The document outlines various methods for blood corpuscle counting and the study of formed cellular elements in blood, detailing the composition of blood, types of blood disorders, specimen collection methods, and laboratory tests. It discusses the interpretation of results from tests like CBC, Hb estimation, and differential counts, as well as specific disorders associated with RBC, WBC, and platelet counts. Additionally, it emphasizes the importance of recognizing anemia and its underlying causes, as well as the methodological approaches to diagnosing blood-related conditions.

1. BLOOD CORPUSCLES COUNTING
METHODS

2. Haematology
• Study of the formed cellular blood elements
• Blood is composed of a Liquid called Plasma
& of cellular elements (RBC’S, WBC’S &
Platelets)

3. Blood plasma is the liquid component of blood, in
which the blood cells are suspended.

4. Blood serum is blood plasma without fibrinogen or the
other clotting factors.

5. How to approach
• Medical history & Physical examination
• Select appropriate tests
– Cost- effective
– Specific test

6. To diagnose various disorders
• Disorder of blood cells
– RBC Eg: anemia, polycythemia
– WBC Eg: leukemia,leukopenia
– Platelets Eg: thrombocytopenia, thrombocytosis
• Disorder of clotting factors
• Infections
– Malaria
– Babesiosis
– Microfilaria

7. Specimen collection
• Phlebotomy
• Anticoagulants –
• EDTA – (Lavender)-CBC
• Sodium citrate- (light blue) - Coagulation
Profile
• Heparin – (Green) – Osmotic fragility

8. Collection of Blood

9. Bedside Lab Tests
• Hb %
• CBC
– Total WBC Count
– Total RBC Count
– Differential Count
– Platelet count
• Hematocrit
• Red cell Indices
• ESR
• Reticulocyte count
• Peripheral smear Examination

10. In cases of bleeding disorder
• Screening tests
1. Platelet count
2.Bleeding Time
3. Clotting Time
• Special tests
1. Prothrombin Time (PT)
2. Activated Partial Thromboplatsin Time (APTT)
3. Thrombin Time (TT)
3. Factor assay

11. Haemoglobin estimation
• Physical – Based on Specific gravity
• Chemical –Based on Iron content of Hb
• Gasometric-Based on O2 combining
capacity of Hb
• Calorimetric-Based on colour
• Photoelectric – Cynmethhemoglobin
method
• Automated methods

12. Sahli’s Haemoglobinometer

13. Photoelectric colorimeter withgreen
filter or at a wavelength of 540nm
• Cynmethaemoglobin method

14. Total WBC count
• Visual haemocytometric method
• Electronic method
• Instruments: WBCpipette,
Neubauerchamber,Lancet, cotton, Micro-
scope, Cover slip and WBC diluting fluid
(Turke’s fluid)

15. Neubauer Chamber - Total WBC
count -Counting areas (Blue)

16. Causes
Exercise
Leukemoid reaction
Bacterial and viral infection
Infectious mononucleosis
Causes
Aplastic anaemia
Typhoid
Drugs
Radiation therapy
Cytotoxic therapy
Subleukemic leukemia

17. Neubauer Chamber - Total RBC
count -Counting areas (Orange)

18. Normal Values
Adult male 4.5 to 5.5 million/cu.mm
Adult female 3.8 to 5.2 million/cu.mm
At birth 4.0 to 6.0million/cu.mm
Increased cell count
Physiological -At birth
Pathological *Haemoconcentration
Diarrhea
Dehydration
*Cyanotic heart diseases
*Chronic lung diseases
*Polycythemia vera
Decreased cell count
-Anaemia
-Haemodilution

19. Differential count

20. Differential count

21. Normal values
• Neutrophils – 50- 75%
• Lymphocytes – 25 – 45%
• Monocytes – 3-8%
• Eosinophils – 1-6%
• Basophils – 0-1%

22. Haematocrit (PCV)
• Percentage of erythrocytes in a known
volume of whole blood
• Useful – Anaemia & Polcythaemia
• Wintrobe’s tube filled with anticoagulated
blood & centrifuged at 3500rpm for 30
minutes.
• The volume occupied by the erythrocytes
is expressed as a percentage of the total
volume.

23. Centrifuge - PCV

24. Centrifuge - PCV
Plasma colour
Clear - Normal
Yellow-Indirect jaundice
Pink -Haemolysis
Turbid –Hyperlipediamias

25. PCV
• Normal values
Males 40 to 55%
Females 35 to 48%
Infants 45 to 60%
• Clinical significance
Increase PCV = Polycythemia, dehydration, burns,
and shock.
Decrease PCV = Anaemia, pregnancy.
• Sources of error
Sample
-Increased amount of EDTA
-Inadequate mixing of blood
Tubes
-Variation in the bore size
-Improper filling of the tube

26. Red cell indices
• MCV
• MCH
• MCHC
Useful for Morphological classification of
Anaemias

27. The Three Derived Indicies
Measurement Normal Range
RBC count 5 million 4 to 6
Hemoglobin(Hb) 15 g% 12 to 17
Hematocrit(Hct) 45 38 to 50
MCV Hct ÷ RBC x 10 = 90 fl
MCH Hb ÷ RBC x 10 = 30 pg
MCHC Hb ÷ Hct x 100 = 33%

28. Normal values
• MCV - 81 to 85 fl
• MCH - 29 to 32 pg
• MCHC - 31 to 33 %
MCV MCH MCHC
Microcytic Hyocromic
anaemia
Macrocytic anaemia
Spherocytosis

29. Erythrocyte Sedimentation Rate
The rate at which the RBC’s settle from the plasma.
• Stage I- Rouleaux formation(Aggregation)
– RBC’s come together like piles of coins, last for 10 mts.
• Stage II- Stage of sedimentation
– RBC’s settle at a constant rate-last for 30 mts.
• Stages III -Stage of packing
– Packing of RBC’s occurs –last for 10 mts.
Methods – Westergren & Wintrobe’s

30. Erythrocyte Sedimentation Rate

31. Procedure
• Take 1.6ml of blood in a vial
• Add 0.4ml of anticoagulant and mix
• Draw the blood up to the 0 mark in the tube
• The tube is set up in the rack perfectly vertical.
• Start a stop watch or clock and take reading
• The reading corresponding to the upper layer of
the RBC column in the pipette is taken.
• Normal
– Male = 0-10mm/hr
– Female = 0-20mm/hr

32. Factors Influencing ESR
• Rouleaux formation -↑ ESR
• Ratio of red cells to plasma
When plasma is more - ↑ ESR
When plasma is less - ↓ ESR
• Length of the tube-
If length of the tube is more –ESR low
If length of the tube is short -ESR ↑
• Bore of the tube
If the bore of the tube is more → ESR will be more
If the bore of the tube is less → ESR will be less
• Position of the tube
Deviation from vertical position = ↑ ESR
• ↑ Plasma globulin and Fibrinogen – ↑ ESR
↓Plasma globulin and Fibrinogen decreased – ↓ESR

33. Interpretation: -
• This is not a specific test.
• An increase in ESR usually seen in
chronic inflammatory conditions like
tuberculosis, rheumatoid arthritis or
malignancy.
• The test is useful in following disease
activity or response to treatment once a
diagnosis is made.

34. Platelet count
• Venous blood(Avoid finger prick
blood),RBC Pipette , Neubauer Chamber
Microscope , Disposable syringe, Spirit ,
Cotton,Cover slip
• Diluting Fluid for Platelet Count:
• 3% sodium citrate containing 1%
formalin also can be used as diluting
fluid. It, however, does not lyse the red
blood cells.

35. Abnormalities Of Platelet Count
• Normal – 1,50,000 – 4,00,000/cu mm (µl).
• Increased Platelet count:
» Chronic Myeloid leukaemia
» Acute hemorrhage
• Decreased Platelet Count:
– ITP , DIC
Autoimmune disease like Systemic Lupus Erythematosis
Viral infecton

36. Peripheral Smear
• First clean the finger with cotton and spirit.
• Puncture the pulp of the index finger.
• Wipe the first drop because it will contain tissue fluid.
• Keep one drop of blood in the end of a glass slide.
• Now spread the blood into a thin smear with a
spreader slide and keeping the spreader slide at 45 °
• Dry the smear and stain with Leishman stain.
A good smear must be tongue shape, should be
evenly spread, should occupy one third of the
slide

37. PS

38. Staining procedure
• Take leishman Stain and Pour drop by
drop and cover the smear.
• Wait for 2 minutes for fixation of smear by
Methyl alcohol.
• Wait for two minutes, add equal amount of
distilled water and wait for 5-10 mins.
• Wash the slide with distilled water or tap
water try the smear and examine it under
oil immersion objective.

39. • A Normal mature Lymphocyte is seen on the left
compared to a segmented PMN on the right. An
RBC is seen to be about 2/3 the size of a normal

40. • Identify the Segmented Neutrophil, Band
Neutrophil, Lymphocyte, Monocyte, Eosinophil,
Basophil, and Platelet in the image below:

41. • The RBC's in the background appear normal. The important finding here
is the presence of many PMN's. An elevated WBC count with mainly
neutrophils suggests inflammation or infection. A very high WBC count
(>50,000) that is not a leukemia is known as a "leukemoid reaction". This
reaction can be distinguished from malignant WBC's by the presence of
large amounts of leukocyte alkaline phosphatase (LAP) in the normal

42. • The RBC's here have stacked together in long chains. This is
known as "rouleaux formation" and it happens with increased
serum proteins, particularly fibrinogen and globulins. Such long
chains of RBC's sediment more readily. This is the mechanism
for the sedimentation rate, which increases non-specifically with
inflammation and increased "acute phase" serum proteins.

43. RBC MORPHOLOGICAL
ABNORMALITIES
SICKLE CELLS
• Morphology:
Sickle
shaped red
cells
• Found in:
Hb-S
disease

44. RBC MORPHOLOGICAL
ABNORMALITIES
TEAR DROP CELLS
• Morphology:
Red cells shaped
like a tear drop or
pear
• Found in:
Bone marrow
fibrosis
Megaloblastic
anaemia
Iron deficiency
Thalassaemia

45. RBC MORPHOLOGICAL
ABNORMALITIES
MALARIAL PARASITE (P. falciparum)
• Morphology:
Ring form of Pl
falciparum in red
cells. Delicate
rings with 1 or 2
chromatin dots.
Often more than
one ring in a red
cell. Accolé forms
are found.
• Found in:
Malaria

46. • The RBC's here are smaller than normal and have an increased zone of
central pallor. This is indicative of a hypochromic (less hemoglobin in each
RBC) microcytic (smaller size of each RBC) anemia. There is also increased
anisocytosis (variation in size) and poikilocytosis (variation in shape).

47. • This Hypersegmented Neutrophil is present along with
Macro-ovalocytes in a case of pernicious anemia. Compare
the size of the RBC's to the lymphocyte at the lower left center.

48. • There are numerous fragmented RBC's seen here. Some of
the irregular shapes appear as "helmet" cells. Such fragmented
RBC's are known as "schistocytes" and they are indicative of
a Microangiopathic hemolytic anemia (MAHA) or other
cause for intravascular hemolysis. This finding is typical for
disseminated intravascular coagulopathy (DIC).

49. • The WBC's seen here are "atypical" lymphocytes. They are atypical
because they are larger (more cytoplasm) and have nucleoli in their nuclei.
The cytoplasm tends to be indented by surrounding RBC's. Such atypical
lymphocytes are often associated with Infectious Mononucleosis.

50. • Here is another example of Sickled Erythrocytes in a patient with Hgb SS
who presented with severe abdominal pain in sickle crisis. The sickled cells
are prone to stick together, plugging smaller vessels and leading to
decreased blood flow with ischemia.

51. WBC MORPHOLOGICAL
ABNORMALITIES
TOXIC GRANULATION
• Morphology:
Increased
granulation.
Granulation more
basophilic and
larger than normal.
• Found in:
Severe bacterial
infection
Non specific finding
- seen in tissue
damage of various
types.
Normal pregnancy
Therapy with
cytokines

52. WBC MORPHOLOGICAL
ABNORMALITIES
DOHLE BODIES
• Morphology:
Small pale blue
cytoplasmic
inclusions, often in
the periphery of the
cell.
• Found in:
Infective and
inflammatory states
Severe burns
Tuberculosis
Post chemotherapy
Pregnancy

53. • ACUTE LYMPHOBLASTIC LEUKEMIA – L1

54. • These mature lymphocytes are increased markedly in
number. They are indicative of Chronic Lymphocytic
Leukemia, a disease most often seen in older adults.
This disease responds poorly to treatment, but it is

55. ACUTE MYELOID LEUKEMIA

56. • There are numerous granulocytic forms seen here, including immature myeloid cells and bands.
This condition is one of the Myeloproliferative states and is known as chronic
myelogenous leukemia (CML) that is most prevalent in middle-aged adults. A useful test
to help distinguish this disease is the leukocyte alkaline phosphatase (LAP) score, which should
be low with CML and high with a leukemoid reaction.

57. PLATELET MORPHOLOGICAL
ABNORMALITIES
GIANT PLATELETS
• Morphology:
Platelet larger
than a normal
red cell.
• Found in:
Increased
platelet
turnover
Myeloproliferati
ve disorders
Myelodysplastic
disorders

58. Anaemia – First Test
RETICULOCYTE COUNT %
Normal
Less than 2%
•Fragments of nuclear material
• RNA strands which stain blue
•New Methylene Blue or Brilliant
cresyl Blue

59. Reticulocyte
No definite nucleus
Reticulum of RNA
Deep blue staining
Light blue cytoplasm
Cell size about 10 µ

60. Reticulocytes
Leishman’sSupravital

61. Reticulocyte Production Index
For example the RPI is calculated as
follows
Reticulocyte count 9%
Hb content 7.5 g%
1. Correction for Anaemia
= 9 x (7.5 ÷ 15) = 9 x 0.5 = 4.5 %
2. Correction for increased life span
4.5 ÷ 2 = 2.25 %
3. Thus, the RPI is 2.25

62. Reticulocytes
Reticulocyte

63. RETICULOCYTOSIS

64. Types of Anaemia

65. Causes of Anaemia
1. Decreased production of Red Cells
- Hypo proliferative, marrow failure
2. Increased destruction of Red Cells
- Hemolysis (decreased survival of
RBC)
3. Loss of Red Cells due to bleeding
- Acute / chronic blood loss
(hemorrhagic)

66. Hypoproliferative Anaemias
Failure of cell
maturation
Nuclear
breakdown
Cytoplasmic
breakdown
Megaloblastic Anaemia
Defective DNA synthesis
Folate or B12 deficiency Haem defect Globin defect
Thalassemia
Sickle cell AFe Phorph
IDA, SA

67. Anaemia
Hypoproliferative Hemolytic
RPI < 2 RPI > 2
Hb% < 12, Hct < 38%

68. Workup – Second Test
• The next step is ‘What is the size of RBC’ ?
• MCV indicates the Red cell volume (size)
• Both the MCH & MCHC tell Hb content of
RBC
• If the RPI is 2 or less
• We are dealing with either
– Hypoproliferative anaemia (lack of raw material)
– Maturation defect with less production
– Bone marrow suppression (primary/ secondary)

69. Red Cell Size

70. Mean Cell Volume (MCV)
• RBC volume (rather) is measured by
• The Mean Cell Volume or MCV and RDW
Microcytic
< 80 fl
MCV
Normocytic Macrocytic
80 -100 fl > 100 fl
< 6.5 µ 6.5 - 9 µ > 9 µ

71. Anaemia Workup - MCV
Microcytic
MCV
Normocytic Macrocytic
Iron Deficiency IDA
Chronic Infections
Thalassemias
Hemoglobinopathies
Sideroblastic Anemia
Chronic disease
Early IDA
Hemoglobinopathies
Primary marrow disorders
Combined deficiencies
Increased destruction
Megaloblastic anemias
Liver disease/alcohol
Hemoglobinopathies
Metabolic disorders
Marrow disorders
Increased destruction

72. Anaemia Workup – 3rd Test
Peripheral Smear Study
• Are all RBC of the same size ?
• Are all RBC of the same normal discoid
shape ?
• How is the colour (Hb content) saturation ?
• Are all the RBC of same colour/ multi
coloured ?
• Are there any RBC inclusions ?
• Are intra RBC there any hemo-parasites ?
• Are leucocytes normal in number and D.C ?
• Is platelet distribution adequate ?

73. Coulter counter

74. Principle of Coulter

75. • This is Normal data from a complete blood
count as performed on an automated instrument,
including an automated WBC differential count.

76. • Here is data from a CBC in a person with Iron Deficiency
Anemia. Note the low hemoglobin (HGB). Microcytosis is
indicated by the low MCV (mean corpuscular volume).
Hypochromia correlates here with the low MCH (mean
corpuscular hemoglobin).

77. • The CBC here shows a markedly increased MCV, typical for
megaloblastic anemia. The MCV can be mildly increased in
persons recovering from blood loss or hemolytic anemia,
because the newly released RBC's, the reticulocytes, are
increased in size over normal RBC's, which decrease in size

78. • The CBC of a patient with Microangiopathic
Hemolytic Anemia (MAHA) demonstrates a markedly
increased RDW (red cell distribution width) due to the
marked variation in size and shape of the RBC

79. • Here is the CBC of a person with a severe anemia who
underwent transfusion. This accounts for the dual RBC
population as seen in the graph at the lower left. A
reticulocytosis in response to anemia has also increased the

80. What is Anaemia ?
Important to remember
• Anemia is a clinical sign of disease
• It is not a single disease by itself
• Need to look for the underlying cause !
• Will we ignore a fever without investigation
?
• Its diagnosis is not that simple !! We’ll
make it
• Its very common and imp. in our practice
• Drug Rx. depends on the cause

81. Definition of Anaemia
• Decrease in the number of circulating
red blood cell mass or Low Hb and
there by O2 carrying capacity
• Most common hematological disorder
• Almost always a secondary disorder
• As such, critical for all practitioners to
know how to evaluate / determine its
cause / treat

83. Bleeding time
• Is the duration of bleeding from a standard puncture
wound in the skin to stop bleeding.
• It is a measure of platelet count, platelet function, as well
as integrity of vessel wall.
• This is one of the common preoperative investigations
done before surgery.
• DUKE’S METHOD
• A small cut is made with a lancet in the Ear lobe or
finger tip. Blood flow from the puncture is and the time
taken for the bleeding to stop is measures with a stop
watch which is taken as BLEEDING TIME

84. Bleeding time-Duke’s
, IVY’s & Template
md
Uses: Diagnosis of Bleeding Disorder.
Before Surgery.
Before any tooth extraction
Before Liver or pleural or Bone marrow Puncture

85. Bleeding time
• Normal BT ;- 1-6 mints.
• Above 10 mints is abnormal
• Prolonged bleeding time
• Thrombocytopenia ( Decreased Platelet count)
• Defect in platelet function( Thromboasthenia)
• Drugs : heparin and Aspirin
• Other method ;- IVY’S METHOD

86. Clotting time
• The time taken for the given blood to clot
is called Clotting time.
• It is a measure of plasma clotting factors.
• It is a screening test for coagulation
disorders.
• Methods ;- 1. Capillary tube method of
Wright
• 2. Lee & White Method

87. Capillary tube method of Wright
• Materials Required:Capillary tube,Spirit,Cotton ,Lancet&Stop
Watch
• Procedure:
• First clean the pulp of the finger
• Prick the Finger
• Once bleeding starts start the stop watch and note the time.
• Collect the bleeding blood in capillary tubes.
• Seal the ends of the capillary tubes with plasticine
• Once in every 30 seconds break one capillary tube until a thread like
clot is seen between the broken ends.
• Now note the time and this gives the CLOTTING TIME
• Normal Value for this method: 5-10 mins

88. Lee & White Method
• Sprit,Cotton ,Lancet,Stop Watch,Test
tubes,Water bath&Disposable syringe
• Principle:- Intravenous blood when
collected and kept out side the body in a
test tube it forms a solid clot. The time
taken to form solid clot is measured as
CLOTTING TIME.

89. Lee & Whites Method
• Take three test tubes .and put 1ml of blood in each test
tube
• Place the test tube in a stand for 10 mins. (Waterbath at
37degree)
• After 10 mins take the first tube and gently tap every 30
seconds to test for clotting
• Once the blood in the first tube is found to be clotted,
note the time
• Now take the second tube and start tapping every 30
seconds, till the blood clots
• Then repeat the same with the third tube. Note the time.
• The time interval is taken as clotting time

90. Lee & Whites Method

91. Normal (4 – 11 minutes)
• PROLONGED CLOTTING TIME
• Hemophilia
• VonWillibrand Disease.
• Vit K Deficiency
• DECREASED CLOTTING TIME
• Corticosteroid treatment
• Drugs like epinephrine

92. THANK YOU