Stool Analysis Practical Flashcards
What are two commonly used feacal analysis techniques?
Faecal pancreatic elastase
Faecal occult blood testing.
Faecal I’m uno chemical test
What is the function of faecal pancreatic elastase test?
Identify exocrine pancreas insufficiency.
Enzyme no arriving in the duodenum, implies problem with acinar cells or pancreatic duct —> malabsorption.
Sign of inadequate digestive function
What are the main clinical manifestations of exocrine pancreatic insufficiency?
Maldigestion and malabsoprtion
What are the main causes of pancreatic insufficiency?
Chronic pancreatitis - can lead to autodigestion
Cystic fibrosis - mucus secretions build up.
Obstructive pancreatic tumours
Pancreatic surgical resection
Zollinger-Ellison syndrome
Coeliac disease
What are some of the symptoms of EPI?
Bloating
Abdominal pain
Foul smelling stool
Staetorrhoea
Diarrhoea
Abnormal stool frequency
Excess flatulence
Weight loss.
What is pancreatic elastase?
Known as E1
Secreted by acinar cells - proteases
Little degradation, travels well through the colon - good marker of exocrine pancreatic function in the stool when quantified.
Normal exocrine sufficiency: >200 E1 ug/g stool
What is normal pnacreatic elastase?
> 200 E1 micrograms/g in stool
Between 200 -100 is a mild exocrine insufficiency.
Below 100 is seen as severe exocrine pancreatic deficiency.
What enzyme is commonly used to test for pancreatic function in stool?
Pancreatic elastase
What is meant by a serial dilution?
A step-wise series of dilutions where the dilution factor stays the same for each step
Typically take from previous (higher conc dilution) to create next lower conc dilution
Exocrine Pancreatic Insufficiency Practical
How do you create standard solutions for creating the calibration curve?
Serial dilution starting with a standrad solution of elastase enzyme (10mg/ml)
Create 2 ml Volumes of dilutions - all will contain 1m Hepes solution and 1ml of the previous conc solution
Once dilutions are made transfer 0.5ml into ependorf tubes
Elastase substrate 0.5ml can later be added and incubates for 5 minutes before recording in the spectrophotometer
Also use a blank of 1ml Hepes - minus from these values for the absorption of enzyme activity only
Exocrine Pancreatic Insufficiency Practical
How do you extract elastase from the stool samples?
Weight a known amount of stool for patient A and B (weigh by difference and aim for 0.1g)
Add each sample to 8ml of extraction buffer and mix vigorously using the vortex until lumps dissolved.
Transfer two lots of 1ml from each sample into eppendorf tubes (total of 4 tubes)
Centrifuge tube - faecal matter goes to bottom and enzyme found in supernatant
Immediatly removed 0.5ml from each tube and add to eppendorf (4 tubes)
Exocrine Pancreatic Insufficiency Practical
How do you test the patient samples of pancreatic elastase to work out the activity of elastase?
Should have 2 0.5ml sample of elastase for each patient
One will act as a blank (when additional 0.5ml Herpes is added) and the other will be tested for activity by adding 0.5ml of synthetic elastase substrate
Incubate for five minutes(optimum temp for enzyme activity) (not blank)
Then put through spectrophotometer and record absorption
later identify absorption and read across for elastase concentration from the calibration curve
Exocrine Pancreatic Insufficiency Practical
Why do you not need to minus the blank absoprtions from the patient samples when using a spectrometer?
put blank in before each relevant non-blank and tear the spectrophotometer - this will minus the blank for you
Exocrine Pancreatic Insufficiency Practical
How do you convert from elastase conc in the supernatant to in the stool sample?
Supernatant concentration - Micrograms per mililitre
Elastase in stool sample - want to micrograms in gram
reverse procedure at end scale up to get the weight in grams.
In this case: x2 (to get 1ml as 0.5ml of elastase used in sample - ignore 0.5ml of Hepes), then multiply by 8 (as diluted by 8 when added to 8ml of extraction buffer), then x10 to get from 0.1grams to 1 gram
How do you complete the foecal occult blood test?
Use a Hema-Screen card for each stool sample
Place two thin smears of stool on the oval test areas at the front of the card
Close the cover and placed the card face down on the bench
Lift the flap at the rear of the card and apply 2 drops of Hema-screen developer to each sample and 1 drop of developer to the control area
Close the flap and start the timer
After 30-60 seconds lift the flat and alayse the results
Blue colour indicates a positive result for blood.
