[IISER Pune][Athale Lab]

LAB- Thin Smear of Peripheral Blood & RBC Counting


First Year Lab for BS/MS students at IISER Pune

26 to 30 Sep 2016 in G1-UG-Bio-Labs, IISER Pune.


Dr. Chaitanya Athale (e m a i l cathale at the rate iiser pune dot ac dot in),

Dr. Krishanpal Karmodiya [website]

Teaching assistants:

Hemlatha Rao, Prabhavati Shinde and Poornima Raveendran


  1. The RBC count data from your practical is being collated. Help us do some interesting statistics by filling out the following form
    [LINK] http://goo.gl/forms/0LtQBGxZ9Q
  2. The sampling error observed in the RBC counting has some statistical background- read more below

Protocol (Procedure)

Click here to download the PROTOCOL (description of materials, methods and observations).

The most interesting images taken by students will be uploaded, if you send them (with your name and batch) to the coordinator of this module Chaitanya Athale (c a th a le at the rate of i i s er pune etc)

Examples of Arthropods

Fig 1: A collage of Arthropods. Copyright Wikipedia.

The colour of blood

Subphylum: Vertebrata has a Fe3-centred Hemoglobin: Red when oxygenated.

Phylum Arthropoda: Cu-centred Hemocyanin: Blue when oxygenated. Not all Arthropods show this, for example those belonging to Class Insecta.

Colour of blood

Red blood from a grouper fish compared to blue blood from a mollusc

Fig 2: Red blood compared to blue blood (Copyright: Stanford University's Hopkins Marine Station, Pacific Grove, CA, USA).


Thigh bone in adult humans contains bone marrow which gives rise to the different lineages of the blood cells found in circulation.


Fig 3: Hematopoesis in adult humans begins with cells exiting the bone marrow (Copyright 2001 Terese Winslow, Lydia Kibiuk)

Blood transfusion and Storage

1914: Albert Hustin & Luis Agote demonstrated Sodium Citrate (Na3C6H5O7) as anticoagulant for blood transfusions

Used at concentrations of 3.2% w/v in H2O

Sodium citrate is a mild irritant

First Recorded Blood Storage

Blood bank: 1935 Dr John S. Lundy, Mayo Clinic, USA.

Citrated blood with glucose stored in an “ice box” for 14 days

Patients given citrated blood helped recovery

Blood Bank Tests

Central Drugs Standards Control Org., Govt of India

Hepatitis B virus (HBV)

Hepatitis C virus (HCV 3.0)

Human Immunodeficiency viruses, Types 1 and 2 (HIV 1,2)

Human T-Lymphotropic virus (HTLV-I/II)

Syphilis (Treponema pallidum)

Anti-Plasmodium antibody

Thin Smear of Blood

Peripheral blood (capillary)

Allows identification of blood types

Blood pathogens

Qualitative measure of infection & inflammation by microscopic examination

Relative proportion of blood cells

Basic and gold standard test in diagnostics

Can be preserved

Cheap and easy to do

1. Making a Thin Smear

Label the slide with your name

Clean with a tissue paper to remove grease, industrial coating

Hold the slides only from the edges

Place on a piece of kitchen towel

Unsheath the lancet from protective pack

Swab the finger to be pricked with using 70% Ethanol which has been soaked in piece of cotton (middle or ring-finger)

2. Smear the Blood Droplet

Smearing the blood droplet- a schematic view

Fig 4: Spreading the blood droplet

3. Discarding Lancet and Swab

Place lancet after use in its original paper cover, and bend it in the middle to ensure no re-use is done

Discard lancet and swab ONLY in designated containers

Use two swabs one before puncturing and one after

4. Fixation & Stain

Air dry (do not heat) blood smear

Dip 5 min in 99% methanol, let it drip dry

Dip 15 min in concentrated Giemsa stain, let it drip dry

Rinse in water, let it drip dry

Observe under a microscope 10x, 40x (air)

5. Clean up

Discard material only in appropriate containers

Clean working area with bleach (or ethanol)

Artefacts in RBC Morphology

Water artifact: humidity or water in fixative

Crenation: due to slow drying

Smudged cells: blood aging


Leishman Stain (Leishman 1901)

Methylene blue: Basic dye for acidic material (e.g. nucleic acids- DNA, RNA). Colour bluish-purple

Eosin-Y: Acidic dye for basic material. Pinkish colour, usually cytoplasmic

Wright-Giemsa Stain (Wright 1902, Giemsa 1904)

Methylene blue: Basic dye for acidic material (e.g. nucleic acids- DNA, RNA)

Azure B: Basic dye for acidic material (e.g. nucleic acids- DNA, RNA)

Eosin-Y: Acidic dye for basic material. Colour pinkish colour, usually cytoplasmic

To Do (and note in Lab-Diaries)

  1. Observe different blood cell types
  2. Note RBC morphologies (ratio bright_diameter:whole_diameter, colour). A ratio of .3-.4 is normocytic
  3. How many morphologically different cell types do you observe?
  4. Draw a schematic figure for each. Note numbers (proportions of different WBC types observed)
  5. Why are acidophilic granules present in some WBCs but not others? What molecules do are stained?


Typically 0.3 times the number of RBCs

Prevent bleeding by aggregation in response to injury

Release potent chemotactic factors to aid wound healing

Originate from megakaryocytes

Comparing Sizes

Ultrastructural image in scanning electron microscope (SEM)

White Blood Cell (WBC) Types

Neutrophil (polymorpho

nuclear leukocytes)

Basophils Eosinophils (Granulocytes) Monocytes Lymphocytes
Neutrophils Basophil Eosinophil Monocyte Lymphocytes
Percentage of total WBCs 40-50% 0.5-1% 1-4% 2-8% 20-40%

Red Blood Cell Count

RBCs do not require staining due to pigmentation. Using citrated-blood the count of RBCs can be made in a calibrated hemocytometer.

High cell densities mean dilution required. Using a Red Blood Cell Dilution pipette we take in 0.5 times volume into the pipette of a lancet-pricked blood droplet. The volume units are arbitrarily selected and the proportionate gradations of 0.1 indicate progression upto 0.5. The remaining pipette volume is made upto 101 (=100 units of volume, marked beyond the bulb) with 3.2% Sodium Citrate. This results in a dilution of the blood by 1:200 (i.e. d f =200).

RBC dilution pipette


A calibrated chamber with grids (XY) and a heavy cover-slip to maintain constant height (Z)- volume constant. The RBC counting region consists of 25 boxes (5x5) bounded by either bold lines (see below left) or triple-lines. The 25 boxes have an area of 1 mm 2 and the z-height is 0.1 mm.

Hemocytometer grid

How to count

Count in each 4x4 box (figure above, right) the SUM OF RBCs

Use L-rule for cells falling on bounding boxes (vertical line in right box, horizontal line in upper box)

Report your values on the board

Use the mean of 25 boxes for a blood cell count per microlitre

To Do (and record in your lab-diaries)

  1. Record your sum value (16 boxes) count
  2. The mean value of 25 boxes for your sample
  3. Calculate blood count in unit: RBCs/ul Discuss deviation from “typical” value
  4. What are the sources of errors?
  5. Quantify the std. dev. of count.

Your images

Image Student Comment
WBCs in a smear (taken from a cellphone down the eyepiece of a microscope 40x*10x) Siddharth Adityhan, 1st yr IISER BS/MS (2-Sep-2014) Spread of WBC's. Zooming in to the interesting areas will however show a rather pixellated view, making unambiguous WBC type identification difficult.

Further reading

Mendiratta et al. (2006) Evaluation of different methods for diagnosis of P. falciparum malaria. Ind. J. Med. Microbio.

Molecular Hematology by Provan & Gribben

Clinical Methods: The History, Physical, and Laboratory Examinations.

Marcinek DJ, Bonaventura J, Wittenberg JB, Block BA. (2001) Oxygen affinity and amino acid sequence of myoglobins from endothermic and ectothermic fish. Am J Physiol Regul Integr Comp Physiol. 280(4):R1123-33. [pubmed]

Stockert et al. (2014) Identifying different types of chromatin using Giemsa staining. Methods Mol Biol. 2014;1094:25-38.

Horobin RW (2011) How Romanowsky stains work and why they remain valuable - including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme. Biotech Histochem. 86(1):36-51.

E Ponder, JF Yeager, HA Charipper (1928) Studies in comparative hematology: I. CAMELIDAE. Experimental Physiology (Physiological Soc.) [Experimental Physiology] This study demonstrated experimentally for the first time (and verified by numerous studies later) that camel RBCs were errorneously thought to be nucleated, due to the dense hemoglobin staining seen in their centres. Infact, RBCs in camels are ellipsoid in shape and have a higher mass in the centre, leading early work (1900's) to suppose it must be due to a nucleus. However even recent textbooks (from 2005) continue to contain inaccuracies in this regard. (PDF of the paper)

RBC counting and statistics of errors

The question of error's in cell-density estimation has been addressed by statisticians. The first person to do so was a statistician William S. Gosset who reported on the errors in cell count estimation using the hemocytometer [1]. The renowned statistician R.A. Fisher in his obituary to the 'Student' (a pseudonym W.S. Gosset took on, to avoid the ire of his superiors) is glowing [2].


[1]. Student. (1907) On the error of counting with a haemacytometer. Biometrika 5: 351-360. PDF

[2] Fisher RA. (1939) Student. Annals of Eugenics 9: 1-9.

Last update: 30-Sep-2016