EBME 306 LECTURES 1-3-FALL-2023-ZIATS.pptx

INTRODUCTION to BIOCOMPATIBILITY Readings for Sept. 15, 18 & 20, 2023 Biomaterials: The Intersection of Biology and Materials Science, J.S. Temenoff and A.G. Mikos, Pearson/Prentice Hall, 2008, pp.7-9, 314-324, 353-365, 393-398, 437-438 Biocompatibility “The ability of a material to perform with...

INTRODUCTION to BIOCOMPATIBILITY Readings for Sept. 15, 18 & 20, 2023 Biomaterials: The Intersection of Biology and Materials Science, J.S. Temenoff and A.G. Mikos, Pearson/Prentice Hall, 2008, pp.7-9, 314-324, 353-365, 393-398, 437-438 Biocompatibility “The ability of a material to perform with an appropriate host response in a specific application” Biomaterials-Tissue Interactions Hemostasis/Thrombosis Design of Replacement Tissues Sequence of Events Intended Application In Vitro, In Vivo, Ex Vivo Testing Tissue Procurement and Material Evaluation Regulatory Issues (Safety) Medical Devices and the FDA Food and Drug Administration (FDA): Regulatory program for devices (not materials) in 1938, with goal of premarket testing, manufacture and postmarket experience of devices (not materials), good manufacturing practices (GMP) Medical Devices and the FDA Medical Device/FDA websites: 1. How to classify devices (a good introductory website): http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/Overview/C lassifyYourDevice/ucm051530.htm 2. 501K and Device definitions: https://www.fda.gov/media/82395/download 3. Another version: Message from Greenlight Guru Medical Devices and the FDA Any instrument, apparatus, implement, machine, appliance, implant, in vitro reagent or calibrator, software, material or other similar or related article, intended by the manufacturer to be used, stored or in combination for human beings for one or more of the following: ► ► ► ► ► ► diagnosis, prevention, monitoring, treatment or alleviation of disease diagnosis, monitoring, treatment alleviation of or compensation for an injury investigation, replacement modification or support of the anatomy or of a physiological process supporting or sustaining life control of conception disinfection of medical devices Medical Device Classification-FDA  Class I  Class II  Class III  Combination Product  When applicant files a Premarket Approval application (called a 501K and includes safety, efficacy, clinical utility of a device), FDA then classifies device. The class of the device determines the level of control and whether or not exemptions can be used. No exemptions for Class III. Medical Device Classification-FDA  Exemptions refer to premarket notification to the FDA and means the manufacturer doesn’t need to inform FDA about launching onto the market, but are NOT exempt from general controls which include:  Manufacturing under quality assurance and GMP  Suitable for intended use  Adequately packaged and labeled Medical Device Classification-FDA  Class I  Low risk. Are devices:     For which certain general controls, are considered to provide reasonable assurance of the safety and effectiveness of the device Devices that are not intended to support or sustain life or cause an unreasonable illness or injury Need not be reviewed by FDA before marketing Examples: tongue depressors, bedpans, elastic bandages, examination gloves, and some hand-held surgical instruments Medical Device Classification-FDA  Class II    Devices that present more risk, special controls needed Class II medical devices are held to a higher level of assurance than Class I medical devices in that they will perform as indicated and will not cause injury or harm to patient or user. Example: x-ray machines, powered wheelchairs, infusion pumps, surgical drapes, surgical needles, suture materials, acupuncture needles, stainless steel bone plate, vascular grafts Medical Device Classification-FDA  Class III   Devices that are intended to support or sustain life Example- heart valve, artificial heart, assist devices Categorization of Medical Devises by Nature of Body Contact IV. Combination Product   A product comprised of two or more regulated components, i.e., drug/device, biologic/device, drug/biologic, or drug/device/biologic, that are physically, chemically, or otherwise combined or mixed and produced as a single entity Device coated or impregnated with a drug or biologic such as drug-eluting stent, pacing lead with steroid-coated tip, catheter with antimicrobial coating; condom with spermicide, skin substitutes with cellular components, prefilled syringes TRANSLATIONAL QUESTION: As a biomaterial scientist describe the sequence of events in taking a raw chemical and making a device for implantation into a human, for example, a new type of vascular graft. What do you need to do to get FDA approval? What would be the estimated cost of getting this device to the bench, through clinical trials and then to the FDA for approval? Biocompatibility Evaluation  Why do biomaterials fail?  Pathogenesis of biomaterial failure Pathogenesis of Biomaterial Failure Sequence of events that lead to the structural or functional abnormalities = The pathogenic mechanism(s) A---- B---- C---- Implant Failure---- Remove Biomaterial Implant Morbidity or Mortality Diagnostic Pathology of Implants  Removal of organ or tissue- actually called an explant  Examination using eyes- pathologic findings- gross & microscopic  Report Diagnostic Pathology of Implants  Pathologic Findings-Morphologic ► GROSS- DESCRIPTIVE- “liver-like, hard, soft, fibrotic, necrotic” ► MICROSCOPIC- DESCRIPTIVE- “fibrotic, calcific, neoplastic, necrotic, apoptotic, pyknotic” ► IMAGING- CT, MRI, Ultrasound, etc ► CLINICAL LAB FINDINGS- QUANTITATIVE- K+ level, cholesterol level, BUN, LDH, serum IgG, urine glucose, CSF, Blood count or CBC, differential blood count etc. Most of this is automated. Histologic Evaluation of Biomaterials & Tissues  Fixation  Processing  Embedding  Sectioning  Staining  Microscopy Gross Examination and Fixation Gross examination consists of describing the specimen and then placing all or parts of it into a small plastic cassette which holds the tissue while it is being processed to a paraffin block. Initially, the cassettes are placed into a fixative, usually 10% buffered formalin. Other fixatives can be used, e.g. 2% buffered glutaraldehyde for SEM Gross Pathology Aortic Stenosis- Calcification Gross Pathology Vascular Graft Failure Tissue Processing Once the tissue has been fixed (formalin), it must be processed into a form in which it can be made into thin microscopic sections. The usual way this is done is with paraffin (wax). Tissues embedded in paraffin, which is similar in density to tissue, can be sectioned at anywhere from 3 to 10 microns, usually 6-8 routinely. The technique of getting fixed tissue into paraffin is called tissue processing. Tissue Embedding Tissues that come off the tissue processor are still in the cassettes and must be manually put into the blocks by a technician who must pick the tissues out of the cassette and pour molten paraffin over them. This "embedding" process is very important, because the tissues must be aligned, or oriented, properly in the block of paraffin. Tissue processing: embedding moulds: (A) paper boat (B) metal bot mould (C) Dimmock embedding mould (D) Peel-a-way disposable mould (E) base mould used with embedding ring (F) or cassette bases (G) Tissue Sectioning Once the tissues have been embedded, they must be cut into sections that can be placed on a slide. This is done with a microtome. The microtome is nothing more than a knife with a mechanism for advancing a paraffin block standard distances across it. It is important that for proper sectioning one has a very sharp knife Sectioning and Slides Sectioning tissues is a real art and takes much skill and practice. Once sections are cut, they are floated on a warm water bath that helps remove wrinkles. Then they are picked up on a glass microscopic slide. Tissue Staining The embedding process must be reversed in order to get the paraffin wax out of the tissue and allow water soluble dyes to penetrate the sections. Therefore, before any staining can be done, the slides are "deparaffinized" by running them through xylenes (or substitutes) to alcohols to water. The staining process makes use of a variety of dyes that have been chosen for their ability to stain various cellular components of tissue. The routine stain is that of hematoxylin and eosin (H and E). Other stains are referred to as "special stains" because they are employed in specific situations according to the diagnostic need. Coverslipping The stained section on the slide must be covered with a thin piece plastic or glass to protect the tissue from being scratched, to provide better optical quality for viewing under the microscope, and to preserve the tissue section for years to come. Histologic Evaluation Of Implants/Explants-Stains             HEMATOXYLIN & EOSIN-general morphology COLLAGEN- Masson’s Trichrome ELASTIN- Verhoff’s GLYCOSAMINOGLYCAN’S- PAS/Alcian blue BACTERIA- Gram’s BLOOD SMEAR- Wright’s FUNGI- PAS or Methenamine silver IRON- Prussian blue CALCIUM (CaPO4)- von Kossa FIBRIN- PTAH AMYLOID- Congo red with polarizing light IMMUNOSTAINING-Antibodies to specific antigens How does a Pathologist/Biomaterial Scientist make a diagnosis?  Careful examination of the gross specimen, which is then sampled for microscopy  Low-power magnification (20-40x)  Organ/tissue site  Basic disease process (e.g. inflammatory, neoplastic)  Specific diagnosis possible?  High-power magnification (200-400x)  Initial impressions correct?  Specific diagnosis?  Pathologists look for patterns  Ancillary testing and/or expert consultation, as needed Hematoxylin and Eosin Hematoxylin- stains cells/tissue blue (basophilia, a basic dye with (+) charge and reacts with anionic components, (phosphates, sulfates, carboxyl groups), stains nuclei Eosin- stains cells/tissues pink (eosinophilia, an acidic dye with (–) charge react with cationic components, cytoplasm and collagens Hematoxylin & Eosin Stain Cytoplasm Cells Nucleus Histologic Evaluation- H & E Comparative Staining H&E Trichrome Wright Stain of a Blood Smear Eos PMN-band Platelet PMNseg Lympho Baso Mono Bacteri and Fungi Gram’s & Gomori’s Silver Stains Gram + Gram - Gomori’s silver Other HistologicTechniques Polyethylene particles from a knee prosthesis Immunochemical markers used in biomaterials research Biological response Biomaterial research applications Specific immunochemical markers Inflammation Inflammatory response to orthopaedic wear debris is believed to be the primary reason for orthopaedic implant failure. Pro-inflammatorycytokines (IL-1, IL-6 TNF- ). Anti-inflammatory cytokines (IL-10) Cell attachment Frequently measured to determine substrate biocompatibility in vitro for a variety of biomaterials and tissue scaffolds. Materials may be seeded with proteins to either aid or inhibit cellular attachment. Integrins and focal contacts.  v 3, vitronectin, focal adhesion kinase (FAK) Cell death (apoptosis or necrosis). Toxic leaching or other material characteristics may cause cell death. Apoptosis (programmed cell death) causes significantly different biological response to necrosis. To asses the “health” of cells exposed to biomaterials, cell stress can be analysed. p53, PARP, BAX, caspases Cell activation Biomaterials may affect the cell cycle, either stimulating or inhibiting cell division. Cell cycle stage influences cell phenotypic responses. Ki-67 Cells that are in active stages of their cell cycle (i.e. not in G0). Bone formation Considerable bone research is focused on generating new bone formation, different materials affect bone nodule formation in vitro and in vivo. Alkaline phosphatase, osteocalcin, Bone morphogenic protein –1 (BMP-1), collagen Angiogenesis (blood vessel formation) Angiogenesis is vital for both bone formation and inflammation and therefore has numerous applications in biomaterials and tissue engineering. Including orthopaedic implant osseointegration and failure, tissue engineering of skin and bone. Pro-angiogeneic vascular endothelial growth factor (VEGF). Angiogeneic inhibitors endothelin, angiostatin Cell stress Heat Shock proteins (HSPs), are expressed by cells undergoing a variety of environmental stresses. Immunohistochemistry Addition of substrate and chromogens (DAB or Alkaline phosphate substrate solution). Enzyme labelled streptavidin conjugate Labelled (biotinylated) primary antibody Antigen A labelled primary antibody (e.g. biotin labelled mouse anti-human antibody) binds to the antigen of interest (an antigen is a “foreign substance”), Followed by incubation with an enzyme labelled conjugate, e.g. streptavidin labelled conjugate and prior to the addition of substrates and chromogens Immunohistochemistry of a Normal Tissue glucagon insulin Estrogen receptor positive breast cancer Immunohistochemistry of Cell/Material Interactions Note the altered morphology of endothelial cells with heavy particulate load (arrows), with a rounded cell shape and reduced elongation. 0.01mm VEGF receptor expression (red) by endothelial cells challenged with CoCr particles retrieved from tissue surrounding hip replacements with alkaline phosphatase conjugate. Immunofluorescence Fluorescent labelled primary antibody Antigen A fluorescent labelled primary antibody (such FITC labelled mouse anti-human antibody) binds to the antigen of interest. Different primary antibodies labelled with different fluorochromes can be incubated together for double/triple immunofluorescence Fluorescence Microscopy Fluorescence Microscopy Fibroblast stained for actin Immunofluorescence Cell-Materials Application Flow Cytometry Defined: A technique in which cells suspended in a fluid flow one at a time through a focus of exciting light, which is scattered in patterns characteristic to the cells and their components, AKA as FACS= Fluorescent activated cell sorting    They are often labeled with fluorescent markers (usually antibodies) so that light is first absorbed and then emitted at altered frequencies A sensor detecting the scattered or emitted light measures the size and molecular characteristics of individual cells Tens of thousands of cells can be examined per minute and the data gathered are processed by computer Sample put here Flow Cell Flow Cytometry Applications  Detection and quantification of cell bound antigens: Surface or Intracellular  Detection and quantification of bead bound antigens  Identification and enumeration of specific cell types within a complex mixture of cells  Separation of a specific cell type within a complex mixture of cells Typical peripheral blood scatter properties granulocytes monocytes Lymphocytes Heterogeneous Cells Are Readily Resolved by Using Flow Cytometry Each dot = one cell These cells are CD8+ and CD3+ CD 8+ cells CD3+ cells The advantages and disadvantages of commonly used immunocytochemistry techniques in biomaterial research Immunocytochemical technique Advantages Disadvantages Applications Immunohistochemistry and immunocytochemistry Specific localisation of antigens in situ. Staining lasts for years. Relatively inexpensive- no specialised equipment required. Not suitable for light impenetrable materials. Qualitative, and subjective without computer analysis. Commonly used for the examination of pathological samples e.g. disease diagnosis in biopsies. Immunofluorescence Has same advantages as immunohistochemistry but also enables the visualisation of cells grown on light impenetrable materials in situ. Can co-localise several antigens on the same cell. Fluorescence fades over time. Requires a relatively expensive fluorescent microscope. Widely used in cellular research both in vivo and in vitro. Western Blotting Sensitive, specific and semiquantifiable depending on band intensity. Cells lysed prior to examination and therefore unable to localise cell specific antigen expression. Commonly used in cell culture research and in vivo digested tissue sections. ELISA Quantifiable detection of antibody or antigen. Relatively quick and enables the detection of soluble factors released by cells. Very sensitive. Relatively cheap equipment. Often limited to commercially available ELISA kits. Does not allow in situ localisation of antigens. Frequently used for the detection of mediators released by cells in cell culture media and for diagnostic detection of antibodies to disease in human serum. Fluorescence activated cell sorter (FACS) Allows the quantification of the proportion cells expressing a certain antigen/antigens. Cells have to be detached from material prior to analysis. Requires expensive equipment. Commonly used in cell culture research and in vivo digested tissue sections. Used clinically Immunoelectron microscopy High magnification allowing subcellular location of antigens. Can be performed on light impenetrable materials Expensive and time consuming requires electron microscopy. Allows the intra-cellular location of antigens. Scanning Electron Microscopy The SEM is an instrument that produces a largely magnified image by using electrons instead of light to form an image. A beam of electrons is produced at the top of the microscope by an electron gun. The electron beam follows a vertical path through the microscope, which is held within a vacuum. The beam travels through electromagnetic fields and lenses, which focus the beam down toward the sample. Once the beam hits the sample, electrons and X-rays are ejected from the sample Surface Contamination of a Biomaterial Identification of a Biomaterial Surface Exposed to Blood surface Blood-Material Interactions PDMS (negative control) Nonactivated platelets DACRON (positive control) vs. Activated platelets Use of SEM to Identify Blood Cells on Materials FXII Immunogold Labelling to Identify Proteins on Surfaces FIB TRANSLATIONAL QUESTION: What are some of the advantages and disadvantages of using immune procedures in biomaterials research and how does this apply to clinical research and development? Describe what SEM is and how it can be used in biomaterials research. How can flow cytometry be used in biomaterials applications as well as translate to clinical research and development? Design of Replacement Tissues Sequence of Events Intended Application In Vitro, In Vivo, Ex Vivo Testing Tissue Procurement and Material Evaluation Regulatory Issues (Safety) Biocompatibility Testing  In Vitro (“In the Test Tube”)  In Vivo (“Inside the Animal”)  Ex Vivo (“Outside the Body”) General Principles of Biological Evaluation of Medical Devices  The following need to be considered for their relevance to the overall biological evaluation of the device        The material(s) of manufacture Intended additives, contaminants or residues Leachable products Degradation products Other components and their interaction with the final product Properties and characteristics of the final product If appropriate, identification and quantification of extractable chemical entities of the final product should preceed biological evaluation General Principles of Biological Evaluation of Medical Devices  Tests to be used in biological evaluation and the interpretation of the results of such tests should take into account:  the chemical composition of the materials, including the conditions of exposure  the nature, degree, frequency and duration of exposure of the device or its constituents to the body General Principles of Biological Evaluation of Medical Devices  The range of biological hazards can be Short term effects- acute toxicity, irritation to tissues, hemolysis, thrombogenicity  Long-term or specific toxic effects- sensitization, genotoxicity, carcinogenicity, and effects on reproduction  General Principles of Biological Evaluation of Medical Devices  All potential biological hazards should be considered for every material and final product, but does not imply that testing for all potential hazards will be necessary or practical  Any in vitro tests shall, be based on end-use applications and require good laboratory practice followed by evaluation by competent people. Positive and negative controls shall be used  Whenever possible in vitro screening should proceed in vivo testing General Principles of Biological Evaluation of Medical Devices  Testing shall be performed on the final product or on a representative sample of the final product or materials used in the final product  Test results cannot ensure potential for biological hazard, therefore, biological evaluations shall be followed by careful observations for unexpected adverse reactions or events in humans during clinical use Biological Evaluation Tests ISO-10993 indicates these are the initial tests          Cytotoxicity Sensitization Irritation Intracutaneous reactivity Systemic Toxicity (acute) Subacute/Subchronic Toxicity Genotoxicity Implantation Hemocompatibility ISO 10993 ISO 101993 document: Use of International Standard ISO 10993-1, "Biological evalu ation of medical devices - Part 1: Evaluation and testing with in a risk management process" - Guidance for Industry and Food and Drug Administration Staff (fda.gov) See pages 25-42 for tests Biocompatibility Testing Biological Evaluation Tests Supplemental Tests  Chronic Toxicity  Carcinogenicity  Reproductive Toxicity  Biodegradation  Toxicokinetics  Immunotoxicity Biocompatibility Testing In vivo vs. in vitro Test/Assay Advantages Disadvantages In vitro Turnover is fast, low cost, screening, standarized with + and - controls Relevance to in vivo In vivo Multisystem interactions, more comprehensive Relevance depends on species & site, controls, Turnover low-weeks, Concerns of using animals, cost!, outcomes more difficult to determine In vitro Tests for Biocompatibility  Agar Diffusion  Direct Contact  Extract Dilution or Elution  Hemolysis  Mutagenicity  Others-research In Vitro Testing How does one get cells? Organ/tissue This is “in vitro”” Dissect Digest Isolated cells or organ culture Agar Diffusion Test Tissue culture cells with an overlayer of agar or agarose gel. Then add test materials which will/will not diffuse through gel and measure uptake of a dye (cell death) over short time Grade Reaction Zone of Reaction 0 None No detectable zone 1 Minimal Few cells dead 2 Mild Reactive zone is adjacent to sample 3 Moderate Reactive zone may extend to 1 cm beyond sample 4 Severe Reactive zone extends beyond 1 cm around sample Direct Contact Test  Tissue culture cells without an overlayer of agar or agarose gel. Then add test materials in direct contact with cells. Depends on diffusion in culture medium. Measure with vital stain over time.  Problems: Movement may cause injury, therefore sensitivity issue and/or misinterpretation of toxic effect. Use with other tests. Extract Dilution (Elution) Test Extract of material is prepared and evaluated for cytotoxicity. Usually done in saline, PBS or culture medium or sometimes solventsDMSO. Then add to cultured cells and determine cell responses over time. Grade Cell Response Comments 0 None No detectable adverse response 1 Minimal >20% response 2 Mild >50% response 3 Moderate >70% response 4 Severe Near 100% adverse response Hemolysis Testing  Exposure of material to animal blood (rabbit) for a period of time, ~1 hour. After centrifugation, read supernatant for hemolysis (545nm). Less than 5% hemolysis indicates compatibility. Hemolysis Testing Test Biomaterial Exposure of material to animal blood (rabbit) for a period of time, ~1 hour. After centrifugation, read supernatant for hemolysis (545nm). Less than 5% hemolysis indicates compatibility. Control Biomaterial Other In vitro Tests Most of these are research assays Cell adhesion and spreading assays  Cell proliferation  Cell number over time, MTT test, Alamar blue test  Radioactive thymidine or chromium  LDH release  Cell Migration Assays  Intracellular Function Assays  Co-Culture Assays  Apoptosis tests  Cell Culture Cell Proliferation Assay Assay: counting number cells per well by enzymatic removal of cells using trypsin solution Test Groups: Culture wells coated with nothing (None), Fibronectin (FN) or Type IV collagen or Type V collagen prior to seeding cells , next day cells then counted at Day 0, then at Days 4, 7 Cell Migration Assay 1. Cell adhesion to beads 3. Migration of cells from beads Protein Coated Surface 2. Cells on Beads 4. Microscopy & Analysis Migration of Cells from Beads onto Coated Surfaces + Control Coated Surface Test Coated Surface Intracellular Function Assay Cell Cytoskeletal Disruption +Fibronectin coating Giemsa Stain CSLM for actin filaments and stress fibers +Test coating Few cells Stress fibers are in disarray (RhoA) Cell Co-Culture Assays Smooth Muscle Cells NO NO NO Culture Medium Millipore Insert NO NO NO NO Endothelial Cells Measure smooth muscle cell function in response to NO In Vivo Testing       Use of healthy animals vs. use of diseased animals (if applicable) Animal species Implant site Surgical technique Implant form- size, conditioning, dose, texture Evaluation of tissue response ► Histology, Immunohistology, Microscopy, Biochemistry, Mechanical testing Implantation of Biomaterials Inflammation and Healing Injury Acute inflammation Chronic inflammation Granulation tissue Foreign body reaction Fibrosis 1. 2. 3. 4. 5. 6. In vivo Biocompatibility”  ► ► Subcutaneous Cage In Vivo Biocompatibility Testing Subcutaneous implant system Subcutaneous Implant Polymer/Material It is most important to have controls! What is a positive control? What is a negative control? Control, + or - Biomaterial Evaluation of In Vivo Testing Fibrous encapsulation 7 days Biomaterial Capsule Underlying tissue 14 days Biomaterial 21 days Biomaterial 28 days Measure the capsule thickness over time In Vivo Analyses H&E Trichrome Material was here In Vivo Biocompatibility Testing Cage Implant System Days 3, 7, 14 & 21 Material Exudates Analyzed for Cell Counts Insert Cage (Empty cage can serve as control or another material as + control) Implantation Surfaces Explanted and Adherent Cells Analyzed By Microscopy-Light or SEM SEM of Cells on Materials in Cage Implant Polyurethane Degradation Adherent macrophages & FBGC release degradative enzymes and radicals. Macrophage adhesion Foreign body giant cells (FBGC) DEVICE FAILURE Explanted pacemaker lead Full thickness cracking Surface cracking Surface pitting  TRANSLATIONAL QUESTION: What is a positive and negative control in an experiment? In an experiment testing Material XYZ, this material is implanted in the back of a rat (subcutaneous tissue). All that you know about the material is that it is a “soft polymeric material that is porous”. What might you use as a positive or negative control? How might you determine what is the positive and negative control and how would you go about getting these materials? Ex vivo Blood Testing Circulate animal blood through tubes in an ex vivo shunt (types of tubes, time course and flow) Remove tubes, cut to 1 mm squares and do protein adsorption tests, thrombosis testing and SEM Data analyses of cell adhesion and protein adsorption Ex Vivo Blood Testing Biomaterial ANIMAL Thrombogenesis Testing  Exposure of material to animal blood and look for formation of blood clotting. Rather crude tests.  Specific coagulation tests, PTT, APTT  Platelet adhesion/activation studies  LDPE and PDMS used as standards In Vitro Blood Testing Blood-Material Interactions OK Adherence Embolization Fragmentation  TRANSLATIONAL QUESTION: How does one use these tests to correlate laboratory findings to clinical findings to eventual development of products? Biocompatibility “The ability of a material to perform with an appropriate host response in a specific application” Summary  Device Classification  Biocompatibility and biomaterial failure  Tissue processing, gross examination, microscopic examination and use of staining  General principles of biological evaluation of medical devices  Testing- general and specific aspects  In vitro, In vivo, Ex vivo testing