Haemoglobin Measurement
Date: 12/01/2016
Written by: Amira Boulos
B00658765
Aim: To estimate haemoglobin concentration in blood using the haemoglobincyanide (HiCN) (cyanmethaemoglobin) procedure and to determine the levels of foetal haemoglobin (Hb F) by alkali denaturation assay.
Abstract:
The haemoglobincyanide procedure is a method used to determine haemoglobin concentration of blood samples. The procedure is important to detect and measure the severity of anaemia or polycythaemia however; E. Lengfelder et al (2015) indicate that it is not usually used to test for polycythaemia as the haematocrit test is more accurate for detecting this. The procedure begins by diluting a sample of blood in Drabkin’s solution which contains potassium cyanide, potassium ferricyanide and a detergent. The procedure uses the presence of the detergent to cause cell lysis to release haemoglobin from erythrocytes. The Haemoglobin (Hb), methaemoglobin (Hi), and carboxyhaemoglobin (HbCO) convert to cyanmethaemoglobin (HiCN) by potassium cyanide and potassium ferricyanide. Absorbance (540nm) of the solution is then measured using a spectrophotometer and the results are used to plot a graph of Hb concentration versus absorbance (540nm). Therefore we can calculate the Hb concentration of an unknown blood sample by interpolating the absorbance (540) value into the graph so that we can analyse the result. Table 1 shows the normal Hb concentration reference ranges and the anaemia and polycythaemia diagnostic values.
Table 1: Table to show the normal Hb concentration ranges and the anaemia and polycythaemia diagnostic values according to B. Blanc et al (1968) and M. F. McMullin et al (2006).
Age and/or Gender Normal (g/lHb) Mild anaemia (g/lHb) Moderate anaemia (g/lHb) Severe anaemia (g/lHb) Polycythaemia
Female ≥120 119-110 109-80 <80 >170
Male >130 129-110 109-80 <80 >180
The alkali denaturation assay is an adaption of the haemoglobincyanide procedure but instead it is used to determine the levels of Hb F in blood. It is a relatively sensitive assay because the levels of Hb F are only slightly elevated with hematological disorders. Even though this test is relatively sensitive it is not diagnostic however it can be used for the evaluation of blood and as a guide for treatment action plans and be used to measure the success or failure of a medication. In order to achieve greater sensitivity the absorbance of the sample is measured at 413nm compared to 540nm which is used in the haemoglobincyanide procedure. This assay works by exploiting the circumstance that Hb F is more resistant to denaturation by strong alkali (sodium hydroxide (NaOH)) than other haemoglobin resulting in only Hb F left in the test solution. Ammonium sulphate ((NH4)2SO4) is then added to precipitate the denatured haemoglobin. The resulting test solution is then filtered and the absorbance (413nm) of the filtrate is measured using a spectrophotometer and the total percentage of Hb F is calculated using the following equation:-
%Hb F= (Absorbance (413nm) of sample × 100)/(Absorbance (413nm) of standard × 20)
Table 2 shows the normal %Hb F reference ranges.
Table 2: Table to show the normal %Hb F ranges.
Age %Hb F
Children at 6 months <2-3
Children at 12 months <1
Adults 0.2-1
Risk assessment:
The appropriate Personal Protective Equipment (PPE) must be worn at all times. B. Mangnall (2013) explains that Drabkin’s solution is a toxic chemical and is fatal if swallowed. Disposable curvettes must be used and it is essential to place them in a container of bleach in the fume cupboard after use. Any glassware or re-useable plastic ware that has been used for Drabkin’s must be soaked in dilute bleach and left overnight in a fume cupboard and washed the following day. Any remaining Drabkin’s must be bleached overnight before disposal. Drabkin’s reagent must be stored at room temperature and protected from light (stored in amber bottle). Powdered Drabkin’s reagent is stable for at least 2 years and prepared Drabkin’s solution is stable for at least 6 months.
Procedure:
The being the haemoglobincyanide procedure, first switch on spectrophotometer and set to 540nm and allow approximately 20mins to warm up. Using water as a diluent, a range of standards from the 180g/L Hb solution where created making up a final volume of 3ml. Table 3 shows the make up of the range of standards.
Sample Hb solution (ml) Water (ml) Hb concentration
(g/lHb)
1 0 3 0
2 0.5 2.5 30
3 1 2 60
4 1.5 1.5 90
5 2 1 120
6 2.5 0.5 150
7 3 0 180
Table 3: Table displaying the range of HiCN standard solutions.
HiCN standards are prepared using the appropriate pipettes to make up the 3ml solutions into separate clean test tubes which were labelled correctly. Once prepared the standard samples are left for 3-5mins to allow complete conversion of Hb to HiCN.
The unknown test blood sample is prepared by initially mixing the blood and then 20μl of the sample is added to 4ml Drabkin’s solution into a correctly labelled test tube. The sample is left for 3-5mins to allow complete conversion of Hb to HiCN. All samples are then transferred into dust-free curvettes which are correctly labelled and the absorbance is measured at 540nm using a spectrophotometer. The results are recorded and a graph is plotted of absorbance (540nm) against Hb concentration. Interpretations could be made based on the graph.
Hb F alkali denaturation assay begins by making up the HiCN solution by measuring 10ml of Drabkin’s solution into measuring cylinder and adding 0.6ml of erythrocyte lysate to the Drabkin’s solution using the appropriate pipette. Leave sample for 3-5mins to allow complete conversion of Hb to Hb F. Using a pipette 2.8ml of HiCN solution is added to a new clean test tube which is correctly labelled along with 0.2ml NaOH. The sample is mixed well and left for exactly 2mins and then 2ml of saturated (NH4)2SO4 is immediately added. Insure the sample is mixed well and leave it for 10mins. Using a funnel the sample is filtered through Whatman No.42 filter paper and collected into a conical flask. Filtration can have duration of 10-20mins.
A standard needs to be prepared by diluting 100μl of the remaining HiCN solution that was prepared previously with 3.4ml Drabkin’s solution into a new clean correctly labelled test tube. The spectrophotometer needs to be zeroed with the Drabkin’s solution with the absorbance 413nm. Transfer the standard and the filtrate into two separate dust-free clean curvettes which are correctly labelled and record the absorbance (413nm).
Results:
Figure 1 is a graph showing Hb concentration versus absorbance (540nm). A calibration curve has been added to the graph which is presented as a straight line. From the straight line we can extrapolate that the absorbance is directly proportional to Hb concentration; this means that as Hb concentration increases the absorbance simultaneously increases. The calibration line is calculated to be γ = 325.02χ – 0.1701. From the equation we can find the Hb concentration of the unknown sample by substituting the absorbance into this equation as follows:-
γ = 325.02(0.501) – 0.1701 = 162.7%Hb
Table 4 displays all the results of Hb concentration versus absorbance in table form.
Table 4: Table to show Hb concentration of each sample after carrying out the haemoglobincyanide procedure.
Sample Absorbance (540nm) Hb concentration (g/lHb)
1 0 0
2 0.094 30
3 0.188 60
4 0.272 90
5 0.371 120
6 0.462 150
7 0.555 180
Unknown sample 0.501 162.7
Table 5: Table to show the absorbance (413nm) of each sample after carrying out the alkali denaturation assay.
Sample Absorbance (413nm)
Standard 2.141
Unknown sample 0.342
Table 5 shows the absorbance results after carrying out the alkali denaturation assay. The absorbance results can be substituted into the following equation in order to work out the percentage of Hb F in the unknown blood sample:-
%Hb F= (Absorbance (413nm) of sample × 100)/(Absorbance (413nm) of standard × 20)
%Hb F of unknown sample = (0.342 × 100)/( 2.141 × 20) = 0.799%Hb F
Discussion:
J. P. Peña-Rosas et al (2015) explains that erythrocytes store and carry oxygen in the blood. Anaemia is a condition caused by a lack of haemoglobin in the body or by a less than normal number of erythrocytes in the blood. The most common causes of anaemia are iron and vitamin deficiency. Folate is important for healthy erythrocyte production. Vitamin B12 is needed for proper absorption of iron. Iron is used by the body to make haemoglobin which is a protein that is in involved with oxygen transport around the body. T. C. Pearson et al (2000) declares that polycythaemia (also known as erythrocytosis) is caused by a high concentration of erythrocytes in the blood. Anaemia negatively affects organs and tissues due to the decreased amount of oxygen being transported around the body. Polycythaemia causes blood to become thicker; this affects the flow of the blood, impedes its ability to travel through the body which can result in blood clots.
Anaemia and polycythaemia are a result of many different conditions that affect Hb concentration. A decreased Hb concentration could be a result of bleeding, kidney disease (due to damage A. Martíez-Castelao et al (2015) states that the kidneys do not make enough erythroprotein (EPO)), inflammation (C. Y. Wang and J. L. Babitt (2016) explains that Inflammation produces a lot of interleukin-6 which stimulates hepcidin production and release from the liver. Hepcidin reduces ferroportin which is a protein that carries iron in circulation), haemolysis, thalassaemia and sickle-cell disease (N. Bianchi et al (2016) and A. Mozar et al (2016) state that these hematological diseases are caused due to mal-functional erythrocytes), bone marrow failure (H. Kook et al (2016) state that this is due to the bone marrow not producing enough erythrocytes), liver cirrhosis (A. Zumrutdal and N. Sezgin (2012) explains that enlargement of the spleen as a result of liver cirrhosis can cause the spleen not to function properly and trap erythrocytes) and cancers affecting bone marrow.
J. H. Lawrence and N. I. Berlin (1962) explains that an increased Hb concentration could also be a result of dehydration (due to an increased concentration of the blood as a result of plasma volume loss), excess production of erythrocytes in the bone marrow, severe lung disease (due to increased EPO production in response to chronic hypoxia) and several other diseases.
The haemoglobincyanide procedure confirms the Hb concentration levels to be normal at 162.7g/lHb (see table 1 for reference ranges and diagnostic values). An Hb concentration below 120 for females and below 130 for males is an indicator of anaemia. An Hb concentration of above 170 for females and above 180 for males is an indicator of polycythaemia. Anaemia is treated depending on the type that has been acquired. Iron and vitamin deficiency anaemia can be treated by supplements. Moderate cases of anaemia can be treated with intravenous iron infusions and severe anaemia can be treated with blood transfusion or EPO injections. Polycythaemia can be treated through venesection, (intravenous removal of blood) medication to reduce erythrocyte production (such as hydroxycarbamide and interferon) and blood thinning medication such as aspirin to prevent blood clots.
Alkali denaturation assay confirmed that the levels of foetal haemoglobin were normal at 0.799%Hb F (see table 2). Foetal haemoglobin is almost completely replaced by adult haemoglobin after 6 months of age except in hematological disorders such as thalassaemia and sickle-cell disease. D. W. Allen, and J. H. Jandl (1960) explains that foetal haemoglobin has a higher affinity for oxygen than adult haemoglobin. Adult haemoglobin interacts with 2,3-bisphosphoglycerate (2,3-BPG) to decrease its affinity for oxygen. The increased affinity for oxygen in Hb F differs to adult haemoglobin because of a single amino acid differentiation of histidine to serine which causes a lack of interaction with 2,3-BPG. Histidine (present in adult haemoglobin) is positively charged and interacts well with the negatively charged surface of the 2,3-BPG in comparison with serine (present in HB F) which is neutrally charged and as a result interacts less efficiently.
Haematological disorders are a result of not being able to produce functional erythrocytes. N. Bianchi et al (2016) explains that thalassaemia is caused by defects in one or more of the haemoglobin chains causing them not to function properly. A. Nakagawa et al (2014) explains that sickle-cell disease is the production of abnormally shaped erythrocytes however does not affect Hb F and they are still able to function correctly. The main function of an erythrocyte is to transport oxygen around the body and this loss in ability for adult haemoglobin can result in a compensatory increase of Hb F due to its higher affinity to oxygen. The increase in Hb F is only slight and cannot work efficiently to mask the symptoms of haematological disorders and proper treatment must be taken. The Alkali denaturation assay is the method discussed here for the evaluation of %Hb F however thalassaemia and sickle-cell disease is more currently detected through genetic prenatal testing using chorionic villi sampling (CVS).
E. A. Rachmilewitz and P. J. Giardina (2011) explains that thalassaemia can sometimes be cured with a bone marrow transplant however if not curable the patient will require episodic blood transfusions. J. B. Segal (2008) tells us that sickle-cell disease can be treated with hydroxycarbamide (also known as hydroxyurea) which is a drug that stimulates production of Hb F, however the patient may still require episodic blood transfusions. Transfusion dependent patients can develop iron overload and will need chelation therapy to remove the excess iron.
The values of the results from both experiments don’t raise any major concerns because they are those expected from a healthy person. However the accuracy of the results may be improved by more precise pipetting to help avoid any errors and using a stopwatch to stay consistent with timing especially during the conversion step of Hb to HiCN. Another way to insure more accurate results is to repeat the experiments and take averages!
Conclusion:
The haemoglobincyanide procedure confirms the Hb concentration levels tested were normal at 162.7g/lHb. Alkali denaturation assay confirmed that the levels of foetal haemoglobin tested were normal at 0.799%Hb F. These results validate that the patient from which the sample was taken is healthy and does not show any signs of having anaemia, polycythaemia or any hematological disorder.
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