Hematology techniques

1. Hematology
techniques

2. Blood
 Plasma
 Formed elements
 Red blood cells
 White blood cells
 Platelets

3. Collection of blood
 Capillary method
 Venous method
 Arterial method

4. Capillary method

8. Venous method

9. IV canula Butterfly needle
When a series of samples needed

11. Arterial method
 For blood gas analysis
 From radial artery usually
 Compress hard after drawing blood

12. Plasma & serum
 Anticoagulated blood allowed to stand –
blood cells sink to the bottom leaving
yellow plasma above
 If fresh blood allowed to stand – clot form
– a pale yellow fluid separates
Serum is plasma devoid of clotting
substances

13. Anticoagulants
 Calcium chelators
 Oxalates
 EDTA
 Trisodium citrate
 Natural anticoagulant
 Heparin

14. XII XIIa
XI
IX
XIIIa
XIa
X Xa
IXa
XIII
VIIIa
VIII
TF
X
VII
VIIa
Va
V
Prothrombin Thrombin
Fibrinogen
Soluble
fibrin
In soluble fibrin
Intrinsic pathway
Extrinsic pathway
Ca++
Calcium
chelators

15. XII XIIa
XI
IX
XIIIa
XIa
X Xa
IXa
XIII
VIIIa
VIII
TF
X
VII
VIIa
Va
V
Prothrombin Thrombin
Fibrinogen
Soluble
fibrin
In soluble fibrin
Heparin
Intrinsic pathway
Extrinsic pathway
Ca++
Antithrombin III
Heparin

16.  Within 2hrs
 Coagulation tests
 Platelet count
 Blood smear examination
 Within 3hrs
 ESR
 Within 24hrs
 RBC count
 WBC count
 Reticulocyte count
 Hb, PCV

18. Hemoglobin
 Complex protein in red blood cells
 Gives red colour to blood
 Consists of
 Iron containing – Heam
 Protein – Globin

19. Normal values
 Men
 15 ±2g/dl
 Women
 13 ± 2g/dl
 Infants
 17-20g/dl
 Adult level by puberty

20. Hemoglobin estimation
 Based on 4 different principles
 Colour – Colourimetric method
 Sp.gravity – Physical method
 Iron content – Chemical method
 Oxygen carrying capacity of blood –
Gasometric method

21. Colourimetric method
 Sahli’s method / Acid hematin method
 CyanmethHb method

22. Physical method
 Specific gravity method
 used in blood donor screening

23. Packed Cell Volume

24.  PCV or Hematocrit
 Defined as
 Volume of packed red cells in a given sample
of blood & is expressed as a % of the total
volume of blood sample

25. Methods
 Macromethod / Wintrobe’s method
 Micromethod

27. Normal values
 Men
 45 ± 5 %
 Women
 41 ± 5 %

28. Buffy coat
 Thickness of buffy coat = number of WBC
& platelets
 0.1mm = 1000WBC/ml
 Normally buffy coat ≤ 1mm thickness

29. Plasma
 Normally 48-52% & yellow coloured
 Dark Yellow – Jaundice
 Red – Hemolysis
 Milky - Lipemia

30. Microhematocrit
 Micro PCV
 Capillary tubes - 75mm length, 1mm
internal diameter
 Tubes centrifuged in microcentrifuge
 12000rpm for 5mts

32. Variations in PCV
 Increased in
 Polycythemia
 Hemoconcentration states – burns, diarrhoea
 Decreased in
 Anemia
 Hemorrhage

33. ESR
Erythrocyte Sedimentation Rate

34. Methods
 Westergren’s method
 Wintrobe’s method
 Capillary method

35. Normal values
 Westegren’s method
 Males
 0-6mm/1st hr
 Females
 0-8mm/1st hr
 Wintrobe’s method
 Males
 0-10mm/1st hr
 Females
 0-20mm/1st hr

36. Pathological variations
 High ESR
 All acute infections
 Chronic infections
 Multiple myeloma
 Anemia
 Low ESR
 Polycythemia
 Congestive cardiac
failure
 Burns

37. Importance of ESR
 Non specific test
 Useful in assessing the prognosis or
progress or course of disease in patients

38. W
W W
W
R
3mm

39. Diluting fluids
 Normal saline
 Formal citrate
 Dacie’s formal citrate
 Hayem’s fluid
 Turks’s fluid – WBC
 Hingleman’s solution – Eosinophils
RBC

40. Calculation
No: of cells/µl =
No: of cells counted
Dilution x chamber depth x chamber area
Commonly expressed as
Cells x Dilution factor x Depth factor
Area counted

41. Hemocytometry
Counting of cells in the blood

42. Apparatus
 Counting chamber
 Dilution pipettes
 Diluting fluids

43. Neubauer counting chamber

44. Counting chamber
Cover slip
Platform
Gutter
0.1mm

45. Diluting pipettes
 RBC pipette
 WBC pipette
 20µl pipette

46. RBC
pipette
WBC
pipette

47. Red Blood Cell count
 Instruments
 Neubauer’s counting chamber
 RBC pipette
 RBC diluting fluids

48. RBC counting
 2 methods
 Using RBC pipette (Thoma RBC pipette)
 Bulk dilution method
 Calculation = N x 10,000

49. Normal RBC counts
 Men
 5.0 ± 0.5 million
 Women
 4.3 ± 0.5 million
 Birth
 6.0 ± 1.0 million

50. Variations in RBC counts
 Increased count
 Newborn
 Polycythemia vera
 Hemoconcentration
 Burns
 Diarrhoea
 Decreased count
 Childhood
 Old age
 Pregnancy
 Anemia

51.  Instruments
 Neubauer’s counting chamber
 WBC pipette
 WBC diluting fluid – Turk’s fluid
 Calculation = N x 50
White Blood Cell count

52. Normal values
 Normal WBC count
 4000 – 10,000/cmm
 Newborns
 10,000 – 12,000

53. Variations
 Leucocytosis
 Bacterial infections
 Leukemias
 Leucopenia
 Typhoid fever
 Malaria
 Certain drugs

54. Eosinophil count
 Fuchs – Rosenthal chamber
 20µl pipette
 Hingleman’s solution
 Calculation = N X 3.125

55. Normal value
 Normal range – 40 - 440/µl
 Eosinophilia
 Parasitic infestations
 Allergic conditions

56. Platelet count
 Formal citrate solution with 1-2drops of 1%
Brillant Cresyl Blue
 Calculation = N X 1,000

57. Normal value
 Normal platelet count
– 1,00,000 - 3,00,000/µl
 Increase – Thrombocytosis
 Decrease – Thrombocytopenia

58. Variations
 Throboctopenia
 Acute leukemias
 ITP
 Aplastic anemia
 Hypersplenism
 Thrombocytosis
 CML
 During infections
 After h’ge

59. Erythrocyte indices
 RBC indices
Or
 Wintrobe’s constants
 Based on
 Hemoglobin concentration
 Hematocrit
 RBC count

60.  MCV
 Mean corpuscular volume
 MCH
 Mean Corpuscular Hemoglobin
 MCHC
 Mean Corpuscular Hemoglobin Concentration
 Colour index
 Mean cell diameter
 RDW
 Red cell Distribution Width

61. Normal value
 Normal MCV = 87± 5fl
 Normal MCH = 27 – 32 pg
 Normal MCHC : 32 – 36 %
 Normal colour index = 0.85 – 1.15
 Normal Mean cell diameter : 7.2 – 7.5µm
 CV : 11.5 – 14.5 %
 SD : 30 – 50 fl

62. Electronic counters
 Largely used now
 Rapid
 Precise
 Many tests simultaneously

63. Bleeding time
 Duration of bleeding from a standard
puncture wound
 It measures
 Function of platelets
 Integrity of vessel wall
 Duke’s method

64. Normal value
 Normal bleeding time : 2-7mts
 BT increases
 Platelet count < 1,00,000

65. Clotting time
 Whole blood when removed from vascular
system & exposed to foreign surface will
form solid clot
 Normal clotting time : 5 – 10mts
 >20 – abnormal

67. Blood smear Preparation
 Thin blood smear
 Thick blood smear

68. Thin smear
 To study the morphology of blood cells
 Use chemically clean slides, free from
grease & oil, wiped free of dust

75. Body
Tail
Ideal smear
Head

76. Ideal thickness : RBCs should just touch throughout
much of the film

77. Good

78. Thick smear
 For detecting presence of parasites like
 Malaria
 Microfilaria

79. Thick smear
Before
staining
After
staining

80. Romanowsky stains
 Various combinations available
 Wright’s stain
 Leishman’s stain
 Giemsa stain
 Jenner’s stain
 Methylene blue / toludine blue – Basic dye
 Eosin / Azur I /II – Acid dye

81. Stained smear

82. RBC – Tannish red
PLT – Bluishpurple
WBC – Distinct

83. Red Blood Cells
 Size
 Shape
 Colour
 Immature forms
 Inclusion bodies
 Arrangements
 Parasites

84. Normocytes

85. Microcytes Macrocytes

86. Normal Spherocyte
Dacryocyte
Tear drop
Micro
spherocyte
Irregularly
contracted
Ovalocyte
Elliptocyte Target cell
Stomatocyte Keratocyte Fragmented
Echinocyte
Crenated
Acanthocyte Sickle cell Boat shaped
cell
S-C Poikilocyte
P
O
I
K
I
L
O
C
Y
T
E
S

87. Hereditary
spherocytosis Micro spherocytes

88. Elliptocytes Ovalocytes

89. Teardrop cells Target cells

90. Stomatocytes Acanthocytes

91. Fragmented cells Sickle cells

92. Normochromic Hypochromic

93. Polychromasia Nucleated RBC

94. Agglutination Rouleaux

95. Malaria Microfilaria

96. White Blood Cells
 Number
 Predominant cell type
 Abnormal or immature forms
 Hypersegemnted
 Blasts

97. WBCs
Eosinophil
Neutrophil
Basophil
Lymphocyte
Monocyte

98. Pelger-Huët anomaly
Hypersegmented
neutrophil

99. Myeloblasts Lymphoblasts

100. Platelets
 Number
 Form
 Abnormal size & shape
 In groups or lying scattered

101. Normal platelets Thrombocytosis

102. Giant platelets Platelet aggregation

103. Differential leucocyte count

104. Neutophilia Eosinophilia

105. Variations in WBC counts
 Neutophilia
 Bacterial infections
 AML & CML
 Neutropenia
 Viral infections
 Lymphocytosis
 In children
 IMN
 ALL & CLL
 Lymphopenia
 A/C infections

106.  Eosinophilia
 Parasitic
infestations
 Allergic conditions
 Eosinopenia
 Cushings syndrome
 ACTH therapy
 Monocytosis
 Kala azar
 Typhoid
 Monocytic leukemia
 Basophilia
 CML

107. Bone Marrow

108. Indications
 Absolute
 Megaloblastic anemia
 Subleukemia or aleukemia
 Diagnostic importance
 Multiple myeloma
 Aplastic anemia
 Gaucher’s disease

109. Methods
 Aspiration / needle biopsy
 Salah’s & Klima needle
 Trephine biopsy
 Jamshidi needle
 Open biopsy

110. Sites
 Adults
 Tibia
 Ant.iliac spine
 Vertebral spinous process
 Post. iliac spine
 Children
 Tibia
 Post.iliac spine
 Calcaneum

116. BM examination
 Naked eye examination to select smear
containing marrow particles

117. Trephine biosy

118. Cellularity
Erythropoiesis
Myelopoiesis
Megakaryocytes
 Metastatic cells
 Parasites

119. Normocellular Hypercellular
Hypocellular

120. Erythropoiesis

121. Myelopoiesis

122. Megakaryocytes

123. URINALYSIS

124.  Indispensable part of Clinical Pathology
 Easily obtained specimen
 First test in identification of pathology of
Urinary tract
 Diagnosis of various systemic diseases

126. SPECIMEN COLLECTION
Types of specimen
 Random specimen
 Early morning specimen
 Post prandial specimen
 24 hour urine specimen
 Catheterised urine specimen

127. Random specimen
 Any fresh specimen of urine
 Adequate for routine examination

128. Early morning specimen
 First morning specimen
 Most concentrated
 Lowest pH
 Preserve formed elements

129. Post prandial specimen
 2hrs after meal
 Best for sugar & protein

130. 24 hour urine specimen
 Total urine excreted in 24 hours is
collected
 For quantitative tests
 For concentration of AFB

131. Catheterised urine specimen
 Taken when difficult to get a proper
sample for culture and sensitivity

132. Method of collection
 Also called Clean catch specimen
 For culture & sensitivity urine is collected
in Sterile bottle

133. PRESERVATION
 Sample should be examined within 1-2
hours
 If delay - preserve urine
 Refrigeration
 Chemicals

134. Preservatives
 Boric Acid
 Toluene
 Conc. HCL
 Thymol
 Chloroform
 Sodium carbonate
 Formalin

135. Urine examination
 Physical Examination
 Chemical Examination
 Microscopic Examination

136. Physical Examination
 Volume
 Colour
 Appearance
 Odour
 Reaction
 Specific Gravity

137. C
H
Y
L
U
R
I
A

140. Chemical examination

141. Protein
 Proteinuria- passage of protein in urine
 Albumin is the chief component –
commonly referred as albuminuria

142. Types of proteinuria
 Accidental
 Functional
 Organic and renal

143. Bence - Jones Protien
 A small globulin – easily filtered & excreted
in urine
 Seen in, multiple myeloma
 Unusual property of precipitation – at 40-
60oC, then redissolve on boiling

144. Heat and Acetic acid test
 Fill 2/3rd of test tube with urine
 Heat the upper 2-3cm to boiling
 White cloud in the heated portion
 Add 2-3 drops of 3% acetic acid
 Cloudiness persist

145. Sugar
 Glycosuria / Glucosuria
 Other sugars
Lactose
Galactose
Pentose

146. Sugar
in
urine
cupric cuprous
Heat
Coloured
compound
Benedict’s test

147. Benedict’s test
0.5-1.0g/dl
1-2g/dl
++++ : brick red ppt (>2.0g/dl)

148. Ketone bodies
 Acetone
 Acetoacetic acid
 Betahydroxybutyric acid
 Rothera’s test

149. Bile
 Bilirubin
 Foam test
 Fouchet’s test
 Urobilinogen
 Ehrlich’s Aldehyde test
 Bile salts
 Hays test

150. Blood
 Hematuria
 Hemoglobinuria
 Benzidine test

151.  Protein
 Sugar
 Ketone bodies
 Bilirubin
 Urobilinogen
 Blood
 Fouchet’s test
 Benzidine test
 Benedict’s test
 Rothera’s test
 Heat & acetic acid
test
 Ehrlich’s Aldehyde
test
Match the following

152. Microscopic examination

153. Unorganised sediments Organised sediments
Urinary sediments
crystals casts
2 broad classes

154. Calcium oxalate crystals Cystine crystals

155. Leucine crystals Tyrosine crystals

156. Triple phosphate crystals Calcium carbonate crystals

157. Ammonium biurate crystals

158. Organised sediments
 Tubular casts
 Epithelial cells
 White blood cels
 Red blood cells
 Spermatozoa
 Bacteria
 Parasites

159. Tubular casts
 Casts Suggest Kidney pathology
 Formed in renal tubules - Cylindrical in
shape
 Due to coagulation of protein - Tamm-
Horsfall protein
 Different types of casts

160. Hyaline casts Waxy casts

161. Granular casts

162. Epithelial cell cast Pus cell cast

163. RBC cast Fatty cast

164. RBCs WBCs

165. Bacteria
 In Urinary tract infection bacteria can be
found in large numbers
 Direct smears prepared & stained
 Gram stain in UTI
 AFB in suspected tuberculosis

166. Spermatozoa
 Occasionally seen in urine of male
patients

167. Parasites
 Protozoal parasite – Trichomonas
vaginalis
 Ova – Schistosoma hematobium
 Larvae - Helminths

168. Pin worm egg