Collection of Blood and Hemoglobin Estimation

Blood: Blood is a fluid connective tissue that consists of two parts: fluid(60%) and cellular
(40%) part different cells which are RBC, W and platelets in a ratio of 50: 1: 30. in mammals
except camel RBC are circular and non nucleated. where as in camels these are elliptical and non
nucleated, while in birds,reptiles,fishes and amphibians these are oval and nucleated.

Collection of Blood: When blood can be collected from different veins in various animals and
detailed below:

● Cow / large animal ● Jagular(preferable), mammary,

coccygeal

● Sheep,pig ● Anterior venae cover, jugular vein

● Dog ● cephalic, external

saphenous,tibial,Sublingual and
femoral.

● Cat ● cephalic vein, jugular, tibial, femoral

● Chicken ● Wing vein

● Monkey ● Antecubital, jugular, femoral

● Small laboratory animals ● jugular, tail vein,retro orbital venous

plexus, marginal ear vein,heart, toe
nail(small puppy)

Requirements:
● Hypodermic needle
● Syringes(2-5m)
● Collection vial/ tube containing anticoagulant
● Blood/ serum/ plasma

Procedure for collection of blood:

I. Clip the hair from the site of blood collection.
 II. Apply antiseptic( ethanol/ tinctures of Iodine) over the site.
III. Raise the wind by pressure.
IV. Insert the needle into the vein and collect the blood in try syringes for required quantities
of blood( 5 mm ml in case of large animal and 2 mL in case of small animals).
V. Disengage the needle And transfer blood from syringe to anticoagulant containing
vial .Mix the blood is using rotational movement in the vials.

Precautions:
I. Used dry syringes and needles.
II. Never shake the blood vigorously which will cause rupture of blood cells.
III. Don’t fill the blood collecting vials up to top and allow adequate space for mixing.
IV. Remove the needle before filling the collection tube, which may cause haemolysis.
V. Blood should be collected when the animal is at rest.
VI. Do not shake too much time while collecting blood otherwise clot may be formed.
VII. Failure to agitate the blood sample immediately, otherwise clot may be formed.
VIII. Blood smear should be made from fresh blood preferably be me or within 15 minutes
collection of blood.

Hematological tests are to be categorised as follows:

● Erythrocytes related tests Hemoglobin estimation

Packed cell volume

Total erythrocyte count

● Leukocyte related tests Total leukocyte count

Differential leukocyte count

● General / non-specific Erythrocytes sedimentation rate
 Anticoagulants for Haematological Examination

Name Concentration/ Mechanism of

ml of blood action

1.Heller's and Pauľ's 2 mg of oxalate/ml of blood Chelates calcium

oxalate mixture
(Ammonium and
potassium oxalate in
60: 40 ratio)

2.EDTA 1-2 mg/ml of blood

(Ethylenediamine
tetraacetate-
dipotassium ---------do---------
salt,sodium salt
sequestrene, versene)

3.Heparin 10 IU/ml of blood Antithrombin and

antithromboplastin

4.Sodium citrate 3.8% solution in Chelates calcium

1:10 ratio

5.Sodium fluoride & 10 mg sodium fluoride Chelates calcium

thymol
 (10:1 ratio) and I mg of thymol/ml of
blood

6.ACD solution (acid 25 ml ACD solution for 100

citrate dextrose) ml of blood (Dextrose 14.7g

trisodium citrate 13.2 g citric

----------do-----------
acid anhydrous 4.4 g and
distilled water q.s -1000 ml-

autoclaved at 15 lb

pressure for 15 minutes)

Estimation of Haemoglobin
Learning Objectives:
● Basic Structure of Hemoglobin.
● Function of Hemoglobin.
● Various laboratory methods for estimation of hemoglobin.
● Enumerate the advantages and disadvantages of each method.

Introduction:
● Hemoglobin is the major constituent of the red cell cytoplasm, accounting for
approximately 90% of the dry weight of the mature cell.
● It is composed of heme and globin.

Hemoglobin and its forms

The principal component of erythrocytes is hemoglobin (Hb),which makes up about one‐third of
the erythrocyte content, the remainder being water and stroma (structural components).The
hemoglobin molecule has a molecular weight of about 67,000 and is composed of four heme
groups combined with one molecule of globin (the protein component). Globin is composed of
four polypeptide chains, each containing one of the heme groups. Each heme group contains an
iron atom that combines loosely and reversibly with one oxygen molecule.Therefore, one
molecule of hemoglobin contains four molecules of oxygen. The iron atom of heme has a
valence of +2 (Fe2+,ferrous) regardless of whether molecular oxygen is combined with it.
Because of the presence of hemoglobin, blood can transport about 60 times more oxygen than
would be possible by its simple solution. Certain conditions cause the ferrous iron of heme to be
oxidized to its ferric state. In one such condition,nitrate poisoning, the hemoglobin formed is
known as methemoglobin,and it cannot transport oxygen. Another abnormal form of hemoglobin
is carbonmonoxyhemoglobin (sometimes called carboxyhemoglobin). As the name implies,
carbon monoxide occupies the site normally occupied by oxygen.Hemoglobin has an affinity for
carbon monoxide that is about 200 times greater than its affinity for oxygen. Thus, small
concentrations of carbon monoxide compete more favorably for sites on Hb than normal
concentrations of oxygen.The hemoglobin of muscle is known as myoglobin. It differs from
hemoglobin in that it has only one polypeptide chain and one associated heme group, so it can
only combine with one molecule of oxygen instead of four. The concentration of hemoglobin in
the blood of domestic animals averages about 12 g/dL.

Function of Hemoglobin:
● Heme has the ability to bind oxygen reversibly and carry it to tissues.
● It also facilitates the exchange of carbon dioxide between the lungs and tissues.
● Thus, hemoglobin functions as the primary medium of exchange of oxygen and
carbon dioxide.

Normal Haemoglobin concentration in various animals & Poultry:

Species Hemoglobin (g/dL)

● Horse* 14.4

● Cow 11.0
 ● Sheep 11.5

● Pig 13.0

● Dog 15.0

● Chicken 9.0

Physiological variations in HB concentrations:

● Elderly-renal insufficiency,inflammation,testosterone deficiency,diminished
erythropoiesis,myelodysplasia reduce Hb concentration
● Exercise - increases Hb concentration
● Posture - lying to sitting increases Hb concentration
● Altitude - increases Hb concentration.

Indications for Hb estimation:

● Determine presence and severity of anemia.
● Screening for polycythemia.
● Response to specific therapy in anemia.
● Estimation of red cell indices.
● Selection of blood donors.

Methods of Estimation of Haemoglobin: There are various methods for hemoglobin estimation
as given below, but for clinical purposes Sahli’s acid haematin method is best.

1. Colour based: - Based on the colour of haemoglobin or a derivative of haemoglobin.

2. Physical method: –Based on specific gravity.
3. Chemical method: –Based on the iron content of Haemoglobin.
4. Gasometric method :–Based on oxygen combining capacity of Haemoglobin.
 5. Spectrophotometric method:-Based on measurements using spectrophotometric devices

I. Direct matching methods A. Tallquist hemoglobin scale.

B. Dare haemoglobinometer.

II. Oxyhaemoglobin method

III. Cyanmethemoglobin method

IV. Sahil’s is acid haematin method

❖ Colorimetric methods: Colour comparison between standard and test sample by

➢ Visual methods
■ Sahli's acid hematin,
■ Tallquist hemoglobin chart,
■ WHO hemoglobin Color scale,
■ Oxyhemoglobin Method
■ Specific gravity method
➢ Photoelectric methods
■ Cyanohemoglobin method
■ Oxyhemoglobin Method
■ Alkaline Hematin Method

❖ Gasometric Method:
➢ Oxygen carrying capacity measured by Van Slyke apparatus.
➢ Based on formula,1 gm of Hb carries 1.34 ml of oxygen
➢ It does not measure carboxyhemoglobin,sulfhemoglobin,methemoglobin.
➢ Time-consuming and expensive.
➢ Result is 2 percent less than other methods.
 ➢ The principle is based on the fact that one molecule of O2 binds to each iron
atom.
➢ So one molecule of haemoglobin binds 4 molecules of oxygen. Thus oxygen
combining capacity thus indirectly measures the amount of Hb.
➢ It is estimated that 1 gram of haemoglobin binds about 1.34 ml of oxygen.

➢ From this the haemoglobin concentration is calculated by using the following

formula.
■ Hb in gm/dl = (O2 binding capacity in ml/dl blood)/1.34
➢ Disadvantages:
■ This method measures only functional
■ Haemoglobin and not Sulfhemoglobin or carboxyhemoglobin, which does
not bind with oxygen.

❖ Visual comparison method:

➢ Tallquist scale method.
➢ Dare’s method.
➢ WHO haemoglobin colour scale.
➢ Spencer method.
➢ Acid haematin or Sahli’s method.
➢ Alkaline haematin method.
➢ Haldane’s carboxyhemoglobin method
 ➢ BMS hemoglobinometer(comparator).

❖ Talliquist scale method:

➢ Talliquist method involves direct visual matching of red colour of a drop of whole
blood on a filter paper with colour standards on paper.The technique is totally
unsatisfactory with a high degree of error ± 20% to 5o%.
➢ Series of lithographed colors said to correspond to Hb values ranging from 10 to
100 percent.
➢ Blood obtained from finger puncture.
➢ Placed on a piece of absorbent paper.
➢ Colour is matched against the colour on the chart.

➢ Corresponding reading taken.

➢ Cheap and simple.
➢ Error-20 to 50 percent.

❖ Dare’s method:
➢ Here undiluted blood is spread in thin films between glass discs for direct
matching.This method is inaccurate.
❖ Specific Gravity method:
➢ Rough estimate is made from specific gravity of blood.
➢ Copper sulfate technique.
➢ Used in mass screening like selection of donors.

➢ Haemoglobin being the largest single constituent, affects the specific gravity of
blood more than other substances.
➢ Serum proteins are the next heaviest constituents of blood.
➢ It is assumed that (which is not always true) the level of serum proteins and other
smaller constituents remain the same, so any change in the specific gravity of
blood is mainly due to change in concentration of haemoglobin.
➢ Use in blood donor screening:
■ It is used in blood banks as a screening procedure to ensure that the donor
is not anaemic.
■ Since there is no need to know the individual's exact Hb value of the
donor, the blood bank sets a cut off value for men and women and copper
sulphate solutions with corresponding specific gravity are prepared.

❖ Chemical methods (estimation of the iron content):

➢ The principle is based on the fact that each molecule of haemoglobin contains 4
atoms of iron or 0.347 grams of iron per 100 grams of haemoglobin.
➢ The iron present is detached from the haemoglobin and measured.
➢ The haemoglobin is calculated by using the formula.
 ■ Hb(gm/dl) =( Blood iron content in mg/dl blood)/3.47
➢ This is a complex method which is difficult and time consuming but very accurate
and is therefore used as a “reference method “especially by those who are
preparing the Cyanmethemoglobin standard.
➢ It is almost never done in routine clinical laboratories.

❖ Sahil’s is acid haematin method:

➢ Principle:
The basic principle involved includes hemoglobin conversion to acid hematin
using dilute hydrochloric acid and matching the developed colour with comprater/
standard colorimeter

.
➢ Requirements: It consists of following:
■ Comparator of brownish/ amber coloured.
■ Sahli’s haemoglobinometer glass tube with making of gram
percent/ Percentage of haemoglobin.
■ 0.1 N(N/10) Hydrochloric acid.

➢ Procedure:
I. Mix the anticoagulant added in the collection vial by rotation.
II. Take in tutube up to mark 10 percentage side/Mark 2 on hemoglobin side.
III. Draw the anticoagulant added blood up to mark 20mm in the sahli’s
pipette .
IV. Wipe the blood from the tip and if blood is over the mark, reduced by
tapping with finger.
A. Transfer the blood directly into Sahli’s graduated tube containing
HCL. Suck the contents in the pipette and expel in the HCL several
times.
B. The content should be mixed by circular movements/ stirrer and
allow to stand for 5 to 10 minutes.
C. The mixture with distilled water drop by drop,mixing with stirrer
till colour matches with colour of standard.
 D. Record the reading is sahli’s tube and record the concentration of
hemoglobin as g/100 ml of blood.

Precautions:
1. Mix the blood sample by rotation.
2. Wipe out extra blood from the external surface of pipette.
3. Allow sufficient time to complete chemical reaction.

Source of error:
➢ Some inactive forms of hemoglobin such as
methemoglobin/sulfhemoglobin/carboxyhemoglobin in acid solutions.

❖ Spencer method:
➢ In this method,the colour of diluted oxyhaemoglobin is matched visually.This
method is less accurate than Sahli’s method because it is more difficult for the
human eye to accurately grade and match small differences in red colour than the
brown colour of acid haematin.

❖ Alkaline Hematin method: In this method the Hb is converted to alkali haematin by the
addition of N/10 NaOH.
➢ The alkaline haematin gives a brown colour that can be read against comparator
standards or in a colorimeter
➢ Apparatus:
■ Photo electric meter with green filter.
■ N/10 NAOH
■ 0.05 ml pipette
■ Standard(Gibson’s and Harrison’s):This is a mixture of chromium
potassium sulphate,cobaltous sulphate and potassium dichromate in
aqueous solution.The solution is equal in colour to 1 in 100 dilution of
blood containing 16.0 Hb per dl.
➢ Technique:
 ■ Add 0.05 ml of blood to 4.95 ml of N/10 NAOH.
■ Mix well and boil for 4 minutes, along with 5 ml standard solution.
■ Cool quickly in cold water,and match the test against the standard using a
colorimeter using a green filter.If the test gives too high a value add 5.0 ml
of water and read again.

➢ Calculation:
■ If the OD of test =21
■ OD of standard =28
■ As the standard is equivalent to 16 gm per 100 ml,the haemoglobin of the
test will be 21/28 X 16 g per 100 ml =12 g per 100 ml.

❖ Acid Alkali method:

➢ In Alkaline haematin method the solution of Hb has to be heated to ensure
complete denaturation.This procedure can be omitted if the blood is collected first
into acid and after standing for 20-30 min,sufficient alkali is added to neutralize
acid and convert acid haematin to alkaline haematin.
➢ Procedure
■ Add 0.05 ml of blood to 4.95 ml of 0.1 N HCL and immediately mix well.
■ After standing for 20-30 mins,add 0.95 ml of 1 N NAOH and the tube is
inverted several times.
■ After standing for not less than 2 mins, the test sample can be matched in
photoelectric colorimeter using a yellow-green filter using Gibson And
Harrison’s standard.

❖ Haldane carboxyhemoglobin method:

➢ The haemoglobin is converted to carboxyhemoglobin by the action of coal gas on
diluted blood.
➢ Apparatus and requirements:
■ Haldane’s graduated tube and standard.
■ Approximately 0.4% ammonia in D/W.
 ■ 0.02 ml pipette.
➢ Technique:
■ Fill the graduated tube upto the 20 mark with ammoniated D/W.
■ Add 0.02 ml of the patient's blood and mix well.
■ Pass coal gas through this solution for 2-3 minutes.By means of rubber
tubing attaching pasteur pipette to the gas supply.Dip the end of the
pipette into caprylic alcohol and gently bubble the gas through the blood
solution.The caprylic acid prevents frothing.
■ Add 0.4% ammonia drop by drop,mixing between each addition,until the
solution,viewed in diffuse daylight,matches the standard
■ Read the amount of solution in the calibrated tube.The calibration gives
the Hb concentration as a percentage.
■ Calculate the amount of haemoglobin in gm per 100ml of blood.
■ Eg: if the colour standard is defined as corresponding to14.6g/100ml and
reading is 95,then Hb content is calculated as:
■ 100 % = 14.6 g/100 ml
■ 95 % =(14.6 X95)/100 = 13.87 g of Hb per 100 ml blood.

❖ Spectrophotometric method:The estimation is based on Beer’s and Lambert’s law i.e.

the optical density (OD) of a coloured solution is directly proportional to the
concentration of the coloured material in the solution and the pathlength.i.e diameter of
cuvette.Here pathlength is constant i.e 1cm.
➢ It is of 2 type:
■ Cyanmethemoglobin method.
■ Oxyhemoglobin method.

❖ Cyanmethemoglobin Method:
➢ Most accurate method for estimation of Hb.
➢ Recommended by International Committee for Standardisation in hematology
because : -
➢ All forms of Hb are converted to cyanmethemoglobin (except sulfhemoglobin)
➢ Stable and reliable standard is available
➢ Principle
■ Blood is mixed with Drabkin's solution.
■ Drabkin's solution –pH 7.0 -7.4
● Potassium ferricyanide(200 mg)
● Potassium cyanide(50 mg)
● Potassium dihydrogen phosphate(140mg)
● Non-ionic detergent(1 ml)
● Distilled water(1000 ml)
● Note - In the place of Non-ionic detergent,Sterox SE - 0.5 ml,
Triton X-100 – 1 ml, or Saponin - 1 ml may be used

➢ Erythrocytes are lysed producing an evenly distributed Hb solution.

➢ Potassium ferricyanide converts Hb to methemoglobin.
➢ Methemoglobin combines with potassium cyanide to form cyanmethemoglobin.
➢ All Hbs present in blood are converted to this form.
➢ Absorbance is measured in spectrophotometer at 540 nm
➢ To obtain an unknown Hb sample,its absorbance is compared with the standard
cyanmethemoglobin solution.
➢ Requirement - Equipment
■ Photoelectric colorimeter or spectrophotometer
■ Sahlis pipette at 20 microlitre
■ Pipette 5 ml
➢ Procedure:
■ Take 5 ml of Drabkin's solution and to it add 20 microlitres of blood
■ Stopper the tube,mix by inverting several times
■ Allow to stand for 5 minutes
■ Transfer the sample to cuvette
■ Read the absorbance in the spectrophotometer at 540 nm
■ Also take the absorbance of the standard solution.
➢ Calculation:

➢ Advantages
■ All forms of Hb except SHb are readily converted to HiCN.
■ Direct comparison with HiCN standard possible.
 ■ Stability of the diluted sample.
■ Easy to perform the test.
■ Reagents are readily available.
■ The standard is stable.
➢ Disadvantages
■ Increased absorbance not due to haemoglobin may be caused by turbidity
due to abnormal plasma proteins,hyperlipaemia, high WBC count or fat
droplets.
■ Potassium cyanide in the solutions is poisonous, though it is present only
in a very low concentration hence the reagents should be handled
carefully.
■ Explosion can occur if undiluted reagents are poured in the sink.
Hydrogen cyanide is released by acidification and the gas if it accumulates
can result in explosion.
➢ Reagents and samples should be disposed of along with the running water in the
sink.
❖ Oxyhemoglobin Method:
➢ Blood mixed with weak ammonia solution
➢ Absorbance compared with the standard
➢ Rapid and simple
➢ No stable solution is available

❖ Automated Blood Count Method:

➢ Modification of cyanmethemoglobin method.
➢ Other chemicals-sodium lauryl sulphate,imidazole,sodium dodecyl sulphate
➢ Measurements are made at various wavelengths depending on the final stable
product.
➢ 2 type- 3 part Differential Analyzers,5 part Differential Analyzers
■ 3 part Differential Analyzers-
 ● MCV,MCHC,RDW,hematocrit and platelet parameters
● Two chambers-
– Hb/WBC chamber
– RBC/platelets chamber

■ 5 part Differential Analyzers-

● Classify cells as neutrophils,eosinophils,Basophils,lymphocytes
and monocytes
● These provide accurate platelet count,red cell parameters including
various reticulocyte parameters,immature platelets
❖ WHO Hemoglobin Colour Scale:
➢ Useful for screening blood donors
➢ Screening women and children in health programmes
➢ Iron-therapy
➢ Devised by Scott and Lewis
➢ Principle is similar to Tallqvist method
➢ Rapid,simple,inexpensive,reliable
➢ 1 gram/dl for diagnosis of anemia
➢ Printed set of colors corresponding to Hb values from 4-14 grams/dl
➢ Efficiency-greater than 90 percent in detecting anemia
➢ 86 percent-in classifying its grade
 Submitted BY: Karan kamboj

Admission No.2017V137B-IIver

Roll no.BA 2088