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Hemoglobin Estimation - Values and Methods

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Hemoglobin is a protein responsible for carrying oxygen from the lungs and returning carbon dioxide to the lungs. Read the article below to know it.

Medically reviewed by

Dr. Mubashir Razzaq Khan

Published At January 3, 2023
Reviewed AtJuly 16, 2023


Hemoglobin has a lot of uses in human blood, the deficiency of which results in various forms of anemia, a severe condition worldwide. Therefore regular blood tests are required to identify hemoglobin levels to avoid conditions like anemia.

What Are the Normal Hemoglobin Values?

The normal level of hemoglobin in the blood is:

  • Adult women 12 to 16 g/dL.

  • Adult men 14 to 18 g/dL.

  • Newborn 14 to 20 g/dL.

How Is Hemoglobin Estimated?

Hemoglobin (Hb) estimation can be divided into two broad categories. It is an invasive technique that involves the collection of blood samples from the patient. Many clinical methods are available to measure hemoglobin, and it depends on the laboratory setup which one it prefers to use. With the introduction of image processing, microscopic photographs of neural networks can be taken and processed for the parameters that are to be determined. This is currently a wide-open field of scientific research. However, only some researchers have reported their work done on regression techniques and artificial models of neural networks. The non-invasive methods are sensor-based. The work in this category involves designing sensor signal conditioning circuits, processors, or controllers for analyzing the observation.

1. Pathology Laboratory Methods:

  • Various clinical methods are approved by the World Health Organization (WHO) to be used in the pathology laboratory to estimate the hemoglobin in the blood. To name some of them are Sahli's method of hemoglobin determination, measurement by hemoglobinometer or HemoCue, colorimetry-hemoglobin cyanide method, or cyanmethemoglobin method.

  • In Sahli's method of hemoglobin estimation, carrying out the test requires dilution of blood and visual color match. Sahli's method is based on converting hemoglobin to acid hematin and then visually matching its color against a solid glass standard.

  • Another method for hemoglobin estimation is the HemoCue method. Blood lysis is required for this. Lysis means the breakdown of a cell, frequently viral, enzymic, or osmotic mechanisms that comprise its integrity.

  • The following method is the hemoglobin cyanide or cyanmethemoglobin method, which is regarded as the most precise technique of hemoglobin estimation and is also considered the standard for reference. The equipment used in this method is also the most complex one among all those utilized in other tests. In this technique, a particular amount of whole blood sample is included in Drabkin's diluting fluid. The erythrocytes in the blood get hemolyzed, and the hemoglobin gets transformed into a stable compound called hemoglobin cyanide.

  • In this method, the blood sample is placed in a colorimeter, and the amount of light absorbed by it at a certain wavelength is proportional to the quantity of hemoglobin in the blood. A known hemoglobin level reference solution is used to standardize the method.

  • Modern analyzers use the SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis method to estimate hemoglobin by breaking the red cell wall and measuring the OD (optical density) of the fluid in a buffered solution by machines.

2. Image Processing and Neural Network Approach-BasedTechnique:

H. Ranganathan and N. Gunasekaran introduced another concept of invasive determination of hemoglobin in blood by color analysis method and an artificial neural network. The researchers have tried to measure the hemoglobin content of the blood by using the color measurement technique. As per the authors, this method is very useful in remote areas with a low resource setting. To avoid errors due to human interpretation, the author utilizes an artificial neural network (ANN) approach for the determination of Hb in the blood.

The procedure includes:

  • Step 1- The blood samples were smeared on the slides by the automated system. They captured high-resolution microscopic color pictures of the blood samples using a digital camera mounted on a microscope.

  • Step 2- Researchers selected a segment of the image of (256 × 256) pixels for further processing.

  • Step 3- The calculations are made to extract color information for each image using MATLAB. Then, they calculated the average intensity for each of the R, G, and B planes, giving each print an average value of the red, green, and blue colors. The size of the generated matrix became 3 x N, where N is the number of images.

  • Step 4- Researchers implement a program for training the neural network.

3. Regression Model-Based Methods:

Saif Al Zahir and Han Donker published a paper on a regression-based model for anemia detection. They have presented an efficient method to find blood hemoglobin values and detect anemia using color data. They have developed a logit regression model using 1000 blood samples. The researchers have compared the output of the proposed method with the standard laboratory results.

  • Blood sample collection.

  • Blood color image analysis.

  • Building the model.

  • Detecting anemia.

The database used by researchers collects 1000 blood samples that are chosen randomly among the crowd. After making the smear of the sample on a glass slide, its microscopic color images are captured with a digital camera attached to a microscope with 10X magnifications.

4. Sensor-Based Approach:

Herlina, Fatimah, and Nasir put forward a non-invasive system to identify hemoglobin in the blood during dengue fever. In this method, the authors have used bioelectric impedance analysis (BIA) to predict the hemoglobin quantity in dengue patients. The authors also suggested that BIA is a single-frequency, inexpensive, swift, and painless mode of determining the body components.


This article briefly describes various invasive, non-invasive, and pathological laboratory methods used for estimating hemoglobin amounts in the blood by implementing several techniques based on different approaches. The diagnostic value of a particular method varies significantly according to its accuracy and reliability. Accuracy can be estimated by comparing the results obtained by a particular method with the results of a standard method. The invasive techniques are more accurate than the non-invasive methods, as the color of the blood directly indicates hemoglobin in the blood. Also, for non-invasive techniques, more accurate mathematical analysis is required to calibrate the data from the sensors, which may lead to a false reading. But looking at today's demand, there is a requirement for a non-invasive online monitoring system in which patients' samples can be monitored continuously without any delay, resulting in better treatment outcomes and recovery.

Frequently Asked Questions


What Are the Methods Available for Hemoglobin Estimation?

- Hemoglobin estimation methods measure the concentration or amount of hemoglobin in the blood. Various methods for hemoglobin estimation are:
- Based on color development:
- Sahli’s or acid hematin method.
- Cyanmethemoglobin method.
- Oxyhemoglobin method.
- Alkaline hematin method. 
- Direct reading hemoglobinometers.


What Are the Two Common Types Used for Hemoglobin Estimation?

Two common types of hemoglobin estimation are:
- Cyanmethemoglobin Method: This method involves converting hemoglobin into a stable-colored compound called cyanmethemoglobin, which can be measured spectrophotometrically.
- Hemoglobinometer Method: This method uses a hemoglobinometer, a portable device that measures the color intensity of a blood sample and provides an estimate of hemoglobin concentration.


What Are the Normal Hemoglobin Values?

The normal range for hemoglobin levels can vary slightly depending on age, sex, and altitude. In general, for adult males, the normal range is around 13.5 to 17.5 grams per deciliter (g/dL), and for adult females, it is around 12.0 to 15.5 g/dL.


What Is Meant by Sahli’s Hemoglobin Estimation Method?

Sahli's method is a colorimetric method used for estimating hemoglobin levels. It involves comparing the color of a blood sample with that of a standard color scale to determine the hemoglobin concentration.


Where Is Hemoglobin Found in the Body?

Hemoglobin is primarily located inside red blood cells (erythrocytes), where it binds to oxygen and transports it throughout the body.


What Is Meant by Hemoglobin and What Are Its Types?

Hemoglobin is a protein present in red blood cells and fulfills the vital role of transporting oxygen from the lungs to various tissues throughout the body. There are different types of hemoglobin, including:
- Hemoglobin A (HbA): The most common type in adults, consists of two alpha and beta globin chains. 
- Hemoglobin A2 (HbA2): Present in small amounts, consisting of two alpha and delta globin chains.
- Hemoglobin F (HbF): Predominant in fetuses and newborns, consisting of two alpha and two gamma globin chains.


What Does the Hemoglobin Do?

Hemoglobin is primarily found inside red blood cells (erythrocytes), where it binds to oxygen and transports it throughout the body. Hemoglobin is a protein present in red blood cells and fulfills the vital role of transporting oxygen from the lungs to various tissues throughout the body.


Which Is the Relevant Method for Hemoglobin Estimation?

The most accurate method of hemoglobin estimation is generally considered the cyanmethemoglobin method, which is based on the conversion of hemoglobin to cyanmethemoglobin and subsequent spectrophotometric measurement.


What Are the Ways to Increase Hemoglobin Levels in a Day?

To increase hemoglobin levels within 24 hours, it is necessary to have a healthy diet rich in iron, folate, and vitamin B12. Foods like red meat, spinach, legumes, nuts, and fortified cereals can help. Additionally, staying hydrated, getting enough rest, and avoiding activities that may cause blood loss can support hemoglobin production.


What Is the Amount of Variation in Hemoglobin Levels in a Day?

Hemoglobin levels typically do not change significantly within a day. However, minor fluctuations can occur due to factors like hydration status, physical activity, and underlying medical conditions.


What Are the Instruments Used in Hemoglobin Estimation?

Instruments used for hemoglobin estimation include:
- Hemoglobinometers: Portable devices that measure the color intensity of a blood sample to estimate hemoglobin levels.
- Automated Hematology Analyzers: Modern machines can provide a complete blood count, including hemoglobin estimation, as part of routine blood testing.
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Dr. Mubashir Razzaq Khan
Dr. Mubashir Razzaq Khan



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