Experiment 5: Determination of Haemoglobin by the alkaline haematin method

Introduction

Alkaline haematin method, a new method for the economical and accurate measurement of
haemoglobin has been developed as an alternative to the conventional cyanhaemiglobin
method. This method involves the conversion of all heme, haemoglobin, and haemiglobin
species into a stable end product by an alkaline solution of a non-ionic detergent (AHD reagent)
(Zander et al. 1984). It is an obsolete colorimetric technique for estimating the blood
haemoglobin concentration based on the formation of hematin from haemoglobin, using 0.1
mol sodium hydroxide (NaOH).

One of the alkaline haematin methods is AHD-575 method. The AHD-575 reagent is non-
toxic and not photosensitive. Also, this method offers the possibility of safe and quality
haemoglobin estimation at a reduced cost (Anchinmane and Sankhe, 2016). It gives a rapid,
accurate and precise comparable haemoglobin estimation results with internationally accepted
reference HiCN (haemoglobincyanide) method. However, the Drabkin reagent used for the
HiCN measurement is more complex and a change in the composition of the reagent causes a
variation in the results of measurement.

A calibration curve is prepared by measuring the absorbance of alkaline haematin solution.

Therefore, with the standard curve provided, the concentration of haemoglobin in blood
samples can be estimated and determined.

Objectives

1. To study the concentration of haemoglobin by alkaline haematin method.

2. To study the mechanism of alkaline haematin method.
3. To study the advantages of alkaline haematin method compared with conventional method.
Results

Table 1 Absorbance value of different tubes with different amount of haemoglobin.

Tube No. Haemoglobin (mg/100ml) Absorbance at 520nm

1 0 0.000
2 3.0 0.425
3 5.9 0.776
4 7.4 0.985
5 8.9 1.143
6 11.8 1.585
7 14.8 1.947

Standard curve of amount of haemoglobin against

absorbance at 520nm
2.5

2
Absorbance at 520nm

y = 0.1313x + 0.0082
1.5

0.5

0
0 2 4 6 8 10 12 14 16
Haemoglobin (mg/100ml)

Figure 1 Standard curve of amount of haemoglobin and its absorbance value at 520nm.
Calculations

From the figure 1 which is the standard curve of amount of haemoglobin against its absorbance
at 520nm, we could use the equation y = 0.1313x + 0.0082 to read off the haemoglobin
concentration in the blood sample.

Absorbance value at 520nm of blood, y = 0.502

0.502 = 0.1313x + 0.0082

x = 3.76

Therefore, haemoglobin concentration in blood = 3.76 mg/100ml

Discussions

In this experiment, a standard curve of optical density readings against haemoglobin

concentrations is plotted. By mixing different volume of alkaline haematin solution with
different volume of 0.1M NaOH as diluent the different concentration of haemoglobin is
obtained. The standard curve shows that when the haemoglobin concentration increases, the
optical density readings will also increase. The absorbance value of blood at 520nm is 0.502.
Using the equation from the standard curve, concentration of haemoglobin for our sample is
calculated and obtained as 3.76 mg/100ml which is also equivalent to 3.76 gm/dL.

Determination of haemoglobin can use to detect disease characterized by deficiency or

excess of haemoglobin and study the changes in haemoglobin concentration caused by loss or
gain of plasma. Under modern condition, adult should have 2 to 12% of his total haemoglobin
circulating in an inactive form. Most method only measure the active haemoglobin which is
the oxygen-carrying haemoglobin and this may give a false picture of pigment metabolism
since they take no account of inactive haemoglobin which may be capable of regeneration to
the active form. By using alkaline haematin method, total pigment can be meausured (Clegg
and King, 1942).

The normal range for haemoglobin concentration in men is 13.5 to 17.5 gm/dL whereas
the normal range for haemoglobin concentration in women is 12.0 to 15.5 gm/dL. Normal
ranges for children vary with age and sex. New born usually contain 17-22 gm/dL, however
children usually contain 11-13 gm/dL (William et al. 2019). The range for a normal
haemoglobin level may differ from one medical practice to another (Anon, 2019). From the
 result we obtained, the haemoglobin concentration is very low compared to any gender and age
group. Therefore the blood sample was suspected is not taken from human but other sources.

In both men and women even with normal haemaglobin level, tertiles of haemaglobin
levels were positively associated with body mass index (BMI), total-cholesterol (TC),
triglyceride (TG), uric acid (UA), diastolic blood pressures (DBP) and fasting plasma glucose
(FPG). Increasing haemoglobin levels, even in subjects with normal level were associated with
increasing prevalence of cardiovascular risk factors, suggesting that a slightly low haemoglobin
level might be beneficial too (Lianxiang, 2014). When the haemoglobin level is low, it may
indicate that the person may have anaemia nutritional deficiency of several nutrients such as
iron, folate or vitamin B12 (Merritt, 2014)

Conclusion

In conclusion, the objective of this experiment is successfully achieved. Different gender

and age group will have different haemoglobin concentration in body. Haemoglobin level may
also differ from one medical practice to another. The haemoglobin concentration of our blood
sample is 3.76mg/100ml.
References

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Lianxiang R., Bing Gu, Yixing D., Xin Wu, Xinjian L., Hui Wang, Li Jiang, Yan Guo, and
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