Basic Investigations in Haematology Lecture 1 - Haemoglobin Estimation Notes

Haemoglobin Estimation

Introduction

Manual, semi-automated, and automated techniques are available for determining full blood count (FBC) or complete blood count (CBC) components. Manual techniques are less expensive concerning equipment and reagents but are labor-intensive. Automated techniques, although capital-intensive, allow for the rapid processing of numerous blood counts with fewer staff, and are generally more precise and accurate than manual methods, provided proper calibration is maintained.

Why Estimate Haemoglobin (Hb) Content of Blood?

• Detect anemia and its severity.
• Monitor Hb levels following treatment to assess patient response.
• Monitor Hb levels in patients undergoing treatment with specific drugs, such as AZT in HIV patients.
• For blood donation purposes.

Haemoglobinometry

Haemoglobinometry refers to the estimation or measurement of haemoglobin content/concentration in the blood. Different methods are available, including:

• Measurement of color.
• Determination of its power of combining with oxygen or carbon monoxide.
• Analysis of its iron content ($0.347g$ iron = $100g$ of haemoglobin).

While all the above can estimate haemoglobin concentration, some are not practical for routine lab use. Common methods involve color or light-intensity matching techniques, measuring methaemoglobin (Hi) or sulphaemoglobin (SHb) to varying extents. Available methods include dilution, non-dilution, and visual color comparison techniques.

Dilution Techniques

• Hemiglobincyanide (Cyanmethaemoglobin) Technique: A filter colorimeter or direct read-out meter (spectrophotometer) is used. This is the recommended method by ICSH because stable haemiglobincyanide (HiCN) standards are available for instrument calibration. It employs a diluting fluid known as Drabkin’s reagent.
• Other methods: Oxyhaemoglobin (HbO2) method, Sahli’s Acid haematin method, and Alkaline Haematin method.

Non-Dilution Techniques

Blood is collected into a single-use microcuvette or other sampling device coated internally with reagent (sodium nitrite and sodium azide) to lyse the blood and convert the haemoglobin to azidemethaemoglobin, enabling direct read-out meter measurement. These methods are particularly useful when dilution is not feasible and are user-friendly for non-lab staff.

Non-dilution techniques are gaining popularity due to their accuracy and ease of use. Examples include the Developing Health Technology (DHT) Haemoglobinometer and the HaemoCue system.

Direct Read-Out Haemoglobin Meters

These meters have built-in filters and a scale for direct reading of haemoglobin concentration in g/dl or g/l. They are generally based on the oxyhaemoglobin (HbO2) method. Many instruments utilize a light-emitting diode of the appropriate wavelength and are standardized to give results comparable to the HiCN method. These are mainly used for bedside or point-of-care testing.

Visual Comparative Technique

This technique is used when photometric techniques are not available (e.g., deprived areas and some district hospital labs). It uses simple clinical devices that compare blood color against a range of colors representing haemoglobin concentrations. It is intended for anemia screening when lab facilities are absent. Examples include the WHO Colour scale, Haemoglobin Colour Scale.

Using the Haemoglobin Colour Scale

• Use only approved test-strips.
• Add a drop of blood to one end of a test strip to cover the aperture in the Colour Scale completely.
• Wait about 30 seconds, then read immediately by comparing the blood stain with the Colour Scale to find the best colour matches.
• Keep the test-sing close to the back of the Colour Scalo.
• Avoid direct sunlight, marked shade or shadows.
• If the lui stain matches one of the sides of red each record the haemoglobin valve. If the colour lies between two sides, record the muda in doubt betwear bo shades, record the lower value.
• Discard the test strip after use. Wipe the back surface of the Scale at the and of each session or if it becomes soiled during.

Haemiglobincyanide/Cyanmethaemoglobin Method

Principle of the Test:

Whole blood is diluted 1 in 201 or 251 in a modified Drabkin’s solution containing potassium ferricyanide and potassium cyanide. Red cells lyse, and the haemoglobin is oxidized by the ferricyanide to methaemoglobin (Hi). The Hi is then converted to stable haemiglobincyanide, HiCN (Cyanmethaemoglobin).

Method

The absorbance of the HiCN solution is read using a spectrophotometer at a wavelength of $540 nm$ or a filter colorimeter using a yellow-green filter (e.g., Ilford 625, Wratten 74, Chance O Gr1). This method measures oxyhaemoglobin (HbO2), Methaemoglobin (Hi), and carboxyhaemoglobin (HbCO) but not sulphaemoglobin (SHb).

The absorbance is then compared with that of a reference HiCN standard solution. Haemoglobin values can be obtained from tables prepared from a calibration graph or, if using a direct read-out haemoglobin meter, from the digital display..

Test Procedure

1. Sample required: EDTA anticoagulated venous blood or capillary blood.
2. Measure carefully $20 {ml}$ of capillary blood or well-mixed venous blood into $4$ or $5 {ml}$ Drabkin’s neutral diluting fluid.
3. Stopper the tube, mix, and leave the diluted blood at room temperature, protected from sunlight, for $4-5$ minutes.
4. Place a yellow-green filter in the colorimeter or set the wavelength at $540nm$.
5. Zero the colorimeter using Drabkin’s fluid and read the absorbance of the patient’s sample.
6. Using the table prepared from the calibration graph, read the patient’s haemoglobin value.

Calculation

The patient’s Hb concentration can be calculated using the formula:

$Hb (g/dl) = (\frac{A540 \text{ of test sample}}{A540 \text{ of standard}}) \times \text{Conc. of standard (mg/l)} \times \frac{\text{Dilution factor (201 or 251)}}{1000}$

Note:

• If written in mg%, multiply the figure by 10 to give mg/l.
• = result g/dl è g/l ÷ 10
• $1000$ = factor to convert mg/dl to g/dl

Precautions and Sources of Error

• Warm up the colorimeter or spectrophotometer for at least 15 minutes before use to ensure galvanometer stability.
• Guard against incorrect pipetting.
• Wipe off blood around the pipette tip to avoid falsely high Hb values.
• Mix blood samples well before pipetting.
• Avoid haemodilution or haemoconcentration.
• Place the cuvette correctly in the cuvette holder of the colorimeter (with the transparent side facing the light source).
• Wipe off any fluid or grease on the outer surface of the cuvette before reading absorbance.
• Avoid air bubbles in the cuvette during reading.
• Mix blood/Drabkin's reagent solution thoroughly before taking absorbance readings.
• Wipe the surface of cuvettes with lint-free cotton material.
• Avoid fluctuating voltage supply (use a stabilizer or UPS).

Quality Control

Daily Quality Control:

• Preserved whole blood (known Hb).
• Stable control haemolysate (Hb known).
• HiCN (in the absence of preserved whole blood or control haemolysate).
• Examine Drabkin’s reagent for color change or turbidity (includes visible and photometric check).

Monthly Quality Control:

• Check the pH of Drabkin’s reagent.

Haemiglobincyanide/Cyanmethaemoglobin Standard

• Needed to calibrate a filter colorimeter or spectrophotometer.
• Usually available commercially in ampoules with a volume of $10 – 25 ml$.
• Has stability for many years ($\geq 2 \text{ years}$).
• The concentration is usually between $550-850 mg/l$ ($55-85 mg/$).
• The specific value will be printed on the label of each ampoule, from which the equivalent Hb value must be determined.

Example:

If a haemoglobin standard is labeled $60 mg/$ ($60 mg/100ml$):

1. Multiply the figure by 10 to convert it to mg/l.
2. Multiply the new figure by the dilution to be applied to the blood sample.

What will the concentration be if you dilute 1 in 200 or 1 in 250?

Ready-to-use diluted HiCN standards come in concentrations of $30 g/l$, $115 g/l$, and $180 g/l$. They are suitable for preparing calibration graphs for use in obtaining patients’ Hb values using $20 ml$ blood in $4 ml$ Drabkins diluting fluid, i.e., 1 in 201 dilution.

Drabkin’s Fluid (Reagent)

• The original reagent had a pH of 8.6.

• ICSH-recommended Drabkin’s reagent has a pH of 7.0 – 7.4. It is less likely to cause turbidity from precipitation of plasma proteins and requires a shorter conversion time ($3-5$ mins) than the original Drabkin’s solution.

• The only disadvantage is that it causes frothing.

• The pH of 7.0 – 7.4 must be checked with a pH meter at least once a month.

• The diluent should be clear and pale in colour and must not be used if it loses its colour or becomes turbid.

Preparation of Drabkin’s Reagent

• When the Drabkin’s solution is measured against water as a blank in a spectrophotometer at a wavelength of $540 nm$, the absorbance must be zero.
• The solution must be stored in a light-opaque container (e.g., brown borosilicate glass bottle or ordinary glass bottle wrapped in silver foil).
• If stored at room temperature in a brown borosilicate glass bottle, the solution is stable for several months.
• If the ambient temperature is higher than $30 ^0C$, the solution should be stored in the refrigerator but brought to room temperature before use.
• The solution must not be allowed to freeze.
• The reagent must be discarded if:
  • it becomes turbid,
  • the pH is found to be out of range,
  • it has an absorbance other than zero at $540nm$ against a water blank.