Laboratory Diagnostics Flashcards
What can erythrocytes by indications of?
Anemia and erythrocytosis
What can leukocyte levels be indicators of?
Inflammatory conditionsNeoplastic conditionsChemotherapy
What can platelet levels be indicators of?
Bleeding disordersDisseminated intravascular coagulation
How are counts and morphology carried out in the laboratory?
Counts done on different cell types by machineMorphology checked by microscope
What is the packed cell volume?
Distance of buffy coat divided by total distance
What is the buffy coat?
White blood cells
What colour should plasma be and what would cause a colour change?
Clear or straw coloured normallyPink if hemolysed
What would contraction of the spleen cause to be dumped into the blood stream?
A large amount of red blood cells
What is contained in a complete blood count?
RBC concentration - no. of red blood cellsHGB - total haemaglobinHCT - haematocrit and spun PCVMCV - mean cell volumeMCH - mean cell haemoglobinMCHC - mean cell haemoglobin concentrationRDW - red cell distribution width
What should total haemoglobin usually correspond to?
Red blood cell concentration
What does the mean cell volume tell you?
How big red blood cells are
What is the mean cell haemoglobin concentration?
How much haemoglobin there is per red blood cell (mg/volume of RBC)Red cells mature when they reach a certain concentration of haemoglobin
What are the 3 classifications of anemia?
Mil, moderate or severe - can give an idea of underlying problem
What features can be used to determine regenerative anemia from non-regenerative?
MCV - increased during regenerative as reticulocytes are larger than mature erythrocytes - normal in non-regenerativeMCHC - decreased in regenerative anaemia as reticulocytes are larger and less packed with haemoglobin - normal in non-regenerative
When are microcytic red blood cells seen and why?
During portosystemic shunt, iron deficiency and hepatic failureIron is needed to build haemoglobin so with less iron get less haemoglobin so RBC divide again as a certain Hb concentration is needed to become more mature
Which breed of dog is microcytic anaemia a normal thing to see?
Akitas - born with smaller RBC
When are macrocytic red blood cells seen?
In regeneration - polychromatophils are larger than mature cells
Which breed of dogs can have a rare case where all RBC are larger?
Poodles
What do hypochromic cells look like?
Lots of cells without Hb concentration
Why are hyperchromic cells not seen unless artificially?
Haemolysis has to occur for them to be seen - not possible naturally
What is the first thing we should classify when identifying anaemia?
Whether it is regenerative or non-regenerative
What are the only 2 reasons for regenerative anaemia?
Haemolysis (destroying blood)Haemorrhage (losing blood)
What generally appears with regenerative anaemia?
Larger RBC appearing as bone marrow isn’t damaged and they still pump out RBCs
What are the three most common non-regenerative causes of anaemia?
Anaemia of inflammatory/chronic disease - mildChronic renal failure - no EPO being producedDecreased production in marrow - not producing RBC
What is the difference between reticulocytes and polychromatophils?
Same cell but different - only reticulocytes when RNA is precipitated
How can we see polychromatophils?
Quik or Giemsa stained smear - younger cells containing ribosomal RNA show up as larger, bluer cells
What do we use to see reticulocytes on a blood smear?
Stain polychromatophils with New Methylene Blue and RNA precipitates forming aggregates - reticulocytes
What, in cats, is the same as reticulocytes in other animals?
Aggregate retics - do mature to punctate retics over time but only really interested in aggregate retics
What does an increase in reticulocyte percentage mean and why can it be misleading?
More young red blood cells meaning anaemia is regenerativeCan be misleading if red blood cell count is not normal
What is the corrected reticulocyte percentage?
(Reticulocyte percentage multiplied by patient PCV)/normal PCV
What are the normal corrected reticulocyte percentages for a dog or a cat?
45% for dogs35% for cats
When can anaemia be categorized as regenerative when regarding corrected reticulocyte percentages?
> 1% corrected in dogs>0.4% corrected in cats
What signs of regeneration can be seen on a blood smear?
Polychromasia - high number of red blood cellsAnisocytosis - variability of RBCMacrocytosis - enlarged RBCnRBCs - nucleated RBCHowell-Jolly bodies - remains of nucleus (more in regen.)Codocytosis - cells with bullseye appearanceBasophilic stippling - small periphery dots
What does a blood smear look like with a lack of regeneration?
Uniform cells - pretty uneventful
What 5 things should be looked for when looking at RBC morphology?
Spherocytes/ghost cellsHypochromasia/leptocytosisShear productsOxidative damageOrganisms
What should be looked for in a total white blood cell count?
NeutrophilsLymphocytesMonocytesEosinophilsBasophils
What should the typical differential of leukocytes be?
80-95%
Where do neutrophils spend most of their time and how do they travel?
In tissues - travel in bloodstream
If different counts are taken of neutrophils throughout the day what will the values look like?
Values will differ
What is an increase in neutrophils called?
Neutrophilia
What is a decrease in the total number of neutrophils?
Neutropenia
What is left shift of neutrophils?
A bigger demand for neutrophils so younger cells begin to come into the circulation
What is the difference between regenerative left shift and degenerative left shift?
Regenerative - neutrophilia - segmented>bandsDegenerative - neutropenia - bands>segmented
What is leukemia?
Neoplastic cells within the circulation
Which lype of leukemia is worse?
Acute - will kill quicklyChronic - bad in long term but in short term they’re alright
Describe an acute leukemia
Blast cells in circulation - can’t identify cell of originMore likely to be lymphoidIf segmentation - myeloidDetect by immunophenotyping using a flow cytometer
Describe chronic leukemia
Lymphoid - persistent high numbers of small, mature lymphocytesMyeloid - persistent high numbers of normal neutrophils
What is a common problem with platelet measurements?
Clumping due to activation of ability to plug holesMachine can’t distinguish platelet from clump
What are five examples of clinical pathology tests?
HaematologyClotting profileBiochemistryUrinalysisCytology
What is biochemistry used for?
Evaluating different organ systems - liver, kidney etc.
What should biochemistry be used with?
Urinalysis and CBC
What is recommended most of the time for biochemistry?
Serum not plasma
What should be used for biochemistry for birds and reptiles and why?
Heparinized plasma - small sample size
What is the difference between serum and plasma?
Plasma contains all clotting factorsSerum is fluid part of blood after clot formation
Describe serum collection
Collect into tubes without anticoagulantUsually have a red or brown topAlso have serum separator tubes with gel layer separating cells and serum
Why do we seperate red cells and serum?
Red cells continue living and can undergo changes so seperate so no more interface between serum and RBC
How is serum seperated?
Allow blood to clot and centrifuge - should be done immediately if possible
What temperature should serum be stored at?
4 degrees C
How is plasma collected?
Blood collected into EDTA, heparin or citrateSeparate using centrifugeStore at 4 degrees C
Give at least 5 examples of things found in a biochemistry profile
Total proteinAlbuminGlobulinsBicarbonateAnion gapCalciumPhosphorusGlucoseElectrolytesUrea nitrogenCreatinineBilirubinCholesterolAmylaseLipaseCK - creatine kinaseALT - alanine transaminaseALP - alkaline phosphataseSDH - saccharopine dehydrogenaseGLDH - glutamate dehydrogenase
Why should caution be taken when looking at an animals biochemistry profile?
Only 95% of animals fall within reference rangesLarge number of things measuredLikely that some of their values fall outside these rangesCould be a problem but sometimes mean nothing
What are alterations to levels of chemicals in the blood always a result of?
Either a change in the amount going in or the amount going out
Describe how total protein is measured and what it is made up of?
Measured on serum or plasma (slightly greater due to fibrinogen) by refractometer or colorimetric methodMade up of albumin and globulins
What are total protein levels falsely increased by?
Icterus, severe haemolysis and lipemia
What can caused increased protein levels?
DehydrationInflammationNeoplasia
What can cause decreased protein levels?
Blood lossDecreased synthesisDilution
How is albumin measured and in what animals is this method unreliable? What method should be used instead?
Dye binding methodUnreliable in birds - use electrophoresis instead
When is increased albumin seen? Decreased?
Increased - dehydrationDecreased - increased blood loss, decreased synthesis and third spacing
How can the different types of globulin be separated?
Electrophoresis
What can cause a selective increase in globulins?
Inflammation (polyclonal)Neoplasia (monoclonal)
What is the difference between polyclonal and monoclonal gammopathy?
Polyclonal - increase in all globulin typesMonoclonal - increase in a single globulin type by a single clone of cells
What three things can be used to evaluate the liver?
EnzymesMetabolitesFunction tests
What can be used to evaluate pancreas function?
Amylase and lipase
Why are measurements of amylase and lipase not specific indicators of pancreatic problems?
Can have increases without pancreatic damage e.g. kidney damage
When are increases in amylase and lipase seen in dogs and cats?
Dogs = 4 to 5 fold increase with pancreatitisCats = not seen with pancreatitis
What can cause increases in amylase and lipase?
Renal insufficiencyPancreatitis - dogs
What do we need to look at to evaluate the urinary system?
Both serum/plasma and urine
What are both indicators of glomerular filtration and where are they produced?
Urea - produced in liver and excreted by kidneyCreatinine - derived from creatinine in muscle
What else can the urinary system be used as a marker for?
Liver and kidney function
What is azotemia? What can cause it?
Increases in nitrogenous wastes in the circulationCaused by dehydration, renal disease and obstruction
What should be checked to determine azotemia?
Urine specific gravity - ability to concentrate urine so should compare with serum/plasma urea and creatinine
What should urine specific gravity be if urea and creatinine are increased?
1.030 in dogs1.035 in cats1.025 in horses/ruminants
What is happening if urine specific gravity is less than the recommended values?
Decreased concentrating abilityRenal failure is present
What isn’t a reliable indicator of renal disease?
Urea - should use creatinine only
What are urea levels influenced by and why may they be increased?
Influenced by protein intakeIncreased by high protein meal or GI bleeding
Why is urea not a reliable indicator of renal disease in ruminants?
Secrete into the saliva and goes into the rumen
What is the main ion in extracellular fluid?
Sodium
What regulates sodium along with water?
Kidney
How can increased sodium occur? Decreased?
Increased - increased intake or water loss, decreased water intakeDecreased - increased loss or water intake
What is the main ion in the intracellular space? What affects its levels?
Potassium - affected by acid-base changes
What regulates potassium levels? What causes increases or decreases?
Regulated by intake and renal functionIncreases - renal failure and hypoadrenocorticism and cell leakageDecreases - loss and decreased intake
What should be considered before investigating an in/out problem with potassium?
Whether an acid-base balance shift that would cause potassium shift
What do changes in chloride usually coincide with?
Sodium
What are changes in chloride without sodium changes associated with?
Alteration in acid/base statusVomiting/abomasal displacement
What two states is calcium present as within the body?
Free - activeBound - bound to albumin
What is calcium affected by?
Albumin levels
What are calcium levels regulated by?
PTH and calcitonin
What is phosphorous regulated by?
PTH and calcitonin
What are increases in phosphorous associated with and when are they often seen?
Increase associated with renal diseaseSeen in growing animals along with elevated calcium and ALP
What three things are looked at in urinalysis?
Gross appearanceChemical analysis - specific gravity and urine stripSediment analysis - cellular elements, crystals, casts and others
What are the advantages of cytology?
Quick, easy and inexpensiveNon-invasiveMinimal risk to patientCan determine what diagnostic procedure should be performed next
What are some limitations of cytology?
Relies on sample quality - skill of collector, smear quality and tissue typeLimited by ability/experience of person examiningLack of information about tissue architectureDiagnostic challenges
What are the advantages and disadvantages of histopath?
-More expensive- Slower turn around time- Poor detail for round cell tumours+Tissue architecture+Tumour grading+Immunohistochemistry more available
What are the two types of samples used for cytology?
Aspiration or imprints- superficial masses, lymph node and organs/deep massesFluids- body cavities, joints, respiratory tract and cerebrospinal fluid
What should fine needle biopsies be used for?
Solid or fluid filled masses
What should fine needle biopsies be done under?
Visual or ultrasound guidance
Describe what is done during a fine needle biopsy
Similar to fine needle aspiration but no negative pressure is applied to the syringeSmall gauge needle (22-24 gauge) inserted into mass several times - also sample wall not just centreUse needle to take sample then air-filled syringe to expel cells onto slide
What is the difference between a fine needle aspiration and a fine needle biopsy?
Aspiration uses negative pressure to draw sample from a mass redirecting needle 2-3 times to ensure a representative sampleBiopsy doesn’t use negative pressure i.e. drawing a syringe back
What needs to be done to ensure cells can be seen on a smear?
Flatten the cells out
Describe smear preparation
Remove needle from syringeDraw air into the syringeReplace needleExpel aspirated fluid onto slide being gentleSmear - don’t spray
What are the three goals with smear preparation?
Thin areas with good cell spreadMinimization of cell damageMinimization of blood content
Describe how to make smears?
Put another slide over the top of the samplePull slides apart - use gentle pressure
What happens if too much or too litle pressure is used when making a smear?
Excessive pressure - rupture cellsNot enough pressure - too thick preparation
What are touch impressions good for?
Evaluation of excised tissue or superficial lesions
When are imprints made?
Before excised tissue is placed in formalin and submitted for histopathology
Describe how to take an imprint
Use fresh cut surface of the tissueBlot until drywith paper towelImprint directly onto glass slide - roll 4 to 5 times then air dry and stain
What normal cells do we expect in various samples?
Tracheal wash - ciliated cellsBAL - alveolar macrophagesJoint fluid - mononuclear cells
What is the difference between inflammation and neoplasia in cytology?
Inflammation - sample dominated by inflammatory cellsNeoplasia - sample dominated by tissue cells
Describe the differences between septic and non-septic inflammation upon cytology
Septic- contains bacteria/organisms, degenerate neutrophils, bacteria must be intracellular within neutrophils to be significant also may be contaminantsNon-septic- no bacteria or organisms seen and non-degenerate neutrophils present
Describe degenerative changes in neutrophils
Nuclear changeNucleus swells, loses lobulation and becomes palerSecondary to release of bacterial toxins
When are increased numbers of macrophages seen in cytology?
Granulomatous inflammationPyogranulomatous inflammation - if neutrophils alsoForeign body reactions
Which tumours present as round cell tumours?
LymphomaHistiocytomaMast cell tumourPlasmacytomaTransmissible venereal tumourMelanoma
Describe epithelial cells upon cytology
Sheets/rafts/clustersLarge cell sizeCell-to-cell junctionsOval to angular in shapeNuclei round, centrally locatedCytoplasm often abundantGood cell yield
Describe mesenchymal cells upon cytology
Individual cells or clumpsSmall to medium size cellsSpindle to fusiform to stellateIndistinct cell bordersElongated nucleusPoor exfoliationProduced in the matrix
How can malignancy be assessed?
Uniformity vs. pleomorphismMonotonyNuclear criteria are the most reliable
What is needed before you can call a tumour malignant?
Need at least three nuclear criteria of malignancy
What are the three cellular criteria of malignancy?
Anisocytosis - variation in cell sizeMacrocytosis - large cellsCell crowding
What are nuclear criteria of malignancy?
Anisokaryosis - variation in nuclear sizeMultinucleation - nuclei vary in size and odd numbersMacrokaryosis - giant nucleiHigh nuclear to cytoplasmic ratioIncreased mitotic figuresAbnormal mitotic figuresCoarse chromatinNuclear moulding - nuclear deformation by other nucleiMacronucleiVarying nucleolar shapeVarying nucleolar size
What are some common problems with cytology samples?
Formalin fumes - destroy blood or cytologyRefrigerating glass slidesBreakage during shippingLack of freshly made smears
What are the two immunodiagnostics used in veterinary medicine?
Serology and immunoassay
What does serology allow?
Evaluation of immune status/function - exposure of animal to infectino, response to vaccination and diagnosis of immune-mediated disease
What does immunoassay allow in veterinary medicine?
Use of labelled antibodies as detection reagents - presence of pathogen in a sample, measurement of a biomarker and immunophenotyping
What two ways can we sample the immune system?
Blood sample and tissue biopsy
What are the two different types of blood sample you can take?
Serum - clotted sampleCells - citrate/heparin anticoagulant
What are the serological markers of innate immunity?
Acute phase proteins - C-reactive protein, serum amyloid A, fibrinogen and haptoglobin
What are the serological markers of adaptive immunity?
Antibodies and cytokines
What two ways can be used to measure total immunoglobulin?
Radial immunodiffusion and serum protein electrophoresis
What three things can measurement of total immunoglobulin be useful in?
Failure of passive transfer in foalsSpecific Ig deficiency syndromesMonoclonal/polyclonal gammopathy
What can antigen-specific immunoglobulin measurement be useful in determining?
Exposure of an antigen to pathogenResponse to vaccinationDiagnosis of antibody-mediated hypersensitivity
Describe serum protein electrophoresis
Various peaks of protein levelsNormally albumin should be dominant peakCan be used to divide albumin and globulins - gamma globulin most important
What is seen in a monoclonal gammopathy on serum protein electrophoresis?
One peak of protein increased
What are the seven ways you can measure antigen-specific antibody?
ELISAImmunofluorescent antibody testVirus neutralisation assayComplement fixation testHaemagglutination inhibitionMicroscopic agglutination testAgar gel immunodiffusion test
What two things can ELISA be used to detect?
Antigen - pathogen presenceAntibody - immune response
What is better to look for if exposure to a pathogen was recent?
Presence of the causative organism in infected tissue or biological fluids
What is it better to look for if exposure occurred greater than seven days ago?
Antigen specific antibody in serum
What would you expect to see in an ELISA with a recent infection?
Greater IgM:IgG ratio and an increasing IgG titre
What is different about an immunofluorescence assay compred to an ELISA?
Destination antibody is labelled with a fluorescent marker rather than an enzyme - fluorescence indicative of a positive result
What is a viral neutralisationg assay more indicative of than an ELISA?
Presence of a biologically active antibody
Describe the basics behind a viral neutralisation assay
Cultured cells deliberately infected with virus either with serum or withoutIn the absence of specific antibody cells are infected
What is the advantage of a virus neutralising antibody over an ELISA?
Indicates presence of biologically active antibody
How can T cell responses be evaluated in vitroor in vivo?
In vitro- cytokine release following stimulation with specific antigenIn vivo- delayed-type hypersensitivity test
Describe the bovine TB gamma-IFN test
Heparinised blood sample sent to AHVLACulture cells in vitro with mycobacterial antigensMeasure IFN-gamma production by ELISAMore sensitive but less specific than tuberculin “skin test”
What should be tested to diagnose immunodeficiency syndromes?
Neutrophil function
What is measured with serology-based tests in allergic disease?
Allergen specific IgE
What are the two types of hypersensitivity that can be measured with intra-dermal skin testing?
Immediate-type - IgE mediatedDelayed-type - T cell mediated
What are intradermal skin tests used a lot for?
Diagnosis of small animal allergic skin disease and immediate-type hypersensitivity
What are three tests used for immunodiagnostics of autoimmunity?
Coomb’s test for IMHA (Immune Mediated Haemolytic Anaemia)Anti-nuclear antibody (ANA) for systemic lupus erythematosus (SLE)Specific autoantibody serology
What three things can antibodies be used as detection reagents for?
Presence of pathogen in biological sampleMeasurement of a biomarkerImmunophenotyping
What three ways can antibodies be used to detect infection within a sample?
Sandwich ELISA (biological fluid)Immunofluorescence (biological fluid and tissue)Immunohistochemistry (tissue)
What are the methods used for measurement of a biomarker using labelled antibodies?
ELISAChemiluminescenceRadioimmunoassay
How can cell type presence be identified within a biological fluid or tissue biopsy?
Antibodies against cell-surface marker
What are the two most common methods of identifying a pathogen’s nucleic acid in a biological sample?
PCR or qPCR
What can molecular techniques be used for in pathogens?
Genotyping the pathogen or epidemiological studies
What things need to be considered when using genetic tests of animals?
Insertion, deletion or substitutionInherited as simple or complex disorderAutosomal or sex-linkedDominant or recessiveCopmlete or incomplete penetrance
What sample would you need for a molecular diagnostic assay for FIA?
EDTA blood
How would you process a sample for a molecular diagnostic assay for FIA?
Extract DNA
What would you need to know to develop a test for molecular diagnosis of FIA?
DNA sequence for the three mycoplasma spp for primers/probesCould you design a test for all or could you do tests for each?
What type of assay would you use as a molecular diagnosis for FIA?
PCR or qPCRqPCR more specific and allows an estimate of pathogen load
