Page 380 - The Veterinary Laboratory and Field Manual 3rd Edition

Clinical chemistry 349

Table 7.6 Preparation of reagents.

reagent blank Standard Sample Sample blank
Test serum - - 200 µl 200 µl
Control serum* - 200 µl - -
Saline 5 ml 5 ml 5 ml 5 ml
Biuret reagent 5 ml 5 ml 5 ml -
Blank diluent - - - 5 ml

Note: *Control serum with total protein 50 g/l.

PrIncIPLE PrEParatIon oF a Standard GraPH
A dilution (1 : 10) of whole blood is made using Owing to the fact that the haemoglobin stan-
a weak solution of sodium hydroxide (0.1 µm) dard is expensive it is useful to prepare a stock
to form haemoglobin, this is measured colori- solution of normal blood with a haemoglobin
metrically against a haemoglobin standard (for concentration of about 11% (11g/dl). This will
example, Gibson-Harrison). require titration of a blood sample with known
haemoglobin concentration which has been
rEaGEntS compared against a standard control such as the
1 Gibson-Harrison Haemoglobin standard – Gibson-Harrison standard. The procedure is as
1
16 g/dl (B.H.D) follows:
2 NaOH 0.1M (100 mmol/l).
• Use any normal blood sample. Take 100 µl of
MEtHod blood, add to 10 ml of 0.1 M NaOH and mix.
Pipette 50 µl (0.05 ml) of whole blood into Wait for the solution to clear.
4.95 ml of 100 mmol/l sodium hydroxide • Pipette 10 ml of the haemoglobin standard
(NaOH), heat for 4–5 min in a boiling water into another tube and place both tubes in a
bath and then cool rapidly in water. boiling water bath for 4–5 min. Remove the
The Gibson-Harrison haemoglobin stan- tubes from the water bath and allow to cool
dard should be boiled and cooled alongside the for 5 min.
test sample. When cold, but within 30 min of • Read the OD at 540 nm.
heating, read the OD using a colorimeter set at • Calculation as before.
540 nm (yellow-green filter) for both samples.
By comparing the OD readings for the standard If the sample has a high haemoglobin concen-
haemoglobin sample and the test sample it is tration it can be titrated to 11 g/dl and stored
possible to calculate the haemoglobin content until required or the OD for a range of dilutions
of the test sample. can be recorded to produce a reference curve.
Other methods of determining the haemoglobin
Calculation: Concentration of test OD of concentration in a sample include the Drabkin’s
blood sample/ OD Gibson-Harrison stan- method (Chapter 5). If available, modern colori-
dard × 16 = g/dl metric techniques can also be used, for example
the Diaspect Tm handheld haemogobinometer
The standard error using this method is +/–5%. described in Chapter 2 (Figure 2.45).

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