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Haemoglobin estimation ,different methods and normal values
 HAEMOGLOBIN IS A COMPLEX PROTEIN PRESENT IN
THE RED BLOOD CELLS
 WHICH GIVES RED COLOUR TO THE BLOOD
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
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
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
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
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.)
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
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)
RESULTS
• The CONCENTRATION OF Hb ESTIMATION ARE
EXPRESSED AS (Eg.12)g/dl OR G%
Haemoglobin estimation ,different methods and 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
Haemoglobin estimation ,different methods and normal values
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
Haemoglobin estimation ,different methods and normal values
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
…..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
….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
…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
….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
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
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
Haemoglobin estimation ,different methods and normal values
Haemoglobin estimation ,different methods and normal values
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
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
• 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
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
…..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)
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
….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
DISADVANTAGES OF THE METHOD
• TURBIDITY INTERFERS WITH THE READINGS
• ANY TURBIDITY DUE TO ABNORMAL PLASMA
PROTEINS
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
Haemoglobin estimation ,different methods and normal values
1.COLORIMETRIC METHODS OF ESTIMATING HAEMOGLOBIN
 TALLQVIST METHOD
 SAHIL’S METHOD OR ACID HAEMATIN
 HADEN –HAUSER HAEMOGLOBINOMETER
OTHER OLD METHODS
 HALDANE METHOD USING CARBOXYHAEMOGLOBIN
 DARE METHOD
 SPENCER METHOD
 PHOTOMETRIC METHODS
 OXYHAEMOGLOBIN METHOD
 CYANMETHAEMOGLOBIN OR HAEMIGLOBINCYANIDE
(HCN)METHOD
 CYANMETHAEMOGLOBIN SOLUTION(DRABKIN’S SOLUTION)
2.PHYSICAL METHOD –SPECIFIC GRAVITY
3.CHEMICAL METHOD- BLOOD IRON CONTENT
4.GASTROMETRIC METHOD – OXYGEN
COMBAINING CAPACITY
CLINICAL CONDITIONS
DECREASED – ANEMIA AND IRON DEFICIENCY
ANEMIA
INCREASED – POLYCYTHAEMIA
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
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
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)
Haemoglobin estimation ,different methods and normal values

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Haemoglobin estimation ,different methods and normal values

  • 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)