Blood sampling and assays

Weeks two ENI - monday content

What is the difference between plasma and serum?

• serum is blood that’s been allowed to coagulate, then centrifuged

  • plasma is blood that we’ve added an anti-coagulant to, then centrifuged

Why may we use serum for a blood sample - 3 reasons

1. chemical constituents in the circulation can be measured

2. no clotting factors

3. antibodies - serology

How do we collect a serum sample?

1. leg coagulation take place

2. centrifuge

3. remove liquid supernatant

• use plain glass/plastic tube OR

• ones with clot activators/gel that forms a barrier between cells and serum (no need for an additonal transfer tube)

Why may we use tubes containing gel for a sample?

• serum/plasma - when centrifuged allowed a barrier between cells and serum/plasma so we don’t need an additonal tube for testing/transfer

Why may use use a whole blood sample?

• counting cells - haematology

  • certain cellular chemistry - GSH-Px

How do we collect a whole blood sample?

• tube that contains anticoagulant to stop blood clotting

• anticoagulant is either dry powder coating/liquid (beware of dilution)

Why may we use a plasma sample?

• test chemcial constituents in circulation e.g. fibrinogen

How do we obtain a plasma sample?

1. use a tube with an anticoagulant (with desired effect)

2. anticoagulant either a powder/liquid (beware dilution)

3. centrifuge and remove liquid supernatant from cells

others:

• tubes with beads to help mix anticoagulant

  • tubes with gel that forms a sealant barrier b/w cells and plasma

What are the 3 layers in this sample, is this plasma or serum?

• plasma, buffy coat, rbcs

• PLASMA sample

What are the three layers here

1. serum

2. gel

3. blood clot

How do (in vitro) anticoagulants work?

• calcium binding

• heparin through anti-thrombin

How does calcium exist in blood?

1. ionised (iCa)

2. Albumin bound

3. complexed

What is EDTA

• Calcium chelator (grabber)

• irreversibly binds to calcium

How does citrate act as a calcium chelator

• forms ionic complexes with iCa

• reduced iCa below that needed for coagulation cascade

• reversible

Give an example of when we use citrate as an anticoagulant

• transfusion - along with glucose and adenine to support RBCs

How can we reverse the effects of citrate?

• add Ca back to sample

How is citrate often found in tubes, what do we therefore need to consider with our sample?

• liquid

• dilution correction factor

How does oxalate work as a calcium chelator?

• most commonly found mixed with fluoride

• traps Na and K - therefore can’t use this anti-coagulant if we want to sample these ions

Outline fluoride as a calcium chelator

• usually with oxalate as the Ca binding anticoagulant

• inhibits glycolysis

• preserves glucose in sample if posting

How does heparin work as an anticoagulant

• indirect anticoagulation

• depends on presence of: anti-thrombin III (AT)

• promotes AT ability to bind Factor Xa

• locks AT and thrombin (IIa) together

Why is heparin often used in blood samples?

• usually Li heparin

  • therefore doesn’t affect ability to measure Na or K without interference

• also recommended for bird/reptile haematology too

What are 3 tube types that promote coagulation

1. plain glass/Z serum

2. clot activator tube (CAT)

3. serum separation tube with gel (SST)

What are 4 tubes that impede coagulation?

1. sodium citrate (fill to line)

2. EDTA (K2/K3)

3. Na/Li heparin

4. NaF - K oxalate

For each of these tube types, why is tube fill important?

1. EDTA

2. Citrate

3. Liquid anticoagulants

1. osmotic effects, under-filled tubes → cell shrinkage

2. need to know how much calcium required to overcome clotting. Citrate in concentration needs to be known precisely. Depends on filling volume expected

3. anti-coagulant solution as a liquid, need to know vol for dilution factors

which sample do we always do last?

EDTA

what are doughnuts/beads used for in blood sampling?

• mixing/even distribution of additives

How do we ensure our sample is mixed properly?

• gentle tipping back and forth - don’t want to cause haemolysis

What sample can’t be analysed by a cell counter?

• clotted haematology

What coagulation sample can’t be analysed?

• clotted citrate coagulation

What anticoagulant/sample do we want to use for:

1. most clinical chemistry

2. haematology of whole blood

3. glucose by mail/delayed analysis

4. coagulation?

1. serum and LiHep

2. EDTA

3. OxF

4. citrate

For each species, what size needle to we tend to use?

1. cat

2. dog

3. horse

1. 23-21G, 5/8 inch

2. 21G - 5/8 or 1 inch long

3. 18-20G - 1 inch

How do we prepare a site for blood withdrawal?

1. clip the area

2. clean with chlorohexidine 4% (hibi scrub)/10% povidone iodine solution

3. squirt 70% surgical spirit onto the site

4. use cotton wool/gauze swabs

5. may use gloves (re-sterilise site each time you touch without gloves)

What are the 3 veins we can use for a blood sample?

1. jugular

2. cephalic

3. saphenous

How do we take a cephalic blood sample?

1. clip fur

2. restrain foot

3. hibiscrub with cotton wool - check to see if dirty

4. surgical spirit

5. feel vein and occlude

6. clean again

7. angle the syringe at 35 degrees, bevel pointed dorsally

8. check blood has entered the needle, then draw back gently

9. place lid back on needle, remove into sharps bin

10. place droplets into each bood sample tube (prevents contamination of any coagulants, therefore affecting future tests)

What angle do we want to enter the jugular at?

• 30 degrees upwards

How do we make the saphenous vein obvious?

• cusp behind the knee and apply downwards pressure - keep leg off ground

• vein should occlude and become visible

For vacutainer collection for a horse:

1. what vacutainer needle size

2. other equipment

3. how does it work?

1. 18-20G

2. needle holder and appropriate vacutainer

Method:

1. attach needle to the vacutainer (has an internal and external bit)

2. hold the vacutainer by the flange with the selected tube

3. insert the needle as usual, once blood shows in the vacutainer, click the tube in place and take sample.

What equipment is needed for a blood sample in horses?

1. 20G needle

2. 10-30ml (usually) syringe

3. appropriate sampling tube/vacutainer

How do we perform a jugular blood sample?

1. locate the jugular groove between the brachiocephalicus and sternocephalicus

2. occlude more caudally

3. avoid the lower third of the area (the carotid artery is also present)

4. enter at a 30 degree angle upwards

Where do we take a blood sample from a cow?

1. jugular - in a crush, either with a halter on or nosing

2. tail vein

How do we take a blood sample from the tail vein?

1. raise tail up (supporting ventrally with hand in a V-shape)

2. palpate the vein in the tail groove

3. insert needle perpendicular to the tail

4. once blood present in needle head, draw back/click tube into vacutainer

When do we recap a needle when taking a sample from a cow?

• at a safe distance

Where can we identify the jugular vein in a fully wooled sheep?

• look for the wool break

what is the grey bit present on some needles?

• haemoguard

what do we need to consider when taking a blood sample, relating to obtaining relevant data?

• TYPE OF DETECTON METHOD USED

• where to get it tested - lab/in house

what are 4 detection methods for assays?

1. colourmetric - measure colour change

2. turbidometric - measure cloudiness

3. fluorometric - light excitement

4. immunoassay - antibody/antigen, RIA/ELISA

How do we ensure we measure the correct substrate in a reaction e.g. A +B → C + D and required E and F (enzyme and cofactor)

• ensure what is being measured is the limiting reagent

• everything else must be in excess

For most routine chemsitries how many calibration points are there?

Are there any concerns with this?

• 1

• yes, not necessarily reliable (if calibration goes wrong, results will be wrong).

  • DOESNT WORK FOR HOMRONE

What are 4 components of immunoassays

1. antibodies

2. tracer

3. detection systems

4. separation

what antibodies are used in immunoassays?

• Polyclonal or monoclonal

• needs to react with hormone in species of interest

what is a tracer in an immunoassay?

• an enzyme/radioactive tag on molecule/antibody

What separation is needed in immunoassays?

• need tracer signal that’s reacted with hormone separated from which hasn’t

What is the dose/response curve for:

1. Radioimmunoassays

2. others e.g. ELISA

1. -ve response curve - inverse relationship

2. +ve response curve

Why may we choose to use an ELISA over an RIA?

• ELISA avoids complexities of handling radiation

What is chemiluminescence?

• enzyme induced light emission

• causes a light induced reaction rather than colour change => non-specialist lab common practice

How many calibration points do there tend to be in reference lab results? Is this good/bad?

• multiple

• improves performance of the test, good thing

How do we know that it’s okay to use results?

• validation

  • imprecision (how closely results match if we keep repeating the test)

  • accuracy (how far away from real result, ours is)

  • species differences

• quality control

How do we measure imprecision?

• Coefficient of variation (CV%) = %SD/mean

• varies across range of concentrations

How does the imprecision of immunoassays compare to colouimetry?

Immunoassays - 3-10% (hormone results just less precise)

Colourimetry: creatinine <2%, electrochemistry for K+, <1%!

What can we use accuracy to compare?

• different results of different methods

  • harder to compare against true gold standard

State advantages and disadvantages of reference lab testing

1. quality assurance and QC taken care of

2. daily or more frequent → cheaper?

3. slower (next day)

4. validated precise and accurate methods

5. inspected annually if accredited

State advantages and disadvantages of in-clinic lab

1. validation check responsible by in-clinic lab

2. may be less precise/accurate

3. QC responsibility

4. interpret, spot errors and understand results yourself

5. quicker

6. more expensive ??

Rules of shipping blood samples?

1. less than 50ml

2. primary receptacle - leak proof

3. secondary packaging - leak proof

4. absorbent material b/w primary and secondary (must be able to absorb contents in whole)

5. outer packing

• must pass 1.2m drop test

• not smaller than 100mm by 100mm

what paperwork should be included with a blood sample?

Submission form:
- who you are

• contact details

• address/contact

• desired tests

• animal ID, species, age, breed

• sample type

• client ID
  dates taken and posted

• maybe some history/reason for sampling

may able to be an electronic transfer of information

What should we use to write on our samples?

• ideally a waterproof marker, easy to read

what is the UN3373 label?

• not known to have hazardous agents in them

• used for transporting blood - P650

what is the number for infectious category A substance?

UN2814 (affect humans) or UN2900 (affects animals)

How do we use lab tests to define a diagnosis?

1. reference intervals

2. interpretative thresholds (diagnostic cut-offs)

3. +ve/-ve tests

what is a false-negative

• a test restuls for a patient that is actually ill, however their measured level of xyz is low enough to clash with healthy levels

What is a false positive result?

• a result where a healthy patient has a naturally higher level of xyz, but is not unwell. Found within the range considered diseased, but not.

What is sensitivity?

• proportion of animals with a disease that yield a positive test result

  • ability to identify diseased individuals

    • high sensitivity = minimise false negatives

What is specificity?

• proportion of animals that don’t have the disease that yield a negative result

  • ability of a test to identify individuals without disease

  • high specificity = good confirmatory test

How are cut-offs determined?

• balance between specificity and sensitivity

  • often sacrifice one for the other

How is specificity and sensitivity determined?

1. one must have the condition - diseased = need a gold standard test to determine SENSITIVITY

2. one group must not have the condition = healthy = needs similar age and presenting features to diseased group = SPECIFICITY

• need lots of animals in the group = more reliable results.

Outline how contigency tables work

• show diseased vs normal animals

• +ve and -ve results for the test

• helps determine specificity and sensitivity

What is a screening test

• apply to a large population for some who may have mild signs/asymptomatic

• don’t mind a few false positives - check further to ensure they do have it

• there’s a HIGH SENSITIVITY (LOW FALSE NEGATIVE RATE)

What is a confirmatory test?

• ‘‘diagnostic test’’

• ensure animals that test positive do have the disease

  • may be some false negatives but HIGH SPECIFICITY (LOW FALSE POSITIVE RATES)

Consequences of bad sensitivty?

1. higher false negatives

2. diagnosis missed

3. may re-present

4. outbreak may worsen in epidemic

5. costs (financial, life, welfare, emotional)

Consequences of bad specificity?

1. higher false positives

2. unnecessary lifelong therapy (e.g. endocrine)

3. unnecessary euthanasia

4. costs (financial, life, welfare, emotional)

What is prevalence?

• proportion of animals in the tested population that have the condition

  • pre-test probability

How can we affect pre-test probability?

• widespread healthy population screen for an infectious disease - low pre-test probability

• only testing in animals for disease that have several relevant clinical circumstances - high pre-test probability

what is LDDST a test for?

— hyperadrenocorticism/Cushing’s

As prevalence increases what happens to:

1. POSITIVE predicted value

1. INCREASES - more likely that a positive result is truly positive

as prevalence falls what happens to negative predicted value?

• increases

• if the disease is rare in the tested group, negative more likely to be true

In terms of positive results:

1. what tells us if we can believe the result?

2. what has influence on PPV?

3. PPV of low specificity tests?

4. PPV of high specificity tests?

5. acronym?

1. PPV

2. specificity and prevalence

3. poor, except when prevalence is really high

4. good PPV even at low prevalence

5. sPin

In terms of negative results:

1. how do we know if we can trust the result

2. what has an influence on NPV

3. NPV of low sensitivity tests?

4. NPV of high sensitivity tests?

5. acronym?

1. look at NPV value

2. prevalence and sensitivity

3. poor except when prevalence is very low

4. good even with high prevalence

5. sNout

what kind of tests use:

1. sensitivity

2. specificity?

1. screening

2. confirmatory

Relevance of specifcity/sensitivity in endocrinology?

• diagnostic tests are poor for endocrine diseases

  • booth physiological and pathological causes for lots of conditions, false positives are common (low specificity)

How can we mitigate problems of diagnostics in endocrinology?

• look at response

  • we can stimulate xyz to produce more hormone

  • we can suppress using principle of negative feedback

  • we can monitor blood concentrations to see if they change to pathological levels

Case 1

A 15yo cat is brought into your practice, as her owners are worried that she has lost weight even though she has a ravenous appetite. They have also noticed her drinking more.

On physical examination she is underweight (BCS 2/5) and has an unkempt coat. She is tachycardic (HR 200 bpm) with a gallop rhythm noted on auscultation. The owner is happy for you to investigate further. 

Your differential diagnoses at this stage include: Hyperthyroidism, diabetes mellitus, renal insufficiency/failure

From the information available and blood tubes on display

1.   Select the appropriate blood tube for collection.

2.   Select the needle and syringe to collect the sample.

3.   Where would you take the sample from?

1. We want to assess biochemistry:

• renal function: serum (plain/gel) or LiHep

• glucose: FOx OR LiHep

• Total T4: plain Z/serum gel

• Haematology: EDTA

2. 23G, 2ml syringe (look at condition of cat, poor, don’t want to take lots of blood from it)

3. jugular (preferred) or cephalic

Case 2

A 10yo FE Lab cross is brought

in by her owners as they are concerned about her increased drinking and bed wetting. She is overweight (BCS 4/5) making abdominal palpation difficult, but the owners say she has been losing weight recently and vomiting. 

 

Clinical examination:  You note bradycardia (HR 56) with weak peripheral pulses and that she is continuously panting. There is mild muscle atrophy, to which her owners say she has been slowing down but they think this is due to her age.

 

Your differential diagnoses at this stage are:

·       Hypercalcaemia

·       Diabetes mellitus

·       Diabetes insipidus

·       Renal insufficiency/failure

·       Cushing’s disease

·       Addison’s disease

·       Pyometra

·       Liver disease

From the information available and blood tubes on display

1.   Select the appropriate blood tube for collection.

2.   Select the needle and syringe to collect the sample.

3.   Where would you take the sample from?

1. Biochemistry:

• renal and liver: serum or LiHep

• glucose: FOx or LiHep

• haematology: EDTA

2. 21G needle and 5ml syringe

3. jugular, cephalic

1. Coughing horse

You are called to an equine livery yard where more than one horse has begun coughing over the past few days. You suspect equine influenza and decide to take acute serum samples for antibody detection from several horses on the yard.

1. which vacutainer tube will you use?

2. how long should you allow blood to clot for serum separation

3. how soon will you expect to return and collect a convalescent serum sample?

1. plain Z or CAT

2. at least 30 mins, the longer the better. Practically, takes longer than 30 mins to get from yard to lab anyway

3. 10-14 days

2. Suspected Equine Pituitary Pars Intermedia Dysfunction (PPID)

You have been called to visit a pony that is suffering from laminitis. The owner has noticed that the pony has begun to drunk more water while it is stabled recently. You decide to check for endogenous ACTH.

 

Which of the tubes will you select in this case?

How will you process the sample?

1. EDTA plasma

2. centrifuge as soon as possible, transfer supernatant and discard red cells

What type of tests requires a citrated blood sample?

Coagulation test e.g. PT and aPTT

what are two things that are vital to do when collecting blood for a citrated sample?

1. fill to indicated mark or allow vacutainer to fill completely if no mark and mix tube after (gentle inversion 5-10x) collection

C. Farm animal blood sampling

 

Case 1

You visit a farm, the Holstein- Friesian Dairy cows have poor fertility as indicated by a declining conception rate.  On inspection you notice the cows have a longer coat that expected with the black hair over the ribs in particular having a reddish hue, the cows also have some hair-loss around the eyes giving a spectacled appearance.  You suspect that this could be a nutritional, mineral issue and wish to investigate further by taking a blood sample.

 

Please consult the given information and determine:

A)  What sample(s) do you need to take?

B) which animals should we sample

C) how many should we sample?

D)what animals should we avoid sampling?

A) heparinised and serum sample. LiHep or CAT tube

B) representative proportion of the herd and across management groupings

C) at least 6 animals or 10% of herd - ideally 4 animals per management group

D) Ill animals

Why do we want to avoid measuring blood of ill animals in a farm sample, especially for copper?

• caeruloplasmin (CP) is used to assess copper - it’s an acute phase protein

• elevated during illness → false results

Case 2

You visit a farm, the Holstein- Friesian Dairy cows have a recent drop in milk yield and quality.  They have recently changed from first cut to 2^nd cut grass silage.  You suspect that the energy of the 2nd cut silage is much lower than the first cut was and the formulated diet is deficient in energy.  You have persuaded the farmer to send a sample of the silage off for analysis and the diet will be reformulated in light of this.  However, this will take a while and you wish to confirm that this is indeed an energy issue and wish to investigate further by taking a blood sample.

 

Please consult the given information and determine:

A)  What sample(s) do you need to take?

B) Which animals do we sample

C) How many animals?

D) what animals should we avoid sampling?

A) serum sample, (EDTA plasma could be used) plain Z or CAT tube

B) representative proportion across management groups

C) 4 per management group, 10% herd ideally, at least 6

D) ill animals - results will show response to illness not nutritional issue

D. Blood sampling problems - Consequences

 

Case 1. Sharpie’s Addison’s diagnosis

 

Results similar to these led to Sharpie being treated with the mineralocorticoid (Fludrocortisone acetate), initially at the recommended dose, due to suspected Addison’s disease. After 2 years of treatment he was referred to a specialist because the treatment appeared ineffective.

 

Why should alarm bells have rung when the vet received this result (or even before the lab issued the results)?

 

The specialist identified the problem as being selection of the wrong blood tube. Can you suggest what type of blood tube had been used by Sharpie’s vet and why this gave the wrong result for his potassium levels?

 

Which type of blood tube should have been used?

• NOT EDTA TUBE - the anticoagulant IS Potassium!!!!!! This is why there is an elevated K+ level.