Haematology Methods and Techniques

Question 1

What are the constituents of blood?

Answer 1

○ Erythrocytes - 45%
○ Platelets - 1%
○ Leucocytes - <1%
○ Plasma - 55%
Water
Ions
Proteins
Nutrients
Wastes
Gases

Question 2

Describe the difference between plasma and serum.

Answer 2

○ Plasma is the liquid, cell free part of blood that has been treated with anticoagulants (when no clot is formed).
○ Serum is the liquid part of blood after coagulation, that lacks clotting factors (ex. Fibrinogen).

Question 3

How is whole blood collected?

Answer 3

○Purple lid - contains K+EDTA (Ethylene dimene tetracetic acid).
○Prevents clotting by binding to calcium.
○Preserve cells well and allows visualisation of cell morphology.
○Can measure most proteins from plasma stored from EDTA blood.
○Can store genetic material from EDTA buffer coats.
○Allows FBC, blood groups, sickle screen, malaria and HbA1C.

Question 4

How is serum collected?

Answer 4

○Yellow/red top.
○Both contain clot activator, but yellow also has gel.
○Centrifugation separates cells, causing serum to be above gel.
○Gel isn’t a complete barrier.
○Allows urine and electrolytes (U&E’s), liver function tests, lipids, cholesterol, thyroid function tests and bone profile.

Question 5

How is plasma collected?

Answer 5

○Blue lid - contains sodium citrate that is used for coagulation testing and platelet function test.
○Removes calcium from blood, but reaction is reversible so if calcium is added blood will clot.
○It shouldn’t be the first tube to be filled after venepuncture as even a few mm of blood will activate it.

○Grey lid - contains fluoride oxalate that is used for glucose and lactate.

Question 6

Describe the order blood components should be drawn.

Answer 6

1) Blood cultures.
2) Citrate tube.
3) Gel separator tube.
4) Heparin tube.
5) Gel separator tube with heparin.
6) EDTA tube.
7) Glucose tube.

Question 7

What does K+EDTA contamination do?

Answer 7

○K+EDTA contamination increases plasma K+ concentration while decreasing plasma concentration of calcium, magnesium and zinc.

Question 8

What does sodium citrate contamination cause?

Answer 8

○Sodium citrate contamination may cause hypernetrimaeia, which is increased sodium in blood.
○Normally, this indicates dehydration or dysfunction of kidneys.
○This has a dilution effect on other analytes.
○Results make patients appear very sick.

Question 9

What does fluoride oxalate contamination cause?

Answer 9

○Fluoride oxalate contamination results in increased Na & K, and decreased Ca.

Question 10

Describe some blood tests that can be performed.

Answer 10

Full blood count (FBC):
○Gives information on erythrocytes, leucocytes and plasma.
○Performed in anti-coagulated tubes that contains Na/K EDTA.

Coagulation tests:
○Performed using sodium citrate, gives information on plasma.

Erythrocyte sedimentation rate (ESR):
○Gives a global score of physical properties of blood.
○Prevented by sodium citrate.

Cytogenetic analysis:
○Heparin.

Question 11

Describe some pre-analytical factors that need to be considered.

Answer 11

○What are appropriate tests?
○Any special preparation for patient to do?
○Does sample need to be protected from light?
○Does sample need to be stored on ice or refrigerated?

Question 12

Describe some factors to consider when performing a venesection.

Answer 12

○Should patient sit or lie down?
○Use of tourniquet.
○Correct sample tube.
○Order and site of draw.

Question 13

Describe some factors to consider when transporting a sample.

Answer 13

○Is it a priority sample?
○Does it require urgent processing?
○Temperature.
○Time.
○Does it need to be transported on ice/heat block?

Question 14

How may a haemolysed sample occur?

Answer 14

○Haemolysed sample can occur due to poor blood collection technique, or if sample is shaken or due to fine bore needle.
○Results in release of potassium and other erythrocyte constituents.
○Breakdown occurs, which interferes with testing, as testing is done using colorimetric tests so breakdown of cells prevents sample from being analysed properly.

Question 15

How may a lipemic sample occur?

Answer 15

○Lipaemic sample may be caused by blood sample being taken soon after food (post prandial).
○Causes abnormally high lipid concentration in blood.
○This interferes with indirect ion sensitive electrode (ISE), by causing a dilution effect and showing falsely low sodium, and also interferes with colorimetric tests.

Question 16

What is an icteric sample?

Answer 16

○Icteric samples is when serum appears dark yellow, brown or greenish.
○Results in increased bilirubin - above 300umol/l.
○Interferes with Creatinine Jaffe method, which is a colorimetric test used to check creatine levels in blood and urine, and shows a falsely low result.

Question 17

Describe some analytical factors.

Answer 17

○Maintenance procedures.
○Are reagents stored and made up correctly, and are they out of date?
○Are there any calibration failures?
○Has assay passed quality assurance checks?
○Have any IQC failures been investigates?
○Are there fliers on results caused by falsely low results as a results of clotted serum samples?

Question 18

Describe how the conveyor belt operates.

Answer 18

1) Sample is picked up by claws in conveyor belt, which knows what sample to pick up when barcode is entered.
2) Sample goes to machine where analysis will occur by moving along conveyor belt.
3) Lid is unscrewed by machine, which also takes required volume of sample.
4) Testing occurs by colorimetric tests or impedance.
5) After testing is complete, sample is once again stored until it is needed again or can be discarded.

Question 19

Describe spectrometry.

Answer 19

○Measures haemoglobin levels.
1) Small blood sample is mixed with a non-ionic detergent, which destroys erythrocyte membrane and leaves a red/pink soup (haemoglobin).
2) The reduction of light beam passage by density of solution is measured by machine, as this is directly proportional to haemoglobin levels in original sample

Question 20

Describe flow cytometry.

Answer 20

○Allows visualisation of erythrocytes and leucocytes, and gives indication of platelets.
○Involves a fine beam of light (laser) which is disrupted.
○Degree of disruption is directly proportional to cell size.
○Degree of light scatter gives information on granularity of cell.
○Frequency of light interruption gives information on number of cells.
○Only the nucleus is analysed as chemicals are used to remove cytoplasm.
○Cells can be identified by adding chemicals, which determines cells that contain enzymes and are able to metabolise the chemical.○

Question 21

Describe impedance FBC.

Answer 21

○Exploits the principle that passage from one electrode to another can be interrupted by particles.
○Blood cells pass through a small pore in chamber.
○Passage of electricity is impeded every time a blood cell passes through pore, as phospholipid membrane is non-conductive. ○This is proportionate to cell size.
○The higher the cell number, the greater the frequency of disturbances on flow of electricity.
○Software converts frequency of disturbances to number of cells, and also tells the size of cells impeding the current.
○Provides an erythrocyte count, leucocyte count, platelet count, and give the mean cell volume (MCV).
○However, doesn’t give information on the different type of leucocytes.

Question 22

Describe the Siemens 21:20 analyser.

Answer 22

○Samples are assigned a barcode and put into racks.
○There is no contamination as it is an enclosed system.
○Sample is drawn into machine, where it is mixed.
○Gives a good proportion of cells in the sample.
○Sample goes through a clot filter.
○ Analyser contains many valves that could get blocked by a fibrin clot.
○If sample clots, it will withdraw from sample and the blood cells will get mixed up in the fibrin clot, thus count cannot be done.
○Any clotted samples that don’t give an accurate count of sample are rejected by machine.
○Sample goes through a shear valve, which sends parts of sample to different chambers to employ different techniques to do the FBC.○

Question 23

Describe the results generated by the analyser.

Answer 23

RBC count:
○Light scatter indicates cell size and absorption.
○Absorption of RBC’s is equivalent of Hb in sample.
○Absorption of platelets shows granulation.
○Normal patient scatter is to the right of figure.
○ Anaemic patient scatter would be to left of figure.

WBC differential:
○Technique removes cells cytoplasm, and nucleus goes through flow cell.
○The different WBC’s are determined, depending on their composition and the different chemicals available.
○Dark blue - lymphocytes.
○Purple - neutrophils and eosinophils.

Question 24

What are the RBC indices?

Answer 24

○Haematocrit (hct) - the amount of RBC’s in the whole blood, 0.42L/L or 42%.
○Hct = (MCV x RBCC)/1000.

○Mean cell volume (MCV) - volume of average RBC, 95fl.
○Mean cell Hb (MCH) - average amount of HB in cell, 29.5pg.
○Mean concentration of HB (MCHC), 330g/L.

Question 25

Describe white cell differential.

Answer 25

○Analyser uses cytochemistry to analyse cells of a particular lineage.
○Determines whether cell is myeloid or lymphoid.
○Myeloperoxidase kills bacteria and catalyses chemical reactions.
○Neutrophils have a high content of this (involved in bacterial destruction).
○Lymphocytes have a low content of this (involved in viral destruction).
○Analyser stains intracellular myeloperoxidase content of cell.
○Neutrophils stain purple while lymphocytes stain blue.

Question 26

Describe light microscopy.

Answer 26

○Allows further analysis of size, shape and maturity of cells by performing a manual FBC.
○Uses Kohler illumination.
○A drop of blood is smeared on the slide and then fixed.
○Stained with Modified Wrights stain.
○Methylene blue is a basic dye that stains acidic components (like nucleus and basophils granules) blue-violet.
○Eosin is an acidic dye that stains HB and eosinophil granules red/orange.

Question 27

Describe the results from light microscopy.

Answer 27

○Check RBC size, shape of centre of pallor.
○Normal RBC is comparable to a small lymphocyte.
○ Check WBC count.
○Manual platelet count.

Question 28

What is rheology?

Answer 28

○Gives information about the physical properties of blood.
○Two examples include: ESR and PV.

Question 29

Describe Erythrocyte Sedimentation Rate (ESR).

Answer 29

○Westergren method: a thin column of anti-coagulated blood in a vertically aligned tube is allowed to settle under influence of gravity.
○After an hour a band of clear plasma will form which sits on top of a column of blood cells, as blood cells separate from plasma.
○The decrease in blood cell levels is recorded at a rate of mm/hour.
○Fall of RBC occurs in three stages:
1) Rouleux occurs, where RBC’s aggregate together.
2) Fall of cells.
3) Sedimentation at bottom of tube.
○High ESR is seen in inflammation, infection or cancers.

Question 30

Describe plasma viscosity (PV).

Answer 30

○Quantify proteins in plasma.
○Shows how thick/thin plasma has become.
○Plasma is drawn through a capillary tube and the run time between two points is calculated.
○A parallel rise of ESR only reflect protein components of blood.
○Not affected by anaemia.
○Normal range - 1.5-1.72mPa.
○Children normal range - <1.5mPa.
○Chronic disorders, cancers etc. - >1.72mPa.

Question 31

Describe the bell shaped curve.

Answer 31

○Shows measurement variation in a healthy individual.
○Indicates the level of abnormality (if any) of the results produced.
○Locally defined.
○Based on a large number of healthy individuals.
○Healthy individuals fall in middle of curve.
○Age and gender needs to be taken into account.