Alkaline Hematin D method for manual Hemoglobin Estimation – The Manual Method Only

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ِAlkaline Hematin D method fo Manual Hemoglobin Estimation Test – 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 hemoglobin in a blood sample is measured electronically using a filter colorimeter or a direct read-out hemoglobin 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 hematin 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, chlorohemin 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 talked before about the Hemiglobincyanide technique (HiCN method) (CLICK HERE to READ IT) and  now we will talk about The Alkaline hematin D methd (AHD method) and we will add the other methods soon in a links connected with page and then publish it

Hemiglobinocyanide method (Drapkin’s method)

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Alkaline Hematin D (AHD) 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 Alkaline Hematin D

==> This reagent is considered a diluting solution and it is contains  Sodium hydroxide (NaOH) 4 g, Triton X-100 (or equivalent) 25g and Distilled water.

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 NaOH to alkaline hematin D-575, which is a stable colored compound that we measure its absorbance and whose concentration equal the concentration of hemoglobin.

==> In this test we dilute hemoglobin in 1 to 151 [1 AHD solution (standard or sample) : 151 AHD reagent] (NOTE: we can use the dilution 1 : 150 >>>> see the methods)

==> Absorbance of the AHD 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 AHD 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 AHD reagent in Your lab? CLICK HERE

1)   Components:

This fluid contains:

==>  Sodium hydroxide (NaOH) …………… 4 g (Role = Formation of Alkaline Hematin D)
==>  Triton X-100 (or equivalent) ………… 25 g (Role = Deterget to dissolve lipid of the RBCs membrane to rupture it and release Hb)
==>  Distilled water ………………………………. 1000ml (Role = solvent for preparation of the reagent)

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2)   Standard solutions used to calibrate the spectrophotometer or colorimeter

==>The standard we use, should be solution of Alkaline hematin D-575 which is prepared in the central lab

 

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 3)  Practical Work 

 Calibration of spectrohpotometer or colorimeter:  

1)  Materials and reagents

_ Spectrophotometer, hemoglobinometer or colorimeter

_ Test-tubes

_ Test-tube racks

_ Corks or rubber stoppers

_ Cuvettes

– Variable pipette

_ Grease pencil

_ Cotton wool or gauze

_ AHD standard (supplied by the central laboratory)

_ AHD reagent 

A 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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Calibration of Spectrophotometer:

2)   Procedure:

1. Note the concentration of the AHD standard indicated on the label, e.g. 160g/l at a 1:151 dilution

(NOTE: this label doesn’t mean that the standard is already diluted but means that the AHD concentration is 160 g/l when it is diluted to 1:151.)

2. Pipette 20μl of AHD standard into a clean test-tube containing 3 ml of AHD reagent (20:3020 = 1:151).

NOTE: If you want to prepare 1:150, put the 3 ml of the AHD reagent in a clean tube then remove 20μl using micropipette to be 2980μl, then add the 20 μl of the standard, THUS, 20: (2980+20) = 20:3000 which means 1:150)

3. Stopper the test-tube using a clean cork or rubber stopper and mix by inversion. Leave the tube to stand for 2–3 minutes.

4. Fill a clean cuvette with the undiluted AHD reagent to Adjust the spectrophotometer or hemoglobinometer to read zero (blank).

Precautions:

Dry the outside of the cuvette with cotton wool or gauze (to avoid false reading by impurities on the outside of the cuvette) and place it in the cuvette chamber.

5. Replace the undiluted AHD reagent in the cuvette with the diluted AHD standard solution; repeat the measurement procedure and adjust the spectrophotometer or hemoglobinometer to read the correct hemoglobin concentration indicated on the label, e.g. 160 g/l.

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Calibration of colorimeter:

2) procedure: 

1. Switch on the colorimeter and set the wavelength to 540nm. Allow the colorimeter to warm up for the time recommended by the manufacturer.

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

– Arrange six test-tubes in a test-tube rack. Label the test-tubes 1, 2, 3, 4, B and N.

– Pipette 5 ml of AHD reagent into the test-tube marked B.

–  Pipette 3 ml of AHD reagent and 20μl of AHD standard into the test-tube marked (N).

at this point you dilute the standard to 1:151 at which the AHD concentration is 160 g/l, and now we will concsider the diluted reagent in the N is the standard that we will make a serial dilution from it

– Dilute the reference solution in test-tube N as described in the following table

 Tube  B  1  2  3  4
 AHD standard (ml)  —  4  3  2 1
AHD reagent (ml)  5  1  2  3  4
Dilution   Blank  4:5  3:5  2:5  1:5

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.

– Pour the AHD reagent from test-tube B into a clean cuvette. Dry the outside of the cuvette with cotton wool or gauze. Place the cuvette into the cuvette chamber, close the cuvette chamber and adjust the colorimeter to read zero absorbance (blank).

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

Replace the AHD reagent in the cuvette with the reference solution from test tube 4. Record the absorbance. Pour the solution back into test-tube 4.

Repeat the procedure using test-tubes 3, 2, 1 and N, respectively in sequence.

 Tube  B  1  2  3  4 N
 Dilution  Blank  4:5  3:5  2:5 1:5  undiluted
Absorbance  
AHD conc. (g/l)(i.e equal to Hb conc.) 160 g/l 

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

 calculation of Hemoglobin concentraiton

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 N is CONSIDERED undiluted and the Hb conc. is equal to the conc. Of AHD standard printed on the AHD standard Kit

How to calculate the dilution factor?

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

dilution-factor

 

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

dilution-factor---Copy

We take example that the standard is 160 g/l >>>> here we don’t need to multiply by 10 or divide by 1000

So,

tube N: 160gHb/l (i.e. it contains the un diluted standard)

tube 1: 160gHb/l 4/5 = 128gHb/l

tube 2: 160gHb/l 3/5 = 96g Hb/l

tube 3: 160gHb/l 2/5 = 64g Hb/l

tube 4: 160gHb/l 1/5 = 32g Hb/l

tube B: 0gHb/l (i.e. it contains only the reagent)

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 as many of the points plotted as possible.

==> 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 hemoglobin 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)

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 Estimation of Hb of blood sample: 

Method using a spectrophotometer or haemoglobinometer

1. Switch on the spectrophotometer or haemoglobinometer. Allow it to warm up for the time recommended by the manufacturer (usually 10 minutes).

2. Arrange the test-tubes in a test-tube rack: one for each sample to be tested, one for the blank and two for the control samples.

3. Using a grease pencil, label the test-tubes with the appropriate laboratory numbers of the samples to be measured, B for the blank, and C1 and C2 for the control samples.

4. Pipette 3 ml of AHD reagent into each test-tube.

5. Pipette 20μl of blood collected in EDTA from a patient into the AHD reagent of the appropriate tube. Flush the pipette carefully five times with the AHD reagent.

6. Pipette 20μl of AHD standard into test-tubes C1 and C2.

7. Plug all the test-tubes with a clean cork or rubber stopper and mix by inversion. Leave the tubes to stand for 2–3 minutes.

8. Pour the AHD solution from test-tube B into a clean cuvette. Dry the outside of the cuvette with cotton wool or gauze. Make sure that there are no air bubbles in the solution. Place the cuvette in the cuvette chamber and adjust the spectrophotometer or haemoglobinometer to read zero.

9. Repeat the procedure with the solution in test-tubes C1 and C2, respectively. If the readings of the two controls differ by less than 2.5%, measure the haemoglobin concentration of all the test samples. Record all the results.

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Method using a colorimeter

The AHD method is also applicable using a colorimeter. The measurement procedure is the same as that described for a spectrophotometer or haemoglobinometer. However, the absorbance in a colorimeter does not increase linearly with haemoglobin at elevated concentrations. Therefore, a calibration curve must be used to relate the absorbance readings to the haemoglobin concentration, as described above.

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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, hemoglobinometer 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 hemoglobinometer 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, hemoglobinometer 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:

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

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Hemiglobinocyanide method (Drapkin’s method)

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