HAEMOGLOBIN ESTIMATION-laboratory practices
- 2. HEMOGLOBIN metallochromoprotein Mol wt- 64, 468 dalton 90% of weight in a mature red cell Each molecule carries 4 02 atoms Each gram transports 1.34ml of o2 6.25 g of hemoglobin is synthesised everyday
- 3. structure Conjugated protein Tetramer; 2 pairs of globin chains – covalent – Heme complex( ferroprotoporphyrin IX) HEME COMPLEX ferrous iron with tetrapyrrole protoporphyrin IX Suspended in between E & F helices of globin B,G,H forms floor of pocket
- 4. Heme forms covalent bond with Imidazole nitrogen of proximal histidine @F8 Vander waals bond with other molecules If heme extracted- C,D,E,F helices of globin chains unfolds- decreased solubility
- 6. DERIVATIVES METHAEMOGLOBIN SULPHAEMOGLOBIN CARBOXYHAEMOGLOBIN METHAEMOGLOBIN iron in ferric state incapable of reversible binding wit O2 dark brown Normal – 1-2%
- 7. SULPHAEMOGLOBIN Green Cannot carries 02 Cant be measured by HiCN method Sulphonamides Irreversible Remains in the carrier state
- 8. CARBOXYHAEMOGLOBIN Formed by CO or CO2 Cherry red Affinity is 200 times than for 02 Reversible NORMAL RANGES General people- 0.16% Smokers & mine workers- 1- 10%
- 9. FUNCTIONS Imparts red color Buffers blood PH delivers O2 to tissues and CO2 from tissues to lungs
- 10. PURPOSE OF ESTIMATION Diagnose anaemia or polycythemia- severity, monitor response to treatment Detects O2 carrying capacity of blood Prior to donation Calculate red indices Detecting certain diseases
- 11. OBJECTIVE OF DONOR TESTING To prevent iatrogenic anaemia To ensure adequate yield and dose of red cell components Of being aware that he/she can become anaemic To provide proper medical guidance if found to be abnormal
- 12. NORMAL VALUES Men- 13- 18 gm/dl Women- 11- 16 gm/dl Full term/ cord blood- 13- 19 gm/dl Children 1yr- 11- 13 gm/dl Children 10 -12 years- 12- 15 gm/dl
- 13. PHYSICAL EXAMINATION Clinical examination Donor is physically fit Pallor – checked by medical officer ( looking onto conjunctiva, tongue and nail bed) Confirmed with various test available
- 14. METHODS OF ESTIMATION Colorimetric method/ Visual comparison Specific gravity method Chemical method Gasometric method Direct Spectrophotometry
- 15. VISUAL METHODS SAHLI’S METHOD DARES METHOD WHO HEMOGLOBIN COLOR SCALe HALDANE’S METHOD TALLQUIST’S METHOD SPENCER METHOD
- 16. SAHLI’S METHOD PRINCIPLE Haemoglobin Acid hematin by hydrochloric acid Acid hematin – diluted until it matches with comparator’s block Concentration of hemoglobin- read from calibration tube
- 17. REQUIREMENTS Haemoglobin meter- comparator, Haemoglobin tube, Haemoglobin pipette, stirrer N/10 Hcl Distilled water Dropper
- 18. Procedure Fill Sahli’s Hb tube up to mark 2 with N/10 HCl. Deliver 20 l (0.02 ml) of blood from a Hb pipette into μ it. Stir with a stirrer and wait for 10 minutes Add distilled water drop by drop and stir till colour matches with the comparator. Take the reading at upper meniscus
- 19. HAEMOGLOBINOMETER COMPARATOR Hb tube – in middle slot Non fading brown tinted glass pieces on either side Opaque white glass at back for uniform illumination Hb PIPETTE Has 20 cumm mark only No bulb
- 20. HAEMOGLOBINOMETER Hb TUBE K/A sahli- adam’s tube 2-24g% graduation on one side 20-140% graduation on other side
- 22. PROCEDURE Clean & dry out all the tube and pipette Fill Hb tube with N/10 Hcl upto lowest mark( 2g ) Prick finger with aseptic precaution DISCARD FIRST DROP NEVER SQUEEZE THE FINGER
- 23. PROCEDURE.. Draw blood up to 20 cumm mark of the pipette PREVENT ENTRY OF AIR BUBBLE if blood – sucked above 20 cumm bring it down by tapping pipette against finger ( not wit h cotton wool) Transfer 0.02ml from pipette to tube by blowing out Rinse it thrice by drawing up and blowing out
- 24. PROCEDURE... NO SOLUTION SHOULD REMAIN IN PIPETTE Allow it to stand – 10 min Dilute the acid hematin drop by drop with water; mix and match with comparator HOLD THE STIRRER ABOVE LEVEL OF SOLUTION NEVER TAKE STIRRER OUT OF THE TUBE
- 25. PROCEDURE Dilute it –until matches with std ; read & express as g% Lower meniscus- taken Add 1 drop of distilled water – observe- has to be lighter than standard - accurate
- 27. SOURCES OF ERROR TECHNICAL ERRORS Improper mixing errors in pipetting Tissue fluid contaminating capillary blood VISUAL ERRORS - reading is subjective
- 28. SOURCES OF ERROR Quality of color comparator Insufficient time Carboxy Hb, methHb, sulphHb cannot be read Protein, liquids & cell stroma- interferes in color Time delay in reading
- 29. ADVANTAGES Quick Easy to perform Cheap No technical expertise Used as bedside procedure
- 30. DISADVANTAGES Less accurate Lack of standard certain Hb cannot be read Color of acid hematin – develops slow
- 31. DISADVANTAGES Subjective reading If matching pt passed- repeat the whole procedure Color of comparator - fades
- 32. ALKALINE HEMATIN METHOD PRINCIPLE Haemoglobin alkaline hematin by adding N/10 NaOH Brown color read against a comparator
- 33. REQUIREMENTS Photoelectric meter with green filter N/1O NaOH 0.05 ml pipette
- 34. STANDARD SOLUTION Mixture of chromium, potassium, cobaltous sulphate & potassium dichromate in aqueous solution Equal to color in1 in 100 dilution of blood – Hb 16g/dl
- 35. METHOD Add o.05 ml of blood to 4.95 ml of NaOH Mix well & boil for 4 min with 5ml of standard solution Cool quickly in cold water, match it against standard with colorimeter using green filter If high value add 5 ml of water – read again
- 36. CALCULATION If OD of test- 21 & standard – 28 Standard – 16 g /100ml Test- 21/28 * 16= 12g/100ml 16 g in 100 ml is 100% So, 12/16* 100= 82%
- 37. Advantages Other Hb derivatives can be studied Determine foetal Hb in blood DISADVANTAGES Has to be heated for complete denaturation Matching – within 30 min after boiling
- 38. ACID – ALKALI METHOD In alkali method – solution of hemoglobin has to be heated for complete denaturation Can be omitted by collecting blood to acid first Stand for 30 min Add alkali- neutralises acid and converts acid hematin to alkali hematin
- 39. PROCEDURE Add 0.05 ml of blood to 4.95 ml of 0.1N Hcl Mix well Stand for 30 min Add 0.95 ml of 1N NaOH, invert it several times Allow standing for less than 2 min read it in photoelectric colorimeter with green yellow filter against Harrison & Gibson standard
- 40. HALDANE METHOD PRINCIPLE Haemoglobin carboxyhemoglobin by coal gas on diluted blood
- 41. REQUIREMENTS Haldane s graduated tube & standard 0.4% ammonia in distilled water Pipette- 0.02 ml
- 42. PROCEDURE Fill the graduated tube with ammoniac distilled water Add 0.02 ml of blood- mix Pass coal gas for 2-3 min by means of rubber tubin to Pasteur pipette for a steady state of gas supply
- 43. PROCEDURE dip the end of the pipette to caprylic acid and bubble the gas thro solution Add 0.4% ammonia drop by drop-mix wit each addition Read in daylight against standard
- 44. CALCULATION Read amount of solution in calibrated tube – as % Calculate amt of Hb in gm per 100 ml of blood Eg- if color standard- 14.6g/ 100ml reading is 95 100% = 14.6 g/ 100 ml 95% = 14.6 * 95/ 100=13.87 g per 100 ml of blood
- 45. DARES METHOD Undiluted blood spread between thin glass disc for direct matching inaccurate
- 46. TALLIQUIST SCALE METHOD Drop of blood- added on a filter paper Compared with standards High error- +20-50%
- 47. SPENCER METHOD Color of diluted oxyhaemoglobin is matched visually Less accurate than sahli More difficult for human eyes to match& grade small differences
- 48. WHO HEMOGLOBIN COLOR SCALE Drop of blood absorbed on a chromatography paper Compared against a printed scale of color Each values corresponds to different levels of Hb from 4- 14 g/ dl
- 49. advantages Simple Reliable for community Easier to use than cuSO4 METHOD
- 50. HAEMOCUE METHOD Quantitative; precalibrated battery operated spectrometer self filling disposable micro cuvette with reagents, control cuvette(verifying calibration of photometer), photometer( calibrated against HiCN method)
- 51. PRINCIPLE Converts Hb to methemoglobin OD of azide meth Hb – read @ 2 wavelength – 575and 880 nm
- 52. PROCEDURE Prepare the pulp of finger Keep the machine ready A microcuvette with the reagents(sodium nitrite & sodium azide) automatically draws a precise volume of blood Insert the cuvette into photometer-absorbance read @ 565 nm- results within 15-45 sec If procedure wrong- displays error
- 53. ADVANTAGES Quick Easy Reliable Cost efficient Used for mass screening in mobile camp Uses a single drop of blood
- 54. Automatically zeroes itself Automatically checks intensity of light & operation of photocells no blood dispensing, pipetting or mixing with reagents Unaffected by bilirubin, lipids or wbc Cuvettes be stored with drying agent in a temp of 15-30* C
- 55. SPECIFIC GRAVITY METHOD Haemoglobin- single large constituent of blood next is serum proteins Assumption is any change in the specific gravity of blood is d/t change in the concentration of hemoglobin
- 56. principle When a drop of blood is added to cuSO4, Copper proteinate is formed around the blood Prevents the drop from dispersing onto the solution sinks/ floats dep upon the specific gravity cuSO4 solutions of different specific gravity- dep upon the desired Hb cut off for donor
- 57. PREPARATION OF STOCK SOLUTION Dissolve 159.63g of pure air dried crystals in distilled water Make it upto 1000ml @ 25* c Specific gravity of solution -1.100
- 58. PREPARATION OF STOCK SOLUTION Prepared solution be stored in tight capped containers- prevent evaporation 30 ml of solution – 10 test Prepare working solution daily
- 59. SPECIFIC GRAVITY STOCK SOLUTION DISTILLED WATER Hb EQUIVALENT 1.052 51ml 100ml 12g 1.053 52 ml 100ml 12.5g 1.054 53ml 100ml 13g 1.055 54ml 100ml 13.4g
- 60. Method- 2 Dissolve 8.33 g of pure dried crystals in 100 ml of distilled water Specific gravity be 1.053 Functional validation of cuSO4 solution has to be done
- 61. PROCEDURE Prepare the pulp of finger Sterile lancet for prick – free flow of blood Drop be collected in a capillary tube or pipette Allow to fall from a height of 1cm above the surface of solution Cu proteinate prevents change in specific gravity for 15 sec
- 62. If drop is @ surface / rises few mm - drop is lighter- Hb is less than 12.5g/dl If it sinks immediately within 15 sec- Hb is higher than 12.5g
- 63. ADVANTAGES Quick, rapid, economical Solution cleanses itself after each test- encased drop settles 2 bottom as precipitate
- 64. SOURCES OF ERROR Taking 1 st drop of blood Squeezing the finger Dirty pipette Chipped delivering end of pipette Addition of > 25 drop- changes specific gravity of the solution NOTE- if plasma protein levels are below normal- donor be rejected- specific gravity is below normal
- 65. CHEMICAL METHOD Based on the fact- each molecule of Hb has 4 atoms of iron or 0.347 g of iron per 100 g of hemoglobin Iron is detached and measured Complex method
- 66. Hb is calculated – blood iron content / 3.47 never done as routine time consuming Accurate, used as REFERENCE method for cyan meth hb method
- 67. GASOMETRIC METHOD Using van slyke apparatus Reference method Very accurate Not done as routine 02 carrying capacity of Hb
- 68. Principle one molecule of hemoglobin binds 4 molecules of o2 Thus O2 carrying capacity indirectly measures amount of hemoglobin 1g of hemoglobin combines with1.34ml of O2
- 69. Hb in gm/dl=O2 binding capacityin ml/dl / 1.34 DISADVANTAGE can measure only functional hemoglobin other derivatives cant be measured
- 70. SPECTROPHOTOMETRY Based on BEER- LAMBERT LAW The optical density of a colored solution is directly proportional to the concentration of the colored material in the solution and the pathlength Path length is diameter of cuvette here It is always constant as 1cm CYANMETHEMOGLOBIN METHOD OXYHEMOGLOBIN METHOD
- 71. CYANMETHEMOGLOBIN METHOD Preferred Most accurate Internationally accepted
- 72. PRINCIPLE Blood is diluted with KCN and potassium ferricyanide ferricyanide oxidises hemoglobin in ferrous state- converts to methemoglobin KCN provides cyanide ions – HiCN Absorbance measured at 540nm or Photoelectric colorimeter wit a green yellow filter
- 73. DILUENT MODIFIED DRABKIN REAGENT KCN- 50 mg Potassium ferricyanide- 200 mg Potassium dihydrogen phosphate- 140 mg Nonionic detergent- 1ml Distilled water-1L NOTE- in place of nonionic detergent, saponin-1ml,triton-X- 100 1ml or steroxSE -0.5 ml can be used
- 74. PROPERTIES Must be clear Pale yellow Ph- 7- 7.4 When measured against water as blank , absorbance must be zero Stored @ room temperature DISCARD – If ph> 7,4,absorbance> 0. turbid Never to be frozen
- 75. ADVANTAGES Less turbidity Lesser conversion time( 10 min to 3 min ) Detergent- complete RBC lysis Ensures complete conversion
- 76. METHOD Make 1 in 201 dilution of blood( 20microliter of blood with 4ml of diluent) Stopper the solution and invert it several times Let it stand @ room temp for 5min Pour into cuvette & read absorbance@ 540nm / photoelectric colorimeter with green yellow filter Measured within 6hrs of initial dilution
- 77. calculation Absorbance of commercially available HiCN standard be compared with reagent blank CALCULATION Hb = A 540 of test sample/A 540 of standard* concentration of standard*dilution factor/ 1000 Standard graph – for many samples Absorbance on Y axis Concentration of Hb on X axis
- 78. ADVANTAGES All forms are convertable except sulphemoglobin Direct comparison with standard Stability of diluted sample Easy to perform Reagents are readily available Stable standard
- 79. DISADVANTAGES Increased absorbance- turbidity due to abnormal plasma protein Increased conversion time- hyperlipidaemia, improper RBC lysis KCN – toxic; sodium azide & sodium lauryl sulphate- used in automated system Explosion- if undiluted reagents poured in sink- always be disposed along with running water
- 80. OXYHAEMOGLOBIN METHOD PRINCIPLE Blood – diluted with weak alkali( 0.04% ammonium hydroxide; sp gravity-0.88) Lysis of RBC’ : oxyhemoglobin is released Complete conversion Immediate reaction Stable color
- 81. STANDARD Hb value of normal anti- coagulated blood- using HiCN method Blood - diluted as 1in 201 ( 20 microliter with 4ml of ammonium hydroxide) Serial dilutions made in ammonia ; absorbance read @ 540nm : plotted as graph Neutral grey screen of 0.475- be used as 14.6g/dl( 100%) standard
- 82. METHOD Add 0.02 ml of blood in a tube with 4 ml of 0.04% ammonia Mix and invert several times Allow it to stand @ room temperature – 10 min Read absorbance value @ 540nm or with colorimeter using green yellow filter If absorbance > 7- blood be diluted again wit equal volume of water
- 83. ADVANTAGES Simple Quick and accurate than visual comparison method Stable color is formed DISADVANTAGES Meth Hb and carboxy Hb cannot be read accurately No standard solution
- 84. DIRECT SPECTROPHOTOMETRY without need of standard be previously calibrated
- 85. METHOD Blood diluted with 1: 250 with cyanide- ferricyanide reagent stopper the tube: invert it Allow to stand @ room temp for 10 min Read absorbance @ 540 nm
- 86. CALCULATION Hb- A540 HiCN* 16114* dilution factor/ 11* d*1000 Where 16114 – mol wt of Hb 11- millimolar coefficient extinction D- layer of thickness in cm 1000- conversion of milligram to gram
- 87. SOURCES OF ERRORS ERRORS IN SAMPLING Inadequate flow of blood Excessive squeezing of finger Prolonged use of torniquet- concentrated red cells Insufficient mixing-forms sediment Adding too little blood to drabkin soluion Air bubbles trapped in pipette
- 88. Sources of error.... Reagents on exposure to extremes of temperature Reference preparation- out of date or detoriated – if left open for a long time FAULTY EQUIPMENTS broken or chipped pipettes, dirty cuvettes,dirty filters Defective spectrophotometer or colorimeter
- 89. Faulty technique Using a dilution factor different from the one proposed Inadequate mixing of treagents Air bubbles in cuvette Using a standard filter from another spectrophotometer
- 90. Faulty tech Non linearity’ Cuvettes- dirty, scratched, improperly positioned Improper calibration Main voltage variation Patient conditions- hyperbilirubinemia, hyperprotenemia, leucocytosis
- 91. WAYS TO MINIMISE Adequate training Adherence to oral and written instructions Familiarity wit equipments Use of automated machines
- 92. QUALITY CONTROL To identify those steps – errors are high and care be taken to minimise Measured followed are Duplicating the samples Hemolysate of known values run with batches of test Hb values – compared with others ; eg- PCV= 3* Hb Check peripheral smear- if values are too abnormal
- 93. ANAEMIA POLYCYTHEMIA Iron deficiency High altitude Lead poisoning Polycythemia vera Drugs Smoking Parasitic infestations copd CKD Pulmonary hypertension Cu deficiency CHF OSAS EPO secreting tumors
Editor's Notes
- #55: Haemoglobin- single lae