Hemoglobin estimation

Hb Estimation by M.Ishaq Wani

1. Hemoglobin Estimation By: M. Ishaq Nims University Rajasthan

2. Hemoglobin (Hb) contains two pairs of polypeptide chains α2 β2 and four haem groups each having an atom of ferrous iron (Fe +2 ) . Hb is the main component of RBCs and accounts for approximately 34% of the RBCs by weight. Iron content of Hb is 0.347 gm /100 g By: M. Ishaq (A.P.) Nims University Rajasthan

3. By: M. Ishaq (A.P.) Nims University Rajasthan Various forms of Hb Oxyhemoglobin (Hb O2) Carboxy hemoglobin (Hb CO) Sulfhemoglobin (SHb) Methaemoglobin Variants of Hb Normal: Hb A, Hb F- gama , Hb A2-delta Abnormal: HbS , HbC , HbD & HbE The measurement of concentration of Hb in the blood is known as HEMOGLOBINOMETRY.

4. By: M. Ishaq (A.P.) Nims University Rajasthan S amples used for Hb estimation Capillary blood from finger prick. Intravenous sample—It should be well anticoagulated, preferably in EDTA. Liquid anticoagulants should not be used at all as these dilute and decrease Hb concentration.

5. By: M. Ishaq (A.P.) Nims University Rajasthan Indications for Hb Estimation To determine presence and severity of Anemia. Screening for polycythemia. To assess the response to a specific therapy in anemia. Estimation of Red C ell Indices Selection of blood donor

6. By: M. Ishaq (A.P.) Nims University Rajasthan METHODS FOR ESTIMATION OF HAEMOGLOBIN - are divided into 4 groups as under: Colorimetric method : Colorimetric method is based on colorimetric measurement of the intensity of color developed on addition of some substance to the blood. Include the following: Visual Methods Photoelectric Methods

7. By: M. Ishaq (A.P.) Nims University Rajasthan METHODS FOR ESTIMATION OF HAEMOGLOBIN Visual Methods A.a. Sahli’s method A.b . Tallquist chart (obsolete) A.c . WHO Hb Color Scale B. Photoelectric Method B.a. Cyanmethemoglobin method B.b . Oxyhaemoglobin method & Alkaline Hematin method B.c. Electronic counter method B.d . Direct reading electronic haemoglobinometer

8. By: M. Ishaq (A.P.) Nims University Rajasthan 2. Gasometric Method o r Measurement of O2 carrying capacity of Hb : Measurement of O2 carrying capacity of Hb can not be used for mass screening but is used in referral or research laboratories only. 3. Chemical Method or Measurement of iron content of Hb : Measurement of iron content of Hb is used only for research purpose 4. Specific gravity method: It is a very rapid method and is useful for screening blood donors for anemia in blood donation program. Normal specific gravity of blood ranges from 1.048-1.066.

9. By: M. Ishaq (A.P.) Nims University Rajasthan C ommonly used methods Sahli’s Method – A C olor Based Method Principle : Hb is converted into acid hematin with the action of dilute hydrochloric acid (N/10 HCl ). The acid hematin is brown in color and its intensity is matched with a standard brown glass comparator in a visual colorimeter called Sahli’s colorimeter.

10. By: M. Ishaq (A.P.) Nims University Rajasthan Sahli’s Method Procedure „ Fill Sahli’s Hb tube upto mark 2 with N/10 HCl . Deliver 20 μl (0.02 ml) of blood from a Hb pipette into it. „ Stir with a stirrer and wait for 10 minutes. „ Add distilled water drop by drop and stir till color matches with the comparator. „ Take the reading at upper meniscus .

11. By: M. Ishaq (A.P.) Nims University Rajasthan Equipment: a. Sahli’s Hemaglobinometer consisting of : 1. Graduated hemoglobin tube 2. Comparator with a brown glass standard b. Hb Pipette c. Stirrer d. Dropper (dropping pipette) REAGENTS: N/10 HCl b. Distilled Water Specimen: a. EDTA anticoagulated venous blood. b. Blood obtained by skin puncture

12. By: M. Ishaq (A.P.) Nims University Rajasthan ADVANTAGES: Simple bedside test. Reagents and apparatus are cheap. DISADVANTAGES There can be visual error. Carboxy , met and sulfhaemoglobins cannot be converted to acid hematin . Comparator can fade over the years. Color attainment of acid hematin takes long time and also fades quickly. Source of light (day light or artificial) influences the color comparison.

13. By: M. Ishaq (A.P.) Nims University Rajasthan Normal Values of Hb Men 15 ± 2 g/dl Women 13.5 ± 1.5 g/dl Infants 16.5 ± 3 g/dl Critical Values Less than 7 gm /dl- S evere Anemia More than 20 gm /dl- Hperviscosity

14. By: M. Ishaq (A.P.) Nims University Rajasthan WHO Hemoglobin Color Scale This is rapid, simple, inexpensive and reliable. Procedure: A drop of blood is placed on strip of chromatography paper and the color developed is matched visually against the printed color scale. Color scale consists of printed set of colors corresponding to Hb values.

15. By: M. Ishaq (A.P.) Nims University Rajasthan

16. By: M. Ishaq (A.P.) Nims University Rajasthan Tallquist Hb Chart This chart consists of a series of lithographic colors said to correspond to Hb values ranging from 10% to 100%. It is obsolete. Photoelectric Method Cyanmethemoglobin method Oxyhaemoglobin method

17. By: M. Ishaq (A.P.) Nims University Rajasthan Cyanmethemoglobin method: This is method of choice for estimation of Hb , recommended by ICSH. EQUIPMENTS & REAGENTS -Colorimeter/ Spectrometer - Sahli’s Pipette marked at 20 microns -5 ml pipette - Drabkins’s Solution -Standard soln. with known Hb Value Pot. Ferricyanide Pot. Cyanide Pot. Di-hydrogen Phosp. Non-ionic detergent Water

18. By: M. Ishaq (A.P.) Nims University Rajasthan Principle & method: Erythrocytes are lysed by non-ionic detergent and Hb released evenly. Pot. Ferricyanide converts Hb to methamoglobin . Methamoglobin reacts with Pot. Cyanide to form cyanmetHb . All forms are converted except SULF- Hb .

19. By: M. Ishaq (A.P.) Nims University Rajasthan Sample: Blood from Skin Puncture or EDTA anti-coagulated blood Take 5 ml of Drabkin solution in two test tubes each. Add 20 micro liter of blood and mix well Allow the tube to stand for at least 5 min. or more. Transfer the sample to cuvette and read the absorbance in Spectrometer at 540 nm. Hb is derived from the formula: Hb in grams/dl =Absorbance of test / Absorbance Of Standard X Conc.of standard X Dilution Factor / 100.

20. By: M. Ishaq (A.P.) Nims University Rajasthan OXYHEMOGLOBIN METHOD In this method blood sample is mixed with a weak ammonia solution. Absorbance of this solution is measured in a spectrophotometer at 540. Absorbance of this solution is compared with that of the standard solution. It is a rapid & simple, however derivatives other than oxyHb are not measured.

21. By: M. Ishaq (A.P.) Nims University Rajasthan Specific Gravity Method T his method gives approximate value of Hb . Method A drop of blood is allowed to fall in copper sulphate solution of specific gravity 1.053 from a height of 1 cm. Specific gravity of 1.053 is equivalent to Hb of 12.5 grams. This drop of blood is covered by CuSo4 and remains discrete for 15-20 seconds. If drop sinks within this time its specific gravity is higher. If it floats- specific gravity is low hence low Hb .

22. By: M. Ishaq (A.P.) Nims University Rajasthan THANK YOU . . .