The pre-analytical question in anatomical pathology. Implications and challenges. for personalized medicine in

Transcription

1 The pre-analytical question in anatomical pathology. Implications and challenges The pre-analytical question in anatomical pathology. for personalized medicine in Implications and challenges for a digital world personalized medicine in a digital world Pedro Soares de Oliveira M.D. Dept. of Pathology, Hospital da Luz, Lisbon, Portugal Pedro Soares de Oliveira M.D. Dept. of Pathology, Hospital da Luz, Lisbon, Portugal

2 Disclosures

3 THE FOLLOWING PRESENTATION IS CONTROVERSIAL AND MAY BE OFFENSIVE TO SOME AUDIENCES VIEWER DISCRETION IS ADVISED

4 BIG MAC

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6 Looks the SAME and taste the SAME everywhere

7 c-erb B2

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9 Does not look the SAME everywhere

10 The McDonnald s around the corner IS BETTER than YOUR Pathology Department! SHAME ON YOU

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12 More and more cancer drugs are pathology dependent (ex. Pembrolizumab for NSCLC) More and more genomic tests are done in FFPE tissue (ex. OncotypeDX) More and more image analysis assessments are made in IHC digitalized slides (ex. Ki-67 scores)

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16 Williams Edwards Deming ( )

17 WORLDWIDE

18 Pay attention to the PRE-ANALYTICAL phase in Anatomic Pathology

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20 Workflow in Anatomic Pathology Tissue Specimen Labeling, Transporting Specimens Specimen Accession Path Lab Receiving Grossing Specimens Slides Delivered Staining Mounting Labelling Embedding Cutting Section Fixing Processing Specimens Examining Interpreting Slides Considering Ancillary Test Others Composing Report Receiving Interpreting Report

21 Workflow in Anatomic Pathology Tissue Specimen Labeling, Transporting Specimens Specimen Accession Path Lab Receiving Grossing Specimens Slides Delivered Staining Mounting Labelling Embedding Cutting Section Fixing Processing Specimens Examining Interpreting Slides Considering Ancillary Test Others Composing Report Receiving Interpreting Report

22 Anatomical Pathology really begins immediately after the tissues have been removed!

23 For small biopsies the process is more or less controlled

24 that s NOT the case for large specimens

25 to a theather near you!

26 FIXATION is the most critical step in the pre-analytical phase. Impact in morphology Impact in IHC Impact in Genomics (Impact in digital image)

27 Modern Pathology (2013) 26, 1 9 & 2013 USCAP, Inc. All rights reserved /13 $ Delay to formalin fixation cold ischemia time : effect on ERBB2 detection by in-situ hybridization and immunohistochemistry Bryce P Portier 1, Zhen Wang 1, Erinn Downs-Kelly 1, Jordi J Rowe 1, Deepa Patil 1, Chis Lanigan 1, G Thomas Budd 2, David G Hicks 3, David L Rimm 4 and Raymond R Tubbs 1 1 Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, USA; 2 Department of Solid Tumor Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA; 3 Department of Pathology and Laboratory Medicine, University of Rochester, Rochester, NY, USA and 4 Department of Pathology, Yale University, New Haven, CT, USA

28 Volume 80, Issue 2, April 2006, Pages Assessment of fixatives, fixation, and tissue processing on morphology and RNA integrity Melissa L. Cox,, Carrie L. Schray, Chandra N. Luster, Zachary S. Stewart, Peter J. Korytko, Kanwar Nasir M. Khan, Joseph D. Paulauskis, Robert W. Dunstan Molecular characterization of morphologic change requires exquisite tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses on fixed tissues, we assessed morphologic and RNA integrity in rat liver when sections were fixed in 70% neutral-buffered formalin, modified Davidson s II, 70% ethanol, UMFIX, modified Carnoy s, modified methacarn, Bouin s, phosphate-buffered saline, or 30% sucrose. Each sample was subjected to standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity. Modified methacarn, 70% ethanol, and modified Carnoy s solution each resulted in tissue morphology representing a reasonable alternative to formalin. Modified methacarn and UMFIX best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was modestly improved. We conclude that modified methacarn, 70% ethanol, and modified Carnoy s solution provided acceptable preservation of tissue morphology and RNA quality using both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens.

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30 Element to Reconcile Cold ischemic time Handling of specimens obtained remotely Fixation time in neutral buffered formalin Optimal sample for testing 2007 HER2 Testing Guideline Recommendation Time from tissue acquisition to fixation should be as short as possible; no specification of time or requirement to document No recommendation 6 to 48 hours in neutral buffered formalin; less fixation time permissible for needle biopsy specimens Resection specimens preferentially recommended for testing because of possible artifacts on core biopsy 2010 ER and PgR IHC Testing Guideline Recommendation Recommends the interval be one hour and requires that the time between tissue removal and initiation of fixation must be recorded to document that tissue is handed from the surgical field and placed in fixative as quickly as possible. Recommends that specimens be bisected through the tumor on removal and that time of removal, fixative type, and time placed in fixative must be recorded 6 to 72 hours in neutral buffered formalin for all specimens Core needle biopsy specimens preferentially recommended for testing to avoid prolonged interval before fixation Reconciled Recommendation for HER2 Testing Follow ER and PgR 1 Testing recommendation Follow ER and PgR 1 recommendation Testing No changes in the recommendation listed in the 2007 HER2 guideline. No change in recommendation. Pathologist to use discretion in selecting sample for testing

31 How can WE take control of FIXATION?

32 Virchows Arch (2008) 452: DOI /s x LETTER TO THE EDITOR Tissue transfer to pathology labs: under vacuum is the safe alternative to formalin Gianni Bussolati & Luigi Chiusa & Antonio Cimino & Giuseppe D Armento Contents lists available at ScienceDirect Science of the Total Environment journal homepage: Vacuum-based preservation of surgical specimens: An environmentally-safe step towards a formalin-free hospital Cinzia Di Novi a, Davide Minniti b, Silvana Barbaro b, Maria Gabriella Zampirolo b, Antonio Cimino c, Gianni Bussolati c, a Dept. Of Public Policy and Choice University of Eastern Piedmont, Italy Received: 16 October 2007 / Accepted: 16 October 2007 / Published online: 21 December 2007 # Springer-Verlag 2007 b Azienda Ospedaliera-Universitaria San Giovanni Battista di Torino, Italy c Dept. Biomedical Science and Human Oncology University of Turin, Italy The time-honoured use of formalin, both as a preserver and fixative for histological processing is encountering increasing criticisms because of toxicity and environmental concerns. Moreover, the declaration recently issued by the International Agency for Research on Cancer [1] which classified formaldehyde as a class 1 carcinogen has definitely increased the request by health authorities, technicians and practicing pathologists to reduce exposure. Such requests contrast with the considerable advantages offered by this safe, chip, reliable fixative. Although it should be acknowledged that in modern pathology laboratories, visitors are not any more met by the permeating flavour of a stingy scent because formalin processing is mostly carried out under aspiration hoods, a critical passage is still represented by the transfer of tissues from the surgical theatre to the pathology lab. Apart from the small biopsies, which are directly collected into pre-filled containers and cause no concern, problems are encountered with the immersion of large specimens and organs into large boxes to be filled with formalin. A list of such problems follows: 1. Plastic containers are large and relatively heavy, while on occasion spilling may occur. 2. Immersion into formalin prevents the collection of fresh material for tissue banking. Fixation starts, but only at the periphery. Discoloration occurs, while a delay in the transfer to pathology labs is somehow justified by the fact that the tissue is already in formalin. G. Bussolati (*) : L. Chiusa : A. Cimino : G. D Armento Department of Biomedical Sciences and Human Oncology, University of Turin, Via Santena n. 7, Torino, Italy gianni.bussolati@unito.it 3. Nurses at the surgical theatre are becoming increasingly concerned for toxicity and cancerogenicity issues because the fluid has to be managed freely and not under hood. 4. When the container arrives at the pathology lab, its opening, extraction of the specimen and reduction of the latter constitutes a major cause of diffusion of formaldehyde fumes. To circumvent such problems, we adopted a transfer under vacuum. We selected a semi-professional machine, of a relatively limited cost and size (Mod. VAC 10, by Milestone, Bergamo, Italy; see medsrl.com) (Fig. 1) and capable of handling large bags. After testing the use of such machine on different tissue specimens and organs (colon, gallbladder, spleen and kidney) and checking the safety of the processing from the point of view of histological preservation, we trans- Fig. 1 The under vacuum machine used in our hospital article info Article history: Received 2 March 2010 Received in revised form 7 April 2010 Accepted 13 April 2010 Available online xxxx Keywords: Formalin Tissue specimens Histopathology Under-vacuum sealing Pathology 1. Introduction abstract Formalin, a 4% solution of formaldehyde in water, is a world-wide and extensively used fixative for histopathological specimens. Since its discovery at the end of 19th Century (Fox et al., 1985), this aldehyde has been universally appreciated as a simple reagent that is a good antiseptic, penetrates tissues quickly (diffusion rate of 1 cm in 24 h) and is easy to handle. In tissues that are formalin-fixed, morphology is well preserved, as is tissue antigenicity, and immunohistochemical procedures of diagnostic interest have routinely been adapted to formalin-fixed tissues (Dabbs, 2008). In addition to multiple industrial applications, the medical use of formalin as a tissue preserver and fixative is extensive, especially in pathology laboratories. In fact, the amount used in public hospitals in the Piedmont region (Italy) alone for the preparation of approximately 300,000 histological exams is in the range of 100,000 liters per year. Tissues preserved in formalin are even sent by post, in the number of several thousands per year. Despite its advantages, formaldehyde has some drawbacks that demand caution: it is allergenic to the skin and produces irritating vapors, causing asthma. The International Agency for Cancer Research Corresponding author. Department of Biomedical Sciences and Human Oncology, University of Turin, Via Santena 7, Turin, Italy. Tel.: ; fax: address: gianni.bussolati@unito.it (G. Bussolati) /$ see front matter 2010 Published by Elsevier B.V. doi: /j.scitotenv Formalin as a fixative has no practical substitutes, but is toxic and potentially carcinogenic, so caution of its use in hospitals and elsewhere is mandatory. In our hospital, preservation of surgical specimens into formalin to be transferred to pathology labs was replaced by under-vacuum sealing (UVS) tissues into plastic bags and preservation at 4 C until transfer. Data analysis showed UVS processing to be superior in terms of staff satisfaction and of gross anatomic preservation; no problems in terms of technical feasibility or histopathologic preservation were encountered. Formalin was confined to pathology labs while its use on hospital premises was vastly reduced Published by Elsevier B.V. (IARC, 2006) has declared formaldehyde to be a Class 1 carcinogenic agent, and statistical evidence has been presented on a possible link between formaldehyde exposure and lymphohematopoietic malignancies (Beane Freeman et al., 2009). Several attempts have been made to find a substitute for formalin, but so far all of the proposed alternatives have failed, being either ineffective or unreliable (Titford and Horenstein, 2005). A more practical approach would be to limit the use of formalin to pathology laboratories, where this toxic agent is carefully handled with hoods and gloves in safe environmental conditions, and to avoid its use in other less-protected areas of the hospital, such as in surgical theaters, where removed tissues are commonly placed in boxes full of formalin until transfer to the pathology labs. In fact, despite the advantages linked to this procedure (fixation and anti-sepsis begin immediately for the removed tissues and organs, and dehydration is avoided) several disadvantages are also recognized (see Table 1). To overcome these problems, we proposed an alternative procedure, which is the under-vacuum sealing of tissues (UVS) in plastic bags inside the surgical theatre immediately after removal, and to keep them cooled at 4 C until transfer to the pathology labs, where they are routinely processed. Safety and advantages linked to this UVS procedure have already been reported elsewhere (Bussolati et al., 2008). This processing was tested for more than two years in a single surgical theater, and it is now being extended to the whole hospital. The present study compares the compliance of personnel and the feasibility of this new procedure in a large regional hospital to the

33 Standardization and documentation of the pre-analytical step MILESTONE H E L P I N G P A T I E N T S FORMALIN-FREE TissueSAFE High vacuum biospecimens transfer system

34 FORMALIN

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36 With fresh specimens received in optimal conditions the AP LAB can really decide and control the FIXATION process and provide a SAFETY environment!

37 SAMPLING

38 Do you know how thick are the samples taken for processing in your lab? 1 mm? 2 mm? 3 mm? 4 mm? 5 mm?

39 Bacon knows

40 Proper fixation and processing are related to the thickness of the samples taken!

41 Cutting board

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43 2 mm 1.5 mm Calibrators

44 Specimen Standardization With Accu-Edge Grossing Tools Cutting-Edge Tools Specially Designed for Uniform Results Grossing board has two adjustable wells to accommodate specimens of varying size and thickness Grossing forks feature two sets of sharp tines separated by a space of 1.5, 2.0, or 2.5 mm for precise grossing of the most difficult tissues Tissue tampers hold specimens firmly in the wells for easier trimming Scalpel and trimming blades provide unsurpassed blade-edge sharpness for maximum cutting efficiency The critical foundation necessary for uniform grossing and optimal processing results. We offer an extensive line of grossing boards and wells: 4800 Tissue-Tek Accu-Edge Grossing Board, with Wells Standard 42.3 (L) x 29.3 (W) x 2.5 (H) cm The enhanced grossing wells allow for either left- or right-hand operation and can even be used on top of traditional grossing boards Tissue-Tek Accu-Edge Grossing Wells 33.0 (L) x 8.6 (W) x 5.7 (H) cm 4801 Tissue-Tek Accu-Edge Grossing Board, with Wells Large 42.3 (L) x 59.9 (W) x 2.5 (H) cm 4812 TissueTek Accu-Edge Grossing Board, Large 42.3 (L) x 29.3 (W) x 2.5 (H) cm Visit our web site at Trusted Innovation TM Sakura Finetek U.S.A., Inc West 214th Street Torrance, CA U.S.A. Phone: (800) Sakura Finetek U.S.A., Inc. All rights reserved Printed in U.S.A.

45 and you will see how EASY it is to standardize sampling thickness!

46 And, what about histological processing?

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49 Anatomic Pathology Labs are NOT YET evaluated by MICHELIN!

50 WE need to STOP using different RECIPES for histological processing and more important, WE need to STOP using HOUSE MADE solutions!

51 Labmade solutions

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54 The New Era in Rapid Tissue Processing

55 Innovative Technology De livers Remarkable Results Absolute Automation and Intelligent Design Novel System of Reagents for Distinct Detail Emphasizes a unique retort design, with advanced robotic and microwave technology Reduces reagent carryover with unique robotic features that vibrate each basket Enables increased tissue size with the use of enhanced reagents up to 33% thicker compared to the previous generation Complete Tissue Processing With Only 2 Reagents 1. Processing reagent #1 fixates, dehydrates, and clears. Highlights intuitive menu screens and software that foster easy access to processing information Supports compatibility with the Tissue-Tek AutoTEC Automated Embedding System Meets objective Lean and Six Sigma criteria. Utilizes a unique blend of low power microwave and traditional technology Groundbreaking Microwave Technology Includes an exclusive paraffin formulation Preserves DNA, RNA, and proteins in the paraffin blocks of fresh tissues or tissues preserved in the Tissue-Tek Xpress Molecular Fixative Eliminates the use of formalin and xylene, minimizing waste management costs Virtually eliminates chemical exposure due to closed system Features easy-to-use processing reagent sets x120 two 3.8 L bottles of reagent #1 and two 3.0 L pouches of reagent #2 x50 one 3.8 L bottle of reagent #1 and one 3.0 L pouch of reagent #2 Conventional Processing Tissue-Tek Xpress x Series Processing 2. Processing reagent #2 promotes paraffin impregnation. Features consistent circular microwave patterns (Fig. 1) Low power and even temperature distribution eliminate hot and cold spots (Fig. 2) Fig. 1. The whispering chamber effect. The Tissue-Tek Xpress x Series technology and reagents provide exceptionally sharp morphology and nuclear detail that pathologists expect and require for accurate examination and diagnosis. Continuous throughput eliminates batching bottlenecks Allows all specimen types to be processed in the same run Fig. 2. Exclusive agitation system. The x120 uses 2 rapid changes of reagents and paraffin

56 The PARAFFIN embedding

57 Welcome to Sakura Americas - Welcome of Sakura-Americas 11/02/16, 10:54 HOME HISTOLOGIC TECH SUPPORT WEBINAR LIBRARY SEARCH: Go GROSSING & TRIMMING PROCESSING PRINT INFO EMBEDDING Tissue-Tek AutoTEC a120 Tissue-Tek AutoTEC Tissue-Tek TEC 5 Tissue-Tek NanoMolds MICROTOMY PRIMARY & SPECIAL STAINING/COVERSLIPPING IMMUNOHISTOCHEMISTRY STORAGE & CASSETTES Tissue-Tek AutoTEC a120 The new AutoTEC a120 remains the industry s first and only fully automated embedder. Enhanced features include a cassette barcode reader for LIS connectivity and traceability of specimen blocks, the new SMART Air Technology to remove excess paraffin, and the larger 15-inch touchscreen monitor and enhanced operating system both deliver improved user convenience. With its smaller footprint, the AutoTEC a120 can be installed in small laboratories, and Sakura s Tissue-Tek isupport program provides remote diagnostics and peace of mind. CYTOLOGY PRINTERS DIGITAL IMAGING VIEW THE ELECTRONIC CATALOG VIEW SAFETY DATA SHEETS Make the move to automated embedding. Sakura Finetek will launch Tissue-Tek AutoTEC a120 Automated Embedder at NSH 2015 in Washington, DC, booth 101. For more information, click on the tabs below, contact your Sakura representative, write to mail@sakuraus.com, or call and press 1 for customer service. Tissue-Tek AutoTEC a120 Automated Embedder The second-generation Tissue-Tek AutoTEC a120 remains the industry s first and only fully automated embedding system that eliminates the tedious and labor-intensive need to manually orient and embed tissue specimens and form tissue or cell paraffin blocks. The AutoTEC technology, combined with the Tissue-Tek Paraform Sectionable Cassette System ensure that the orientation of specimens, determined by pathologists and pathologist assistants, is locked from grossing to microtomy for all routine tissue types, thereby eliminating the risk of orientation mistakes and tissue loss for increasing patient safety. New enhanced features The new enhanced features include the incorporation of a cassette bar code reader for future LIS connectivity and traceability of specimen blocks, and the new SMART Air Technology to remove excess paraffin. A larger 15-inch touchscreen monitor and the enhanced operating system both deliver improved user convenience. Redesign of the input and output doors, the paraffin reservoir and access to the base molds provides improved ergonomics while requiring minimal user maintenance and further increasing up-time. With its smaller footprint, the AutoTEC a120 can be installed in small laboratories, and Sakura s Tissue-Tek isupport program provides remote diagnostics and peace of mind. Increased productivity with predictable performance in TAT and block quality Automatic Embedding Página 1 de 2

58 Nano Technology Coated Base Molds

59 Save Time, Energy and Money Faster Block Release Eliminates Need For Using Mold Release Solution Less Cleaning Available In 5 Popular Mold Sizes Specifications (Dimensions sizes in mm s) #4215 NanoMold, 9 x 9 x 4 #4216 NanoMold, 15 x 15 x 4 #4217 NanoMold, 20 x 20 x 5 #4218 NanoMold, 20 x 26 x 5 #4219 NanoMold, 20 x 33 x 5 Tissue-Tek NanoMolds produce uniform paraffin blocks much quicker and release easier than traditional methods all without the use of messy chemical mold release solutions. NanoMold stainless steel base molds are manufactured with an exclusive and patented Nano Technology coating that speeds up the embedding process helping laboratories save time, labor and increase efficiency. Even more time savings are achieved because residual paraffin is no longer left in the molds, drastically reducing the amount of cleaning necessary. Tissue-Tek NanoMolds are available in five popular sizes to fit all of your embedding needs. Sakura Finetek USA, Inc th Street Torrance, CA Phone: (800) Sakura Finetek USA, Inc Rev. A Printed in USA

60 What about MICROTOME SECTIONING?

61 SECTIONING has strong implications in the pre-analytical phase. Impact in morphology Impact in staining Impact in IHC (Impact in digital image)

62 Image analysis

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64 and AutoTrim technologies The Next Level in Precision Sectioning Optional and included accessories Specifications Fully Automated SMART Microtomy Item Code Included 5000 Optional Description AutoSection AutoSection Automated Microtome Low-Profile Blade Holder Wireless Remote Control Waste Collection System Foot Pedal AutoSection High-Profile Blade Holder Waste Collection Bags 250/pcs Related Products Tissue-Tek Cold Plate 24/case Tissue-Tek TEC Plus, Cryo Module Tissue-Tek SmartWrite Color Slide Printer Blades Accu-Edge Paraform Blades Accu-Edge High-Profile Disposable Blades Accu-Edge Low-Profile Disposable Blades Tissue-Tek Feather Low-Profile Blades Function Administrator access Section thickness Trimming thickness Retraction distance Sectioning speed Trimming speed Return speed Maximum specimen size Maximum vertical stroke Maximum horizontal stroke Number of presets Number of macro program steps Number of user preferences Specifications Password protected microns microns No retraction, microns mm/second mm/second mm/second 60 x 60 mm 122 mm 10 mm Up to 16 different presets Up to 16 steps Up to 32 different preferences Power Supply Requirements single electrical design accommodates the following voltages: 115 VAC ± 10%, 50/60 Hz, single phase, 7.1 amps 230 VAC ± 10%, 50/60 Hz, single phase, 4.1 amps 100 VAC ± 10%, 50/60 Hz, single phase, 8.2 amps Dimensions Centimeters: Inches: Weight: 38 (W) 67 (D) 44 (H) 15 (W) 26.4 (D) 17.4 (H) 55 kg/121 lb Microtome Specifications Section thickness: microns Trim thickness: microns Sectioning speed: mm/second Maximum specimen size: mm External Agency Approval UL, CSA, CE, IVD, ISTA Visit our web site at Trusted Innovation TM Sakura Finetek USA, Inc West 214th Street Torrance, CA U.S.A. Phone: (800) Bluetooth is a registered trademark of Bluetooth SIG Sakura Finetek USA, Inc. All rights reserved Rev. A Printed in U.S.A.

65 STAINING

66 Question #1: How good is your H&E?

67 Question #2: How consistent is your H&E?

68 FADDING GLORY

69 Integrated drying, staining, coverslipping and curing of H&E specimens CareGiver remote system monitoring Discover a new world of H&E staining with the VENTANA HE 600 system Large, easy to see, user interface with intuitive software Automated system reports Individual slide staining; fresh reagents on every slide Glass coverslips No xylene or alcohol Recyclable consumable packaging Limited daily maintenance Error proof reagent exchanges DRAFT

70 The VENTANA HE 600 system Average quality score 3.8 Traditional dip and dunk stainer Average quality score 3.0 White paper How Dr. Mueller Comparative analysis of H&E stain quality between the defined each 204 VENTANA HE 600 system and a traditional linear stainer rating: Excellent 63 Excellent Excellent Virtually flawless in every way; photographic quality stain. Good Tissue was interpretable but minor defects were visible. Fair Defects distract from main event when diagnosing. 138 Good Poor Severe defects lead to frustration and extreme eye fatigue. 32 Good 4 Fair 23 Fair 16 Poor

71 In summary

72 Too many rough spots on the pre-analytical phase

73 How to fix them: technology new NORMS

74 Thanks