Clinical Biochemistry Flashcards
Define clinical biochemistry
Clinical analysis of bodily fluids for diagnosis, therapy and prevention of disease
What are typical panels measured in biochemistry?
Fluids from serum, urine and joints Liver- ALT, GLDH, ALP. GGT, bilirubin, bile acids Kidney- urea, creatinine Proteins- TP, albumin Electrolytes- Na+, K+, Cl-, Ca2+, PO4- Glucose and lipids
What quality control measures for biochemistry should be made?
System set up Maintenance Cleaning Storing samples Interpretation of results Control tests
What is plain tubes used for and some examples?
For samples that are allowed to clot and have serum separated
Bile acids, protein electrophoresis
Dont use for fibrinogen
What are heparin tubes used for and how do they work?
Plasma
Stops clot formation by increasing action of antithrombin III
What is OxF tube used for?
Measuring exact glucose levels
What are citrate tubes used for and how do they work?
Haemostasis
Anticoagulant binds to calcium
What are EDTA tubes used for and how do they work?
Haematology
Contains potassium and uses up calcium
What are examples of biochemistry analysers?
Glucometers
Wet/dry biochemistry analysers
Electrolyte analyser
Blood gas analyser
What are biological factors that affect results?
Interindividual- differences between groups of animals
Intraindividual- differences within one individual, should be minimised as much as possible
What pre analytical factors affect results?
Poor sampling Haemolysed, lipemic or icteric plasma Wrong container Wrong anticoagulant Storage of sample
What is lipemia and what effects does it have on samples?
Lipids in serum causing milky with visible turbidity
Can dilute other substances and interferes with haematology and spectrophotometric assays
What is icterus in samples?
Jaundice/increased bilirubin so yellow serum
What is haemolysis of samples and what effects does it have on analysis?
Red plasma from free haemoglobin
Interferes with spectrophotometric assays, haematology and chemical interactions
How can haemolysis be prevented?
Good sampling technique
Avoid delays
Keep refrigerated
Separate serum
What are reference intervals?
Normal value for 90% of healthy population
What causes high levels of bilirubin?
RBC breakdown, liver disease, bile duct obstruction
What causes high levels of bile acids?
Decreased hepatic function and bile flow from portosystemic shunt
What liver enzymes increase with liver damage?
ALT
AST
GLDH
Cholestatic enzymes
What substances show liver function?
Urea Glucose Albumin Bile acids Bilirubin
How to run bile acid stimulation test?
Take fasted sample
Feed
Collect sample 2 hours later
What do different pancreatic enzymes suggest is a cause?
Pancreatic lipase immunoreactivity- pancreatic injury
Trypsinogen like immunoreactivity- exocrine pancreatic functional mass
What is azotaemia?
Increased serum urea
What are the different types of azotaemia?
Prerenal- decreased renal perfusion, shock, CV disease, dehydration
Renal- renal disease
Post renal- urinary tract obstruction causes accumulation and rupture so waste enters abdomen
What are clinical causes of high blood glucose and most common causes of low blood glucose in samples?
High- stress, diabetes, steroids
Low- incorrect sample handling, insulinoma tumour, insulin overdose
What causes increased creatine kinase?
Skeletal muscle injury
What do urea and creatinine levels measure?
GFR
What causes changed levels of urea and creatinines?
Increased- dehydration, renal disease, urinary obstruction, heart disease
Decreased urea- liver failure
Decreased creatinine- muscle wasteage
What are the proteins known as total proteins and what are their purpose?
Albumin and globulins
Maintain colloid osmotic pressure
What are consequences of changed levels of total proteins?
Increase- lipemia (false), dehydration, inflammation
Decrease- haemorrhage, GI disease, renal disease, hepatic disease
What causes increased and decreased albumin?
Increased- dehydration
Decrease- inflammation, liver disease, kidney disease, GI disease, haemorrhage
How does serum protein electrophoresis work?
Elevates groups of protein in serum showing fraction of albumin and globulin
Define haemostasis
Ability to stop bleeding
What are the stages of haemostasis?
Primary- rapid unstable response, platelets form plug which helps activate other platelets
Secondary- fibrin mesh formation to stabilise platelet plug
Tertiary- break down of clot to return normal vascular flow by preventing overclotting
What are the consequences of haemostasis disorders?
Defective- haemorrhage
Excessive- thromboembolism
List lab tests can assess haemostasis
Buccal mucosa bleeding time Platelet numbers Clotting time Fibrinolysis tests Platelet function tests Genetic tests Individual clotting factors Thromoelastography
Explain the buccal mucosal bleeding time test and what it tests for
Cut MM
Collect blood with filter paper until bleeding stops
Normal time takes 3.3 minutes or less
Tests primary haemostasis
Explain how clotting times can be tested and what the normal values can be
Collect whole blood and allow to clot
Dogs- less than 90 seconds
Cats- less than 60 seconds
What are clinical disorders of haemostasis?
Primary or secondary- GI bleeding, epistaxis, haematuria
What are the most common causes of haemostasis disorders?
Low platelets
Low production, over use due to haemorrhage or increased destruction
Define thrombocytopenia
Low platelets
Define haematogram
Shows erythrocyte leukocyte and platelet parameters
Platelets should be double checked
How does haematology analyser generate parameters?
Laser- RBC count and mean cell volume
Lyses cells- haemoglobin
Calculated- haematocrit, mean corpuscular haemoglobin and concentration
How to store blood samples?
Fill EDTA tube to line
Invert and roll to prevent clotting
Store in fridge
Prepare and dry smears but dont put in fridge
Define haematocrit
Proportion of blood that is red blood cells
What are evaluations that can be made about RBC from a sample?
Circulating RBC mass- haematocrit, PCV, RBC count
What RBC looks like- mean corpuscular volume, haemoglobin and conc
RBC morphology- peripheral blood smear
Define PCV
Packed cell volume
Percentage of red blood cells in blood
How do you read PCV?
Centrifuge whole blood
Read as % of column
Shows plasma, buffy coat of WBC and platelets, packed red cells
What are suffixes associated with haemostasis?
Philia/cytosis- increase in number
Penia- decrease in number
(Philia- granulocytes
Cytosis- all other cells)
Describe flow cytometry in haematology
Individual cells pass through laser, absorbing and scattering light
Light interruptions count cells
Scattering determines size and complexity
Describe impedance in haematology
Cells pass in isotonic solution between electrolytes and are poor electrical conductors
Change in electrical impedance is proportional to cell size
What interferes with haematology analysers?
Clots Platelet clumps RBC agglutination Nucleated RBC Lipemia Leukocyte agglutination Handling delays
What are the different areas of a blood smear?
Base- not examined
Monolayer- main bulk with RBC not overlapping
Feathered edge
How do you carry out a leukogram?
Identify cell types
Count number
Smear exam of monolayer
Why do you need to do a blood smear?
Automated analysers dont pick up morphology changes
Help make quick clinical choices
How to analyse a blood smear?
Low magnification at feathered edge
Move into monolayer
Increase to oil layer at monolayer
Move to lateral edge and count 100 leukocytes into types and observe abnormal forms
What RBC observations can you make from blood smear?
Number
Normality
Evidence of regeneration
What are normal morphology RBC in dogs and cats?
Dogs- uniform, pale central 1/3 of cell
Cats- small, uniform, no central colour
What platelet observations can you make from blood smear?
Number
Morphology
What do platelets look like in a blood smear?
Size of RBC
No nucleus
Granulation
Can be in clumps
What observations can you make for WBC from blood smear?
Number
Type
Morphology
What are different types of WBC and what do they look like?
Neutrophils- segmented ribbon shaped nucleus, pale cytoplasm
Eosinophils- pink granules
Basophils- purple granules
Lymphocytes- round with round nucleus and little cytoplasm
Monocytes- granular appearance, some have vacuoles
How do you estimate platelet counts?
Count platelets in 10 fields using oil immersion lens in monolayer
Calculate average and multiply by 15 or 20 then x10^9
What is the normal average number of platelets counted in monolayer?
15-30
What are the problems of automated platelet counts?
Commonly lead to artefactual thrombocytopenia due to clumping or macroplatelets
RBC and platelets are same size so can be miscounted
When should you do a blood smear for platelet counts?
Signs of haemorrhage
Low automated counts
What is observed about RBC morphology for the different types of anaemia?
Non-regenerative- few normal looking RBCs
Regenerative- few abnormal looking RBCs
What are causes of anaemia?
Reduced RBC count
Reduced haemoglobin concentration
Low PCV
What are clinical signs of anaemia?
Pale/yellow MMs Lethargy Tachycardia Tachypnoea Collapse GI blood Pica
How can you classify anaemia?
RBC indexes
Regenerative or non-regenerative
Severity
When does regeneration of RBC naturally take place and how is it determined?
Low oxygen
Reticulocyte concentration
What are reticulocytes?
RBC precursors, 2 types
What are the types of reticulocytes?
Aggregate- get counted, immature, released by bone marrow in response to anaemia, have lots of dots in cytoplasm
Punctate- matured, stay in blood so unreflective of current status, counted as % in blood smear
What are types of anaemia when based off mean cell volume?
Normocytic- normal sized erythrocytes
Microcytic- low MCV, iron deficiency causes division into small RBCs
Macrocytic- high MCV, immature RBC in circulation larger than mature RBCs
What are types of anaemia based off haemoglobin levels?
Normochromic
Hypochromic- iron deficiency, immature RBCs
Hyperchromic- usually are arterfact
What are common morphological changes of RBCs?
Anisocytosis- different cell sizes
Polychromasia- indicate regeneration, RBC look purple
Hypochromasia- low haemoglobin so larger pale area
Spherophytes- small, round, dark
Ghost cells- only membrane of RBC present
What are signs of thrombocytopenia?
Petechiae/pinpoint haemorrhage
Bruises
GI bleeding
Spontaneous haemorrhage
How is thrombocytopenia diagnosed?
Blood smear
Rerun haematology
Define polycythaemia and what is its causes
Increased red cell mass due to increased haemoglobin, PCV
What are the types of polycythaemia?
Erythrocytosis- relative due to plasma loss/dehydration
True polycythaemia- actual increased mass
What are signs of polycythaemia?
High PCV Dark MM Sneezing Nose bleeds Neurological signs
How is polycythaemia diagnosed?
Check hydration Lab tests Blood gas Ultrasound Radiography
What are common changes causing WBC disorders?
Stress Inflammation Adrenaline Neoplasia Inverted stress
Define neutrophilia
Increased neutrophils due to infection
Define neutropenia
Decrease of neutrophils due to bone marrow destruction or suppression
How do you count WBCs from blood smears?
Take at edge of monolayer and move up and down
Count percentage of types of leukocyte
What are the morphologies of lymphocytes?
Normal- round, double size of RBC, little cytoplasm
Reactive- smaller nucleus and larger cytoplasm
Lymphoblast- large nucleus
Define lymphocytosis and when is it seen?
High lymphocyte counts
Normal in young for immune development
Epinephrine release, cell mobilisation, antigenic stimulation, cancer
What is monocytosis and when is it seen?
Increased demand and production of monocytes
Infection, inflammation and trauma
What is eosinophilia and what are causes?
Increased demand of eosinophils
Allergy, inflammation, parasites, eosinophil leukaemia
What is lymphopenia and what are caused?
Loss of or decreased production of lymphocytes
Virus or immunodeficiency
Define urinalysis
Physical, chemical and microscopic analysis of urine to diagnose and manage disease
What are benefits of urinalysis?
Rapid in house test, transport effects sample
Cheap and basic equipment
What are the stages of urinalysis?
Physical exam
Chemical analysis
Sediment analysis
Uroculture
What equipment is needed for urinalysis?
Urinary strips Refractometer Centrifuge Tubes Microscope Sediment stain Slides Cytological stain
How can you collect urine for sampling?
Off floor Free catch Bladder squeeze Catheterisation Cystocentesis
How to store urine samples, including the tubes used for free catch and cytology samples?
Free catch- boric acid tube to prevent growth of contaminant bacteria
Cytology- EDTA tube
Store in fridge if analysis takes longer than 30 minutes, but does cause crystal precipitation
Avoid direct sunlight
Return to room temperature before analysis
Describe the process of sediment analysis
Mix well before analysis
Centrifuge 5mls
Decant supernatant leaving 0.5ml for resuspension
Stain when needed
Resuspend until well mixed
Add to slide and examine under microscope
What should be seen under a microscope in urine?
Less than 5 erythrocytes and leukocytes per 500x field
Squamous epithelial cells in free catch, large flat cells
Transitional epithelial cells, large cells
Crystals
What are different urine crystals?
Struvite- rooftop shaped Calcium oxalate- square with cross Bilirubin- branched Biurate- round with spikes Calcium carbonate
What gross appearance should be noted in urine samples?
Turbidity
Colour
Odour
How do you measure specific gravity of urine?
Refractometer, quality control with water
Measure at room temperature
What are the interpretations of urine specific gravity?
Hypersthenuria- concentrated urine, normally hydrated animals
Isosthenuria- not concentrated or dilute, investigate when persistent
Hyposthenuria- diluter than plasma, investigate when persistent
What parameters are measured on urine dipstick tests?
Reliable- pH, protein, ketones, bilirubin, blood
Unreliable- specific gravity, urobilinogen, nitrate leukocytes
What effect does storage have on pH of urine?
Rapidly makes more alkaline
What needs investigating regarding protein levels in urine?
Proteinuria in dilute samples or >1 when concentrated sample
What are causes of proteinuria (pre- post- and renal)?
Pre-renal- hyperproteinaemia/high protein in the blood, hyperthermia, seizures, venous congestion
Renal- glomerular, tubular
Post-renal- inflammation, haematuria
What causes false negative bilirubin and what causes high bilirubin?
False negatives- light
High- liver disease, haemolytic anaemia
What causes ketones to be present in the urine?
Diabetic ketoacidosis
Ketosis
Starvation
What are causes of glucose in the urine?
Hyperglycaemia
Renal tubular disorders
False positives- bleach, hydrogen peroxide
What does nitrate in the urine suggest?
UTI
What are the benefits of in house labs?
Fast turn around Rapid treatment Monitoring Smaller volumes required Cheaper after initial investment
What are post regulations of sending samples?
50ml limit Sealed container Padding enough to absorb entire contents Leak proof bag No class 4 pathogens
What needs to be included when sending samples to external lab?
ID of patient, owner and vet practice
Sample type
Tests required
Any history
How to avoid haemolysis when collecting blood samples?
Clean stick
No vacuum created
What are consequences of stressful samples?
Neutrophilia Lymphocytosis Elevated PCV Elevated creatine Kinase with restraint Haemolysis
How do serum samples differ to plasma samples?
Serum- long time to clot, need spinning later
Plasma- need anticoagulant, spin and separate immediately
What happens if EDTA tubes get contaminated?
Alters electrolyte levels and some enzymes
What is the best collection for cytology?
Fine needle aspirate biopsies
Fluids in EDTA and plain tube for culturing
