Determination of haemoglobin

Determination of haemoglobin and PCV

1. DR. ANINDITA SAHA DETERMINATION OF HAEMOGLOBIN

2. Haemoglobin It is an iron-containing oxygen-transport  metalloprotein  in the red blood cells. Haemoglobin in the blood carries oxygen from the respiratory organs (lungs) to the rest of the body (i.e. the tissues). There it releases the oxygen to permit aerobic respiration to provide energy to power the functions of the organism in the process called metabolism.

3. Structure of haemoglobin: Haemoglobin is a conjugated protein. Haemoglobin = Heme + globin (Conjugated protein) (prosthetic group) (protein)

4. Heme contains ferrous ion (Fe 2+ ) in the centre of a large heterocyclic ring called porphyrin ring made up of 4 pyrrole group. It gives red colour to the blood.

5. Different Hbs are produced during different phases of life like embryonic , fetal and adult. Each consists of tetramer of globin polypeptide chain. . Adult Hb – alpha 2 beta 2 ( Hb A ) alpha 2 delta 2 ( Hb A 2 ) Fetal Hb - alpha 2 gamma 2 ( Hb F)

6. METHODS OF HAEMOGLOBIN ESTIMATION Haemoglobin concentration can be estimated by Measurement of its colour( colorimetric method ) Property of combining with oxygen Property of combining with carbon monoxide Its iron content Measurement of specific gravity

7. COLORIMETRIC METHOD This method is based on colorimetric measurement of the intensity of colour developed on addition of some substances to the blood.

8. This method includes the following : 1) cyanmethaemoglobin method(most accurate) 2) oxyhaemoglobin method 3) electronic counter method 4) direct reading electronic haemoglobinometer 5) Sahli’s method

9. SAHLI’S METHOD EQUIPMENTS : 1) Sahli’s haemoglobinometer comparator 2) Sahli’s haemoglobinometer tube 3) Sahli’s pipette or haemoglobin pipette 4) glass dropper 5) glass rod for stirring

10. Sahli’s haemoglobinometer

11. Sahli’s haemoglobinometer tube I t is a graduated tube marked on ascending order on both sides. one side – shows grams per 100 ml (gm%) opposite side – percenatage 100% = 14.5gm%

12. Haemoglobin comparator It is used for matching the colour of acid haematin formed in the haemoglobinometer tube.

13. Haemoglobin pipette It is a slender special pipette with a single mark 20 cumm . USE: used for quantitative estimation of haemoglobin in blood.

14. Haemoglobin pipette

15. REAGENTS : 1) N/10 HCl 2) distilled water

16. PRINCIPLE : Hb N/10 HCl acid haematin (present in blood) (brown in colour) This brown colour is diluted with distilled water to match that of brown glass standard and Hb value is read directly in grams from the scale of haemoglobinometer .

17. PROCEDURE: Fill sahli’s Hb tube upto mark 2 with N/10 HCl Put 20 ul (0.02 ml) of blood from Hb pipette Stir with stirrer 10 min Add distilled water drop by drop and stir till colour matches with comparator and take reading at lower meniscus

18. Advantages: 1)Simple bedside test 2) reagents and apparatus are cheap.

19. Disadvantages: There can be visual error Colour of acid hematin is not stable . It fades after reaching its peak. Comparator can fade over years. Carboxy , met and sulfhaemoglobin cannot be converted to acid haematin. This method is not suitable for infants as fetal haemoglobin is also not converted to acid haematin.

20. ELECTRONIC COUNTER METHOD It is a multiparameter determining electronic equipment. Instrument is sysmex series of instrument. Hb is measured in a designated channel using sodium lauryl sulphate (SLS)method. Blood is diluted using reagent diluent (isotonic conductive) and then SLS haemoglobin reagent is added.

21. PRINCIPLE: works on electrical impedence principle. Surfactants within SLS reagent lyse RBCs and release Hb and reduce turbidity developing from cell membrane and plasma lipids. SLS converts Fe 2+ to Fe 3+ forming methaemoglobin . Methemoglobin combines with SLS to form SLShemichrome molecule. The absorbance of this molecule is measured at 555nm to determine Hb concentration(gm/dl) using Beer’s law.

22. ADVANTAGES: Free Hb is rapidly converted to detectable chromogen , decreasing measurement time. Reagent is cyanide free(toxic chemical – environmental hazard) DISADVANTAGE: 1) High WBC count (> 30,000/ ul ) causes false elevation of Hb .

23. Normal haemoglobin values: At birth – 13.5 to 19.5 gm/dl Children (2-5 yrs) – 11 to 14 gm/dl Children (6 – 12 yrs) – 11.5 to 15.5 gm/dl Adult female – 13 to 15 gm/dl Adult male – 14 to 16 gm/dl

24. Conditions of decreased haemoglobin: Anaemia Pregnancy Conditions of increased haemoglobin: Physiological: 1)new borns and infants Pathological : 1) polycythemia ( polycythemivera,kidney tumours,high altitude) 2) dehydration 3 ) hypoxia(COPD, congenital heart disease)

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26. DETERMINATION OF PCV

27. DEFINITION: Packed cell volume is defined as ratio of volume of packed RBCs to that of whole blood . It is the volume occupied by red blood cells when anticoagulated blood is centrifuged. Expressed in percentage.

28. ESTIMATION OF PCV IS HELPFUL FOR: 1) determination of red cell indices like MCV , MCHC 2)Diagnosis of anaemia or polycythemia 3)To counter check the value of Hb in the reported sample as Hb in gm/dl x 3 = PCV. 4) Useful as a screening test for anaemia.

29. METHODS: Macro method ( Wintrobe’s tube method ) Micro method ( capillary tube method ) Automated method

30. WINTROBE’S METHOD: Apparatus required: 1) wintrobe’s tube 2)Pasteur pipette with a rubber teat sufficiently long to reach the bottom of the wintrobe tube. 3)Centrifuge machine

31. Wintrobe’s tube: Special thick walled glass tube Length – 110 mm Internal diameter – 3 mm Flat inner base It is graduated from 0 ( bottom ) to 10 (top ) for PCV and 0 ( top ) to 10 ( bottom ) for ESR. The markings are at an interval of 1mm each and holds 1 ml of blood.

32. USES OF WINTROBE’S TUBE : 1) Determination of PCV. 2)Determination of ESR. 3)Buffy coat smear preparation for LE cell phenomenon and studies of abnormal cells in aleukemic leukemia .

33. Wintrobe’s tube

34. Pasteur pipette

35. Procedure: Fill the pasteur pipette with very well mixed anticoagulated blood . Introduce the pipette at the bottom of the wintrobe tube and slowly fill it upto 10 mark. Tip of pipette should remain below the rising meniscus of blood to avoid foaming. Centrifuge the tube at 3000 rpm for 30 min. After centrifugation layers are noted.

36. Upper most layer of PLASMA (yellow) Middle narrow layer of BUFFYCOAT comprised of WBCs and platelets Lower most layer of PACKED RBCs(red) Note the lower most height of coloumn of packed RBC layer and express in percentage.

37. AUTOMATIC ELECTONIC ANALYSER In multi channel coulter counter , PCV can be estimated with other hematologic indices in 40 – 50 seconds.

38. NORMAL VALUE: Adult male – 40 – 50% Adult female – 38 – 45% New born - 44 – 60%

39. CAUSES OF INCREASED PCV : polycythaemia dehydration due to severe vomiting , diarrhoea burns shock CAUSE OF DECREASED PCV: anaemia pregnancy

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