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72 Section Three: Haematology
- In anaemia^ due^ to^ acute^ blood^ loss^
and haemolvti.
anaemias, MCV, MCH and MCHC are all within 1ormal limits.
Table 14.2 Causes of abnormal Pcv
DISEASES CAUSING RAISED PCV DISEASES CAUSING LOW PCV 3. In megaloblastic anaemias, MCV is raised above tha i. Polycythaemia (^) i. Anaemia normal range. ii. Dehydration due to severe vomitings, diarrhoea, profuse
sweating
i. Pregnancy
Key Questions for Viva Voce
Q.1. What are^ the^ three^ phases^ in^ ESR^ in^ sequence? ii. Burns (^) Ans. i) Phase of rouleaux formation, i) phase of settling, ii) phase
of packing. iv. Shock Q.2. Why is ESR reading expressed as 'first hour and not 'per hour? value. Table 14.2 lists the conditions causing raised and
lowered PCV.
Ans. ESR has three sequential phases (rouleaux formation, settling and packing) which are completed in one hour. If blood is allowed to stand for longer duration than one hour, it is only the last phase of packing which will be proceeding at a very slow pace and the
reading in^ the^ second^ hour^ will^ not^ be^ equal^ to^
that of first hour
hence, it^ is^ expressed in^ first^ hour^ and^ not^ per^ hour.
ABSOLUTE VALUES (RED CELL INDICES)
Based on normal values of RBC count, haemoglobin and PCV, a series of absolute values or red cell indices can be derived
Q.3. How do we distinguish between Westergren's pipette and
which have diagnostic importance in various haematologic
disorders. These are as under
Wintrobe's tube? Ans. Westergren's is a pipette (i.e. it is open at both ends), is longer (30 cm) and has markings from 0 at bottom to 200 at the top. Wintrobe's is a tube (i.e. closed at one end), is 11 cm long and has markings on two sides of the tube: 0-10 cm on one side and 10-0 cm on the other side.
1. Mean Corpuscular Volume (MCv)
PCV (%)
10 RBC count (millions)
Normal value= 85 +8 f (77-93 f1)* Q.4.^ Which^ anticoagulants^ are^ used^ in^ Westergren's^ and^ Wintrobe's
method?
- Mean Corpuscular Haemoglobin (MCH) Ans. For Westergren's method, 3.8% trisodium citrate in the ratio of 14 is used, while for Wintrobe's method, EDTA or double oxalate
Hb (g dl) is ised.
RBC count (millions) (^) Q.5. Which side of markings in Wintrobe's tube is used for reading Normal range = 29.5 # 2.5 pg (27-32 pg)* haematocrit? Ans. On the side where it is marked 0 at the bottom and 10 at the
3. Mean Corpuscular Haemoglobin
Concentration (MCHC)
top. Q.6. How do we fill the Wintorbe's tube free of air bubbles? Hb (g/dl) Ans.^ Wintrobe's^ tube^ is^ filled from^ below-upwards^ with a Pasteur
pipette having long capillary. Alternatively, blood can be flledin
the narrow tube from below-upwards with a
needle. syringe^ having^ a^ long
PCV (%)
The normal value is 32.5 + 2.5 g/dl (30-35 g/dl). Since MCHC is independent of red cell count and size, itis
considered to be of greater clinical significance as compared
to other absolute values. It is low in iron deficiency anaemia but is usually normal in macrocytic anaemia.
Q. 7. Out of Westergren's and Wintrobe's method, which is à
preferred method?
Ans. Wintrobe's method is
for preferred^ because^ it^ can^ be^ used^ nrst
ESR and subsequently PCV can be done; moreover EDTA blood
4. Red Cell Distribution Width (RDw) drawn^ for^ CBC^ can^ be^ used^ in^ this^ method.
Q. 8. Which of the absolute values
RDWis an assessment of varying volume of red cells based on reliable and why? (or red cell^ indices)^ is^ mo
size (^) of red cells. For (^) example, Iragmented red cells have (^) a (^) tiny size while the macrocytes and reticulocytes have large size.
Ans. MCHC is based on Hb and
haematocrit (^) and is (^) more (^) reliable while MCV (^) and (^) MCH (^) are (^) based on (^) RBC count accurate. which^ is^ not^ alway
Significance of Red Cell Indices
1. In iron deficiency and thalassaemia, MCV, MCH and
MCHC are reduced
O
For conversions, the multiples used are as follows: "deci (d) =
10, milli (m) = 10 , micro (u) =10-5, nano
10 (n) = 10, pico (p) =1012, femto
Exercise 14: ESR, PCV (Haematocrit) and Absolute Values
10
Plasma
Buffy coat
(platelets+
Leucocytes)
Packed RBCs
2
19D 99 A A
10 STOP
FIGURE 14.2 Haematocrit by Wintrobe's tube method.
FIGURE 14.3 Microhaematocrit method for PCV. (Photograph Cenurifuge the tube at 2000-2300 g for 3t minutes. courtesy of Hetlich, Germany through Global Medical System, Delhi). After centrifugation, layers are noted in the Wintrobe tube
as under (Fig. 14.2):
Uppermost layer of plasma.
ii Thin white layer of platelets.
ii (^) Greyish-pink layer of (^) leucocytes v Lowermost is the layer of RBCs.
Grey-white layer of leucocytes and platelets interposed
berween plasma above and packed RBCs below is called
buffy coat.
Note the lowermost height of column of packed RBC layer
and express it as percentage.
Seal the empty end by plastic seal or by heating on flame.
Centrifuge it in microhaematocrit centrifuge at 10,000 g
for 5 minutes (Fig. 14.3).
Measure the blood column by using a reading device
which is usually a part of centrifuge.
Advantages of micro method i. Less amount of blood is required. ii. Results are available within 5 minutes.
Advantages of macro method
. PCV and ESR can be measured simultaneously. . Buffy coat can be prepared for other tests. . By noticing the colour of plasma, we can know about some of the pathological conditions, eg. in jaundice, it is yellow; in haemolysis, it is pink; in hyperlipidaemia, it is milky.
ii. Method is more accurate, trapping of plasma is less.
Sources of errors in macro and micro methods i. Improper handling of sample. ii. Calibration error. ii. Unclean and contaminated tube.
iv. Improper centrifugation time.
- Microhaematocrit Method (^) 3. Electronic Method
s method,^ a^ capillary tube^70 mm^ long with^ an^ internal e ol mm is used and blood from skin puncture is directly
aken into
heparinised (^) capillary tube.
Electronic methods employ automated counters where derivation of RBC count, PCV and MCV are closely interrelated.
Procedure Take aheparinised capillary tube.
Clinical Significance of PCV
PCV reflects the concentration of red cells and not the total Fit with blood^ by capillary action^ leaving^10 mm
unfilled. red^ cell^ mass.^ PCV^ is^ generally^ three^ times^ the^ haemoglobin
Exercise 14: ESR, PCV^ (Haematocrit)^
and Absolute^ Values
20 40 60 80 (^100 2 ) 120
20
6 4 (^160 ) 80 2 1 200
A. WESTERGREN'S PIPETTE B,^ WINTROBE'S^ TUBE^ AND^ PASTEUR^ PIPETTE^ C,^ MICR0-ESR^ PIPETTE
FIGURE 14.1^ Westergren's pipette,^ Wintrobe's^ tube^ with^ Pasteur^ pipette,^ and^ micro ESR (^) pipette.
Disadvantages
i. Requires more amount of blood.
ii. Dilution^ of^ blood^ in^ anticoagulant^ affects^ ESR.
i. Filling of blood by mouth pipetting should be stricty
discouraged.
- Westergren's method
Owing to^ its^ simplicity,^ this^ method^ used^
io se the most
commonly employed^ standard^ method^ pricr^ t^
the AIDS- era. Westergren's pipette is a straight pipette 30 crm ioag open
at both^ ends^ with^ internal^ bore^ diameter^ of^ 2.5^ mn^ and^ is
calibrated from 0-200 mm from top to bottom (Fig. 14.1, A). 2. Wintrobe's Method
Anticoagulant Trisodium^ citrate^ as^ 3.8^ g/dl liquid^ anticoagu-
ant is used. It is used in the^ concentration^ of^ 1:4^ between^ Wintrobe^ tube^ is^ a^ glass^
tube closed at^ one^ end.^ The^ tube^ is
110 mm^ long and^ has^ an^ internal^ bore^ diameter^ of^ 2.5^ mm.
The tube is^ graduated on^ both^ sides:^ from^0 to^10 cm^ on^ one
side and 10 to 0 cm on the other side (Fig. 14.1, B).
anticoagulant and blood.
Procedure
Patient is advised^ to^ come^ after^ overnight^
fast in the
morning fasting (as^ heavy protein^ diet^ affects^
concentra- tion (^) of (^) plasma (^) proteins).
lake 1.6 ml of patient's blood and mix^ it^ with^ 0.4^ ml^ of
e as^ anticoagulant^ already^ put^
in a tube.^ The^ test
SIould be^ done^ within^ two^ hours^ of^ taking^
blood.
the pipette^ up^ to^ mark^0 with^
citrated blood^ with^ the
p of^ rubber^ teat^ by^ vacuum^ filling^
and fix^ it^ in^ a^ rack vertically away from^ sunlight or^ vibrations.
etlt Stand for one hour after which reading is^ taken^ at^ the
upper meniscus of the RBCs.
Normal values
Anticoagulants Either of the following two anticoagulants can be used: i. Ethylene diamine tetra-acetic acid (EDTA) solid crystals 1-2 mg/ml.
ii. Double oxalate (solid) 2-3 mg/ml (ammonium oxalate
and sodium or potassium oxalate in the ratio of 3:2; the
former causes swelling and the latter causes shrinkage
of RBCs and hence RBC shape is retained).
Procedure
The patient is called in the morning fasting.
Draw 2 ml of blood into the anticoagulant.
Fill Wintrobe tube up to mark 0 with anticoagulated blood
with the help of a Pasteur pipette having a long stem so as
to fill^ the^ tube^ ftree^ of^ air^ bubbles^ (Fig. (^) 14.1,B).
Place the tube vertically in a stand and note the ESR after
Males 3-5 mm 1st hour Females 4-7^ mm^ lst^ hour
Advantages Itis a more sensitive method. (^) one hour. easy to fill^ and^ clean^ the^ Westergren's^
pipette.
Exercise
14
ESR, PCV (Haematocrit)
and Absolute alues
Objectives To learn the principle, technique and interpretation of erythrocyte sedimentation rate (ESR) and packed
cell volume (PCV or haematocrit).
To understand the method (^) of finding absolute^ haematological^ values^
and their^ significance.
negative charge of the RBCs that tends to keep them apar
thus promoting rouleaux formation. Albumin retards the
ESR; thus conditions where albumin is low, ESR is more.
ERYTHROCYTE SEDIMENTATION RATE
Erythrocyte sedimentation rate (ESR) is used as an index for
presence of an active disease which could be due to many
iv) Ratio of red cells to plasma The change in the ratio of RBCs
to plasma affects ESR. When plasma is more, ESR will be
increased, and vice versa.
causes.
Principle
When well-mixed anticoagulated blood is placed in a vertical v)Length of the iwce li iength of the tube or pipette is more
tube, the eryhrocytes tend to fall towards the bottom of the RBCs will have to travel a longer distance and thus ESR s
rube pipette till they form a packed column in the lower part
of the tube in a given time.
lower than when length of the tube is short, and vice versa.
vi) Bore^ of the tube If bore of the tube is more, the negative
charge which keeps the RBCs apart will be less and ESR will
be more, and vice versa.
Mechanism of ESR
Fall of RBCs depends upon the following factors:
i Rouleaux formation vii) Position^ of^ the^ tube^ If^ the^ tube^ or^ pipette^ is not^ vertical,^ ne
ii. Concentration of RBCs^ will^ have^ to^ travel less distance and ESR will be more.
fibrinogen in^ plasma
ii. Concentration of a and Bglobulins
iv. (^) Ratio of (^) red cells (^) to (^) plasma v. Length of the tube vi. Bore of the tube
Phases in ESR
ESR takes place in the
following three (^) phases which^ d
carried out in
sequence within^ one hour:
Phase (^) of rouleaux (^) formation: In (^) the initial (^) period
minutes, the process of rouleaux formation
there is occuls
little (^) sedimentation.
vii. Position of the tube
of 0
i) Rouleaux formation The erythrocytes sediment in the tube/
pipette because their density is greater than that of plasma.
When a number of erythrocytes aggregate in the form of
rouleaux and settle down, their area is much less than that of
the sum of the area of constituent corpuscles. The rouleaux
formation is very important factor which increases the ESR.
and
Phase (^) of settling: In^ the^ next (^40) minutes, settlingO
ofRBC
Occurs at a constant
rate. Phase (^) of packing: In^ the^ last (^10) minutes, sedime
entation
slows and
packing of (^) the RBCs (^) to the (^) bottom Ihat is (^) why (^) ESR by all oci ii) Concentration^ of (^) fibrinogen It leads to colloidal (^) changes methods is (^) expressed (^) a
in plasma which causes increased viscosity of plasma.
Concentration of fibrinogen parallels ESR. If concentration of fibrinogen is raised, ESR is increased. In defibrinated blood, ESR is very low.
mmi
hour rather than
per hour.
Methods of ESR
Westergren's method Wintrobe's method
ii) Concentration of a and Bglobulins'These protein molecules
have a greater effect than other proteins in decreasing the
Micro ESR method
- Automated methods
6 Section Three: Haematology
Table13.6 Causes^ of^ eosinophilia^ and^ eosinopenia. EOSINOPHILIA EOSINOPENIA
- Allergic disorders Steroid^ administration
i. Bronchial asthma ii. Urticaria ii. Hay fever iv. Drug hypersensitivity Parasitic infestations i. Roundworm i. Hookworm ii. Tapeworm
iv. EchinococcosisS Leishman's, X1000 Oil
- Skin diseases FIGURE 13.7 Eosinophilia in PBS
i. Pemphigus ii. Dermatitis herpetiformis i. Erythema multiforme 4 Puimonary diseases Löefler's syndrome i. Tropical eosinophilia
- Haematopoietic diseases
i. Chronic myeloid leukaemia ii. Polycythaemia vera i. Hodgkin's disease iv. Pernicious anaemia
- Miscellaneous conditions
i. Rheumatoid arthritis ii. Polyarteritis nodosa ii. Sarcoidosis iv. irradiation (^) Leishman's, X1000 oil
FIGURE 13.8 Basophil in PBS.
Key Questions for Viva Voce
Q.1. Which part of the PBS
should be (^) selected for (^) correct (^) a
Table 13.7 Basophilia. assess*
ment of various blood i. Chronic myeloid leukemia cells? Ans. An area on the
blood smear should be selected
wne theres slight (^) overlapping of (^) RBCs
but neither there is
rouleauinda rmation
nor the cells are
totally (^) scattered. (^) Such (^) an (^) area is
genetay
ii. Polycythaemia vera ii. Myxoedema the (^) junction of (^) the iv. Ulcerative colitis body and (^) the (^) tail.
Q.2. In what
sequence are the
DLC in percentage? lecucocytes (^) expressed v. Hodgkin's disease writing vi Urticaria pigmentosa
Ans.
Generally (^) accepted sequence (^) of (^) writing DLC IS
DLC is: P, L,^ M,^ E,^
B.
Exercise 13:^ Differential^ Leucocyte^
Count
Table 13.^
Causes of lymphocytosis^ and^ lymphopenia. LYMPHOPENIA LYMPHOCYTOSIS
- Acute infections
i. Aplastic anaemia
i. Pertussis ii.^ High^
dose of^ steroid
ii. Infectious mononucleosis administration ii. Viral hepatitis ii.^ AIDS
- Chronic infections
iv. Hodgkin's disease
i. Tuberculosis v.Irradiation i. Brucellosis ii. Secondary syphilis
Q
- Haematopoietic disorders . CLL
Leishman's, X1000 Oil ii. NHL
FIGURE 13.5^ Lymphocytosis^ in PBS.
Lymphocytes Eosinophils
When the^ absolute^ lymphocyte^ count^ increases^ to^ more^ than
4.000 ul^ it is^ termed^ /ymphocytosis^ (Fig.^ 13.5)^ while^ absolute
Nmphocyte count^ below^ 1,500/ul^
is caled lymphopenia;
causes tor^ these^ are^ given^ in^ Table^ 13.4.
Increase in the absolute^ eosinophil count^ above^ 400/ul^ is
termed eosinophilia (Fig. 13.7)^ while^ the^ fall^ in^ number^ is
called eosinopenia; the^ causes^ for^ abnormal^ eosinophil^ count
are given in Table 13.6.
Monocytes sogh
A Tise in absolute^ monocyte count^ above^ 800/ui^ is^ called
monocytosis (Fig. 13.6).^ Causes^ of^ monocytosis^ are^ given^ in
Table 13.5.
Basophilia refers^ to^ an^ increase^ in^ the^ absolute^ basophil
count above 100/ul (Fig. 13.8). Causes of basophilia are given in Table 13.7.
Table 13.5 Monocytosis.
- Bacterial infections i. Tuberculosis i. SABE ii. Syphilis
- Protozoal infections i. Malaria ii. Kala azar ii. Trypanosomiasis
- Haematopoietic disorders i. Monocytic leukaemia ii. Hodgkin's disease
ii. Multiple myeloma
iv. Myeloproliferative disorders
Leishman's, X1000 Oil
- Miscellaneous conditions
PIGURE (^) 13. Monocytes in^ PBS.
i. Sarcoidosis ii. Cancer of ovary, breast, stomach
6 Exercise 13:^ Differential^ Leucocyte^
Count
Table13.1 Normal^
values for^ leucocytes^ in^ health^ in^ adults
NORMAL RANGE ABSOLUTE^ VALUE
Polymorphs (P) 40-75%^ 2,000-7,500/ul
Lymphocytes () 20-40%^ 1,500-4,000/u!
Monocytes (M) 2-10% 200-800/u
Eosinophils (E) 1-6%^ 40-400/ul Basophils (B) 0-1% 10-100/ul
C (^) Monocyte The monocyte is the largest mature leucocyte in the peripheral
blood measuring 12-20^ um in^ diameter.^ It^ possesses^ a
large, central,^ oval,^ notched^ or^ indented^ or^ horseshoe
shaped nucleus^ which^ has^ characteristically^ fine^
reticulated
chromatin network.^ The^ cytoplasm is^ abundant,^ pale^ blue
and contains many fine granules and vacuoles.
FIGURE 13.3 Counting of WBCs for DLC in DLC counters with pressing Eosinophil kevs Photograph courtesy of Yorco Sales Pvt Ltd, New Delhi).
Eosinophil is similar to segmented neutrophil in size (12-
um in^ diameter)^ but^ has^ coarse,^ deep^ red^ staining granules^ in
the cytoplasm and has usually two nuclear lobes in the form
Lymphocyte of^ a^ spectacle.
Majority of lymphocytes in the peripheral blood are small asophii
(9-12 um in diameter) but large lymphocytes (i2 if pm in
diameter) are also found. Both smailand large t; mphocytes Basophil resembles the other mature granulocytes but is
tave round or slighly indented nuclei with coarsely disiinguished by coarse, intensely basophilic granules which
ciumped chromatin and scanty basophilic and agranular usualy fil the cytoplasm and often overlap and obscure the
CVTOplasm. nucleus.
Table13.2 Morphology of mature leucocytes
FEATURE NEUTROPHIL LYMPHOCYTES MONOCYTE EOSINOPHIL BASOPHIL
(SMALL AND LARGE)
Morphology
Cell diameter SL:9-12 um 12-20 umn 12-15 um 12-15 umn
LL: 12-16 um
12-15 um
Nucleus Large nucleus, round to indented, fills the
Large, lobulated, Bilobed,
indented, with fine clumped chromatin
Bilobed,
clumped
chromatin
2-5 lobed,
clumped
chromatin cell,^ clumped^ chromatin chromatin Cytoplasm Peripheral^ rim^ of
basophilic cytoplasm,
no granules
Light basophilic,
may contain fhine granules or vacuoles
Coarse Large coarse
purplish granules
obscuring the
nucleus
Pink or violet granules crimson red granules
Normal% (^) 40-75 20-40 -10 1-6 (^) 0- iDsolute count (^) per l 2,000-7,500^ 1,500-4,^
Exercise
13
Differential Leucocyte Count
Objectives
To learn^ the method (^) ofexamination of blood smear^ for^ differential^
leucocyte count^ (DLC)^ and^ morphologic
features of mature leucocytes.
To know^ various^ techniques of DLC and^ to^ perform^ DLC^ by^
manual methods.
lo know the normal range of^ mature^ leucocytes in^ blood^ and^
various conditions^ producing^ their^ variatione
in diseases.
of a characteristic dense nucleus, having 2-5 lobes and pale
cytoplasm containing^ numerous^ fine^ violet-pink^ granules.
EXAMINATION OF PBS FOR DLC
Choose an area near the junction of body with the tail of the smear where there is slight overlapping of RBCs, i.e. neither rouleaux formation which occurs in the head and body, nor totally scattered RBCs as occurs at the tail. By moving the slide in horizontal directions under oil immersion (Fig. 13.), start counting the types of WBCs and go on entering P, L, M, E, B in
a box having 100 cubes as shown in Figure 13.2. Alternatively,
100 leucocytes can be counted by pressing the keys of the
automated DLC counter (Fig. 13.3). Zigzag counting of WBCs
is discouraged. WBCs are then expressed as percent in the
folowing sequence: polymorphonuclear leucocytes (P),
iymphocytes (L), monocytes (M), eosinophils (E), basophils
(B), i.e. P L, M, E, B. Invariably, normal range is expressed alongside the results (Table 13.1).
L P P
P
P P P L P
P P (^) L P (^) P L
L
P L (^) M (^) P M
B (^) E (^) P E
MORPHOLOGIC IDENTIFICATION OF MATURE
LEUCOCYTES
M (^) P (^) P
Polymorph (Neutrophil) P^ E^ P P P L L
A polymorphonuclear neutrophil (PMN), commonly called
polymorph or neutrophil, is 12-15 um in diameter. It consists P^ P^ P
Result of DLC
M E
FIGURE 13.1 Counting of cells in PBF by moving in horizontal direction at the junction of the body with the tail of PBF.
FIGURE 13.
Counting (^) of (^) WBCs for (^) DLC in
result of DLC. squares a
expressing
60 Section Three: Haematology
Methylene blue^ is^ the^
basic dye and^ has^ affinity^ for
component of^ the^
cell (i.e. nucleus)^
and (^) eosin/azure
acidic dye and^ has^ affinity^
for basic^ component^ of^ celu
cytoplasm). Most Romanowsky^ stains^
are prepared in^ methyl alee
so that^ they combine^
fixation and^ staining. Various stains^ included^
under Romanowsky gro
dyes are^ as^ under:
i. Leishman's stain
acidic
Head Body Tail is^ th
ie
alcohol
roups of
ii. Giemsa stain
FIGURE 12.2 Parts of a thin blood smear.
ii. Wright stain iv. Field stain V. Jenner stain vi. JSB stain.
Cover Glass Method
Staining of^ Thin^ Blood^
Smear
Procedure Leishman's Stain
Take a number1 (22 mm square) clean cover glass. Touch it on to the drop of a blood. Place it on another similar cover glass in crosswise direction with side containing drop of blood facing down. Pull the cover glass quickly. Dry it and stain it. Mount it with a mountant, film side dowm on a clean glass slide.
Preparation Dissolve^ 0.2^ g^ of^ powdered^ Leishman's^ dve^ in
100 ml of acetone-free methyl alcohol in a conical flask. Warm it to 50°C for half an hour with occasional shaking. Cool it and filter it. Procedure for staining
Pour Leishman's stain dropwise (counting the drops) on
the slide and wait for 2 minutes. This allows fixation of the
PBF in methyl alcohol.
Acdd double ihe qu1antity of buffered water dropwise over
the stide (i.e. iruble the number of drops of stain).
Mix by rocking or blowing for 8 minutes. Wash in water for I to 2 minutes.
Dry in^ air^ and^ examine under oil immersion lens of the
microscope.
Spin Method
This is an automated method.
Procedure
Place a drop of blood in the centre of a glass slide. Spin at a high speed in a special centrifuge, cytospin. Blood spreads uniformly. Dry it and stain it.
Giemsa Stain
Preparation Stock^ solution of (^) Giemsa stain is (^) prepared mixing 0.15 g of Giemsa powder in 12.5 ml of glycerine andby
12.5 ml of methyl alcohol. Before
use, (^) dissolve one (^) volune
of stock solution in nine
volumes of (^) buffered water (^) (diludol
THICK BL0OD SMEAR
This is prepared for detecting blood parasites such as malaria
and microfilaria.
Procedure Procedure
Place a (^) large drop of blood in the (^) centre (^) of a clean (^) glass (^) Pour
diluted stain over slide or
immense (^) blood (^) sinear staining trough. Wait (^) for 15-60 (^) minutes. Wash in water. Dry it^ and (^) examine (^) under oil (^) immersion (^) lens microscope.
slide.
Spread it ina circular area of 1.5 cm with the help ofa stick
or end of another glass slide. Dry it and you should be able to just see the printed matter
through the smear, when kept on printed paper. f^ the
STAINS FOR BLOOD SMEAR
Staining of^ Thick (^) Blood
Romanowsky stains are universally employed for staining Smear
of blood smears. All Romanowsky combinations have two
essential ingredients, i.e. methylene blue and eosin or azure.
It can be stained with
any of the
Romanowsky (^) std
listed
above except that
before (^) staining, the smear is^ a
haemo globinised (^) by (^) putting it (^) in
distilled water for 10
minute
Exercise Preparation and^ Staining
of
Exercise
Peripheral Blood Smear
Objectives
To learn the technique of making thin and thick blood smear and theirsignificance
To know and perform various routine stains used for staining blood films.
The peripheral blood smear (PBS) is of two types:
. Thin^ blood^ smear
- Thick^ blood^ smear.
Move the spreader backward so^ that^ it^ makes^ contact^ with
drop of blood.
Then move^ the^ spreader rapidly forward^ over^ the^ slide.
A thin^ peripheral^ blood^
smear is thus prepared (Fig. 12.1).
Dry it^ and^ stain^ it.
THIN BLOOD SMEAR
Thin PBS^ can^ be^ prepared^ from^ anticoagulated^ (EDTA)^ blood
obtained by venipuncture or from free fowing finger prick
blood by any of the following three^ techuicques
- Slide method
Qualities ofa Good Blood Smear i. It should not cover the entire surface of slide.
i. it should have smooth and even appearance.
- Cover glass method
- Spin method.
ii. It should be free from waves and holes. iv. It should not have irregular tail.
Slide Method Parts of a Thin Blood Smear
A PBS consists of 3 parts (Fig. 12.2):
1. Head, i.e. the^ portion of^ blood^ smear^ near^ the^ drop of
blood.
Procedure
Place a drop of blood^ in^ the^ centre^ of^ a^ clean^ glass^ slide
I to 2 cm from one end.
Place another^ slide^ (spreader)^
with smooth^ edge at^ an
angle of^ 30-45°^ near^ the^ drop^ of^ blood.
- Body, i.e. the main part of the blood smear.
- Tail, i.e. the tapering end of the blood smear.
Spreader
12.1 Method ofmaking thin^ PBS^ by^ slide^
method.
FIGURE
(59)
Exercise 11:^ Counting of Blood^ Cells
WBC counting
RBC counting
Platelet counting
FIGURE 11.2 Improved Neubauer's 0.1 mm. Counting areas for WBCS, RBCS and platelets are depicted by different colours diagrammatically though the improved Neubauers chamber does not have any such colours.
mber. Each corner and central large square have an arm of 1 mm while the chamber has depth of
Number ofWBCs in 0.4 mm3 the light and convert bya detector into
pulses proportionate
to the size of the cells, which are then counted electronically.
A lysate is used to lyse red cells so as to count WBCs.
n nx 10
Number of WBCs in 1 mm
4 Dilution factor (^20)
Advantages
Number of WBCs per mm* (ul) = (^) X 20 4 i.^ Easy^ and^ rapid^ method. ii. Time saving method.
ii. Very large number of cells is counted rapidly.
iv. There is high level of precision.
= nx 50 Where n is the total number of WBCs counted in 4 corner Squares.
Precautions Disadvantages i. Costly equipment.
i The workbench must be free of vibrations and chamber ii. Calibration error.
should not be exposed to heat.
L The cover glass^ should^ be^ of^ special^ thickness^ and
should have perfectly flat surface.
L. The chamber area should be completely filled leaving Normal Range for WBC Count no air bubbles or debris in the chamber area.
iv. The fluid should not overflow to the moat
surrounding Infants at birth
ii. Nucleated RBCs are counted as
leucocytes.
iv. Platelet clumps counted as
leucocytes.
Adults (^) :4,000-11,000/ul :10,000-26,000/ul
Children under 1 year : 6,000-18,000/ul
the ruled area on the chamber.
Electronic Method (^) Causes of (^) Increased WBC Count (^) (Leucocytosis)
Electronic counter is based on the principle of aperture
npedance method, or light scattering technology, or both. In
S,particles passing through a chamber in single file scatter
i. Microbial infections e.g. bacterial, viral,
ii. Leukaemias parasitic
ii. Leukaemoid reactions
Exercise
11
Counting
of Blood^
Cells
To learn the^ principle, techniques^ and^ interpretation^ of^
counting ofWBCs,^ RBCs,^ platelets and^ eosinophils
To know^ their^ normal^ values^ and^ conditions^ producing^
abnormal counts^ of these^
blood cells.
Objectivess
Counting of^ circulating blood^ cells^ is^ a^ basic^ screening^
blood
test and includes^ counting of^ leucocytes^ (total^ and^ differential,
i.e. TLC.^ DLC), red^ cells^ and^ platelets,^ and^ sometimes,
eosinophils. However,^ the^ term^ complete^ blood^ counts^ (CBC)
is commonly used^ these^ days for^ the^ following^ panel^ of^ tests
determined by electronic particle counter:
- Evaluation of WBC: TLC, DLC
2. Eraluation of
haematocrit (Hct), red cell indices (MCV, MCH, MCHC,
red cell distribution width or RDW)
- Evaluation of platelets: Platelet count, mean platelet
volume (MPV) and platelet distribution width (PDV).
A, WBC PIPETTE
RBCs: RBC count, haemoglobin,
B, RBC PIPETTE
0.02 ml
WBC COUNT This is determination of number of white blood cells per pl of C.^ HAEMOGLOBN^ PIPETTE
blood. FIGURE 11.1^ Pipettes^ for^ WBC^ (A)^ and^ RBC^ counting^ (B)^ contrasted
with haemoglobin pipette (C).
Methods
Draw diluting fluid up to mark 11 in the WBC pipette to
get dilution of 1:20.
Mix well by rotating the pipette for 2-3 minutes.
Charge the Neubauer's chamber (haemacytometer)after
discarding1-2 drops of the mixture from the WBC pipette
Allow the cells to settle down for 2 minutes.
Count the WBCs under low power (10X) in 4 large corner
squares (Fig. 11.2). Count the cells lying on left and lower
lines (^) while (^) ignoring those on its (^) right and (^) upper (^) lines
There are two methods:
1. Visual haemacytometer method
- Electronic method
Visual Haemacytometer Method
Principle This is counting of WBCs in a calibrated chamber
by diluting of^ blood^ to^ 1:20^ dilution^ with^ diluent^ which^ causes
lysis of^ RBCs^ and^ stains^ WBCs.
Diluting fluuid Turk's fuid is used which has the following
composition:
Glacial acetic acid
Calculations*
3.0 ml
1% Aqueous gentian violet : 2.0 ml Volume of^ area^ in^ which^ WBCs
Distilled water
counted in 4 corner squares = [(1x1x0.1) x 41 mm
= 0.4 mms
195 ml
Procedure
Suck anticoagulated blood or blood from finger prick up
to mark 0.5 in WBC pipette (Fig. 11.1, A).
Wipe tip and outside of the pipette.
For calculation of count of WBCs, RBCs and
platelets s
Neubauer's chamber, please remember the dimensions ot
squares of the chamber are 1 mm each side and depth 0.1 mm.co
Volume = Length x Breadth x
Depth
of colours^ which^ represent^ haemoglobin^
concentration Disadvantages
(Fig. 10.2).^ Thus,^ no^ calculations are (^) required to^ be^ made.^ i. Since it is manual^ comparison^ of colour^ change, thor
can be visual^ erroor.
here
Disadvantage ii.^ Carboxy-,^ met-^
and sulthaemoglobins^ cannot^ he
converted to^ acid^ haematin.
Calibration of the instrument can be faulty.
ii. Glass^ comparator can^ fade^
over the years.
iv. Colour^ of^ acid^ haematin^
also fades^ quickly. Sahli's Method
Principle Hb^ is^ converted^ into^ acid^ haematin^ with^ the^
action
of dilute^ hydrochloric acid^ (N/10 HCI). The^ acid^ haematin^ is brown in colour and^ its^ intensity is^ matched^ with^ a^ standard brown (^) glass comparator in^ a^ visual^ colorimeter^ called^ Sahli's
colorimeter.
Procedure
Fill Sahli's Hb tube up to mark2 with N/10 HCI.
Deliver (^20) l (0.02 ml) (^) of blood from^ a^ Hb^ pipette into^ it. Stir with a stirrer and wait for 10 minutes.
Add distilled^ water^ drop (^) by drop and^ stir^ till^ colour
matches with the comparator
Take the reading at upper meniscus (Fig. 10.3).
Advantages
i. Simple bedside test.
ii. Reagents and apparatus are cheap. (^) FIGURE (^) 10. Apparatus used for Sahli's^ haemoglobinometry.
