College of Health Sciences
Department of Medical Laboratories
Second Year – Second Term
Hematology – 1
Practice NO (2)
SERUM FERRITIN
With the recognition that the small quantity of ferritin in human serum
(15–300 μg/l in healthy men) reflects body iron stores, measurement
of serum ferritin has been widely adopted as a test for iron
deficiency and iron overload. The first reliable method to be
introduced was an immunoradiometric assay in which excess
radiolabelled antibody was reacted with ferritin, and antibody not bound
to ferritin was removed with an immunoadsorbent. This assay was
supplanted by the two-site immunoradiometric assay, which is sensitive
and convenient. Since then the principle of this assay has been extended
to nonradioactive labelling, including enzymes (enzyme-linked
immunosorbent assay, or ELISA). Most current laboratory
immunoassay systems for clinical laboratories include ferritin in the
assay repertoire. Factors to be considered when selecting an
immunoassay system are discussed later. The method described in the
next section is an ELISA. The most sophisticated equipment required is
a microtitre plate reader.
IMMUNOASSAY FOR FERRITIN
Reagents and Materials
Ferritin
Ferritin may be prepared from iron-loaded human liver or spleen
obtained at operation (spleen) or postmortem. Ferritin is purified by
methods that exploit its stability at 75°C Human ferritin may be stored at
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4°C, at concentrations of 1–4 mg protein/ml, in the presence of sodium
azide as a preservative, for up to 3 years. Such solutions should not be
frozen. Ferritin, from human liver or spleen, may be obtained from
several suppliers of laboratory reagents. This may be used as a standard
after calibration against the international standard
Antibodies to Human Ferritin
High-affinity antibodies to human liver or spleen ferritin are suitable.
Polyclonal antibodies may be raised in rabbits or sheep by conventional
methods and the titre can be checked by precipitation with human
ferritin. An immunoglobulin G (IgG)–enriched fraction of antiserum is
required for labelling with enzyme in the assay. The simplest method is
to precipitate IgG with ammonium sulphate. Monoclonal antibodies that
are specific for “L” subunit–rich ferritin (liver or spleen ferritin) are also
suitable. Suitable antibodies (including a preparation labelled with
horseradish peroxidase) may be obtained from Dako Ltd., High
Wycombe, Bucks, UK.
Conjugation of Antiferritin IgG Preparation to Horseradish
Peroxidase
1. Dissolve 4 mg of horseradish peroxidase in 1 ml of water and add
200 μl of freshly prepared 0.1 mol/l sodium periodate solution.
The solution should turn greenish-brown. Mix gently by inverting
and leave for 20 min at room temperature, mixing gently every 5
min. Dialyse overnight against 1 mmol/l sodium acetate buffer, pH
4.4.
2. Add 20 μl of 0.2 mol/l sodium carbonate buffer, pH 9.5, to a
solution of antiferritin IgG fraction (8 mg in 1 ml). Add 20 μl of
0.2 mol/l sodium carbonate buffer, pH 9.5, to the horseradish
peroxidase solution to increase the pH to 9.0–9.5 and immediately
mix the 2 solutions. Leave at room temperature for 2 hours and
mix by inversion every 30 min.
3. Add 100 μl of freshly prepared sodium borohydride solution (4
mg/ml in water) and let it stand at 4°C for 2 hours. Dialyse
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overnight against 0.1 mol/l borate buffer, pH 7.4.
4. Add an equal volume of 60% glycerol in borate buffer to the
conjugate solution and store at 4°C.
Buffer A
Phosphate-buffered saline, pH 7.2, containing 0.05% Tween 20.. Store
at 4°C for up to 2 weeks.
Buffer B
Prepare by dissolving 5 g of bovine serum albumin in 1 litre of buffer A.
Store at 4°C for up to 2 weeks.
Buffer C
Carbonate buffer, 0.05 mol/l, pH 9.6. Dissolve sodium carbonate, 1.59 g
and sodium bicarbonate, 2.93 g in 1 litre of water and store at room
temperature.
Buffer D
Citrate phosphate buffer, 0.15 mol/l, pH 5.0.
Substrate Solution
Prepare immediately before use by adding 33 μl of hydrogen peroxide,
30%, to 100 ml of buffer D and mixing well. Add 1 tablet containing 30
mg of o-phenylenediamine dihydrochloride (Sigma P 8412) and mix.
Sulphuric Acid
Purchase as a 4M solution.
Preparation and Storage of a Standard Ferritin Solution
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Dilute a solution of human ferritin to approximately 200 μg/ml in water.
Measure the protein concentration by the method of Lowry after diluting
further to 20–50 μg/ml. Then dilute the ferritin solution (approximately
200 μg/ml) to a concentration of 10 μg/ml in 0.05 mol/l sodium
barbitone solution containing 0.1 mol/l NaCl, 0.02% NaN3, and BSA (5
g/l) and adjusted to pH 8.0 with 5 mol/l HCl. Deliver 200 μl into 200
small plastic tubes, cap tightly, and store at 4°C for up to 1 year. For use,
dilute in Buffer B to 1000 μg/l, then prepare a range of standard
solutions between 0.2 and 25 μg/l. Calibrate this working standard
against the World Health Organization (WHO) standard for the assay of
serum ferritin 94/572, recombinant human L type ferritin.
Coating of Plates
Microtitre plates (96-well) for immunoassay are required. Do not use the
outer wells until you have established the assay procedure and can check
that all wells give consistent results. Coat the plates by adding to each
well 200 μl of antiferritin IgG preparation diluted to 2 μg/ml in Buffer C.
Cover the plate with a lid and leave overnight at 4°C. On the day of the
assay, empty the wells by sharply inverting the plate and dry them by
tapping briefly on paper towels. Block unreacted sites by adding 200 μl
of 0.5% (w/v) BSA in Buffer C. After 30 min at room temperature, wash
each plate three times by filling each well with Buffer A (using a syringe
and needle) and emptying and draining as described earlier. Plates may
be stored, dry, at 4°C for up to 1 week.
Preparation of Test Sera
Collect venous blood and separate the serum. Samples may be stored for
1 week at 4°C or for 2 years at -20°C. Plasma obtained from
ethylenediaminetetra-acetic acid (EDTA) or heparinized blood is also
suitable. For assay, dilute 50 μl of serum to 1 ml with Buffer B. Further
dilutions may be made in the same buffer if required.
Assay Procedure
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The use of a multichannel pipette for rapid addition of solutions is
recommended. Standards and sera, in duplicate, should be added to each
plate within 20 min.
- Add 200 μl of standard solution or diluted serum to each well.
- Cover the plate and leave at room temperature on a draught-free
bench away from direct sunlight for 2 hours.
- Empty the wells by sharply inverting the plate and drain by
standing them on paper towels with occasional tapping for 1 min.
- Wash three times by filling each well with Buffer A, leaving for 2
min at room temperature, and draining as described earlier.
- Dilute the conjugate in 1% BSA in Buffer A.
- Add 200 μl of diluted horseradish peroxidase conjugate to each
well and leave the covered plate for a further 2 hours at room
temperature.
- Wash three times with Buffer A.
- Add 200 μl of substrate solution to each well.
- Incubate the plate for 30 min in the dark.
- Stop the reaction by adding 50 μl of 4M sulphuric acid to each
well.
- Read the absorbance at 492 nm within 30 min, using a microtitre
plate reader. Alternatively, transfer 200 μl from each well to a tube
containing 800 μl of water and read the absorbance in a
spectrophotometer.
Calculation of Results
Calculate the mean absorbance for each point on the standard curve and
plot against ferritin concentration using semilogarithmic paper. Read
concentrations for the sera from this curve. If results are captured on a
file and calculated with a computer program, the log-logit plot provides
a linear dose response. For serum ferritin concentrations greater than 200
μg/l, reassay at a dilution of 100 times or greater. Control sera should be
included in each assay.
Interpretation
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The use of serum ferritin for the assessment of iron stores has become
well-established. In most normal adults, serum ferritin
concentrations lie within the range of 15–300 μg/l. During the first
months of life, mean serum ferritin concentrations change considerably,
reflecting changes in storage iron concentration. Concentrations are
lower in children (<15 years) than in adults and from puberty to middle
life are higher in men than in women.
In adults, concentrations of less than 15 μg/l indicate an absence of
storage iron. The interpretation of serum concentration in many
pathological conditions is less straightforward, but concentrations of
less than 15 μg/l indicate depletion of storage iron. In children, mean
levels of storage iron are lower and a threshold of 12 μg/l has been
found to be appropriate for detecting iron deficiency.
Iron overload causes high concentrations of serum ferritin, but these
may also be found in patients with liver disease, infection,
inflammation, or malignant disease. Careful consideration of the
clinical evidence is required before concluding that a high serum ferritin
concentration is primarily the result of iron overload and not a result of
tissue damage or enhanced synthesis of ferritin. A normal ferritin
concentration provides good evidence against iron overload but does not
exclude genetic haemochromatosis. This is because haemochromatosis
is a late-onset condition and iron stores may remain within the normal
range for many years.
Serum ferritin concentrations are high in patients with advanced
haemochromatosis, but the serum ferritin estimation should not be used
alone to screen the relatives of patients or to assess reaccumulation of
storage iron after phlebotomy. The early stages of iron accumulation
are detectable by an increased serum iron concentration, a
decreased unsaturated iron-binding capacity, and increased
transferrin saturation; the serum ferritin concentration may be
within the normal range. In this situation, the measurement of
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serum iron and total iron-binding capacity provides useful clinical
information not given by the ferritin assay.
In patients with acute or chronic disease, interpretation of serum ferritin
concentrations is less straightforward and patients may have serum
ferritin concentrations of up to 100 μg/l despite an absence of stainable
iron in the bone marrow. Ferritin synthesis[30] is enhanced by
interleukin-1—the primary mediator of the acute-phase response. In
patients with chronic disease, the following approach should be adopted:
low serum ferritin concentrations indicate absent iron stores, values
within the normal range indicate either low or normal levels, and high
values indicate either normal or high levels. In terms of adequacy of iron
stores for replenishing haemoglobin in patients with anaemia, the degree
of anaemia must also be considered. Thus a patient with an Hb of 100 g/l
may benefit from iron therapy if the serum ferritin concentration is less
than 100 μg/l because below this level there is unlikely to be sufficient
iron available for full regeneration. Here measurement of serum
transferrin receptor concentration may be of value .
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