Hemoglobinometry- Flashcards by Kyra Catedral

• Done separately or as part of routine CBC
• Relatively simple
• Performed quickly by the laboratory

Hemoglobin determination

How well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

WHO defines anemia as Hb levels of_____ in women and_____ in men

< 12 g/dL

<13 g/dL

How well did you know this?

Not at all

Perfectly

Physiological Factors

•Age
•Gender
•Pregnancy
•Exercise
•Posture (from lying to sitting/standing)
•Altitude
•Smoking

How well did you know this?

Not at all

Perfectly

Pathological Factors

• Testosterone deficiency
• Renal deficiency
• Myelodysplasia
• Diminished erythropoiesis
• BM suppression
• Blood loss

How well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

Pregnant women -
1st trimester
2nd trimester
3rd trimester

124-135 g/l

110-117 g/l

106-109 g/l

How well did you know this?

Not at all

Perfectly

• 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

How well did you know this?

Not at all

Perfectly

Blood can be collected from 3 different sources:

Capillary blood
Venous blood
Arterial blood

How well did you know this?

Not at all

Perfectly

Methods of Hb Estimation

Visual Methods
Photoelectric Method
Gasometric Method

How well did you know this?

Not at all

Perfectly

Visual Methods

• Specific Gravity
• Sahli’s Acid Hematin
• Tallquist Hemoglobin Color Chart
• WHO Hemoglobin Color Scale

How well did you know this?

Not at all

Perfectly

Photoelectric Method

• Cyanmethemoglobin method

How well did you know this?

Not at all

Perfectly

• Most accurate method for estimation of Hb.
• Recommended by _______

Cyanmethemoglobin Method

International Committee for Standardisation in Hematology (ICSH)

How well did you know this?

Not at all

Perfectly

Why is cyanmethemoglobin method recommended

All forms of Hb are converted to cyanmethemoglobin (except sulthemoglobin)
Stable and reliable standard is available.

How well did you know this?

Not at all

Perfectly

Cyanmethemoglobin Method - Equipment

• Photoelectric colorimeter or
spectrophotometer
• Sahlis pipette at 20 micro litre
• Pipette 5 ml

How well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

DRABKIN’S SOLUTION

› Solution should be_____ in color

clear and pale yellow in color

How well did you know this?

Not at all

Perfectly

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%

How well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

Cyanmethemoglobin

MECHANICAL SOURCES OF ERROR:

• Pipetting error.
• Use of dirty or scratched cuvettes.
• Use of deteriorated reagent.

How well did you know this?

Not at all

Perfectly

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 1. hypergammaglobulinemia (e.g. multiple myeloma) 2. air bubbles 3. 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

1. hypergammaglobulinemia (e.g. multiple myeloma) 2. air bubbles 3. 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