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2.  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%

12. 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

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

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

25. 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

26. 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

27. • 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

28. 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

29. …..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)

30. 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

31. ….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

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

33. 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

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

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

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

38. CLINICAL CONDITIONS
DECREASED – ANEMIA AND IRON DEFICIENCY
ANEMIA
INCREASED – POLYCYTHAEMIA

39. 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

40. 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

41. 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)