Hemoglobin Standard

A protein can be tracked down in mature Red Blood Cells/RBCs and assumes the focal part as a transporter of oxygen from lungs to the tissues and different materials, for example, carbon dioxide from the tissues to the lungs during inward breath. 

Need of the Article? 

As for the technical support for the possible suspicion in regard to the quality of the result of the ‘Hemoglobin Estimation’ procedure, as well as for the identification of the causes of inaccuracy of this or that indicator. A hemoglobin standard refers to normative data giving the range of values of hemoglobin including adult, child, pregnant, athlete, and traveler lines, and it is crucial as any deviation from the standard can be indicative of a major medical problem. 

Muzzmal Ahtisham Tahir

Preparation of Hemoglobin Standard

As a reminder before starting the equiped method procedure it is important to prepare a hemoglobin standard. It is used as a basis to compare the intensity of color of the test sample with the standard solutions so as to arrive at the best estimation of the hemoglobin percentage thereof. The following are necessary for preparation of hemoglobin standard:  

• Hemoglobin Powder: Purified hemoglobin, normally from bovine or human fresh, as well as to be exactly known its concentration. 

• Distilled Water: For preparation of dilution of the solution and the hemoglobin powder. 

• Buffer Solution: A particular buffer solution (for example the phosphate buffer) to regulate the acidity of the hemoglobin solution.

• Graduated Cylinders: Measuring cylinders are utilized for liquid measurement and are precise in this regard.

• Pipettes: Used in dispensing small quantities of a liquid for an exact application in a laboratory. 

• Volumetric Flasks: Criticcal in preparing the standard solution at a particular concentration. 

• Sodium Hydroxide (NaOH) or Potassium Hydroxide (KOH): To be utilised when necessary, to alter the pH of the arrangement if necessary. 

• Spectrophotometer: This instrument is used to measure the hemoglobin solution's absorbance and calibrate the densitometer. Standard hemoglobin concentration is calculated using the substance's known concentrations and a prepared calibration curve to compare absorbance and determine substance concentration. 

• Gloves and a lab coat: These items of protective clothing are used to handle materials and reagents during the experiment. 

• Clean Glassware: Guarantee that all the china that you will utilize is spotless and it contains no material which might influence your readiness. 

• Analytical Balance: Used throughout the experiment to weigh hemoglobin powder and other reagents. 

• Attractive Stirrer and Mix Bars: Gear for basic mixing of the hemoglobin arrangement. 

• Refrigerator: To prevent hemoglobin degradation, store the prepared hemoglobin standard solution at a suitable temperature and away from light. 

• Materials for Labeling: Stoppers for marking the standard hemoglobin solution with the concentration, date, and other relevant information on the top of the test tubes.

Isolation of Hemoglobin 

1. Collection of Blood Sample

Use of Anticoagulant Vials: To avoid clotting by collecting the blood sample in a tube containing an anticoagulant like EDTA or heparin. Typically, 5 to 10 mL of blood is sufficient for the yield of hemoglobin when the system is performed.

Centrifugation: To separate the plasma from the RBCs that are contaminating the blood, spin down the sample of blood at 1500-2000g for ten minutes at 4°C. Take a few minutes to spin again and pipet out off the plasma and the buffy coat (the white blood cells and the platelets) down to the packed RBCs. 

2. Washing of the Red Blood Cells

Washing with Saline: After this wash the RBCs need to be resuspended in the same volume of cold isotonic saline (0.9% NaCl). Stir gently in order not to harm the cells. 

Centrifugation: Once more spin the suspension through 1500 -2000 g for 5 minutes with 4°C on centrifuge. Suction off the liquid above and rewash the pellets 2-3 more times to eliminate the traces of plasma and other debris. 

3. Hemolysis or Lysis of Red Blood Cells 

Addition of Distilled Water: Wash the RBCs by spinning down and then resuspending the washed RBCs in four to five times its volume in cold distilled water. The hemolysis is induced by this hypotonic solution that causes the RBCs to swell and burst and the hemoglobin is released into the solution. 

Incubation: After, let the suspension to be incubated on ice for 10-15minutes to ensure that there is hemolysis completely. In the process the cell membranes disintegrate and out of the disintegrated mass the hemoglobin finds a passage to the surrounding water. 

4. Removal of Cell Debris 

Centrifugation: The hemolysate can be further centrifuged at 10 000 g, 15 minutes at 4°C for the removal of abundant proteins. This step will precipitate the cell membranes along with other debris to the bottom and the hemoglobin will remain in the super natant. 

Collection of Supernatant: Sparely transfer the liquid above the pellet which contains the hemoglobin. 

5. Purification of Hemoglobin 

Ammonium Sulfate Precipitation: To precipitate hemoglobin again, decant the supernatant and gradually add the solution's previously saturated ammonium sulfate until it reaches 60-70% saturation. While many other contaminants remain dissolved in the solution, this step sets free hemoglobin. 

Centrifugation: Twist down the combination at 10, 000 rpm for 15 minutes at a temperature of 4 °C. Concentrate the hemoglobin precipitate in a small amount of cold distilled water before discarding the liquid above the pellet by centrifuging it. 

Dialysis: To get rid of ammonium sulfate and other small interferences, wash the resuspended hemoglobin with cold distilled water or a buffer (like phosphate buffer, pH 7.4).

6. Quantification and Storage 

Quantification: Using a spectrophotometer, measure the amount of the substance at a wave length of 540 nM for the hemoglobin form. Select the right eradication coefficient to make the grouping of the arrangement wanted. 

Storage: For those that will be used quickly, the purified hemoglobin solution should be stored at 4°C, while those that will be stored for a long time should be stored at -80°C. To prevent the protein from denatured during storage, include a buffer or other stabilizing agent.

 

Preparation of Hemoglobin Standard Solution

1. Obtain Pure Hemoglobin

Begin with a natural hemoglobin source starting with blood, or with synthetic production within the laboratory. The hemoglobin should be of a known concentration and free from such substances as ATP, 2,3 BPG, Protons and HCO3-. Separate Hemoglobin using the above described procedures. 

2. Dissolve in Acid

To perform this test, dissolve the hemoglobin in a 0. Concentration of 1 N hydrochloric acid {HCl) solution. The acid will oxidize the hemoglobin to form acid hematin just as it is in the Sahli’s method. 

3. Adjust Concentration

Place the hemoglobin solution to a precisely known standard concentration about this value of 14. 8 g/dL to be the specific concentration of Hemoglobin in human blood, the average value. 

 4. Store Properly

It is advisable to store the prepared hemoglobin standard at a dark, cool place so that it does not get degraded. Take measures to guarantee that the container is airtight and does not allow exfiltration or infiltration of moisture in the solution. 

5. Use in Calibration

It is required to standardise the Sahli’s hemoglobinometer with the help of the standard before each estimation of the hemoglobin level. To get calibrated properly, match the color with that of the standard solution with that of the color comparator. 

6. Routine Checks

Make it a practice to use the standard to assess the quality of the procedure being followed in the organization. This makes the hemoglobinometer to be consistent in that it is accurate always no matter the time of the day.

Importance of Hemoglobin Standard 

The hemoglobin standard is a prepared known concentration of hemoglobin normally obtained in the laboratory or purchased from some company. This standard is essential for:

1. Calibration: It assists in calibrating the device since the level of haemoglobin known in the subjects assists in confirming that the readings are correct. 
2. Color Comparison: This standard is used as a basis to compare the intensity of the colour of the acid hematin solution that forms during the test. It is necessary to match the color of the control and the blood sample as close as possible to get the correct hemoglobin concentration. 
3. Quality Control: Using a standard make it possible to use routine to check on how accurate the procedure is and if there are any deviations or errors in the test process. 

 

Conclusion

Both specificity and reliability of the hemoglobin estimation procedure are underlined by the use of the preparation of the hemoglobin standard into the procedure. This step of the equiped method guarantees credible and reliable results so as to improve clinicians’ decision making. The general method used to obtain hemoglobin from blood is through several processes that have very definitive procedures usually done at the laboratory. Preparing blood samples, breaking apart red blood cells from the samples to extract hemoglobin, and purifying hemoglobin are all part of this procedure. The detailed instructions for separating hemoglobin from blood can be found here. The method given below explains procedures for purifying hemoglobin from blood and guaranteeing the purity of the compound in further use of standard preparation and research activity. This process has to be done carefully to not alter the properties of the purified hemoglobin or to get contaminated.

FAQs About Hemoglobin Standard

1. What is a hemoglobin standard, and why is it important?

A hemoglobin standard is therefore a solution of known concentration of hemoglobin to be used in the determination of the unknown solution. It is applied in many haemoglobin assays to fix the standard equipment and establish the standard curves for precise and accurate measurement of haemoglobin concentration in blood samples.  
2. A hemoglobin standard should be prepared?
Hemoglobin standard is made via the determination of the mass of highly purified hemoglobin powder and dilution of it with buffer solution or distilled water to obtain the predetermined concentration of the analyte. The solution is then mixed properly and the concentration of the solution is measured with the help of spectrophotometer with the help of calibration curve.  

3. Can a hemoglobin standard degrade over time?

Yes, unfortunately, spread hemoglobin standards do deteriorate with time particularly if they are exposed to unfavourable environmental conditions. . This is a case where exposure to light and temperature variation can affect the state of the product, and contamination. The standard should be kept in a refrigerator and should be shielded from light to remain accurate all the time. 
 
4. What measures do you take so as to get a correct hemoglobin standard? 
A hemoglobin standard is weighed carefully to get the correct weighing of the substance, calibrated instrumental procedures and equipment are used and the concentration is checked using a spectrophotometer. It can also be occasionally checked against a known reference or a freshly prepared standard for accuracy’s sake. 
 
5. How often should a user check the hemoglobin standard they are using? 
A hemoglobin standard should be replaced in a regular basis depending on the stability and storage of the formerly used hemoglobin standard. If there are the sign of degradation such as colour change or precipitation or if the standard has been in use for sometime one should replace it to get the accurate hemoglobin volumes. Standard solution preparation should preferably be prepared on a new basis every 3-6 months or based on the set laboratory SOP.