tests with the objective of screening and monitoring diseases in neonates, especially those who have the need for hospitalization, it is necessary to deepen solutions that optimize the volume of blood samples collected, aiming to reduce the spoliation and risks caused by the wide blood collection during hospitalization. Thus, the objective of this study is to investigate the possibility of performing biochemical tests on diluted serum and plasma samples.

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2. MATERIALS AND METHODS 2.1 Subjects

Twenty volunteers over the age of 18 years of both sexes, including students, professors and employees of the Federal University of Rio Grande do Norte (UFRN), who did not present any previous diagnosed metabolic disease, were invited to participate in the study. Exclusion factors were diagnosis of anemia and/or other hematological diseases.

The study protocol was approved by the Research Ethics Committee of the University Hospital Onofre Lopes (HUOL)/UFRN under the number 3.617.017. All the individuals who participated in the study signed the Written Informed Consent Form before the blood collection.

2.2 Sample collection, processing and dilution

Blood samples (8mL) were collected after a 12-14 h fasting, in 2 tubes of 4 mL, one tube with clot activator and separator gel and another tube with tetra-acetic ethylenediamine anticoagulant (EDTA). The tubes were wrapped in aluminum foil in order to avoid light interference in the determination of bilirubin.

After collection, the samples were centrifuged at 3,000 rpm for 10 minutes. Then, the serum (for the tubes containing the clot activator and separator gel) and plasma (for the tubes containing the anti-coagulant EDTA) were separated to proceed with the dilutions. No hemolysis was observed in any of the samples.

Each serum and plasma sample were diluted with 0.85% saline solution, according to the information provided by the manufacturer's determination kits, in the proportions of 1:2, 1:3, 1:4 and 1:5.

Blood samples collection, processing and dilution were carried out in the Clinical Biochemistry laboratory of the Department of Clinical and Toxicological Analyses of the UFRN.

2.3 Biochemical evaluation

The concentrations of uric acid, albumin, calcium, creatinine, direct bilirubin, total bilirubin, total cholesterol, HDL cholesterol, glucose, total proteins, triglycerides and urea and the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST),

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6 alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH) were determined. The values of globulin and indirect bilirubin were calculated.

All analytes that were performed in serum were also performed in plasma, except calcium. The samples were processed using LABTEST Kits (Lagoa Santa, Minas Gerais, Brazil), according to the methodology of the manufacturer, and the equipment LABMAX PLENNO (LABTEST, Lagoa Santa, Minas Gerais, Brazil). The values of the linearity of the tests is described in the supplementary table 1.

Biochemical determination occurred in the Clinical Analyses Integrated Laboratory of the Department of Clinical and Toxicological Analyses of the UFRN.

2.4 Statistical analysis

Laboratory values for each parameter are expressed as mean (m), standard deviation (SD), coefficient of variation (CV=m/sd x 100), estimated concentration (Ce) in diluted samples (Ce = Concentration x dilution factor) and confidence interval 95% (IC95%). For analysis of the effect of dilution on the values in the samples, linear regression of random effects was used. The concentration of the parameter is considered the dependent variable, the dilution factors the independent variable of fixed effect and the intraindividual variability as a random component of the model. The statistical program used was Stata version 15. p values below 0.05 were considered significant. The variation of the parameter concentration in relation to the basal values was expressed as a percentage of accumulated loss with the dilution.

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7

3. RESULTS

The parameters that were stable in the serum dilutions were uric acid (p = 0.198; CV = 0.035), albumin (p = 0.336; CV = 0.015), AST (p = 0.523; CV = -0.43), total bilirubin (p = 0.062; CV = 0.014), calcium (p = 0.449; CV = -0.075), ALP (p = 0.5; CV = 0.52), glucose (p = 0.353; CV = 0.73), globulin (p = 0.850; CV = -0.004), LDH (p = 0.372; CV = 6.98) and total proteins (p = 0.706; CV = 0.011). While in plasma dilutions, uric acid (p = 0.908; CV = -0.005), albumin (p = 0.237; CV = 0.018), ALT (p = 0.719; CV = 0.33), AST (p = 0.583; CV = 0.41), total bilirubin (p = 0.216; CV = 0.01), globulin (p = 0.105; CV = 0.03), LDH (p = 0.111; CV = 13.36) and total protein (p = 0.061; CV = 0.047) were stable, as shown in Table 1.

Among the unstable parameters in serum (Table 2), according to their respective coefficients of variation, we can highlight total cholesterol (p = 0.004; CV = -3.31), triglycerides (p < 0.001; CV = -5.74) and urea (p < 0.001; CV = -5.85) as those that varied most between dilutions. In plasma (Table 2), total cholesterol (p = 0.031; CV = -2.44), GGT (p = 0.000; CV = -3.14), triglycerides (p < 0,001; CV = -3.92) and urea (p < 0,001; CV = -5.19) were the ones that changed more. In which urea and triglycerides varied the most, with urea presenting the highest coefficient of variation regardless of the type of sample.

The percentage of variation for unstable parameters was significant (Table 3), demonstrating the behavior of each parameter in each dilution. Thus, this instability was statistically significant (p < 0.005), indicating that the dilution for analysis of these biochemical parameters would possibly not be a viable methodology. Some of them had their result below methodological sensitivity, such as creatinine, GGT, triglycerides and urea for plasma dilution samples and ALT, GGT and urea in serum dilutions (Supplementary Table 1).

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8 Table 1 - Statistical result of stable parameters in dilutions in serum and plasma

Serum

Parameter Mean ± SD CV p IC95%

No dilution Dilution 1:2 Dilution 1:3 Dilution 1:4 Dilution 1:5

Uric acid (mg/dL) 4.0 ± 1.6 4.2 ± 1.6 4.4 ± 1.6 4.2 ± 1.6 4.2 ± 1.6 0.035 0.198 -0.018 0.087 Albumin (g/dL) 4.5 ± 0.3 4.7 ± 0.4 4.6 ± 0.3 4.7 ± 0.3 4.6 ± 0.4 0.015 0.336 -0.016 0.046 AST (U/L) 19.4 ± 6.7 22.8 ± 10.2 18.8 ± 10.3 20.8 ± 11.2 18.3 ± 14.8 -0.430 0.523 -1.748 0.888 T-BIL (mg/dL) 0.6 ± 0.4 0.7 ± 0.5 0.7 ± 0.5 0.7 ± 0.5 0.7 ± 0.5 0.014 0.062 -0.001 0.029 Calcium (mg/dL) 8.7 ± 0.6 8.3 ± 0.5 8.4 ± 1.3 8.1 ± 1.3 8.4 ± 2.6 -0.075 0.449 -0.269 0.119 ALP (U/L) 81.5 ± 23.6 87.4 ± 25.0 80.9 ± 23.0 82.0 ± 28.0 86.8 ± 21.1 0.520 0.500 -0.992 2.032 Glucose (mg/dL) 93.6 ± 8.2 92.3 ± 9.0 92.1 ± 9.9 95.2 ± 12.0 95.8 ± 19.1 0.730 0.353 -0.811 2.271 Globulin (g/dL) 3.2 ± 0.3 3.2 ± 0.4 3.2 ± 0.4 3.3 ± 0.5 3.1 ± 0.5 -0.004 0.850 -0.043 0.036 LDH (U/L) 281.8 ± 37.5 282.1 ± 66.9 285.5 ± 96.0 252.4 ± 122.2 331.5 ± 180.3 6.980 0.372 -8.337 22.297 Total protein (g/dL) 7.7 ± 0.4 7.8 ± 0.5 7.8 ± 0.4 7.9 ± 0.6 7.7 ± 0.8 0.011 0.706 -0.047 0.070 Plasma

Parameter Mean ± SD CV p IC95%

No dilution Dilution 1:2 Dilution 1:3 Dilution 1:4 Dilution 1:5

Uric acid (mg/dL) 4.7 ± 1.5 4.7 ± 1.8 4.7 ± 1.8 4.8 ± 1.7 4.6 ± 1.6 -0.005 0.908 -0.090 0.080 Albumin (g/dL) 4.4 ± 0.3 4.5 ± 0.4 4.5 ± 0.5 4.5 ± 0.4 4.5 ± 0.3 0.018 0.237 -0.012 0.048 ALT (U/L) 21.3 ± 15.4 20.4 ± 17.3 20.0 ± 16.8 22.2 ± 18.5 22.0 ± 23.0 0.330 0.719 -1.469 2.129 AST (U/L) 18.8 ± 8.3 16.8 ± 7.2 17.4 ± 11.3 18.4 ± 13.6 20.0 ± 15.2 0.410 0.583 -1.053 1.873 T-BIL (mg/dL) 0.7 ± 0.4 0.7 ± 0.4 0.7 ± 0.4 0.7 ± 0.5 0.7 ± 0.5 0.010 0.216 -0.006 0.027 Globulin (g/dL) 3.3 ± 0.3 3.4 ± 0.3 3.4 ± 0.4 3.4 ± 0.5 3.4 ± 0.4 0.030 0.105 -0.006 0.065 LDH (U/L) 282.6 ± 47.6 274.0 ± 67.1 307.2 ± 125.7 350.8 ± 194.5 311.0 ± 115.3 13.360 0.111 -3.076 29.796 Total protein (g/dL) 7.7 ± 0.4 7.9 ± 0.4 7.9 ± 0.7 7.9 ± 0.6 7.9 ± 0.4 0.047 0.061 -0.002 0.097 AST, Aspartate aminotransferase; ALT, Alanine aminotransferase; T-BIL, Total Bilirubin; ALP, Alkaline phosphatase; LDH, Lactate dehydrogenase; SD, Standard deviation; CV, Coefficient of variation; IC95%, Confidence range 95%.

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9 Table 2 - Statistical result of parameters without stability in serum and plasma

Serum

Parameter Mean ± SD CV p IC95%

No dilution Dilution 1:2 Dilution 1:3 Dilution 1:4 Dilution 1:5

ALT (U/L) 22.6 ± 16.6 24.0 ± 18.1 20.1 ± 16.2 16.2 ± 14.7 16.5 ± 18.3 -2,00 <0.001 -3.118 -0.882 D-BIL (mg/dL) 0.2 ± 0.1 0.3 ± 0.2 0.4 ± 0.2 0.4 ± 0.2 0.4 ± 0.2 0.039 <0.001 0.025 0.053 IN-BIL (mg/dL) 0.4 ± 0.3 0.4 ± 0.3 0.4 ± 0.3 0.3 ± 0.3 0.3 ± 0.3 -0.023 <0.001 -0.034 -0.011 HDL (mg/dL) 48.7 ± 11.0 52.3 ± 12.3 56.6 ± 12.9 55.2 ± 11.9 53.5 ± 16.1 1.250 0.011 0.283 2.217 Total cholesterol (mg/dL) 184.7 ± 42.1 184.7 ± 43.7 181.8 ± 39.9 180.4 ± 47.3 170.3 ± 55.8 -3.310 0.004 -5.544 -1.076 Creatinine (mg/dL) 0.9 ± 0.2 1.0 ± 0.2 1.0 ± 0.3 1.0 ± 0.2 1.2 ± 0.4 0.055 0.001 0.024 0.086 GGT (U/L) 27.9 ± 24.6 24.1 ± 24.4 20.4 ± 24.3 18.0 ± 24.6 16.0 ± 23.4 -2.980 <0.001 -3.748 -2.212 Triglycerides (mg/dL) 107.3 ± 59.8 98.9 ± 57.3 95.7 ± 51.2 96.6 ± 60.2 79.8 ± 49.2 -5.740 <0.001 -7.654 -3.826 Urea (mg/dL) 26.2 ± 10.2 18.4 ± 12.2 12.0 ± 9.5 4.2 ± 8.8 4.0 ± 9.5 -5.850 <0.001 -6.695 -5.005 Plasma

Parameter Mean ± SD CV p IC95%

No dilution Dilution 1:2 Dilution 1:3 Dilution 1:4 Dilution 1:5

D-BIL (mg/dL) 0.3 ± 0.1 0.3 ± 0.2 0.4 ± 0.2 0.4 ± 0.2 0.4 ± 0.2 0.032 <0.001 0.020 0.045 IN-BIL (mg/dL) 0.4 ± 0.2 0.4 ± 0.3 0.3 ± 0.2 0.3 ± 0.3 0.3 ± 0.2 -0.022 <0.001 -0.033 -0.011 HDL (mg/dL) 47.3 ± 11.0 50.8 ± 10.0 52.8 ± 11.0 52.2 ± 12.8 55.8 ± 12.4 1.840 <0.001 1.164 2.516 Total cholesterol (mg/dL) 183.4 ± 38.9 180.8 ± 44.2 174.9 ± 41.2 175.6 ± 45.7 173.8 ± 46.2 -2.440 0.031 -4.661 -0.219 Creatinine (mg/dL) 0.8 ± 0.1 0.9 ± 0.1 0.9 ± 0.2 0.9 ± 0.2 1.0 ± 0.3 0.040 <0.001 0.019 0.061 ALP (U/L) 1.1 ± 2.5 1,8 ± 2.0 0.9 ± 1.5 1.6 ± 2.8 6.5 ± 7.1 1.060 0.004 0.329 1.791 GGT (U/L) 24.6 ± 22.9 20.9 ± 24.9 17.0 ± 23.5 11.6 ± 23.7 13.5 ± 21.5 -3.140 <0.001 -4.184 -2.096 Glucose (mg/dL) 93.7 ± 6.3 93.9 ± 6.5 90.6 ± 11.6 84.0 ± 18.9 88.3 ± 6.3 -2.080 0.001 -3.357 -0.803 Triglycerides (mg/dL) 108.5 ± 54.7 104.8 ± 56.1 96.8 ± 53.7 95.0 ± 50.6 93.8 ± 57.5 -3.920 <0.001 -5.861 -1.980 Urea (mg/dL) 25.1 ± 10.1 18.0 ± 13.2 12.0 ± 13.2 8.2 ± 12.1 4.0 ± 8.7 -5.190 <0.001 -6.297 -4.083 ALT, Alanine aminotransferase; D-BIL, Direct Bilirubin; IN-BIL, Indirect bilirubin; HDL, High density lipoprotein;ALP, Alkaline phosphatas;GGT, Gamma glutamyl transferase;SD, Standard deviation; CV, Coefficient of variation; IC95%, Confidence range 95%.

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10 Table 3 - Percentage of variation of parameters

Serum

Parameter Variation % p

No dilution Dilution 1:2 Dilution 1:3 Dilution1:4 Dilution 1:5

Uric Acid 0.0 5.2 -8.2 5.7 4.0 0.198 Albumin 0.0 4.2 -3.0 3.8 1.8 0.336 ALT 0.0 6.2 12.4 -28.3 -27.0 <0.001 AST 0.0 17.5 3.5 7.2 -5.9 0.523 D-BIL 0.0 34.8 -34.3 65.2 69.6 <0.001 IN-BIL 0.0 -7.3 7.9 -17.1 -24.4 <0.001 T-BIL 0.0 7.8 -13.5 12.5 7.8 0.062 Calcium 0.0 -4.6 4.1 -7.4 -3.0 0.449 HDL 0.0 7.4 -13.9 13.3 9.9 0.011 Total Cholesterol 0.0 0.0 1.6 -2.3 -7.8 0.004 Creatinine 0.0 8.0 -9.3 10.2 30.7 0.001 ALP 0.0 7.3 0.7 0.7 6.5 0.500 GGT 0.0 -13.5 36.5 -35.4 -42.5 <0.001 Glucose 0.0 -1.3 1.6 1.8 2.4 0.353 Globulin 0.0 -0.3 0.6 2.5 -2.2 0.850 LDH 0.0 0.1 -1.3 -10.4 17.7 0.372 Total Protein 0.0 2.3 -1.7 3.4 0.3 0.706 Triglycerides 0.0 -7.8 12.1 -10.0 -25.7 <0.001 Urea 0.0 -29.6 117.9 -83.9 -84.7 <0.001 Plasma Parameter Variation % p

No dilution Dilution 1:2 Dilution 1:3 Dilution 1:4 Dilution 1:5

Uric Acid 0.0 0.0 -1.7 2.4 -1.6 0.908 Albumin 0.0 3.0 -2.2 2.3 2.3 0.237 ALT 0.0 -4.0 6.5 4.5 3.5 0.719 AST 0.0 -10.4 7.8 -1.9 6.7 0.583 D-BIL 0.0 28.0 -28.6 48.0 56.0 0.000 IN-BIL 0.0 -9.5 23.5 -21.4 -21.4 0.000 T-BIL 0.0 4.5 -2.9 4.5 7.5 0.216 Calcium 0.0 -100.0 -100.0 -100.0 -100.0 0.502 HDL 0.0 7.5 -10.5 10.5 18.0 0.000 Total Cholesterol 0.0 -1.4 4.8 -4.2 -5.2 0.031 Creatinine 0.0 6.1 -11.8 7.3 23.2 0.000 ALP 0.0 63.6 22.2 45.5 490.9 0.004 GGT 0.0 -14.9 44.8 -52.7 -45.0 0.000 Glucose 0.0 0.2 3.4 -10.4 -5.8 0.001 Globulin 0.0 2.7 -2.4 2.7 4.4 0.105 LDH 0.0 -3.0 -8.0 24.1 10.0 0.111 Total Protein 0.0 3.0 -2.3 2.6 3.3 0.061 Triglycerides 0.0 -3.4 12.1 -12.4 -13.6 0.000 Urea 0.0 -28.1 108.8 -67.3 -84.0 0.000

ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; D-BIL, Direct Bilirubin; IN-BIL, Indirect bilirubin; T-BIL, Total Bilirubin; HDL, High density lipoprotein; ALP, Alkaline phosphatase GGT, Gamma glutamyl transferase; LDH, Lactate dehydrogenase.

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11

4. DISCUSSION

In this study, uric acid, albumin, AST, total bilirubin, calcium, ALP, glucose, globulin, LDH and total proteins were stable in all dilutions performed in serum, as well as, uric acid, albumin, ALT, AST, total bilirubin, globulin, LDH and total protein in plasma. This stability is an important indication that serum or plasma dilution may possibly be an option for determining these parameters when the volume of the sample collected is not sufficient for biochemical measurements, which would optimize the laboratory routine of neonatology tests. We estimate that about 250 μL was used to perform the tests on the undiluted samples, while in 1:5 dilutions only 50 μL were used.

In plasma dilutions, some parameters that did not have indication for performance in the presence of EDTA, such as ALP and total cholesterol, presented instability, as expected [21,22]. However, it is worth noting that other parameters that also did not have that indication, such as albumin, LDH and total protein, demonstrated stable values in our study both in the undiluted sample and in its dilutions [23,24,25].

The importance of testing plasma collected with EDTA sample is due to some situations, as emergencies or post-surgical, that requires both biochemical and hematological tests [26]. So, if the same collection tube (containing EDTA) can be used, a smaller quantity of blood could be collected to perform the tests. Moreover, the use of plasma as a matrix for biochemical analysis can be of great advantage, especially in the case of neonatology, since it has a volume yield of 15-20% more than in serum for samples of the same volume collected [27], as well as allows for faster results [28].

The instability of direct bilirubin and creatinine parameters in plasma and serum dilutions may be due to the low analytical concentrations that may have suffered interference in the presence of dilution.

The lack of stability in ALT, GGT and urea in serum, and creatinine, GGT, triglycerides and urea in plasma probably is due to the fact that some of their values after dilution were below the detection capacity of the method. Moreover, for some parameters such as direct bilirubin, creatinine and triglycerides in serum, results at the thereshold were obtained [29,30,31,32,33,34]. Nevertheless, for these parameters, the method used was still reproducible.

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12 To the best of our knowledge, this is the first study that tested the dilution of serum and plasma samples in humans as a methodology for biochemical analyses, however studies that evaluated the effect of dilution in serum/plasma in other species are described in the literature. Johns et al. [35] conducted a study on the effect of pre-dilution in serum of rats and mice, using ultrapure water with dilution factors from 2 to 10 as thinner. In their findings, it was observed that many of the biochemical parameters tested suffered dilution-induced error and even for electrolytes, calcium, albumin, total bilirubin, creatinine and uric acid, this error was found even in low dilutions. The present study found similarity in relation to creatinine values, however, the results obtained in this study for calcium, albumin, uric acid and total bilirubin were stable, with no significant variation between dilutions.

A pre-dilution study of rodent plasma for biochemical determinations, using water for injection as diluent and evaluating dilution up to 10 times, showed good correlation of ALP, total proteins, albumin, urea, cholesterol, triglycerides, calcium and phosphorus values of diluted samples with undiluted ones. Creatinine and total bilirubin values did not have a good correlation. Glucose showed a good correlation only up to a dilution of 5 times [36]. However, the findings of this study do not corroborate the data presented by Goyal et al. [36], since the results obtained in this study shown a significant percentage of variation for ALP, urea, total cholesterol, triglycerides, creatinine and glucose parameters. Nevertheless, these data differences may be due to the use of different diluents, analyzers, methods and/or kits adopted.

Waldoch, Wack and Christopher [37] performed a plasma collected with heparin dilution study in parrots, using sterile water as diluent, in which they observed significant differences between diluted and undiluted samples, except for AST, creatine kinase and glucose. For uric acid, statistical differences were not displayed until dilution 1:3. The study obtained the coefficient of variation higher than the limits established for most of the analyzed parameters, indicating that the precision decreased with dilutions. In the present study, creatine kinase determination was not performed, however, the data obtained confirm those observed for AST. Poitout-belissent et al. [38], in their work, reported that albumin and total bilirubin dilutions should not exceed 50%, at the risk of decreasing the concentration of parameter at levels that compromise detection in the sample. However, these findings are not in agreement with the present study, in which both parameters remained stable in the dilutions for the two matrices analyzed.

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13 Evaluating the parameters tested in serum and plasma with diseases that frequently affect newborns, it is seen that many markers of liver function remained stable, one of them, total bilirubin, which may be useful in the diagnosis and follow-up of neonatal jaundice. Glucose and calcium were also stable in serum, and may aid the diagnosis and monitoring of metabolic disorders that involve them or that are related to the differential diagnosis of other events, such as convulsion and respiratory disorders.

As limitations of this study, we can mention the non-evaluation of neonatal blood samples, as well as the analysis of the behavior of the parameters in the dilutions performed only in healthy patients, not being evaluated this standard for the presence of pathologies, conditions that can change the values of biochemical parameters.

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14

5. CONCLUSION

Dilution (up to 1:5) in serum uric acid, albumin, AST, total bilirubin, calcium, ALP, glucose, globulin, LDH and total protein and plasma uric acid, albumin, ALT, AST, total bilirubin, globulin, LDH and total protein did not interfere in the sensitivity of the determination in this study, indicating that the pre-dilution of serum and plasma samples for these parameters may bring benefits to clinical practice, especially neonatal, because it allows biochemical analysis in small volume samples, and may prove to be a promising option for performing these tests in the neonatal population, reducing blood spoliation and the consequent risks of blood spoliation. However, further studies involving dilutions of serum and plasma samples in humans are necessary to validate this methodology, ensure greater reliability of the method and confirm the findings by this study.

DECLARATION OF INTEREST

The authors report that there was no conflict of interest.

FUNDING SOURCES

This work was funded by UFRN.

AKNOWLEDGMENTS

We are thankful to the Research Group on Metabolic Diseases (GPDM) of the Faculty of Pharmacy of the Federal University Federal do Rio Grande do Norte (UFRN) and to the volunteers who participated in the study.

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15

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19 Supplementary table 1 - Methodological sensitivity values

Parameter Method sensitivity

Albumin (g/dL) 0.015 ALT (U/L) 1.750 ALP (U/L) 0.125 AST (U/L) 1.750 Calcium (mg/dL) 0.100 Creatinine (mg/dL) 0.140 D-BIL (mg/dL) 0.030 T-BIL (mg/dL) 0.090 GGT (U/L) 2.480 Glucose (mg/dL) 1.770 HDL (mg/dL) 0.400 Total cholesterol (mg/dL) 1.040 LDH (U/L) 8.900 Total protein (g/dL) 0.0168 Triglycerides (mg/dL) 3.000 Urea (mg/dL) 0.940 Uric acid (mg/dL) 0.020

ALT, Alanine aminotransferase; ALP, Alkaline phosphatas; AST, Aspartate aminotransferase; D-BIL, Direct Bilirubin; T-BIL, Total Bilirubin; GGT, Gamma glutamyl transferase; HDL, High density lipoprotein; LDH, Lactate dehydrogenase.

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