Hemoglobin estimation dr sandeep

basic of hb estimation all under graduate student

1. Hemoglobin Estimation

2. (RBC) 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 3.47 mg /100 g. Main role to transport oxygen.

3. Various forms of Hb Oxyhemoglobin ( Hb O2) Carboxy hemoglobin ( Hb CO) Sulfhemoglobin ( SHb ) Methaemoglobin - Fe 3+  (ferric)

4. Variants of Hb Normal : Hb F , Hb A , Hb A2-delta Abnormal: HbS , HbC , HbD & HbE Embryo are two zeta and two epsilon chains Fetal hemoglobin Haemoglobin F ( α 2 γ 2 ) . adult - Haemoglobin A (α 2 β 2 ) – Has two alpha chains and two beta chains, Haemoglobin A 2  (α 2 δ 2 ). 97% HBA adult

5. Samples 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.

6. Normal Values of Hb   Men 13.0 to 16.0 g/dl Women 11.0 to 15.0 g/dl Infants 16.0 to 19.0 g/dl   Critical Values-   Less than 7 gm/dl- Severe Anemia More than 20 gm/dl- Hperviscosity

7. METHODS FOR ESTIMATION OF HAEMOGLOBIN The measurement of concentration of Hb in the blood is known as HEMOGLOBINOMETRY. Colorimetric method Gasometric method. Specific gravity method Chemical method.

8. Colorimetric method: Colorimetric method is based on colorimetric measurement of the intensity of color developed on addition of some substance to the blood. values are measured by comparing with known standard. Include the following: A. Visual Methods B. Photoelectric Methods

9.   Visual Methods   Sahli’s method (Acid haematin method) Tallquist chart ( color comparison) obsolete Hemoglobin Color Scale obsolete Photoelectric Method Cyanmethemoglobin . Oxyhaemoglobin & Alkaline Hematin

10. Commonly used methods   Sahli’s Method – A Color Based Method Visual 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.  

11. Equipment : Sahli’s Hemaglobinometer consisting of : Graduated hemoglobin tube Comparator box with a brown glass standard Hb Pipette Stirrer Dropper (dropping pipette) REAGENTS : a. N/10 HCl b. Distilled Water Sample: EDTA anticoagulated venous blood. Blood obtained by skin puncture

12. 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.

13. ADVANTAGES: Simple bedside test no colorimeter is required. 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 appearing of acid hematin takes long time and also fades quickly. Source of light (day light or artificial) influences the color comparison.

14. 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. 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 . B. Photoelectric Method Alkaline haematin Oxyhaemoglobin Cyanmethemoglobin

16. Alkaline haematin N/10 NaOH solution is used to convert alkaline haematin read under Spectrometer Advantages- Stable solution reched . Disadvantage- Not reliable in infant and newborn. Alkalin resistant. Less accurate.

17. Oxyhaemoglobin method Hb is converted into oxyhaemoglobin by 0.04% ammonia solution . Measured by spectrophotometer at 540 nm. Advantage- Reading done in few seconds. And stable for 3 days. Disadvantage - derivatives other than oxyHb are not measured.

18. Cyanmethemoglobin method:This is method of choice for estimation of Hb , recommended by ICSH (International committee for standardisation in haematology ) .   Principle – Blood is diluted in a solution called drabkin’s fluid containing   Pot. Ferricyanide converts Hb to methamoglobin . Methamoglobin reacts with Pot. Cyanide to form cyanmetHb . All forms are converted except SULF- Hb .

19. 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. Read in Spectrometer at 540 nm .  

20. Advantage- Less error, all forms of hemoglobin estimated except sulphaemoglobin . Disadvantage- Hyperbilirubinemia affect value. Turbidity effect.

21. Gasometric method It is indirect method, which estimates the amount of haemoglobin from the amount of oxygen it will absorb. It is accurate method but too complicated for clinical work.

22. Specific Gravity Method This 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 . If drop sinks within this time its specific gravity is higher. If it floats- specific gravity is low hence low Hb . Normal specific gravity of blood ranges from 1.048-1.066.