Hemoglobin Estimation Test Manually (by Drapkin’s method) or hemiglobinocyanide method-The method only


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Hemoglobin Estimation Test Manually by Drapkin’s method (i.e. Hemoglobinocyanide method)- The Method only

We will talk about the technique only and we will talk about result interpretations in another sections

Introduction

1) Measuring units:

==> Hemoglobin values are expressed in grams per liter (g/l) or grams per deciliter (g/dl)

==> To convert from g/l to g/dl divide by 10 (i.e. 10 g/l = 1 g/dl) because 1 dl = 100 ml and 1 Liter = 1000 ml thus 1 liter = 10 dl, so, to convert from liter to dl you should multiply by 10 and because L and dl is in the denominator, we said that you should divide by 10 to convert from ((g/l)) to ((g/dl))

==> g/dl may be written g/100ml in some books because (1 dl = 100 ml)

==> Grams/litre is the recommended way of expressing the mass concentration of hemoglobin. Some countries however continue to express hemoglobin in g/dl and most visual comparative techniques use g/dl.

==> Some labs and kits use mmol/l, but i believe that is not accurate because the mmol depends on the molecular weight of hemoglobin and this is varies according to the type of hemoglobin

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2) Techniques for measuring hemoglobin and assessing anemia:

hemoglobin is measured by:

  • Photometric techniques       or

  • Visual comparative Techniques

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A) Photometric techniques

==> In photometric techniques the absorbance of haemoglobin in a blood sample is measured electronically using a filter colorimeter or a direct read-out haemoglobin meter.

==>  It can be classified into:

Techniques include dilution techniques, and non dilution techniques which do not require prior dilution of the blood.

 1) DILUTION TECHNIQUES 

“In which blood is measured into a measured volume of diluting fluid”

These include:

-      Hemiglobincyanide (cyanmethemoglobin)

==> It is a technique using a filter colorimeter or direct read-out meter.

==> It is the recommended technique because stable hemiglobincyanide (HiCN) standards are available to calibrate instruments.

==> It has however, several disadvantages when used in tropical countries.

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-      Alkaline haematin D-575

==> It is a technique using a filter colorimeter or direct read-out meter.

==> It is as accurate as the hemiglobincyanide technique but less expensive and uses a diluting reagent that does not contain toxic potassium cyanide.

==> A crystalline compound, chlorohaemin is used to calibrate the method (requires preparation in the Central or Regional Laboratory).

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-      Using a direct read-out meter

Developing Health Technology (DHT) Hemoglobinometer

==> Such as the Developing Health Technology (DHT) Hemoglobinometer which is a hemoglobin meter with specifications and technique suitable for use in district laboratories in tropical countries.

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 2) NON-DILUTION TECHNIQUES 

which do not require prior dilution of the blood where Blood is collected directly into a specially designed single-use microcuvette or other sampling device which is internally coated with reagent to lyse the blood and convert the hemoglobin to a form which can be read in a direct read-out meter

While the cost of direct read-out meters for use with non-dilution systems is becoming less, the cost of the sampling devices remains high compared with most dilution systems, particularly the DHT Hemoglobin system.

These include:

HemoCue system-      HemoCue system

==> This non-dilution system is becoming more widely used in tropical countries because of the lower cost of the new hemoglobin meter, its accuracy, and simplicity of use.

==> It is particularly useful when a dilution technique cannot be used, e.g. in a clinic with nursing staff performing hemoglobin tests.

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B) Visual comparative technique

When it is not possible to measure hemoglobin accurately using a photometric technique, e.g. in a health center or antenatal unit, a visual comparative technique such as the Hemoglobin Colour Scale, can help to detect anemia and assess its severity.

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 We now will talk only about the  Hemiglobincyanide technique (HiCN method) but we will add the other methods soon in a links connected with page and then publish it

Alkaline Hematin D method

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Hemiglobincyide (cyanmethemoglobin) method

Category: photometric techniques>>> dilution techniques

“In which blood is measured into a measured volume of diluting fluid”

 1) Prinicple of the test 

First: 

We can’t measure hemoglobin concentration while it is contained inside the RBCs, so to measure the hemoglobin concentration you should lyse the RBCs, so you can measure it

Second:

What is meant by broad absorbance maximum? and why is the compound of broad absorbance maximum? CLICK HERE

==> To measure hemoglobin photometrically, you should convert Hemoglobin to a colored compound that has a broad absorption maximum (i.e. broad range of wavelength that give the same absorbance and one concentration reading) to be measured easily.

==> So, We should use a reagent that contain chemicals that react with hemoglobin and form a colored compound

The reagent we use is called Drapkin’s solution

==> This reagent is considered a diluting solution and it is contains  potassium ferricyanide, potassium cyanide and a detergent.

How this reagent form colored compound with hemoglobin?

Why detergents dissolve  the red cell memebrane??? CLICK HERE

=> The red cells are hemolyzed by the detergent (by dissolving the lipids in the RBC membrane) and hemoglobin is oxidized by the ferricyanide to methemoglobin.

==> Then methemoglobin  is converted by the cyanide to stable haemiglobinocyanide (HiCN) which is the colored compound that we measure its absorbance and whose concentration equal the concentration of hemoglobin.

==> In this test we dilute hemoglobin in 1 to 251 [1 HiCN solution (standard or sample) : 251 Drapkin’s solution]

==> Absorbance of the HiCN solution is read in a spectrophotometer at wavelength 540 nm or in a filter colorimeter using a yellow-green filter.

==> The absorbance obtained is compared with that of a HiCN standard solution.

==> Hemoglobin values are obtained from tables prepared from a calibration graph or if using a direct read-out hemoglobinmeter, from the digital display.

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 2) Reagent 

Drabkin’s solution is neutral diluting fluid, pH 7.0–7.4

How to prepare Drapkin’s solutions in Your lab? CLICK HERE

1)   Components:

This fluid contains:

-      Potassium ferricyanide (hexacyanoferrate 111) . . 200 mg (role = Oxidation of Hb to methemoglobin)

-      Potassium cyanide* . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 mg (Role = Formation of Hemiglobincyanide)

-      Potassium dihydrogen phosphate . . . . . . . . . . . . . . 140 mg (Role = buffer to adjust the Ph to 7.2)

-      Non-ionic detergent (e.g. Nonidet, Triton-X-100) . . . 1 ml (Role = Detergent to disolve the lipid in the RBCs membrane to rupture it and release of Hb)

-      Distilled or deionized water. . . . . . . . . . . . . . . . . . to 1 litre (Role= solvent)

Precautions CLICK HERE TO OPEN

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2)   Standard solutions used to calibrate the spectrophotometer or colorimeter

==>The standard we use, should be solution of HiCN because this is the colored compound we measure its absorbance

==> HiCN solutions are stable for long periods (2 years or longer).

Types of Haemiglobinocyanide (HiCN)  Standard solutions:

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1) Ready to use diluted hemiglobincyanide (cyanmethemoglobin) standards

Ready to use diluted hemiglobincyanide (cyanmethemoglobin) standards equivalent to hemoglobin values:

30 g/l (3.0 g/dl)

115 g/l (11.5 g/dl)

180 g/l (18.0 g/dl)

The diluted HiCN solutions are suitable for preparing a calibration graph for use in obtaining patients’ haemoglobin values. In this type you should to know the dilution factor by which the standards are prepared and you should to dilute the sample by the same dilution factor

For example: if the diluted standards are prepared by add 20 μl HiCN to 4 ml Drabkin’s diluting fluid, then the dilution factor equals 20 μl blood and 4 ml Drabkin’s diluting fluid, i.e. 1 in 201 dilution, (see Method A in following text).

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2) undiluted Hemiglobincyanide (cyanmethemoglobin) standard

you may buy Undiluted Hemiglobincyanide (cyanmethemoglobin) standard solution in ampule  and the concentration is written on the label of the ampule.

From this, the equivalent hemoglobin value needs to be determined.

Note: Preparing a calibration graph from this HiCN reference standard is more complex than when using the previously described ready to use HiCN standards

3) A blood sample of known hemoglobin concentration.
you may use a blood sample of known hemoglobin concentration to make from it a calibration curve

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 3)  Practical Work 

 Calibration of spectrohpotometer or colorimeter:  

1)  Materials and reagents

_ Spectrophotometer (or colorimeter)

_ Cuvettes

_ Test-tubes

_ Test-tube rack

_ Calibrated Blood (Sahli) pipettes, 0.2ml or variable micropipette but in case of the calibrated pipettes, a safe pipette/capillary filler should be used to aspirate and dispense the blood. This can be a simple bulb filler or a pi-pump

blood-sucking-system
_ Drabkin diluting fluid.

_ Reference solution, which may be:

==> The fresh hemiglobincyanide reference solution used to calibrate the instrument,

==> a reference solution previously calibrated against the hemiglobincyanide reference solution, or

==> a blood sample of known hemoglobin concentration.

add-bulbA calibration curve must be prepared before the spectrophotometer (or colorimeter) can be used for hemoglobin estimation. From such a curve a graph can be prepared and a table made for the hemoglobin values.

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2)   Procedure:

there are 3 methods of calibrations.

Calibration using ready to use diluted HiCN standards

30 g/l (3 g/dl) ,      115 g/l (11.5 g/dl),          180 g/l (18 g/dl)

1)   Allow the standards to warm to room temperature.

2)  Place a yellow-green filter in the colorimeter or set the wavelength to read 540 nm.

3)  Zero the colorimeter or the spectrophotometer with Drabkin’s neutral diluting fluid.

4)  Read the absorbance of each standard, beginning with the lowest.

5) Record the readings in the next table:

Standard 

30 g/l (3 g/dl)

115 g/l (11.5 g/dl)

 180 g/l (18 g/dl)

Absorbance

precaution:

Don’t forget to wipe the tip of the pipette after each single pipetting and before you insert the pipette inside another solution 

6) Take a sheet of graph paper and plot the absorbance of each standard (vertical axis) against its concentration in g/l (horizontal axis).

7)   Draw a straight line from zero through the points plotted. Extend the line to obtain readings up to 200 g/l (20 g/dl).

Example of an HiCN haemoglobin calibration graph using commercially produced HiCN standards: 30g/1, 115g/1, 180g/l

Example of an HiCN haemoglobin calibration graph using commercially produced HiCN standards: 30g/1, 115g/1, 180g/l

8) From the graph, make a table for Hb values from 20–200 g/l (2–20 g/dl).

==> Draw up a table of absorbance readings starting from 0.00, 0.01, 0.02 and ending at 1.50.
==> Determine the haemoglobin concentrations for each of the absorbances from the graph.

Absorbance

Hb concentration (g/l)


B: Calibration Using undiluted HiCN standard solution:

1)   First, Prepare the serial dilutions from the HiCN as follow:

-      ­Take 6 tubes and label them B (Blank), 1, 2, 3, 4, 5.

-      Pipette into each tube as follows:

 Tube  B  1  2  3  4  5
 HiCN standard (ml)  –  4  3  2 1  5
Drapkin’s solution (ml)  5  1  2  3  4
Dilution   Blank  4:5  3:5  2:5  1:5  Undiluted

precaution:

Don’t forget to wipe the tip of the pipette after each single pipetting and before you insert the pipette inside another solution 

==> Stopper each tube and mix.

The blank is to zero the spectrophotometer, you may not prepare a blank solution to save the reagent and just Fill a matched cuvette with Drabkin diluting fluid and place it in the spectrophotometer (or colorimeter)

2)   Second: Adjust the spectrophotometer or colorimeter

-      Place a yellow-green filter in the colorimeter or set the wavelength at 540 nm.

-      Zero the colorimeter or the spectrophotometer using the Drabkin’s neutral fluid in tube B or just Fill a matched cuvette with Drabkin diluting fluid and place in the spectrophotometer (or colorimeter).

3)   Third: Read the absorbances and record it in the next table

Make sure the needle or the digital screen return to zero between each reading with Drabkin diluting fluid.

 Tube  B  1  2  3  4  5
 Dilution  Blank  4:5  3:5  2:5 1:5  undiluted
Absorbance  
HiCN conc. (g/l)(i.e equal to Hb conc.)

4)   Fourth: Calculate the haemoglobin (Hb) equivalent in g/l of solutions in tubes 1–5 as follows:

 

If you buy the Standards solution of concentration in:

-      ((g/dl)) then you will not divide by 1000 and multiply by 10 (i.e. remove 1000 from equation)

-      ((mg/l)) then you will divide by 1000 and don’t multiply by 10 (i.e. remove 10 from equation).

Not that tube no. 5 is not diluted and the Hb conc. is equal to the conc. Of HiCN standard printed on the HiCN standard Kit

How to calculate the dilution factor?

We want to calculate the conc. Of HiCN (which is equal to Hb conc.) after dilution so,

Calculate HiCN conc. (i.e. Hb conc.) as follows and write the results in the previous table

5)   Fifth: plot the calibration curve:

==> Take a sheet of graph paper and plot the absorbance of each standard (vertical axis) against its concentration in g/l (horizontal axis).

==> Draw a straight line from zero through the points plotted.

==> Extend the line to obtain readings up to 200 g/l (20 g/dl).

==> From the graph, make a table for Hb values from 20–200 g/l (2–20 g/dl).

  • Draw up a table of absorbance readings starting from 0.00, 0.01, 0.02 and ending at 1.50

  • Determine the haemoglobin concentrations for each of the absorbances from the graph.

Absorbance

0.00 0.01 0,02 0.03 0.04 0.05 0.06 0.07 0,08 1.5

Hb concentration (g/l)


C) Using a blood sample of known hemoglobin concentration

1)  Obtain a sample of blood of known hemoglobin concentration (e.g. 200 g/l).

2) Switch on the spectrophotometer (or colorimeter) and set to wavelength 540nm.

3) Pipette 5 ml of Drabkin diluting fluid into a test-tube.

4) Add 0.02ml (20 μl) of well-mixed blood from the known sample to the diluting fluid.

==> Be sure to wipe the outside of the pipette beforehand to avoid adding excess blood.

==> Mix the haemiglobinocyanide solution by inverting several times.

==> Leave to stand for 10 minutes.

5) Zero the spectrophotometer using Drabkin diluting fluid.

6) Read and record the absorbance of the hemiglobincyanide solution prepared above then calculate the concentration of the HiCN solution as follow:-

(N × V) before dill. = (N × V) after dill.

Assume that the concentration is 168 g/l

7) Prepare a series of dilutions of the hemiglobincyanide solution in four test tubes (labelled 1– 4) as shown in the next Table and calculate the concentration of each dilution from the previous equations

Tube 1 = 168 (g/l) x (4/5) = 134.4 g/l = 13.44 g/dl

Tube 2 = 168 (g/l) x (3/5) = 100.8 g/l = 10.08 g/dl

Tube 3 = 168 (g/l) x (2/5) = 67.2 g/l = 6.72 g/dl

Tube 4 = 168 (g/l) x (1/5) = 33.6 g/l = 3.36 g/dl

 Tube  1  2  3  4
 HiCN standard (ml)  4  3  2 1
Drapkin’s solution (ml)  1  2  3  4
Dilution   4:5  3:5  2:5  1:5
Hb Conc. (g/l)  134.4  100.8  67.2  33.6
Absorbance  

8)   Read and record the absorbances of the diluted solutions in the previous table

9)   Plot a graph of absorbance against hemoglobin concentration, using ordinary graph paper.

==> Draw a straight line starting at the origin passing as close to each point as possible.

==> Extend the line so that you can read absorbances for hemoglobin values greater than 168 g/l.

10)   A reference table of values is prepared using the graphs obtained from as shown in one of the above methods:

==> Draw up a table of absorbance readings starting from 0.00, 0.01, 0.02 and ending at 1.50.

==> Determine the hemoglobin concentrations for each of the absorbances from the graph.

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 Estimation of Hb of blood sample: 

1) Pipette 5 ml of Drabkin diluting fluid into a tube.

2) Draw free flowing capillary blood from lancet puncture or well-mixed anticoagulated venous blood to the 0.02-ml mark of a blood (Sahli) pipette or by the variable micropipette.

Precautions:

==>  Do not allow air bubbles to enter.

==> With venous blood ensure, it should be well mixed with anticoagulant by inverting the tube  repeatedly for about 1 minute immediately before pipetting it.

==> Some micropipettes are marked 0.02ml or 20cmm. These volumes are the same as 20μl.

4) Wipe the blood from around the tip of the pipette. To do this without withdrawing blood from the pipette, draw the blood further up the pipette, out of the tip.

5) Return the blood to the mark. Re-check the blood is still on the 20μl mark before dispensing it pipette by drawing up and expelling the fluid in the tube three times.

6) Dispense the blood into the diluting fluid at the bottom of the tube. Rinse the pipette in the diluting fluid.

7) Stopper the tube, mix, and leave the diluted blood at room temperature, protected from sunlight, for 4–5 minutes*

8) Place a yellow-green filter in the colorimeter or set the wavelength at 540 nm.

9) Zero the colorimeter using Drabkin diluting fluid.

10) Read the absorbance of the Patient’s diluted blood in the spectrophotometer cuvette.

Precaution

If cloudiness appears in the diluted blood, this may be attributable to abnormal plasma proteins or to a high concentration of white cells, so, Centrifuge the diluted blood at 2000 rpm for 5 minutes before measuring the absorbance.

10. Using the table prepared from the calibration curve, record the concentration of haemoglobin in g/l.

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 4)  Quality Control of HiCN technique 

How to prepare Hemolysate and Stabilized Whole Blood Control? CLICK HERE to see preparation of control materials

For the daily control of hemoglobin tests, district laboratories should be provided with a preserved whole blood control or a stable control hemolysate prepared and analyzed in the Regional or Central Hospital Laboratory.

The control hemolysate should be used each day to ensure the clorimeter or hemoglobin meter is functioning satisfactorily, and Drabkin’s diluting fluid has not deteriorated.

A quality control chart should be prepared and the hemoglobin values of the control hemolysate entered daily on the chart. This will detect any drift of values upwards or downwards indicating that the results of hemoglobin tests are becoming unreliable, and there is a problem which must be investigated.

When a control hemolysate or preserved whole blood control is not available, the minimum control of hemoglobin tests using the HiCN technique must include:

==> Daily use of a HiCN reference standard (as used to calibrate the colorimeter) to check instrument performance.

==> Visible and photometric check of Drabkin’s diluting fluid for signs of deterioration, particularly turbidity which is a common problem in tropical countries.

==> When measured against a water blank with a yellow-green filter in place (wavelength 540 nm), Drabkin’s fluid should give a zero reading. The pH of the fluid should be pH 7.0–7.4.

==> If deterioration is indicated, the fluid should not be used.

==> Fresh Drabkin’s reagent must be prepared. During the ‘hot’ season, Drabkin’s fluid is best stored refrigerated.

==> It must be allowed to warm to room temperature before being used.

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 5) Precaution 

==> Potassium cyanide is very poisonous. It must be kept in a locked cupboard at all times when not in use. Wash your hands immediately after handling it.

==> Store Drabkin diluting fluid in a brown reagent bottle because it decomposes on exposure to light. If a brown reagent bottle is not available, use a clear glass bottle carefully wrapped in silver foil.

==> Drabkin diluting fluid should be clear and pale yellow. If it becomes turbid, or loses its colour, it should be discarded. The clarity of the diluting fluid can be checked by measuring its absorbance in a spectrophotometer at 540 nm against water as a blank. The absorbance must read zero.

==> Once the hemiglobincyanide solution has been prepared, the hemoglobin estimation must be carried out within 6 hours.

==> Drabkin diluting fluid remains stable for several months when stored at cool temperatures. If the room temperature exceeds 30 °C, store it in a refrigerator at 4–6°C. Do not freeze, as this may cause decomposition of the compound. Always allow the diluting fluid to warm to room temperature before use.

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 6) Errors in hemoglobin estimation 

Errors in sampling:

— inadequate flow of blood from the finger prick;

— excessive squeezing of the finger after pricking;

— prolonged use of a tourniquet when collecting venous blood, which leads to concentration of blood cells;

— insufficient mixing of venous blood, which has sedimented after collection;

— small clots in venous blood due to inadequate mixing with EDTA after collection;

— adding too little or excess blood to Drabkin diluting fluid;

— air bubbles trapped in pipettes.

Faulty or dirty equipment, such as:

— broken or chipped pipettes;

— dirty pipettes;

— dirty cuvettes;

— use of unmatched cuvets; (i.e. Use different cuvettes that is not for the spectrophotometer model)

— dirty filters;

— a defective spectrophotometer, hemoglobinometer or colorimeter.

— Inaccurate micropipette (if you don’t use Sahli pipette),so, a good grade of pipette with a guaranteed accuracy of greater than 99% is desirable. Calibration of pipets will lessen errors.

Faulty technique:

— using a dilution factor different from the one for which the spectrophotometer, haemoglobinometer or colorimeter was calibrated;

— inadequate mixing of reagent;

— placing the cuvette in the chamber with the frosted sides facing the lightpath;

— air bubbles in the cuvette;

— using a standard filter from another spectrophotometer or haemoglobinometer for adjustment;

— using the wrong filter for the colorimeter.

Human errors:

— Poor training, and poor understanding of the clinical significance of the test and the necessity for a dependable method,

— Failure to follow oral and written instructions, and unfamiliarity with the equipment and with the sources of error.

— The chemist who become fatigue cause errors especially at the end of the day

— Lack of patience cause errors, A technologist who is patient and critical by nature and by training and who is interested in the work is less prone to make errors

Note:

-      If the spectrophotometer, haemoglobinometer or colorimeter requires frequent recalibration, e.g. every 2–3 days, change the bulb and repeat the procedure for internal quality control. If the problem of frequent recalibration persists, send the machine to a servicing agent.

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References:

==> Monica Clinical Laboratory part 2

==> HENRY’S Clinical Diagnosis and Management by Laboratory Methods.

==>  Manual of basic techniques for a health laboratory by the World Health Organization.

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Other methods

Alkaline Hematin D method

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