Hemoglobin

Examination
Dr.dr I Wayan Putu Sutirta Yasa, M.Si

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 Structure of hemoglobin
 “Globin" consists of four polypeptide chains:
two alpha chains, each with 141 amino
acids and two beta chains, each with 146
amino acids.
 The α and β globin chains are very similar in
structure and each one of them is linked
with a heme molecule. Each heme group
can combine with 1 molecule of oxygen or
CO2.
 A heme group is a flat ring molecule
containing carbon, nitrogen and hydrogen
atoms, with a single Fe2+ ion at the center.
Without the iron, the ring is called a
porphyrin.
 Theoxygen-binding site of Hb is the heme
pocket present in each of the four
polypeptide chains; a single atom of
oxygen forms a reversible bond with the
ferrous iron at each of these sites, so a
molecule of Hb binds four oxygen
molecules; the product is oxyhemoglobin
(O2Hb).

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HEMOGLOBIN FUNCTION
 The oxygen delivery function of Hb, that is its ability
to "pick up" oxygen at the lungs and "release" it to
tissue cells is made possible by minute
conformational changes in quaternary structure that
occur in the hemoglobin molecule and which alter
the affinity of the heme pocket for oxygen. Hb has
two quaternary structural states: the deoxy state
(low oxygen affinity) and the oxy state (high oxygen
affinity).

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A range of environmental factors determine
the quaternary state of Hb and therefore its
relative oxygen affinity. The
microenvironment in the lungs favors the oxy-
quaternary state, and thus Hb has high
affinity for oxygen here.

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 By contrast, the microenvironment of the
tissues induces the conformational change
in Hb structure that reduces its affinity for
oxygen, thus allowing oxygen to be
released to tissue cells.

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What is a Hemoglobin Test?
 A hemoglobin test measures the levels of hemoglobin in your blood.
Hemoglobin is a protein in your red blood cells that carries oxygen from your
lungs to the rest of your body. If your hemoglobin levels are abnormal, it may
be a sign that you have a blood disorder.
What is it used for?
 A hemoglobin test is often used to check for anemia, a condition in which your
body has fewer red blood cells than normal. If you have anemia, your cells
don't get all the oxygen they need. Hemoglobin tests are also frequently
performed with other tests, such as:
 Hematocrit, which measures the percentage of red blood cells in your blood
 Complete blood count, which measures the number and type of cells in your
blood

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Why do I need a hemoglobin test?

 Your health care provider may have ordered the

test as part of a routine exam, or if you have:
 Symptoms of anemia, which include weakness,
dizziness, pale skin, and cold hands and feet
 A family history of thalassemia, sickle cell
anemia, or other inherited blood disorder
 A diet low in iron and minerals
 A long-term infection
 Excessive blood loss from an injury or surgical
procedure
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What happens during a hemoglobin
test?
 A health care professional will take a blood sample from a vein
in your arm, using a small needle. After the needle is inserted,
a small amount of blood will be collected into a test tube or
vial. You may feel a little sting when the needle goes in or out.
This usually takes less than five minutes.
Will I need to do anything to prepare for the test?
 You don't need any special preparations for a hemoglobin test.
 If your health care provider has also ordered other blood tests,
you may need to fast (not eat or drink) for several hours before
the test. Your health care provider will let you know if there
are any special instructions to follow 9
Are there any risks to the test?

 There is very little risk to having a blood

test. You may have slight pain or bruising at
the spot where the needle was put in, but
most symptoms usually go away quickly.
What do the results mean?
 There are many reasons your hemoglobin
levels may be outside the normal range
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Low hemoglobin levels may be a sign of:
 Different types of anemia
 Thalassemia
 Iron deficiency
 Liver disease
 Cancer and other diseases
High hemoglobin levels may be a sign of:
 Lung disease
 Heart disease
 Polycythemia vera, a disorder in which your body makes too
many red blood cells. It can cause headaches, fatigue, and
shortness of breath.
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Lower than normal results
 If your hemoglobin level is lower than normal, you have anemia. There
are many forms of anemia, each with different causes, which can include:
 Iron deficiency
 Vitamin B-12 deficiency
 Folate deficiency
 Bleeding
 Cancers that affect the bone marrow, such as leukemia
 Kidney disease
 Liver disease
 Hypothyroidism
 Thalassemia — a genetic disorder that causes low levels of hemoglobin
and red blood cells
 If you've been previously diagnosed with anemia, a hemoglobin level
that's lower than normal may indicate a need to alter your treatment 12

plan.
Higher than normal results
If your hemoglobin level is higher than normal, it may be the result of:
 Polycythemia vera — a blood disorder in which your bone marrow makes too
many red blood cells
 Lung disease
 Dehydration
 Living at a high altitude
 Heavy smoking
 Burns
 Excessive vomiting
 Extreme physical exercise
 If you've been previously diagnosed with polycythemia vera, an elevated
hemoglobin level may indicate a need to alter your treatment plan.
 If your hemoglobin level is below or above normal, your doctor may want to
evaluate the hemoglobin test results along with those of other tests, or 13
additional tests may be necessary, to determine next steps.
 If any of your levels are abnormal, it does not
necessarily indicate a medical problem needing
treatment.
 Diet, activity level, medications, a women's menstrual
cycle, and other considerations can affect the results.
In addition, you may have higher than normal
hemoglobin if you live in a high altitude area. Talk to
your health care provider to learn what your results
mean.

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Results

 The normal range for hemoglobin is:

 For men, 13.5 to 17.5 grams per deciliter
 For women, 12.0 to 15.5 grams per deciliter
 Normal ranges for children vary with age and sex.
The range for a normal hemoglobin level may
differ from one medical practice to another.

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Why it's done
You may have a hemoglobin test for several reasons:
 To check your overall health. may test your hemoglobin
as part of a complete blood count during a routine
medical examination to monitor your general health and
to screen for a variety of disorders, such as anemia.
 To diagnose a medical condition. may suggest a
hemoglobin test if you're experiencing weakness, fatigue,
shortness of breath or dizziness. These signs and
symptoms may point to anemia or polycythemia vera. A
hemoglobin test may help diagnose these or other medical
conditions.
 To monitor a medical condition. If you've been diagnosed
with anemia or polycythemia vera, may use a hemoglobin 16

test to monitor your condition and guide treatment.

 hemoglobinometry

A hemoglobinometry is a medical measuring device of hemoglobin blood

concentration such as
Sahli's Hemoglobinometer

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 Sahli's Hemoglobinometer

Sahli's Hemoglobinometer aims to estimate the

haemoglobin content in blood by Sahli's method.
Principle of the experiment
The principle in Sahli's method involves the
conversion of haemoglobin to acid hematin by the
action of HCl.

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 Materials of the experiment

Sahli's haemoglobinometer consists of:

Standard color tube.
A graduated mixing tube.
A capillary measuring tube.
In addition to:
Hydrochloric acid dilute (0.1 N)
Distilled water
Blood lancet -Glass rod
pipette -Cotton and alcohol 19
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 Procedure Sahli's test
1- Place into the graduated mixing tube 5 drops of HCL
(0.1 N) by pipette, until the mark 2
2- By blood lancet, obtain a good sized drops of blood
from finger. Suck up the blood by the capillary tube till
the 0.02 ml (20 cmm) tube mark, and transfer the blood
into the mixing tube.

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 Procedure Sahli's test
3- Put the pipette into HCL solution and suck up a
little of the acid and let it flow down in the
mixture.
4- By the pipette Suck up a little destilate water
and add to the mixture. Repeat 2 to 3 times for
washing the pipette.
5- The mixture after about 2 minutes will be dark
brown and clear.
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 Procedure Sahli's test
6- Add dist water drop by drop, mixing well by a glass rod and
compare the color of this tube with the standard tube.
7- After 5 minutes, read the haemoglobin content from the scale
on the graduated mixing tube.

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 Result

The haemoglobin content= gm/ 100ml of blood

Level of haemoglobin
Normal level of haemoglobin:
In male 14- 16 gm/100ml of blood
In female 12- 14 gm/100ml of blood

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less room lighting
less room lighting
less room lighting

weakness of the sahli method

1, the tool / reagent is less than perfect,

2, the standard color sometimes gets paler,
3, when the person who takes sample is not right,
4, the person is not right to read the results
5, less room lighting
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 Measurement of hemoglobin
 The Cyanmethemoglobin Method for Hb determination is the reference method.
 Principle:
• Whole blood is diluted in a solution of potassium Ferricyanide and potassium
cyanide. (reagent drabkin)
• The Hb is oxidize to methemoglobin by the potassium Ferricyanide.
• The potassium cyanide then converts the methemoglobin to
cyanmethemoglobin.
• The absorbance of the cyanmethemoglobin at 540 nm is directly proportional to
the Hb concentration.
• Sulfhemoglobin is not converted to cyanmethemoglobin; therefore, it can not be
measured by this method.
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  Hb (Fe++) K3Fe (CN)6 methemoglobin (Fe+++ )

KCN Cyanmethemoglobin

Hb (Fe++) K3Fe (CN)6 Methemoglobin (Fe+++ ) KCN

Cyan-Methemoglobin

The principle of Cyanmethemoglobin Method are :

whole blood is diluted in a solution of potassium Ferricyanide and potassium cyanide,
the Hb is oxidize to methemoglobin by the potassium Ferricyanid,
the potassium cyanide then converts the methemoglobin to cyanmethemoglobin,
and the absorbance of the cyanmethemoglobin at 540 nm
is directly proportional to the Hb concentration. 27
Procedure of standard curve

1. Create a standard curve, using a

commercially available
cyanmethemoglobin standard which, has
constant concentration 25g/dl, the following
dilutions should be made to get the line
between the concentration & the
absorbance of the standard using also
drabkin reagent as shown:
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Procedure

Hb concentration Absorbance reading Volume of St / ml V of Drabkin

g/dl reagent / ml

0 0 0 5
5 0.125 1 4
10 0.250 2 3
15 0.375 3 2
7.5 0.188 2.5 2.5

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Procedure

2. Transfer the dilutions to cuvettes. Starting with the blank,

measure the absorbance on a spectrophotometer at 540
nm.
3. Plot absorbance on the y-axis and the Hb concentration
on the x-axis. The Hb concentrations of the patients’
samples and controls can be read from this standard
curve.

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Standard Curve calculation

∆Y
∆X

Concentration of Standard 31
Calculation

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 Discussion

 mechanical sources of error:

 Pipetting error.
 Use of dirty or scratched cuvettes.
 Use of deteriorated reagents.

 Before the test sample is read, the solution should be clear:

A high WBC count: centrifuge specimen and use the supernatant for reading.
 Hemoglobin S (HbS) and Hemoglobin C (HbC), dilute the mixture 1:1 with
distilled water and then read in the colorimeter; multiply the reading by 2.
 Lipemia can also interfere, and a false result can be corrected by adding 0.02 ml
of the patient’s plasma to 5 ml of the cyanmethemoglobin reagent, this solution
being used as the reagent blank. 33
 Drabkin’s reagent is sensitive to light. It should be stored in a brown
bottle or in dark place.
 Carboxyhemoglobin takes up to 1 hr to convert to cyanmethemoglobin
and therefore, theoretically could cause erroneous results in the samples
from heavy smokers. However the degree of error is probably not
clinically significant.
 Because Drabkin’s reagent contains cyanide, it must be used cautiously; a
minimum of four L of reagent is lethal.
 Acid free sinks should be used for disposal of reagent and samples,
because acidification of cyanide releases hydrogen cyanide gas. Copious
amounts of water should be used to flush the sink after disposable.

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The normal ranges

Newborns 17-22gmdl
One (1) week of age 17-22gm\dl
One (1) week of age 15-20gmdl
One (1) month of age 11-15gm\dl
Children 11-13gm\dl
Adult men 14-18gm\dl
Adult women 12-16gm\dl
Men after middle age 12.4-14.9gm\dl
Women after middle age 11.7-13.8gm\dl
• Normal values in an adult are 12 to 18 grams per deciliter
(100 milliliters) of blood. 35
1. Strengths of the Cyanmeth Method
a. Accurate inspection.
b. Reagent and a device for measuring hemoglobin levels can be controlled
with a stable standard solution.
2. Lack of the Cyanmeth Method
a. Tools for measuring absorbance (spectrophotometers or photometers)
are expensive and require electricity
a. Reagent drafkin solution containing cyanide, is toxic

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37
Acuan

 Medline plus; https://medlineplus.gov/lab-tests/hemoglobin-test/

 Berg, Biochemistry (6th Ed) and Campbell Biology (5th Ed)
Johnson RA, Lavesa M, Askari B, Abraham NG, Nasjletti A (February 1995). "A
heme oxygenase product, presumably carbon monoxide, mediates a
vasodepressor function in rats"
http://sickle.bwh.harvard.edu/hemoglobinopathy.html

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39
Hb examination according to Sahli was
classified
into the colorimetry
Basic consept method
In principle, blood Hb is converted to Hematin chloride,
whose color becomes dark brown (tengguli).
the color that occurs is diluted with aquadest to the standard col
of Hematin chloride

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 Instrument : HAEMOMETER"
1, A pair of glass cylinders contain a standard color solution,
we call it a color comparator.
2, Measuring tube (thinner tube) which has lines,
a scale that shows Hb level, The lowest scale is number 2.
3, Capillary blood pipette (Hemoglobin Pipette)
about the size that has a volume of 20 mm3 on the boundary line.
4, Pipette pasteurs for distilled water
5, Stirring glass stems

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Material : HCl 0,1N and Aquadestilata
Examination Method :
1, Prepare the tube and fill it with 0.1 N HCl to the lowest line (in
number 2)
2, With a pipette Hb suction of blood until the number 20 cm do not let
there be air bubbles who was sucked in
3, Pour blood into the diluent tube, rinse with HCl if there is still blood
in the pipette
4, Leave for 1 minute
5, Add the aquadest drop by drop, stirring with a stirring glass rod
6, Compare the solution in the diluent tube with the standard solution
color
7, the color is the same the aquadest addition is stopped, read the Hb
level on 42

that scale in the thinner tube

Pemeriksaan Hb menurut Sahli
digolongkan kepada metoda colorimetri
Dasar
 Prinsipnya, Hb darah diubah menjadi Hematin chlorida, yang warnanya menjadi coklat tua
(tengguli). warna yang terjadi diencerkan dengan aquadest sampai dengan warna standart
Hematin chloride
Alat :
Alatnya disebut "yang HAEMOMETER" terdiri dari :
1. Sepasang cylinder glass berisi larutan standart warna, kita sebut saja pembanding warna.
2. Tabung pengukur (tabung pengencer) yang mempunyai garis-garis, skala yang menunjukan
kadar Hb. Skala yang terendah adalh angka 2.
3. Pipet darah kapiler (Pipet Hemoglobin) seukuran yang mempunyai volume 20 mm3 pada
garis batasnya.
4. Pipet pasteur untuk aquadest
5. Batang glas pengaduk

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Bahan
 Reagen yang diperlukan : HCl 0,1 N dan Aquadestilata
Cara Pemeriksaan
1. Siapkan tabung dan isilah dengan HCl 0,1 N hingga garis yang terendah (pada angka 2).
2. Dengan pipet Hb hisap darah sampai angka 20 cmm jangan sampai ada gelembung udara
yang ikut dihisap
3. Tuang darah ke dalam tabung pengencer, bilas dengan HCl bila masih ada darah dalam pipet
4. Biarkan selama 1 menit
5. Tambahkan aquadest tetes demi tetes, aduk dengan batang kaca pengaduk
6. Bandingkan larutan dalam tabung pengencer dengan warna larutan standart
7. Bila sudah sama warnanya penambahan aquadest dihentikan, baca kadar Hb pada skala yang
ada di tabung pengencer.

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