Methodologies in Disease Investigation (VET S2007) PDF

METHODOLOGIES IN DISEASE INVESTIGATION (VET S2007) Dr Noman Naseem [email protected] Disease and the Diagnostic Toolbox Clinical Pathology TOPIC OUTLINE Anatomic Pathology Molecular Biology Microbiology Helminthology CONTEXT: WHY DO WE CARE? Treatment proceeds from diagnosis Knowledge...

METHODOLOGIES IN DISEASE INVESTIGATION (VET S2007) Dr Noman Naseem [email protected] Disease and the Diagnostic Toolbox Clinical Pathology TOPIC OUTLINE Anatomic Pathology Molecular Biology Microbiology Helminthology CONTEXT: WHY DO WE CARE? Treatment proceeds from diagnosis Knowledge about diagnostic methodologies will let you choose the best approach for diagnosis Appropriate diagnosis = Early control of disease = good reputation LEARNING OBJECTIVES An overview of the diagnostic toolbox available to a veterinarian Understanding the importance of selecting an appropriate method for early disease diagnostics DISEASE ILLNESS OR SICKNESS • What is the diagnosis? • More challenging with non-human species • Disease diagnosis = puzzle • Vet assembles all the pieces of the puzzle • How many more diseases for all non-human species? • Technology advances = increase our ability to diagnose a specific disease www.vat19.com Depending on the specialty, there are many ways to diagnose disease THE DIAGNOSTIC TOOLBOX • Radiography • Ultrasonography • Endoscopy • Urinalysis • Biochemistry • Cytology • Parasitology • Histopathology • Immunohistochemistry • Molecular diagnostics • Toxicology • Virology • Microbiology • Electron microscopy In Vet Clinic Clinical Pathology Anatomic Pathology C L I N I C A L PAT H O L O G Y - C Y T O L O G Y • Quick, easy, cheap! Advantages • Rapid analysis of a variety of lesions (Skin scrapings, Ear swabs, Masses, Cavity effusion) • Assess for cell types and presence of pathogenic organisms 1 min 1 min 1 min Disadvantages 1. No tissue architecture 2. Low cellularity 3. Low number of cells Ex: Feline sporotrichosis, www.researchgate.net Sometimes very straightforward…. CY TOLOGY Mast cell tumour (toluidine blue staining) CYTOLOGY Sometimes pretty confusing…. C L I N I C A L PAT H O L O G Y – COMPLETE BLOOD COUNT (CBC) • Again, rapid and relatively cheap • Includes: WBC cell count, RBC cell count, haematocrit, haemoglobin, MCV, MCH, MCHC, platelet count, mean platelet volume HAEMATOLOGY (VLS) RBC COUNT HAEMOGLOBIN HAEMATOCRIT PCV (manual) MCV MCH MCHC RETIC % RETICULOCYTE ABSOLUTE PLATELET COUNT (MACHINE) WHITE CELL COUNT NEUTROPHIL BAND NEUTROPHIL LYMPHOCYTE MONOCYTE EOSINOPHIL BASOPHIL PLASMA PROTEIN PLASMA APPEARANCE DOG Units Flag 1.06 36 0.11 0.17 104 34 195 45.0 399.70 X 1012/L g/L L/L L/L fL pg g/L % X 109/L L L L L H H L H H 315 X 109/L 76.14 66.93 6.84 6.1 6.76 0.00 0.00 X 109/L X 109/L X 109/L X 109/L X 109/L X 109/L X 109/L 72 REFERENCE INTERVAL 5.4 – 8.5 125 – 201 0.35 – 0.58 0.34 – 0.58 7 58 – 80 20 – 27 280 – 400 0.00 – 1.50 10 – 110 200 – 520 H H H H H 6 – 17 3.5 – 12 0 – 0.3 0.9 – 3.5 0 – 1.1 0 – 1.4 0–1 g/L 62 – 85 TRACE HAEMOLYSIS BIOCHEMISTRY DOG Units Flag REFERENCE INTERVAL GLUCOSE 2.56 mmol/L L 3.3 – 6.8 UREA 2.8 mmol/L L 3.4 – 10.8 CREATININE 40 umol/L L 50 – 130 TOTAL CALCIUM 2.7 mmol/L 1.6 – 2.8 PHOSPHOROUS 1.05 mmol/L 0.7 – 2.8 TOTAL PROTEIN 61 g/L 50 – 72 ALBUMIN 29 g/L 22 – 38 GLOBULIN 32 g/L 20 – 42 ALT 443 IU/L ALP 233 IU/L 7 – 369 TOTAL BILIRUBIN <2 umol/l 0–4 CHOLESTEROL 4.83 mmol/L 2.9 – 9.7 AMYLASE 340 IU/L 305 – 960 H 11 – 161 C L I N I C A L PAT H O L O G Y – B L O O D B I O C H E M I S T RY Can give a great deal of information, and helps to pinpoint which body system is affected A 25-month-old 2.2-kg castrated male Chihuahua dog was referred to the Veterinary Medical Teaching Hospital for a routine medical examination. Blood screening revealed: Parameter value Reference range Aspartate transaminase (AST) 53 U/L 14 to 44 U/L Alanine aminotransferase (ALT) 169 U/L 14 to 68 U/L Alkaline phosphatase (AP) 1934 U/L 47 to 254 U/L Gamma-glutamyl transpeptidase (GGT) 424 U/L 2 to 15 U/L WHAT WOULD BE THE MOST LIKELY AFFECTED ORGAN? LIVER C L I N I C A L PAT H O L O G Y U R I N A LY S I S URINALYSIS DOG SPECIMEN TYPE Units 1ml Yellow TURBIDITY Can give information on renal disease USG pH (DIPSTICK) • • • H 9.0 H “Dipstick” is immersed in urine, colorimetric reaction WBC (SEDI) BACTERIA (SEDI) Present determines the degree of change. CRYSTALS (SEDI) Large (>10) Give quick assessments of biochemical parameters pH, glucose, protein, cells Assess urine concentration Cytology: Assess for WBC, RBC, pathogens, casts, Crystals 4+ (HGB) H /hpf H <5 5-20 /hpf H <5 NEG KETONES (DIPSTICK) NEG BILIRUBIN (DIPSTICK) /lpf <1 <1 NEG EPITHELIAL CELLS (SEDI) Rare (0-2) /hpf CASTS (SEDI) Rare (0-2) /lpf LIPID (SEDI) Present PROTEIN (DIPSTICK) 5.5 – 7.5 5-20 GLUCOSE (DIPSTICK) COMMENTS • 1.031 BLOOD (DIPSTICK) • • Grossly turbid Easy to perform in the clinic RBCs (SEDI) REFERENCE INTERVAL Ultrasound guided cystocentesis VOLUME COLOUR • Flag 2+ H On sediment exam: Transitional and squamous epithelial cells, granular casts, triple phosphate crystals and cocci bacteria A N AT O M I C PAT H O L O G Y -WORKFLOW Jubb, Kennedy, and Palmer, Pathology of domestic animaly,Vol 1 (2015) P O S T M O RT E M E X A M ( AU T O P S Y / N E C R O P S Y ) • Only get one chance! • Often required for legal cases • The ability to make a gross diagnosis at autopsy is one of the more difficult and most important skills in pathology Collect samples of ALL the tissue for: • Histology • Bacteriology • Virology • Toxicology P O S T- M O RT E M E X A M ( AU TO P S Y / N E C RO P S Y ) Lamb: thyroid hyperplasia (goiter) Post-Mortem Exam/Autopsy/Necropsy Liver (dog) Dd: tumour (hepatic carcinoma, bile duct carcinoma) > metastatic tumours > granulomatous hepatitis >>>>> nodular hyperplasia H I S T O PAT H O L O G Y • Is the study of changes in tissues caused by disease • Tissues can be acquired during surgery, endoscopically, cutaneous punch biopsy, necropsy, etc… • Tissues are typically fixed in 10% buffered formalin (1:10 ratio) and prepped for microscopic analysis CANCER (Adenocarcinoma of the apocrine glands of the anal sac, dog) INFECTIOUS DISEASES – FUNGI (Aspergillosis) INFECTIOUS DISEASES - PARASITES (coccidiosis) INFECTIOUS DISEASES – VIRUS (poxvirus) • IHC combines three disciplines: Histology, Immunology, and Chemistry • The purpose of the IHC technique is to identify specific ANTIGENS and EPITOPES in tissues via their interaction with ANTIBODIES I M M U NO H IS TO C H E M I S T RY (IHC) Wikipedia.org IMMUNOHISTOCHEMISTRY (IHC) Every cell type has a unique array of proteins: • • • Cytoplasmic membrane Nuclear Cytoplasmic • There are three main cell types that we see in biopsies • Epithelial • Mesenchymal • Round cell • We can exploit our knowledge of the specific proteins expressed by each cell type and use it to help us identify the specific cell types in our biopsy (and our skin lump!) EXAMPLE: LYMPHOMA IN DOGS CD3+ neoplastic cells (T-cell lymphoma) CD79+ neoplastic cells (B-cell lymphoma) Immunohistochemistry (IHC) An Example: • CD18, is a membrane marker that is highly expressed on histiocytic (macrophage type) cells • Antibodies directed against CD18 should definitively identify tumours which originate from histiocytic precursor cells. • IHC results - Strong positive (brown) signal is present in the cytoplasm of all the cells of this tumor = Histiocytic sarcoma! http://pathologyoutlines.com/caseofweek/case200781.htm www.pathpedia.com • Excellent for detecting a variety of pathogens in tissues Viruses are almost always visually undetectable in H&E slides (Except if you’re lucky and find an inclusion body!) • IHC – NOT JUS T FOR TUMOURS! IHC is excellent for helping to identify viral antigen in tissues Ex: Feline Infectious Peritonitis virus, lymph node animalpetdoctor.homestead.co m http://www.askjpc.org/wsco/wsc_showconference.php?id=531 PATHOGEN ISOLATION • Can see the footprint of pathogenic organisms in tissue histo: Gram stain or other special stains • Types of inflammation • Necrosis But often require further work up to identify the genus and species of the organism • • • Fresh samples are always best • We don’t always know what agents to test for at the beginning of an investigation, clinical history can be vague • More is better! • Take multiple individual samples of key tissues store frozen: • Liver, lung, spleen, kidney, brain, any lesions • Liver often excellent for toxin testing (b/c of metabolism!) • Hair can be useful for genetic testing Can test later! COLLECTION OF TISSUE S A M P L E S F O R PA T H O G E N I D • Sample type, Date, species MOLECULAR BIOLOGY - PCR • • • • Indispensable to modern diagnoses Targets and amplifies small, specific segments of DNA (viruses, bacteria, fungi) Requires a very small amount of fresh or frozen tissues Cons: nuclei acid detection not localised with a lesion or with a particular cellular location (homogenised samples) en.wikipedia.org VIROL OGY • Lesions of viral infection can be subtle/non-specific on Post-Mortem and Histo • Can use IHC as previously discussed • PCR for the detection of viral nucleic acids is excellent • Frozen tissues (aseptic technique!), RNAlater, Trizol What if you are sure it’s a virus but none of the above techniques work? • • Virus isolation: Grown in cell culture, genome sequenced, view with TEM etc. BACTER IO L O GY/MYCO L O GY • • • • • Bacteria are common causes of death in veterinary species Tissues collected during post-mortem or in the clinic (urine, blood, biopsy) Post-mortem tissues need to be as aseptic as possible • Collect large volume specimen • Submit fresh if possible • Freeze samples for later Traditional culture on a variety of media (blood agar, LB etc..) Collect cultured microbes and identify species with Maldi Tof Mass spectrophotometry PARASITOL OGY • Gross examination of the parasite • Histopathology > different structures • Cytological evaluation of scrapings > diagnosis of arthropod infestations of the skin • Foecal flotation exam > parasitic ova • Cytology of a fecal smear > detection of organisms that may not float as well as parasitic eggs (e.g., coccidian) TOXICOL OGY • WHEN? • Unexpected (and not readily explained) illness or death • Environmental change • Multiple animals in a group affected simultaneously