Hemoglobinometry- Flashcards
• Done separately or as part of routine CBC
• Relatively simple
• Performed quickly by the laboratory
Hemoglobin determination
Indications for Hb estimation
• Determine presence and severity of anemia
• Screening for polycythemia
• Response to specific therapy in anemia
• Estimation of red cell indices
• Selection of blood donors
WHO defines anemia as Hb levels of_____ in women and_____ in men
< 12 g/dL
<13 g/dL
Physiological Factors
•Age
•Gender
•Pregnancy
•Exercise
•Posture (from lying to sitting/standing)
•Altitude
•Smoking
Pathological Factors
• Testosterone deficiency
• Renal deficiency
• Myelodysplasia
• Diminished erythropoiesis
• BM suppression
• Blood loss
Normal Haemoglobin concentration in humans
• Men -
• Women(non pregnant) -
150-20 g/l OR 13-18 g/dL
135 +/- 15 g/l OR 11-16 g/dL
Pregnant women -
1st trimester
2nd trimester
3rd trimester
124-135 g/l
110-117 g/l
106-109 g/l
• Birth-______ OR _____
Lower up to______ by 2mos of age
and _______10 yrs of age
180 - 40 g/I OR 15-20 g/dL
10-14 g/dL
12-15 g/dL
Blood can be collected from 3 different sources:
- Capillary blood
- Venous blood
- Arterial blood
Methods of Hb Estimation
Visual Methods
Photoelectric Method
Gasometric Method
Visual Methods
• Specific Gravity
• Sahli’s Acid Hematin
• Tallquist Hemoglobin Color Chart
• WHO Hemoglobin Color Scale
Photoelectric Method
• Cyanmethemoglobin method
• Most accurate method for estimation of Hb.
• Recommended by _______
Cyanmethemoglobin Method
International Committee for Standardisation in Hematology (ICSH)
Why is cyanmethemoglobin method recommended
- All forms of Hb are converted to cyanmethemoglobin (except sulthemoglobin)
- Stable and reliable standard is available.
Cyanmethemoglobin Method - Equipment
• Photoelectric colorimeter or
spectrophotometer
• Sahlis pipette at 20 micro litre
• Pipette 5 ml
Hb Estimation by Cyanmethemoglobin Method
Reagents required
› pH of the solution-______
› Unstable if_____
› Stored at _____temperature in_____
DRABKIN’S SOLUTION
7.0-7.4
exposed to light
room temp
brown borosilicate bottles
Hb Estimation by Cyanmethemoglobin Method
Reagents required
› pH of the solution-______
› Unstable if_____
› Stored at _____temperature in_____
DRABKIN’S SOLUTION
7.0-7.4
exposed to light
room temp
brown borosilicate bottles
DRABKIN’S SOLUTION
› Solution should be_____ in color
clear and pale yellow in color
Hb Estimation by Cyanmethemoglobin Method
› Available commercially at a specific concentration
› Stored in a______ as it is photosensitive
› Exposure to light causes deterioration in the strength of the standard
› The concentration of the standard used in this demonstration is_____
Hb Standard solution
brown bottle
14.8 gm%
- The method starts with hemoglobin, which is in its ferrous (Fe²⁺) state in red blood cells.
- _______ is added to the sample, causing oxidation of hemoglobin. This oxidation converts hemoglobin (Fe²⁺) into_______, which has iron in the ferric (Fe³⁺) state.
- _______ is then added to the methemoglobin, forming_______. This compound is stable and has a distinct color.
- The concentration of cyanmethemoglobin in the sample is measured using a colorimeter or spectrophotometer at a specific wavelength (usually_____). The absorbance is directly proportional to the hemoglobin concentration in the sample.
K+ Ferricyanide; methemoglobin (Fe+3)
Potassium cyanide (K+ Cyanide); cyanmethemoglobin (HiCN)
540 nm
Cyanmethemoglobin
MECHANICAL SOURCES OF ERROR:
• Pipetting error.
• Use of dirty or scratched cuvettes.
• Use of deteriorated reagent.
Conditions
may affect Hemoglobin
Determination
- Specimen:
-turbidity
-hemolysis
-lipemic - Reagent precipitation due to abonormal proteins
- Hemoglobin type
Cyanmethemoglobin Method
Advantages
• All forms of Hb except_______ are converted to hemiglobincyanide/cyanmethemoglobin (HiCN).
•_____ error is not there as no color matching is required.
• Cyanmethemoglobin solution is____ and it’s color does not fade with time so readings may not be taken immediately.
• Absorbance may be measured soon after dilution.
• A reliable and stable reference standard is available from World Health Organisation for direct comparison
sulphemoglobin
Visual
stable
Cyanmethemoglobin Method
Disadvantages
• Diluted blood has to stand for a period of time to ensure complete conversion of Hb.
•______ is a poisonous substance and that is why Drabkin’s solution must never be pipetted by mouth.
• The rate of conversion of blood containing______ is slowed considerably. Prolonging the reaction time to_____ can overcome this problem.
• Abnormal plasma proteins cause turbidity when blood is diluted with
_______.
• A high leukocyte count also causes_____ on dilution of blood.
•______ the diluted blood can help overcome the turbidity.
Potassium cyanide
carboxyhemoglobin; 30min
Drabkin’s solution
turbidity
Centrifuging
Gasometric Method
• Oxygen carrying capacity measured by_______
Van Slyke apparatus
• Oxygen carrying capacity measured by Van Slyke apparatus
Gasometric Method
Gasometric method
• Based on formula,____ of Hb carries____ of oxygen
It does not measure (3)
• Time-consuming and expensive.
• Result is____ percent less than other
methods.
1 gm; 1.34 ml
carboxyhemoglobin
sulfhemoglobin
methemoglobin
2
Gasometric method
• Based on formula,____ of Hb carries____ of oxygen
It does not measure (3)
• Time-consuming and expensive.
• Result is____ percent less than other
methods.
1 gm; 1.34 ml
carboxyhemoglobin
sulfhemoglobin
methemoglobin
2
• Rough estimate is made from specific gravity of blood
Specific Gravity method
SG Method aka
• Copper sulfate technique.
• Used in mass screening like selection of donors.
SG Method
• Used in mass screening like selection of donors.
SG Method
• Rapid and simple
• Commonly used in blood donor selection
Specific Gravity Method
SG Method
• A drop of blood is allowed to fall in______ of specific gravity of_____ from a height of____
• Specific gravity is equivalent to______
• Drop gets covered with_____
• If drop sinks,specific gravity is higher than copper sulfate
copper sulphate solution; 1.053; 1 cm
12.5 grams/dl
copper proteinate
Common Sources of Error in Specific Gravity Method
- hypergammaglobulinemia (e.g. multiple myeloma)
- air bubbles
- Use of inadequate height for dropping
= falsely high Hb level
= falsely low Hgb
= underestimating the Hgb concentration
Common Sources of Error in Specific Gravity Method
- hypergammaglobulinemia (e.g. multiple myeloma)
- air bubbles
- Use of inadequate height for dropping
Acid Hematin Method
APPARATUS
Sahli apparatus (haemoglobinometer
or haemometer) which is composed of :-
Sahli tube
Pippette
Colour comparator
Acid hematin
Hemolysis of RBC releasing
hemoglobin which is then converted to
acid hematin (Ferriheme chloride)
ACID HEMATIN
STEPS
Put _____ till the mark _____
Add_____ blood (using the micro tube of the pipette)
Shake for_____
HCl + Hb →_____(brown in color)
Add____ to the acid hematin drop by drop
Match colors at full arm length and against light
0.1 HCl; 10%
0.02 ml
15 minutes
acid hematin
distilled water
ACID HEMATIN
Adding____ to the blood causes :-Hemolysis of RBCs
Formation of_____
[HCl + Hb → acid hematin (brown in color)]
The intensity of color is_____ to hemoglobin content in blood.
HCl
acid hematin
proportional
Role of HCl
• As it is_____ results in the hemolysis of RBCs.
• Convert the hemoglobin to a brownish____
hypotonic
acid hematin
PRECAUTIONS
1. Be sure that there is no_____ in the blood column.
2. Clean the tip of pippette from any blood to avoid _____results.
3. Withdraw the blood_____ from the finger
> Ensure alcohol is completely dried
Avoid compression of the finger
air bubbles
false high
horizontally
Advantages of Acid Hematin Method
• Easy to perform
• Quick
• Inexpensive
• Can be used as a bedside procedure
• Does not require technical expertise
Disadvantages of Acid Hematin Method
• For maximum color,______ is required
•_____ with brown glass standard is not possible
• (3) are not converted to acid hematin
• Development of color is slow and____ is not stable
•_____ will influence the comparison of colors
longer time
Perfect matching
Carboxyhemoglobin,methemoglobin and sulfhemoglobin
acid hematin
Source of light
• Series of lithographed colors said to correspond to Hb values ranging from 10 to 100 percent
• Blood obtained from finger puncture
• Placed on a piece of absorbent paper
• Color is matched against the color on the chart
• Corresponding reading taken
• Cheap and simple
• Error ______percent
Tallquist Hemoglobin Chart
20 to 50
WHO Hemoglobin Colour Scale
• Devised by
Scott and Lewis
• Principle is similar to Tallquist method
WHO Hemoglobin Colour Scale
WHO Hemoglobin Colour Scale
• Rapid,simple,inexpensive,reliable
• _______ for diagnosis of anemia
• Printed set of colors corresponding to Hb values from______
• Efficiency-greater than _____percent in detecting anemia
•____ percent-in classifying its grade
1 gram/dl
4-14 grams/dl
90
86
Useful for screening blood donors
Screening women and children in health programmes
Iron-therapy
WHO Hemoglobin Colour Scale
• Modification of cyanhemoglobin method
• Other chemicals:
-SLS (Sodium Lauryl Sulfate)
-imidazole
-SDS (Sodium Dodecyl Sulfate)
Automated Method
• Measurements are made at various wavelengths depending on final stable product
Automated method
• Absorbance measurements are made at a set time interval after mixing the blood and the active reagents, but before the reaction is complete.
Automated Method
• The cyanide-free reagent is ideal for use on an automated hig
through-put clinical hematology analyzer.
Automated method
