Red Cell Disorders

Question 1

How are indices in a full blood count affected by IDA?

Answer 1

MCV/MCH: reduced in relation to severity of the anaemia
Serum iron: reduced 
Total iron binding capacity: raised (INDICATES THE MAXIMUM AMOUNT OF IRON NEEDED TO SATURATE TRANSFERRIN)
Serum ferritin: reduced
Bone marrow iron stores: absent
Erythroblast iron: absent
Hb electrophoresis: normal 
Platelet count: normal/elevated
MCHC: low
Transferrin saturation: >16%

Question 2

How is the FBC reflected visually on a blood film?

Answer 2

Depending on the degree of anaemia see

• Anisocytosis (different shapes and sizes = increase RCW)
• poikilocytosis (pencil like shape)
• Microcytic (very small)
• Hypochromic (very unpigmented)

Question 3

What are the follow-up tests for diagnosing iron-deficient anaemia, and what would these be expected to show?

Answer 3

Perl’s iron stain:
- marrow smear is treated with Prussian blue which stains the iron
within the reticuloendothelial cells (macrophages) blue
- though a bone marrow biopsy is very unlikely in IDA diagnosis as
it is overkill! May be done if you were checking orogression of
leukaemia patients

Serum ferritin:
- can be measure with ELISA (immunochemiluminescence)
- patient serum containing antigen (ferritin) is added to microwell
strips containing the antibody
- second antibody containing the complex is then added
- substrate (chromates solution) is added and will be cleaved by the
enzyme to form a coloured product
- measured quantitatively by spectrometer
- extent of reaction is directly proportional to the concentration of
antigen

Serum Iron Spectrophotometry:

• iron is removed from transferrin by acidification (HCl)
• Fe3+ are reduced to Fe2+ by ascorbate
• Fe2+ is complexed with a colour reagent
• measure spectrophotometrically

TIBC:
- transferrin can be nearly 1/3 saturated with iron
- TIBC is the max iron needed to saturate serum/plasma transferrin
- TIBC is routinely determined by saturation of TRF with an excess
predetermined amount of iron (Fe3+)
- removal of unbound iron (by the precipitant MgSO3) and
measurement of iron that is dissociated from the TRF

Percentage transferrin saturation:
- TIBC is used to calculate this:

                             total iron (ug/dL) - % saturation = ———————  x 100
                                TIBC (ug/dL)

• transferrin saturation of <20% indicate IDA, while >50% indicate iron
  overload

Zinc protoporphyrin (ZPP):
- normal metabolite formed in trace amounts during haem biosynthesis
- there are two pathways to make haem, this is the minor, with fe being
incorporated under normal circumstances
- in cases of age deficiency (and lb poisoning) zinc is incorporated into
protoprphorin X instead of iron and zinc protoporphyrin is produced as
the major product instead of haem
- in patients with IDA, ZPP levels are increased
- a haematofluorimeter is used to measure the ZPP levels
- fluorophore reagent added to whole blood in EDTA and converts the
sample to a stable/standard derivative, this is put on a
coverslip. Standards are prepared the same way
- light of 415 nm is passed through sample and standard, ZPP is
excited
- light is emitted at 519 nm, filtered through a photomultiplier
tube(PMT), current produced equals the ZPP in the sample
- must be performed on FRESH samples
- interfering substances: bilirubin, some drugs and haemolysis (raise)

Question 4

What is the reference range for diagnosing anaemia with Hb?

Answer 4

Below 130 g/L in men over 15 yrs

Below 120 g/L in non-pregnant women over 15

Below 110 g/L in pregnant women