Neisseria gonorrhoeae (NG) har successivt utvecklat resistens mot många antimikrobiella medel och betraktas som ett av de tre reella hoten bland antibiotikaresistenta bakterier. Ciprofloxacin är ett bredspektrum-antibiotikum tillhörande gruppen kinoloner som, förutom att behandla urinvägsinfektioner, används mot NG och infektioner i mage och tarm. Dock har det rapporterats att ca 30 % av NG-isolat som samlats in genom gonokock-isolatövervakningsprojekt (GISP) under 2017 var resistenta mot ciprofloxacin. På molekylnivå är resistens mot ciprofloxacin starkt associerad med en enda mutation i kodon 91 i gyras-genen (gyrA). Detta projekt har undersökt om det går att använda molekylärbiologiska metoder för att detektera NG-isolat med gyrA mutationen. Analysen gjordes med två olika PCR-system, ”7500 Fast Real-Time PCR System” från Applied Biosystems (ABI) och Panther Fusion från Hologic. Proberna som användes designades för påvisning av vildtyp gyrA (ciprofloxacin-känslig) och mutant gyrA (ciprofloxacin-resistent) hos NG. I projektet analyserades 50 NG-positiva prov (analyserade med screeningtest APTIMA COMBO2 från Hologic), från 43 patienter som provtagits under januari-februari 2020 i Region Skåne. Några patienter testades flera gånger vid olika tillfällen. NG-odling hade utförts parallellt från motsvarande tagna prov från patienterna. ABI-metoden påvisade genen hos 90 % (45/50) av NG-positiva prover (APTIMA COMBO2) medan endast 24 av de 49 proven (49 %) kunde odlas med traditionell metodik för att därefter resistensbestämmas. Av de 45 prov där gyras-genen kunde detekteras med ABI-metoden, uppvisade 28 (62 %) av proven en muterad gen och därmed en potentiell resistens för ciprofloxacin. Panther Fusion-metoden påvisade genen hos 80 % (40/50) av NG-positiva prover (APTIMA COMBO2), och såsom tidigare nämnts, kunde endast 24 av de 49 proven (49 %) odlas med traditionell metodik för att därefter resistensbestämmas. Av de 40 prov där gyras-genen kunde detekteras med Panther Fusion-metoden, uppvisade 26 av proven (65 %) en muterad gen och därmed en potentiell resistens för ciprofloxacin. En jämförelse mellan resultaten från PCR-metoderna och odlingarna visar att av de 24 odlingarna som kunde resistensbestämmas fick ABI-metoden resultat för 23 och Panther Fusion för 22. PCR-metodernas resultat överensstämde perfekt med resultaten från odling med samma 8 känsliga och 15 respektive 14 resistenta NG-isolat som odling. De båda PCR-metoderna och traditionell odling uppvisade jämförbara resultat. Av de 24 prov som kunde odlas och därmed resistensbestämmas, detekterades med ABI-metoden gyras-genen i 23 av dessa prov och i 22 av proven med Panther Fusion-metoden. Resistens mot ciprofloxacin uppvisades genom odling i 16 av de 24 odlingsbara prov, och av dessa 24 odlingsbara prov uppvisade ABI-metoden en muterad gen i 15 av proven och Panther Fusion-metoden en muterad gen i 14 av proven. Traditionell odling kunde bara genomföras på 24 av proven och PCR-metoderna identifierade signifikant fler prov innehållande vildtyp eller muterad gyras-gen, 45 respektive 40 prov. Projektet visade tydligt att PCR-metoderna kan identifiera fler prov än genom traditionell odling och kan därmed upptäcka fler prov med förväntad ciprofloxacin-resistens än vad som kan bestämmas genom traditionell odling. / Neisseria gonorrhoeae (NG) has been developing a resistance towards several different antibiotics and is viewed as one of the three real threats among resistant bacteria. Ciprofloxacin is a broad-spectrum-antibiotic belonging to the group quinolone antibiotics which, in addition to being used to treat urinal infections, is used to treat NG and infections in the stomach and intestines. However, it has been reported that 30 % of NG-isolates that have been gathered through the Gonococcal Isolate Surveillance Project (GISP) throughout 2017 were resistant to ciprofloxacin. On a molecular level, resistance to ciprofloxacin is strongly associated with a single mutation in kodon 91 in the gyras-gene (gyrA). This project sought to examine if it is possible to use methods from molecular biology to detect which NG that have the gyrA-mutation. The test was done using two different PCR-systems, ”7500 Fast Real-Time PCR System” from Applied Biosystems (ABI) and Panther Fusion from Hologic. The probes used were designed to show wild type gyrA (ciprofloxacin sensitive), and mutated gyrA (ciprofloxacin resistant) in NG. In this project 50 NG-positive samples (analysed with screentest APTIMA COMBO2 from Hologic), from 43 patients that had been tested during January-February 2020 in Region Skåne, were analysed. Some patients were tested several times, within the time period. NG-cultivation had been done in parallel from corresponding samples taken from the patients. The ABI-method showed the gene in 90 % (45/50) of NG-positive samples (APTIMA COMBO2) while only 24 of the 49 samples (49 %) could be cultivated by traditional methodology, and then tested for resistance. Of the 45 samples where the gyras-gene could be detected with the ABI-method, 28 samples (62 %) exhibited a mutated gene and thus a potential resistance to ciprofloxacin. The Panther fusion-method showed the gene in 80 % (40/50) of NG-positive samples (APTIMA COMBO2), and as mentioned earlier, only 24 of the 49 samples (49 %) could be cultivated by traditional methodology to then be tested for resistance. Of the 40 samples where the gyras-gene could be detected with the Panther Fusion-method, 26 samples (65 %) exhibited a mutated gene and thus a potential resistance to ciprofloxacin. The two PCR-methods and traditional cultivation exhibited comparable results. Of the 24 samples that could be cultivated and thus tested for resistance, the ABI-method detected the gyras-gene in 23 of these samples and the Panther Fusion-method detected the gene in 22 of the samples. Cultivation exhibited resistance to ciprofloxacin in 16 of the 24 samples that could be cultivated, and of these 24 cultivatable samples the ABI method exhibited a mutated gene in 15 of the samples and the Panther Fusion-method exhibited a mutated gene in 14 of the samples. Traditional cultivation could only be done on 24 of the samples and the PCR-methods could identify significantly more samples containing either wild type or mutated gyras-gene, 45 and 40 samples, respectively. The project clearly showed that more samples can be identified with the PCR-methods than through traditional cultivation, and thereby discover more samples with expected ciprofloxacin-resistance, than can be determined through traditional cultivation.

Högerkammare (RV) har en komplex anatomi, spelar en viktig roll för blodsyresättning och kan påverkas av fler patofysiologiska tillstånd. Utvärdering av RV:s funktion är viktig för överlevnad och har prognostiskt värde vid hjärt-och lungsjukdomar. Transthorakal ekokardiografi (TTE) används för RV:s storlek- och funktionsbedömning. Tricuspid annular plane systolic excursion (TAPSE) och annulus tricuspid peak systolic velocity (RVs´) är två vanliga metoder för bedömning av RV:s funktion. Båda metoderna har bra reproducerbarhet och är enkla att utföra.  Syftet med detta arbete är att utreda interobservatörvariation, sambandet och överensstämmelse mellan TAPSE och RVs´. Studien är en tvärsnittsstudie av 53 friska testpersoner 18-60 åringar. Mätningen baserades på en blind dubbelbestämning av två biomedicinska analytiker studenter. Analysen genomfördes med programmet IBM SPSS Statistics.  Interobservatörvariationsanalys visade ingen signifikant skillnad i mätosäkerheten mellan studenterna, (PTAPSE=0,568 och PRVs´=0,548). Enligt regressionsanalysen hade RVs´ något mindre mätosäkerhet än TAPSE. Ett svagt positivt samband hittades mellan RVs´ och TAPSE och 100% överenstämmelse avseende utfall påvisades, Kappavärdet blev 1. Båda metoderna har bra interobservatörvariation hos oerfarna undersökare. Hos hjärt- och lungfriska ser sambandet svagt positivt ut mellan TAPSE och RVs´. Dock kunde tidigare studier identifiera starkare positivt samband. Skillnaden i resultatet kan bero på erfarenhetsbrist hos studenterna och lågt antal deltagare. / The Right ventricle (RV) has a complex anatomy, plays an important role in blood oxygenation and can be affected by several pathophysiological conditions. Evaluation of RV function has prognostic value in heart and lung diseases. Tricuspid annular plane systolic excursion (TAPSE) and annulus tricuspid peak systolic velocity (RVs´) are two common methods for assessing RV function in Transthoracic echocardiography.  The study aimed to investigate interobserver variation, the correlation and agreement between TAPSE and RVs´. The study is a cross-sectional study of 53 healthy participants aged 18-60. The measurement was based on a blind double determination by two biomedical analyst students. The analysis was implemented with the program IBM SPSS Statistics. Interobserver variation analysis showed no significant difference between the two students, (PTAPSE =0,568 and PRVs´=0,548). Regression analysis showed RVs´ had slightly less measurement uncertainty than TAPSE. A weak positive correlation was found between RVs´ and TAPSE and 100% agreement regarding outcome was demonstrated, Kappa value was 1. Both methods have good interobserver variation in inexperienced examiners. In people with healthy heart and lungs, the relationship between TAPSE and RVs looks weakly positive. Previous studies identified stronger positive association. Differences in the results may be due to a lack of experience on the part of the students and a low number of participants.

The engraftment of haemopoietic stem/progenitor cells (HSPCs) from umbilical cord blood (UCB) into adult recipients, although advantageous in terms of sourcing units, the decreased need to match donor and recipient and reduced risk of graft versus host disease (GvHD), is delayed compared to grafts using HSPCs from mobilised peripheral blood (MPB) or bone marrow (BM). One reason for this is the limited number of HSPCs (CD34+/CD133+ cells) in a unit of UCB compared to MPB or BM. The CXCR4-CXCL12 axis is widely recognised as a key player in the bone marrow homing, retention, and engraftment of HSPCs. The aim of this thesis was to investigate whether the engraftment of HSPCs from UCB into the bone marrow could be improved. Firstly, a novel in vitro 3D time-lapse chemotaxis assay to assess the homing capacity of human UCB CD133+ HSPCs, towards the chemokine CXCL12 was developed. One advantage of this assay was that it distinguished cell chemotaxis from chemokinesis and allowed these parameters to be quantified. Human UCB CD133+ HSPC chemotaxis towards CXCL12 was inhibited by the CXCR4 antagonist, AMD3100. Importantly, the presence of CXCL12 or AMD3100 had no affect on cell chemokinesis. To complement the in vitro chemotaxis assay, a short term in vivo homing assay in NSG mice was successfully established. The effect of siRNA silencing of the CXCR4 co-receptor, CD164, which is also expressed on CD133+ HSPCs, on cell migratory and homing ability was investigated. CD164 knock-down using siRNA in human UCB CD133+ HSPCs did not demonstrate an effect on homing to NSG bone marrow in vivo or chemotaxis to CXCL12 in vitro. However, homing to NSG mouse spleen was significantly reduced in cells silenced for CD164. Following this, an 8 day HSPC expansion system using nanofibre scaffolds (Nanex) and differing cytokines was investigated. These serum and feeder free conditions yielded a significant expansion of cells that retained CD133+CD34+ expression and their in vitro chemotactic ability to CXCL12. Time constraints did not permit the engrafting ability of these cells to be analysed in an in vivo HSC reconstitution assay that was initiated. However these studies will provide the basis to support future related research in this laboratory.

Acute Myeloid leukemia (AML) is an aggressive haematological malignancy that mainly affects the elderly. Relapse is common and is thought to be due to the presence of chemotherapy resistant leukemic stem cells (LSC). Within the CD34+ disease (>5% of the blast cells expressing CD34) , two subtypes have been identified; an LMPP/GMPlike expanded type and a MPP/CMP-like expanded type, the former is the most common, accounting for around 80% of CD34+ AML. Both the GMP-like and LMPPlike expanded populations show LSC activity. To improve our understanding of the disease and gain better insight in to how to develop treatments, the molecular basis of the disease needs to be investigated. I investigated miRNAs in the GMP/LMPP-like expanded AML. miRNAs are small non-coding RNAs involved in the regulation of mRNA. In recent years miRNAs have been shown to be implicated in many different diseases. To investigate the role miRNAs play in AML, miRNA expression was profiled in leukemic and normal bone marrow. Bioinformatic analysis was then used to examine the different miRNA expression profiles between normal and leukemic marrow. Our study showed that miRNAs are dysregulated in AML. miRNAs from the miR-17-92 and its paralogous cluster miR-106b-92 were amongst the miRNAs to be found down regulated in AML As had been seen previously at an mRNA level, on an miRNA level the LSC populations more closely resembled more mature progenitor populations than HSC and MPP populations, however the LSC populations did display an aberrant stem cell-like miRNA signature.

Bedömningen av höger kammares systoliska funktion kan med fördel göras med ekokardiografi, bl.a. med tekniker som tvådimensionell ekokardiografi, motion-mode (M-mode) och Tissue Doppler Imaging (TDI). Mätvärden som kan fås ut av dessa tekniker är Tricuspid Annular Plane Systolic Excursion (TAPSE) och s`. TAPSE beskriver annulus tricuspidalis longitudinella rörlighet. s´ beskriver den maximala systoliska hastigheten i annulus tricuspidalis. Syftet med examensarbetet var att undersöka sambandet mellan TAPSE och s´ vid högerkammarfunktionsbestämning. Studien är en retrospektiv tvärsnittsstudie och består av 99 deltagare i åldrarna 18–90 år. Både friska och hjärt-och kärlsjuka deltagare inkluderades, men inte patienter med förmaksflimmer eller individer som var yngre än 18 år. Deltagarna undersöktes med transthorakal ekokardiografi (TTE) vid Länssjukhuset Ryhov i Jönköping, vid undersökningen mättes TAPSE och s´. Det var samma biomedicinska analytiker (BMA) som utförde undersökningarna. McNemars test utfördes och ett Kappa-värde räknades ut. Resultatet visade signifikant skillnad och Kappa-värdet var lågt vilket talar för dåligt samband mellan de båda variablerna. Utfallet i studien visar att fler deltagare klassas med normal högerkammarfunktion mätt med TAPSE, medan samma individer klassas med högerkammardysfunktion mätt med s´. Orsaken till det dåliga sambandet mellan parametrarna är okänt och bör studeras vidare i kommande studier. / The assessment of right ventricular systolic function is done by echocardiography, with techniques as twodimensional echocardiography, motion-mode (M-mode) and Tissue Doppler Imaging (TDI). Measurements ​​obtained from these techniques are Tricuspid Annular Plane Systolic Excursion (TAPSE) and s`. TAPSE describes the longitudinal movement of annulus tricuspidalis. s´ describes annulus tricuspidalis maximal systolic velocity. The purpose of this essay was to explore the relationship between TAPSE and s´. The study consists of 99 participants between the ages of 18 and 90. The population was consisted of individuals with or without cardiovascular disease, but not individuals with atrial fibrillation. Participants were investigated with transthorakal echocardiography (TTE) at Länssjukhuset Ryhov in Jönköping. At the examination both TAPSE and s´ was measured. McNemar's test was performed and a Kappa value was calculated. The result showed a significant difference and the Kappa value was low which indicates a low relationship between the variables. The outcome of the study shows that several participants are classified with normal right ventricular function measured with TAPSE, but the same individuals are classified with right ventricular dysfunction measured by s'. The reason for the poor relationship between the parameters is unknown and should be studied further in future studies.

The alternative road map describes the separation of lympho-myeloid and myeloid-megakaryocyte-erythroid (myeloid-Mk-E) lineages as the earliest haematopoietic commitment event. However, a number of aspects of this lineage restriction process remain poorly understood. Herein this work identified a lympho-myeloid restricted progenitor in the embryo, which resembles the adult LMPP, and demonstrated that lymphoid lineage restriction is initiated prior to definitive haematopoiesis, much earlier than previously appreciated. In vivo fate mapping showed that lympho-myeloid progenitors significantly contribute to steady state myelopoiesis in the embryo. The early thymic progenitor (ETP) as most primitive cell in the thymus was characterised and demonstrated to sustain B, T and myeloid but not Mk potentials at the single cell level. The ETP therefore largely resembles the cellular properties of lympho-myeloid progenitors in bone marrow and foetal liver, which points to these cells as candidate thymus seeding progenitors (TSP). Furthermore the existence of a putative Mk progenitor was explored within the LSKCD150⁺CD48⁺Gata1^pos compartment of a Gata1 reporter mouse providing the basis for a future prospective characterisation. Finally, this work evaluated the earliest lineage restriction of von Willebrand factor (Vwf)-EGFP⁺ and EGFP^- haematopoietic stem cells (HSCs) through in vitro paired daughter fate mapping. Single Vwf⁺ HSCs showed heterogeneous Mk priming and more frequently sustained Mk potential after cell division. Moreover, analysis of lineage priming between daughter cells revealed the asymmetric expression of key lineage determinants and stem cell regulators, which might be employed as reporters for future fate mapping studies.

As more patients with large body surface area burns are surviving and requiring reconstructive surgery, there is a necessity for advances in the provision of bioengineered alternatives to autologous skin cover. The aims of this Thesis are to identify feasible source tissues of Endothelial Colony Forming Cells and Mesenchymal Stem/Stromal Cells for microvascular network formation in vitro with three-dimensional dermal substitute scaffolds. The working hypothesis is that pre-vascularised dermal scaffolds will result in better quality scarring when used with split thickness skin grafts. Human umbilical cord blood, peripheral blood and adipose tissue were collected and processed with ethical approval and informed consent. Samples were cultured to form endothelial outgrowth colonies and confluent Mesenchymal Stem/Stromal Cells, which were characterised using flow cytometry and expanded in vitro. Mesenchymal Stem/Stromal Cell multipotency was confirmed with tri-lineage mesenchymal differentiation. Primary cells were tested in a two-dimensional tubule formation co-culture assay and differences assessed using a proangiogenic antibody array. Tubule formation was tested in four different acellular dermal substitute scaffolds; Integra® Dermal Regeneration Template, Matriderm®, Neuskin-F® and De-cellularised Human Cadaveric Dermis. Umbilical cord blood was the most reliable source of Endothelial Colony Forming Cells, the yield of which could be predicted from placental weight. Microvasculature dissected free from adipose tissue was a reliable source of Mesenchymal Stem/Stromal Cells which supported significantly more tubule formation than Mesenchymal Stem/Stromal Cells from whole adipose tissue. Microvasculature Mesenchymal Stem/Stromal Cells secreted significantly higher levels of the proangiogenic hormone leptin, and addition of exogenous leptin to the tubule formation assay resulted in significantly increased tubule formation. Microvasculature was cultured in all four of the scaffolds tested, but depth of penetration was limited to 100µm. The artificial oxygen carrier perfluorocarbon was shown to increase two-dimensional tubule formation and may be useful in further three-dimensional scaffolds studies to improve microvascular penetration.

During embryonic development, precursor cells commit to specific cell fates in response to environmental cues through the establishment of lineage-specific gene expression programmes. Transcription factors are important downstream effectors of signalling pathways that initiate and maintain cell fate decisions. The haematopoietic transcription factor SCL (TAL-1) is an essential regulator of embryonic blood development. However, the exact stage at which SCL is required, its mechanisms of action, and its genomic targets are poorly understood. Characterising, jiow SCL functions - , during haematopoietic development will provide insights into how stern cells are specified. Using the embryonic stem cell/embryoid body (ES/EB) system to model early mouse development, we describe a critical role for SCL in mesoderm patterning. SCL is first expressed in PDGFRa+ FLK1+ mesoderm populations which contain lateral, paraxial and cardiac precursors. Through loss- and gain-of-function studies, we show that SCL drives lateral mesoderm specification and activates the haematopoietic programme in a direct DNA-binding independent manner, while actively repressing alternative mesodermal fates, specifically cardiac development, in a DNA-binding dependent manner. At a molecular level, we have identified direct genomic targets of SCL in Flk-1 + mesoderm populations. These include haematopoietic and cardiac transcription factors, cardiac-specific structural proteins, signalling proteins and general transcriptional repressors; thereby strengthening the dual function of SCL in mesoderm patterning. Finally, we have shown that the cardiac transcription factor GATA4 acts in a reciprocal manner, specifying cardiac precursors while repressing a lateral mesoderm fate. Collectively, this implicates SCL as a critical transcriptional regulator of cell fate decisions in early mesodermal precursors, employing distinct molecular mechanisms to impose a blood programme. Moreover, and extending earlier reports, we document the existence of an antagonistic cross-talk between haematopoietic and cardiac lineages during mesoderm patterning. In conclusion, this work offers a cellular and molecular platform to begin to dissect the network of genetic interactions involved in these developmental processes.

ATRX is a chromatin remodeling protein associated with X-linked Alpha-Thalassemia Mental Retardation syndrome and cancers that use the Alternative Lengthening of Telomere pathway. In the absence of ATRX there is a DNA damage response associated with telomeres and the expression of certain genes are perturbed. Recent findings (Law et al, 2010 Cell) have shown that ATRX is preferentially enriched at GC-rich tandem repeats in the genome. The mechanism for this localisation is unknown but may be related to the potential for these GC-rich tandem repeats to adopt non-B form DNA structures; ATRX has been shown to bind such structures (G4) in vitro. This study aims to understand the specific factors of the repeats that signal ATRX targeting. To address the research questions, an experimental system was developed, in which known targets, the ψζ VNTR and telomere repeats, were inserted into an inducible ectopic gene in the 293T-Rex cell line by site-directed recombination. ATRX was found to be enriched at the ectopic repeats compared to an endogenous negative control suggesting that it is recruited by the repeats independent of its original context. Furthermore, ATRX enrichment increased upon transcription of the ectopic gene, and this was dependent on the orientation of the repeat with the non-template strand being G-rich. Interestingly, when the repeat was transcribed, the distribution of ATRX across the repeats was asymmetrical with most ATRX binding downstream of the repeat. Moreover, there was a direct correlation between the repeat size and level of ATRX bound: the longer the repeat the higher the increase in ATRX enrichment. To determine the signal for ATRX binding, assays were performed to look for features which reflected the distribution of ATRX including H3K9me3, RNA polII, G4, R loops and DNA supercoiling. R loops look to be a strong candidate for the signaling of ATRX binding.

Introdução: A comunicação na saúde pública é uma ferramenta de promoção da saúde e acesso à informação. A melhoria da comunicação entre o laboratório clínico e os usuários constitui um direito do usuário do serviço público de saúde e também apresenta potencial para a diminuição de erros de análise provenientes da fase pré-analítica e consequente melhoria da segurança do paciente. Este trabalho teve o objetivo avaliar a adequação da linguagem escrita utilizada na comunicação entre o laboratório clínico e os usuários bem como sua influência da qualidade da fase pré-analítica do processo laboratorial. Materiais e Métodos: Foram analisadas instruções de coleta (IC) da Divisão de Laboratório Clínico (DLC) do Hospital Universitário da USP (HU/USP), de setembro de 2014 a agosto de 2016. Na primeira fase, os parâmetros de linguagem e legibilidade das IC foram avaliados pelos índices Fernández-Huerta (IFH), Flesch-Szigriszt (IFS), utilizando os programas de informática TextMeter® e INFLESZ®. Os textos de IC foram traduzidos para o espanhol e utilizados para validação dos índices IFH e IFS para a língua portuguesa. Na segunda fase, as IC foram reestruturadas e os parâmetros de linguagem e legibilidade foram analisados e comparados com as IC anteriores. O perfil sócio educacional dos usuários e uma lista dos problemas relacionados obtenção de amostras de biológicas foram obtidos na DLC. Resultados: Quanto aos parâmetros de linguagem, o número de linhas por IC, de palavras proparoxítonas e polissilábicas foi similar entre as IC anteriores e novas (p>0,05). Enquanto que o número de termos técnicos e de palavras por frase foi menor, e número de orações subordinadas e coordenadas e ordem inversas das palavras foi maior nas IC novas (p<0,05). O escore do IFH (58,4 ± 8,8) foi menor que o do IFS (62,3 ± 6,8, p=0,019) para 27 IC anteriores, mas não diferiu para as 40 IC novas (p=0,162). Os escores de IFS e IFH das IC novas em português foram similares aos das traduzidas para o espanhol, o que é indicativo de que esses índices são validos para a língua portuguesa. Os índices IFH e IFS de textos didáticos usados para avaliar o grau de escolaridade foram similares para o ensino fundamental (EF) I e II, mas foram menores para os dois últimos anos do ensino médio (EM). O número de IC com índices de legibilidade correspondentes aos textos didáticos destinados ao o 3º ano do EF II (32,7%, n=10) foi menor que o número de IC novas (52,5%, n=21), mas a diferença não foi significativa (p=0,280). Os percentuais de gênero e a cor autodeclarada foram similares entre as duas fases do estudo. Entretanto menos jovens (<34 anos), mais idosos (>50 anos) e mais usuários com EF, EM e ensino superior completo foram atendidos, na segunda fase do estudo (p<0,05). Foram identificadas mais falhas de orientação na primeira fase (n=11) que na segunda (n=22) mas sem diferença significativa (p=1,000). Conclusão: Os parâmetros de linguagem e legibilidade melhoraram após a reestruturação das novas IC. A análise de legibilidade contribui para a comunicação do laboratório clinico com os usuários e pode influenciar positivamente a qualidade da fase pré-analítica do processo laboratorial. / Introduction: Communication in public health is a tool to promote health and access to information. The improvement of communication between the clinical laboratory and the users constitutes a right of the public health service user and it is presents potential for the reduction of analytical errors from the pre-analytic phase and consequent improvement of patient safety. This work aimed to evaluate the adequacy of the written language used in the communication between the clinical laboratory and the users as well as its influence on the quality of the pre-analytical phase of the laboratory process. Materials and Methods: Collection instruction leaflet (CIL) from the Clinical Laboratory Division (DLC) of the University Hospital of USP (HU/USP) were analyzed from September 2014 to August 2016. In the first phase, the language and readability parameters of the CIL were evaluated by the Fernández-Huerta indexes (IFH), Flesch-Szigriszt (IFS), using the TextMeter® and INFLESZ® software programs. CIL texts were translated into Spanish and used for validation of the IFH and IFS indexes for Portuguese language. In the second phase, the CIL were restructured and the language and readability parameters were analyzed and compared with the previous CIL. The socioeconomic profile of the users and a list of problems related to obtaining biological samples were obtained in the DLC. Results: Regarding the language parameters, the number of lines per CIL and the proparoxytone and polysyllabic words was similar between the previous and new CIL (p> 0.05). While the number of technical terms and words per sentence were smaller, and number of subordinate and coordinated sentences and inverse order of words was higher in the new CIL (p<0.05). The IFH score (58.4 ± 8.8) was lower than the IFS score (62.3 ± 6.8, p = 0.019) for 27 previous CIL, but did not differ for the 40 new CIL (p = 0.162). The IFS and IFI scores of the new CIL in Portuguese were similar to those translated into Spanish, which is indicative of the fact that these indices are valid for the Portuguese language. The IFH and IFS indexes for didactic texts used to assess the educational level were similar for elementary education (EF) I and II, but were lower for the last two years of secondary education (MS). The number of CIL with readability indexes corresponding to the didactic texts for the 3th year of EF II (32.7%, n=10) was lower than the number of new CIL (52.5%, n=21), but the difference was not significant (p=0.280). The percentages of gender and self-reported color were similar between the two phases of the study. However, young adults people (<34 years), older people (>50 years) and more users with EF, MS and complete higher education were attended in the second phase of the study (p<0.05). More guiding failures were identified in the first phase (n=11) than in the second (n=22) but no significant difference (p=1,000). Conclusion: The language and readability parameters improved after the restructuring of the new CIL. The readability analysis contributes to the communication of the clinical laboratory with the users and can positively influence the quality of the pre-analytical phase of the laboratory process.