Haemoglobin estimation ,different methods and normal values

health science courses

1. HAEMOGLOBIN ESTIMATION, DIFFERENT METHOD, NORMAL VALUES, MERITS AND DEMERITS

2. HAEMOGLOBIN ESTIMATION HAEMOGLOBIN IS A COMPLEX PROTEIN PRESENT IN THE RED BLOOD CELLS WHICH GIVES RED COLOUR TO THE BLOOD

3. SAHLI’S METHOD or ACID HAEMATIN METHOD EQUIPMENT REQUIRED SAHLIS HAEMOMETER GRADUATED DILUTION TUBE(PRESENT IN HAEMOMETER) SPIRIT OR 70% ALCOHOL N/10 HCL (1ml + 9 ml distilled water) Cotton Glass rod(stirrer) EDTA Distilled water

4. PRINCIPLE WHEN THE BLOOD IS MIXED WITH N/10 HCL, THE RED BLOOD CELLS GETS RUPUTRED AND THE HAEMOGLOBIN CONTENT IN THEM COMES OUT INTO N/10 HCL . THE HAEMOGLOBIN GETS CONVERTED INTO ACID HAEMATIN WHOSE COLOUR IS COMPARED WITH NON-FADING COLOURED TUBES PRESENT IN SAHLIS HAEMOMETER. THE MOST APPROPRIATE MATCHING COLOUR INDICATES THE HAEMOGLOBIN CONTENT OF BLOOD SAMPLES THIS IS KNOWN AS COLOUR INDEX METHOD

5. PROCEDURE PLACE N/10 HCL INTO THE TUBE UP TO THE LOWEST MARK DRAW BLOOD UP TO THE 20 µL MARK IN THE SAHIL PIPETTE, AND AFTER WIPING ALL TRACES OF BLOOD FROM THE OUTSIDE OF THE PIPETTE TRANSFER THE BLOOD INTO THE HCL IN THE TUBE

6. PROCEDURE RINSE THE PIPETTE WELL BY DRAWING UP SOME OF THE ACID AND THEN RE-EXPRESSING IT MIX THE ACID AND BLOOD BY SHAKING THE TUBE WELL AND THEN ALLOW THE TUBE TO SIT FOR ATLEAST 10 MINUTES ALLOW THE BROWN COLOUR TO DEVELOP

7. PROCEDURE (MAXIMUM COLOUR IS REACHED AFTER ABOUT 1 HOUR 95 % OF THE COLOUR BY THE END OF 10 MINUTES . IT IS IMPORTANT TO WAIT AT LEAST 10 MINUTES BEFORE PROCEEDING.)

8. PROCEDURE THEN DILUTE THE SOLUTION IN THE TUBE BY ADDING A FEW DROPS OF DISTILLED WATER AT A TIME MIXING AND THEN COMPARING THE COLOUR OF THE FLUID WITH THE COLOUR OF THE GLASS BLOCKS, UNTIL THE COLOUR MATCHES BE SURE TO MIX THE SOLUTION WELL AFTER EACH ADDITION OF WATER USING THE SMALL GLASS ROD WHICH IS PROVIDED

9. PROCEDURE THE ROD SHOULD BE LIFTED UP OUT OF THE SOLUTION WHEN THE COLOUR COMPARISON IS MADE THE MATCHING SHOULD BE ONLY AGAINST NATURAL LIGHT (OR THE LIGHT FROM FLUORESCENT TUBE LIGHTS)

10. RESULTS The CONCENTRATION OF Hb ESTIMATION ARE EXPRESSED AS (Eg.12)g/dl OR G%

11. NORMAL VALUES

12. NORMAL VALUES THE NORMAL HAEMOGLOBIN CONTENT OF BLOOD FROM INDIVIDULAS TO INDIVIDUALS MEN -13 TO 15 g%(AVERAGE -14.5%) WOMEN – 12 TO 15 g% (AVERAGE -13.5%) AT BIRTH THE HAEMOGLOBIN CONCENTRATION OF CORD BLOOD IS HIGH- 17-20g/dl

13. MERITS AND DEMERITS OF SAHLI’s METHOD

14. MERITS AND DEMERITS OF SAHLI’s METHOD 1.ACID HAEMATIN METHOD(SAHLI’S METHOD) MERITS RECOMMENDED FOR PLACES WHERE COLORIMETER OR SPECTROPHOTOMETER IS NOT AVAILABLE BLOOD + N/10 HCL = ACID HAEMATIN DEMERITS ALL TYPES OF HB NOT CONVERTED IN ACID HAEMATIN THE RESULTS NOT VERY ACCURATE COLOUR OF ACID HAEMATIN IS NOT STABLE IT FADES,READINGS TAKEN LITTLE LATE ARE NOT ACCURATE VISUAL ERRORS DUE TO VISUAL COMPARISON OF ACID HAEMATIN WITH COMPARATOR

15. SOURCE OF ERROR

16. SOURCES OF ERROR A . TECHNICAL ERRORS: THESE ARE ERRORS INHERENT IN HANDLING THE SAMPLE IMPROPER COLLECTIONS IMPROPER VENIPUNCTURE TECHNIQUE MAY PRODUCE HEMOCONCENTRATION RESULTING IN HIGH HEMOGLOBIN VALUE IN CASE OF FINGER PRICK COLLECTIONS, IMPROPER TECHNIQUES OR EXCESSIVE SQUEEZING RESULTS IN TISSUE FLUID CONTAMINATING CAPILLARY BLOOD WITH CORRESPONDING ERROR IN THE HB VALUE

17. …..continued IMPROPER MIXING OF BLOOD SAMPLE IF BLOOD IS TAKEN FROM EDTA VIAL IT SHOULD BE MIXED PROPERLY BEFORE FILLING THE PIPETTE. IF NOT, DEPENDING ON WHETHER THE PLASMA OR THE SEDIMENTED RBCS HAVE BEEN ASPIRATED IN THE HB PIPETTE,IT MAY GIVE RISE TO FALSE LOW OR HIGH HEMOGLOBIN VALUE

18. ….continued INSUFFICIENT TIME FOR FORMATION OF HEMATIN A MINIMUM OF 10 MINUTES IS REQUIRED FOR ALMOST COMPLETE CONVERSION OF HB TO ACID HEMATIC IF READING IS TAKEN BEFORE,IT CAN GIVE FALSE READING

19. …continued TIME DELAY BROWN COLOR OF ACID HEMATIN IS NOT STABLE AND THE COLOR GRADUALLY STARTS FADING DELAY IN TAKING THE READING RESULTS IN ERRORS B.VISUAL ERRORS COMPARISON OF COLORS IS VERY SUBJECTIVE AND CAN VARY FROM PERSON TO PERSON & ALSO ON THE SOURCE OF LIGHT (I.E DAY LIGHT /ARTIFICAL) THE RESULTS MAY NOT BE ACCURATE

20. ….continued c. ERRORS INHERENT IN THE EQUIPMENT ACCURACY OF DIFFERENT EQUIPMENTS IS NOT UNIFORM IF HB PIPETTE HAS NOT BEEN ACCURATELY CALIBRATED AT 0.02ML IT CAN GIVE RISE TO WRONG VALUES CALIBRATION OF PIPETTES WILL REDUCE THE ERRORS COLOR COMPARATORS CAN AFFECT THE READING. IF THE GLASS BLOCKS OF THE COMPARATORS ARE OLD OR FADED,IT MAY GIVE AN INACCURATE VALUE

21. CYANMETHMOGLOBIN (HEMIGLOBIN CYANIDE HICN) METHOD CYANMETHEMOGLOBIN IS THE MOST ACCURATE, CONVENIENT,READILY AVAILABLE AND PREFERRED METHOD FOR ESTIMATION OF HEMOGLOBIN CONCENTRATION IT IS THE STANDARD METHOD USED IN MOST OF THE CENTERS

22. PRINCIPLE BLOOD IS DILUTED IN A STABLE STANDARD SOLUTION OF POTASSIUM CYANIDE.HEMOGLOBIN IS FIRST OXIDIZED TO METHEMOGLOBIN(HI) BY THE POTASSIUM FERRICYANIDE THE CYANIDE IONS OF POTASSIUM CYANIDE CONVERT METHEMOGLOBIN (HI) TO STABLE CYANMETHEMOGLOBIN (HICN) THE CYANMETHEMOGLOBIN HAS A BROAD ABSORPTION,MAXIMUM BEING AT A WAVELENGTH OF 540 nm THUS, THE COLOR OF THIS SOLUTION IS COMPARED AGAINST A STANDARD HICN SOLUTION OF KNOWN HB VALUE IN A SPECTROPHOTOMETER OR PHOTOELECTRIC COLORIMETER AT 540 nm

23. APPARATUS PHOTOELECTRIC COLORIMETER WITH A GREEN FILTER OR SPECTROPHOTOMETER AT A WAVELENGTH OF 540nm IS USED OTHER EQUIPMENTS REQUIRED ARE SAHLI’S PIPETTE (MARK OF 20NM) & 5 ml PIPETTE

24. REAGENTS DILUENT IS DETERGENT –MODIFIED DRABKIN SOLUTION CONSTITUENTS OF DRABKIN SOLUTION POTASSIUM FERRICYANIDE -400mg POTASSIUM CYANIDE (KCN) -100mg POTASSIUM DIHYDROGEN PHOSPHATE (ANHYDROUS) – 280 mg NON –IONIC DETERGENT (STEROX SE) -1 ml DISTILLED WATER – 2 Liters

25. DRABKIN SOLUTION SHOULD BE CLEAR AND PALE YELLOW WHEN MEASURED IN THE PHOTOELECTRIC COLORIMETER SHOULD GIVE ZERO READING AT 540 nm AND WATER BLANK THE DETERGENT ENHANCES LYSIS OF RBCS AND DECREASES TURBIDITY DUE TO PRECIPITATION OF PROTEINS

26. Techniques TAKE 5 ml OF DRABKIN SOLUTION IN A TEST TUBE BLOOD SAMPLE REQUIRED IS EITHER BLOOD COLLECTED IN EDTA OR FROM SKIN PUNCTURE TAKE TWENTY MICROLITER OF BLOOD IN A HEMOGLOBIN PIPETTE & ADD IT TO THE ABOVE TEST TUBE RINSE THE HB PIPETTE AT LEAST TWICE BY DRAWING IN DRABKIN SOLUTION

27. …..continued STOPPER THE TEST TUBE AND THOROUGHLY MIX THE BLOOD WITH DRABKIN SOLUTION ALLOW IT TO STAND FOR 5 ( AT LEAST 3) MINUTES AT ROOM TEMPERATURE THIS TIME IS ADEQUATE FOR CONVERSION OF HEMOGLOBIN INTO CYANMETHEMOGLOBIN POUR TEST SOLUTION INTO THE CUVETTE READ THE ABSORBANCE OF THE TEST SAMPLE IN THE PHOTOELECTRIC COLORIMETER AT 540NM OR WITH AN APPROPRIATE FILTER (GREEN FILTER)

28. ADVANTAGES OF THE METHOD Hb VALUE OBTAINED IS ACCURATE SINCE ALMOST ALL FORMS OF HEMOGLOBIN(HEMOGLOBIN,OXYHEMOGLOBIN,METHMOGLOBIN,CARBOXYHEMOGLOBIN,BUT NOT SULPHEMOGLOBIN) ARE CONVERTED INTO CYANMETHMOGLOBIN BEING A COLORIMETRIC METHOD THERE IS DIRECT COMAPRISON WITH HICN STANDARD VISUAL ERROR DURING MATCHING THE COLOR LIKE IN SAHLI’S METHOD IS ELIMINATED

29. ….CONTINUED CYANMETHEMOGLOBIN IS A STABLE COMPOUND AND COLOR DOES NOT CHANGE WITH TIME SO READINGS CAN BE MADE AT THE OPERATOR’S CONVENIENCE DELAY IN TAKING READING DOES NOT ALTER THE VALUE EASY TO PERFORM THE TEST STANDARD IS STABLE AND CERTIFIED CYANMETHEMOGLOBIN STANDARDS ARE AVAILABLE REAGENTS ARE READILY AVAILABLE

30. DISADVANTAGES OF THE METHOD TURBIDITY INTERFERS WITH THE READINGS ANY TURBIDITY DUE TO ABNORMAL PLASMA PROTEINS

31. Automated analyzer (cell counter) INSTRUMENTS MEASURE NOT ONLY HEMOGLOBIN ALSO VARIOUS PARAMETERS LIKE CELL COUNTS,(PACKED CELL VOLUME (PCV) AND ABSOLUTE VALUES MEAN CORPUSCULAR VOLUME – MCV MEAN CORPUSCULAR HEMOGLOBIN – MCH MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION – MCHC

32. DIFFERENT METHOD

33. DIFFERENT METHOD 1.COLORIMETRIC METHODS OF ESTIMATING HAEMOGLOBIN TALLQVIST METHOD SAHIL’S METHOD OR ACID HAEMATIN HADEN –HAUSER HAEMOGLOBINOMETER OTHER OLD METHODS HALDANE METHOD USING CARBOXYHAEMOGLOBIN

34. DIFFERENT METHOD DARE METHOD SPENCER METHOD PHOTOMETRIC METHODS OXYHAEMOGLOBIN METHOD CYANMETHAEMOGLOBIN OR HAEMIGLOBINCYANIDE (HCN)METHOD CYANMETHAEMOGLOBIN SOLUTION(DRABKIN’S SOLUTION)

35. DIFFERENT METHOD 2.PHYSICAL METHOD –SPECIFIC GRAVITY 3.CHEMICAL METHOD- BLOOD IRON CONTENT 4.GASTROMETRIC METHOD – OXYGEN COMBAINING CAPACITY

36. CLINICAL CONDITIONS DECREASED – ANEMIA AND IRON DEFICIENCY ANEMIA INCREASED – POLYCYTHAEMIA

37. PREVIOUS YEAR QUESTIONS NAME THE DIFFERENT METHODS OF HEMOGLOBIN ESTIMATION.DESCRIBE SAHLI’S METHOD IN BRIEF ?(5 MARKS)APRIL 2017 PRINCIPLE OF CYANMETHEMOGLOBIN METHOD OF HAEMOGLOBIN ESTIMATION ?(3 MARKS ) APRIL 2015

38. PREVIOUS YEAR QUESTIONS NAME THE VARIOUS METHODS USED IN ESTIMATION OF HAEMOGLOBIN .DESCRIBE IN DETAIL ABOUT SAHLI’S METHOD OF Hb ESTIMATION ?(5 MARKS) AUGUST 2013 METHODS OF Hb ESTIMATION?(3 MARKS ) MARCH 2013

39. PREVIOUS YEAR QUESTION PAPER NAME DIFFERENT METHODS OF HAEMOGLOBIN ESTIMATION .DESCRIBE SAHLI’S METHOD(AUGUST/SEPTEMBER 2011) NAME DIFFERENT METHODS OF HAEMOGLOBIN ESTIMATION .(FEB/MARCH 2011)

40. COMPLETED HAEMOGLOBIN ESTIMATION,DIFFERENT METHOD, NORMAL VALUES,MERITS AND DEMERITS