Alkaline Hematin D method

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 it is not accurate because the mmol depends on the
molecular weight of hemoglobin and this is varies according to the type of hemoglobin
10 g/dl g/l .. (g/dl) ( g/l)
liter = 10 dL liter = 1000 ml 1dL = 100ml .
.g/dl g/l 10 10 L 10
dl = 100 mL g/100ml g/l
g/dl g/l
.
mmol mmol/l kits

2) Techniques for measuring hemoglobin and assessing anemia:

hemoglobin is measured by:

Photometric techniques

or

Visual comparative Techniques

standard

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.

standard curve

==> 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:
.
)Hemiglobinocyanide (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.

absorbace
) hemiglobincyanide (HiCN
standard

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).

absorbace
reagent hemiglobinocyanide
chlorohaemin

Using a direct read-out meter

==> 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.

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.

 cuvette




DHT hemogloinometer
These include:

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.

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.

photometric technique

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

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 cant 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:
broad absorption maximum
==> 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 hematinD (AHD)

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

broad absorption maximun

 reagent

AHD reagent
NaOH

How this reagent form colored compound with hemoglobin?

reagent

=> 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.

2) Reagent
Drabkins solution is neutral diluting fluid, pH 7.07.4
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)

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

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
_ 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.

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 doesnt 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 20l 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 20l
using micropipette to be 2980l, then add the 20 l 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 23 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.

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 20l 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
AHD standard (ml)
AHD reagent (ml)
Dilution

precaution:

5
Blank

1
4
1
4:5

2
3
2
3:5

3
2
3
2:5

4
1
4
1:5

Dont 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
Dilution
Absorbance
AHD conc. (g/l)(i.e equal to Hb conc.)

B
Blank

1
4:5

2
3:5

3
2:5

4
1:5

N
undiluted
160 g/l

4) Fourth: Calculate the hemoglobin (Hb) equivalent in g/l of solutions in tubes 15 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 dont 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,

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

We take example that the standard is 160 g/l >>>> here we dont 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 20200 g/l (220 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.
0.00

0.01

0,02

0.03

0.04

0.05

0.06

0.07

0,08

1.5

Absorbance
Hb concentration (g/l)

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 20l 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 20l 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 23 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.

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.

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 dont 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 23 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.

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

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 it is not accurate because the mmol depends on the
molecular weight of hemoglobin and this is varies according to the type of hemoglobin
10 g/dl g/l .. (g/dl) ( g/l)
liter = 10 dL liter = 1000 ml 1dL = 100ml .
.g/dl g/l 10 10 L 10
dl = 100 mL g/100ml g/l

 g/l g/dl
.
kits mmol/l mmol

2) Techniques for measuring hemoglobin and assessing anemia:

hemoglobin is measured by:
Photometric techniques

Visual comparative Techniques

or

standard

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.

standard curve

==> 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:
.

Hemiglobinocyanide (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.

absorbace
hemiglobincyanide (HiCN)
standard

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).

absorbace
reagent hemiglobinocyanide
chlorohaemin

Using a direct read-out meter

==> 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.

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.

cuvette




DHT hemogloinometer
These include:

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.

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.

photometric technique

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
Hemiglobincyide (cyanmethemoglobin) method

Category: photometric techniques>>> dilution techniques

In which blood is measured into a measured volume of diluting fluid
hemiglobinocyanide
1) Prinicple of the test

First:
We cant 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:
broad absorption maximum
==> 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 Drapkins solution

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

broad absorption maximun

reagent

Drapkins solution reagent
potassium cyanide potassium ferricyanide

How this reagent form colored compound with hemoglobin?

reagent

==> 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 in Drapkins solution to form HiCN 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.

detergent
methemoglobin ferricyanide
hemiglobinocyanide (HiCN) methemoglobin

HiCN 251 1
colorimeter nm 540 HiCN

 HiCN
calibration curve

2) Reagent
Drabkins solution is neutral diluting fluid, pH 7.07.4

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)

-----
==> Potassium cyanide is highly poisonous. It must be stored securely in a locked cupboard. Because of
the hazard associated with the storage and use of this chemical and often the difficulty in obtaining it and
other chemicals in Drabkins fluid, many laboratories buy Drabkins fluid in a ready-prepared form to
which water is added.

.


==> Drabkins fluid must be stored in a light opaque container, e.g. brown glass bottle or ordinary glass
bottle wrapped in silver foil.

.. foil
==> It is a pale yellow clear fluid and must not be used if it loses its colour or becomes turbid (see later
text Quality Control).

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.

) (standard solution HiCH

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

HiCN
Types of Haemiglobinocyanide (HiCN) Standard solutions:
HiCN
)1
Ready to use diluted hemiglobincyanide (cyanmethemoglobin) standards equivalent to hemoglobin
values:

hemiglobinocyanide

)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 l HiCN to 4 ml Drabkins diluting fluid,
then the dilution factor equals 20 l blood and 4 ml Drabkins diluting fluid, i.e. 1 in 201 dilution, (see
Method A in following text).

) (calibration curve
. dilution factor
. 20 4

 1 : 201 20 : 4020
4 20
2)
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

HiCN
HiCN
before dil = N V after dilution (N V)
( calibration curve)
3)
you may use a blood sample of known hemoglobin concentration to make from it a calibration curve

calibration cuve

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

_ 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.

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.

calibraiton curve 3

2) Procedure:

There are 3 methods of calibrations.

3
A) -----

A: 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 Drabkins 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:
Dont forget to wipe the tip of the pipette after each single pipetting and before you insert the
pipette inside another solution
( 1
540nm ( 2
( 3
3 ( 4
( 5
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).

 ( 6
. absorbance .
200g/l ( 7

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 20200 g/l (220 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.

. g/dl 20-2 g/l 200-20 ( 8

1.50 0.02 0.01 0
Absorbance

Hb concentration (g/l)

B)
B: 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
HiCN standard (ml)
Drapkins solution (ml)
Dilution

5
Blank

1
4
1
4:5

2
3
2
3:5

3
2
3
2:5

4
1
4
1:5

5
5

Undiluted

precaution:
Dont 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.

:
balnk,1,2,3,4,5 6

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)

cuvette
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 Drabkins neutral fluid in tube B or just Fill
a matched cuvette with Drabkin diluting fluid and place in the spectrophotometer (or colorimeter).

540nm

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.

absorbance

Tube
Dilution
Absorbance
HiCN conc. (g/l)(i.e equal to Hb conc.)

B
Blank

1
4:5

2
3:5

3
2:5

4
1:5

5
undiluted

4) Fourth: Calculate the haemoglobin (Hb) equivalent in g/l of solutions in tubes 15 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 dont 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

.. g/l
g/dl 1000 1000 mg g 10
dl L g/l 1000 10
mg/l 1000 mg g mg/dl
10 1000 dilution factor

?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 20200 g/l (220 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.

absorabance

Excel
20g/dl
absorbance 0.0 0.1 0.02 1.5
absorabance
1.5

0,08

0.07

0.06

0.05

0.04

0.03

0,02

0.01

0.00

Absorbance

)Hb concentration (g/l

------ )C
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 4 ml of Drabkin diluting fluid into a test-tube.

4) Add 0.02ml (20 l) 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 after dilution 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

.. 200g/l
4000 4 .. 540nm
20:4020 20
1:201

HiCN 10
. <<<< HiCN
4 168g/l
1000 g/l
. 10
Tube
HiCN standard (ml)
Drapkins solution (ml)
Dilution
Hb Conc. (g/l)
Absorbance

1
4
1
4:5
134.4

2
3
2
3:5
100.8

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

3
2
3
2:5
67.2

4
1
4
1:5
33.6

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.

absorbance
absorbance

Estimation of Hb of blood sample:

1) Pipette 4 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.

.. 4
tips 20
Precautions:
==> Do not allow air bubbles to enter.
==> With venous blood ensure, it is should be well mixed with the 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 20l.




0.02ml 20cmm 2 20 cmm
cubic millimeter
ccm
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.

20
.. 20

5) Return the blood to the mark. Re-check the blood is still on the 20l mark
before dispensing it .

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
45 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 Patients 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.

540nm 5 4
absorbance .
calibration curve

5 2000

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 Drabkins diluting fluid has not deteriorated.

( quality control )
hemolysate

hemolysate
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.

hemolysate hemolysate
.
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 Drabkins 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),
Drabkins fluid should give a zero reading. The pH of the fluid should be pH 7.07.4.
==> If deterioration is indicated, the fluid should not be used.
==> Fresh Drabkins reagent must be prepared. During the hot season, Drabkins fluid is best stored
refrigerated.
==> It must be allowed to warm to room temperature before being used.

HiCN hemolysate

7 pH
FRESH . .. 7.4
.

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 46C. Do not freeze, as this may cause
decomposition of the compound. Always allow the diluting fluid to warm to room temperature before use.

6 30
HiCN

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 dont 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 23 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.

..

References:

==> Monica Clinical Laboratory part 2

==> HENRYS Clinical Diagnosis and Management by Laboratory Methods.
==> Manual of basic techniques for a health laboratory by the World Health Organization.