hematology procedure and various errors occurred.

1. 1
Presented by
Dr. Shrikant Sonune
Guided by
Dr Ashok Patil,
Dr Shilpa Kandalgaonkar,
Dr Suyog Tupsakhare,
Dr Mahesh Gabhane.
Dr. Gaurav Agarwal
Investigations & Artifacts
in Hematology

2. The study of blood components and coagulation. It
includes,
1) Analysis of the concentration, structure and functions
of the cells and their precursors in the bone marrow.
2) Analysis of chemical constituents of plasma or serum
intimately, linked with blood cell structure and
functions.
3) Study of functions of the platelets and proteins
involved in blood coagulation.

3. A. Complete Blood Count
B. Complete Hemogram
C. Tests of Hemostatic Function

4. 1. Hemoglobin (Hb) concentration.
2. Total erythrocyte count (RBC).
3. Total leukocyte count (WBC).
4. Examination of blood film (smear) for,
- Assessment of red cell morphology.
- Differential white cell count.

5. 1. Packed cell volume (PCV).
2. Mean corpuscular volume (MCV).
3. Mean corpuscular hemoglobin concentration
(MCHC).
4. Platelet count.
5. Erythrocyte sedimentation rate (ESR).

6. 1. Bleeding Time.
2. Clotting Time.
3. Prothrombin time.
4. Partial Thromboplastin Time.
5. Determination of Thrombin Time.
6. Determination Fibrinogen.

7. Sound knowledge
Advance planning
Collection of adequate and appropriate specimens
Sufficient documentation
Biosafety and decontamination
Correct packaging
Timely assessment of results

8.  The procedure in which an operator bleeds a specific
amount of blood of the subject for a particular investigation
can be termed as Collection of blood.
 Samples can be collected from:
1. Veins : Most commonly used.
2.Capillaries.
3.Arteries: in case of arterial blood gas analysis and
sometime in pediatric patients.

9. Pricking the skin with needle or lancet.
Site usually ring finger, prick should be 2-4mm deep
to ensure free flow of blood.
Only few drops are collected by this method.
Recommended for the Bleeding & clotting time.

10. Blood is obtained by Venepuncture.
 More reliable results are obtained.
With single prick- multiple tests can be done
Test can be repeated, as the sample is available.
Recommended for all hematological investigation
except few e.g. BT, CT, BLOOD GAS LEVEL.

11. 1. Anatomy and physiology
2. The criteria for choosing a vein
3. The device to use
4. Skin preparation
5. Personal safety – infection control policy

12.  The superficial veins of the
upper limbs are most
commonly used for
Venepuncture.

13.  Sterile gloves
 Syringes and needles
 Tourniquet
 Specimen containers
(Bulbs)
 70 % alcohol or 0.5%
chlorhexidine
 Sterile gauze swabs
 Adhesive dressings
 Rack to hold the specimen
containers.

15.  A vacutainer blood collection tube is a sterile glass or
plastic tube with a closure that is evacuated to create a
vacuum inside the tube facilitating the draw of a
predetermined volume of liquid.
 Most commonly used to draw a blood sample directly
from the vein.
 Vacutainer tubes may contain additives designed to
stabilize and preserve the specimen prior to analytical
testing.

17. Ash fluoride

18. Gauge of needle (22 gauge)
Squeezing of finger.
Initial drops - Discarded.
Ring finger is preferred.
No liquid anticoagulant.

19. 1. Whole blood – for glucose, urea, pH estimation
2. Serum – Total protein, Albumin, Globulin, Bilirubin,
cholesterol,
3. Plasma- chloride, fibrinogen, ascorbic acid.

20.  Clotted specimen.
 Improperly labeled or unlabeled specimen.
 Specimen too old.
 Failure to meet volume criteria.
 Improperly collected (diluted) capillary specimen.
 Leaking tube.
 Delay in transport.
 Collection of specimen in wrong tube.
 Hemolyzed smear

21. Granular fluid
Colt consume platelets & other cells hence reduces
the count.
Basically arise from inadequate
quantity of anticoagulant.
Not mixing the anticoagulant properly.

22. Specimen Collection:
An improper choice in the Venepuncture site, such
as drawing from a distal site to the antecubital
region of the arm rather than drawing from an
antecubital site, has been shown to result in more
hemolysis.

23. Prolonged tourniquet time causes the interstitial fluid
to leak into the tissue and cause hemolysis.
Cleansing the Venepuncture site with alcohol and
not allowing the site to dry may cause hemolysis.

24. An improper Venepuncture, indicated by a slow
blood flow, may indicate occlusion due to the lumen
of the needle being too close to the inner wall of the
vein, causing hemolysis.
The use of a small-bore needle, resulting in a large
vacuum force applied to the blood, may cause shear
stress on the red blood cells, causing them to
rupture.

25. The use of a large bore needle may result in a much
faster and more forceful flow of blood through the
needle, resulting in hemolysis.
Syringe Draws
Pulling the plunger of a syringe back too far while
using a large bore needle, may cause enough
pressure for hemolysis to result during collection.
The pressure may be greater than a standardized
evacuated tube.

26.  Transferring into a tube by pushing down on the syringe
plunger in order to force blood into a tube may cause
hemolysis.
 Vigorous mixing or shaking of a specimen may cause
hemolysis.
 Prolonged contact of serum or plasma with cells may
result in hemolysis.

27. Exposure to excessive heat or cold can cause RBC
rupture and hemolysis.
Specimen Transport:
Mechanical trauma during transport may occur.

28.  Redraw the specimen.
 The most common sites to draw from are the median
cubital, basalic, and cephalic veins from the antecubital
region of the arm.
 Avoid using a needle that is too small or too large.
 The tourniquet should be released after no more than
one minute, and excessive fist clenching should be
avoided.

29. Without touching, allow the Venepuncture site to air
dry before collecting blood.
Avoid drawing the syringe plunger back too
forcefully when collecting blood with a needle and
syringe.

30. Avoid pushing the plunger too forcefully when
transferring to a tube.
Ensure all blood collection assemblies are fitted
securely.
Gently invert the blood collection tube and mix
additive specimens thoroughly according to
manufacturer’s recommendations.

31.  Almost 1hr not much changes takes place regarding
cell morphology.
By 3hr changes may be discernible.
By 12-18 hrs striking changes that includes
Neutrophils nuclei stain homogenously
Nuclear lobes may become separated
Vacuole appear in cytoplasm
Ragged cytoplasmic membranes
RBC’s show progressive crenation & sphering.

32. Neutrophils nuclei stain homogenously
Nuclear lobes may become separated
RBC’s show progressive
crenation & sphering
Homogeneous
staining

33. Cell counts are affected by
The state of the blood circulation,
Nutrition
Time & condition of pt
If strictly comparable values are required, there should also be half an
hour of bed rest before the sample is drawn,
Color Atlas of Hematology Practical Microscopic and Clinical Diagnosis
Harald Theml,M.D.

34.  Agents which prevent the coagulation of blood are called as
anticoagulants.

35.  EDTA (Ethylene di amine tetra Acetic acid)
 Tri-sodium Citrate
 Heparin
 Double oxalate
 Sodium fluoride

36.  It is the most commonly used anticoagulant in routine practice.
 The potassium & sodium salts of EDTA are powerful anticoagulants.
Mechanism Of Action:
 It acts by chelating the calcium molecules in the blood.
 1.2 mg of EDTA is required for each ml of blood to get the desired
results.
 EDTA is used for mainly Blood counts.

37. COMPOSITION :
EDTA Dipotassium salt -44.5 g
EDTA Disodium salt – 41.0 g
1mmol NaOH-75 ml
Water – 1 liter.

38. ADVANTAGES :
 It is the anticoagulant of choice in routine hematological work.
 Best for platelet counts.
DISADVANTAGES:
 RBC morphology is hampered if the concentration is more than the
required.
 Not good for coagulation studies.

39. It is used for coagulation studies.
Mechanism of action :
It also works on the principle of calcium chelation.
For ESR estimation, 4 volumes of blood are added
to 1 volume of sodium citrate solution & well mixed.

40. Having pH – 6.9.
 Trisodium citrate, dihydrate( 102 mmol / l) - 30 g
 Sodium dihydrogen phosphate,
monohydrate ( 1.08 mmol / l ) - 0.15 g
 Dextrose ( 11 mmol / l ) - 2 g
 Water - 1 litre

41. ADVANTAGES :
 Tri-sodium citrate is used for coagulation studies
 Used in blood banking and blood transfusion purposes.(if
citrate phosphate dextrose is not available )

42.  It is used for chemistry , blood gas analysis & emergency
tests.
 It is the best anticoagulant for osmotic fragility tests &
immunophenotyping.
 The lithium or sodium salt of heparin at a concentration of
10- 20 IU / ml blood is used generally.
 It is not suitable for blood counts as it induces platelet &
leucocytes clumping & gives faint blue color on Peripheral
Smear.

43.  It acts by forming complex with calcium i. e. calcium
fluoride.
 It prevents glycolysis by blocking phosphorylase enzyme in
red cells.
 It is the ideal additive used for blood sugar estimation.
 Precaution – should not delay the investigation.

44. 1. Loss of CO2- it diffuse from plasma to air.
2. Conversion of glucose into lactic acid by
glycolysis.
3. Formation of NH3 from nitrogenous substance like
urea.
4. Passage of substances through the red cell
membrane.
5. Conversion of pyruvate to lactate.

45. RBC morphology is hampered if the concentration is
more than the required in case of EDTA.
Heparin is not suitable for blood counts as it induces
platelet & leucocytes clumping & gives faint blue
color on Peripheral Smear.
Incorporation of dust particles may takes place if the
anticoagulants are not stored properly.

46. Clinical Significance:
 Hemoglobin concentration is always as a part of routine
blood test.
 Part of complete blood test
 Increase in amount of Hb is Polycythemia
 Decrease in amount of Hb is Anemia

47. 1. Acid- Hematin (Sahli) method
2. Colorimetric or Spectrophotometric method
3. Oxyhaemoglobin (HbO2) Method
4. Talliqvist method.
5. Chemical method.

48. One of the most commonly used technique.
Simple, cost effective.

49. Why so Hemoglobinmeter is used ???
1. Ability of Hb to combine with oxygen.
2. Presence of known amount of iron in each gram of
Hb.
3. Ability of a solution of a derivative of Hb to refract
specific wavelengths of light , thus giving typical
absorption bands

50.  The Hb present in a measured amount of blood is converted
by dilute HCL into acid hematin.
 Which in dilution is golden brown in color .
 The intensity of color depends on the concentration of acid
hematin.
 Which in turn depends on concentration of Hb.
 The color of solution is matched against golden brown tinted
glass rods by direct vision.
 The readings are obtained against in gm%.

51. Hb
Acid
Hematin
Acid
Hematin –
brown color
Standard
HCl

52. Capillary blood
Thoroughly mixed anticoagulated (EDTA or Double
oxalate) venous blood.
Amount 0.02ml.

54. Pour 1/10 HCl into Hb tube.
Specimen collected by Hb pipette.
Pour into Hb tube.
Wait for 10 min
Add distilled water drop by drop
Till it mach with standard.
The reading on Hb tube is considered as
concentration of Hb.

55. Normal Values : Hb, g/dl
- MEN 13-18
- WOMEN 12-16.5
- CHILDREN (up to 1 year) 11.0-13.0
- CHILDREN (10-12 years) 11.5-14.5

56. Simple fairly accurate.
Less time consuming
Cost effective

57. Acid hematin is not a true solution, some turbidity
may occur.
Measures only oxy Hb & reduced Hb. Other forms
such as carboxy Hb & met Hb are not established.

58. 1. Faulty instruments
Hb pipette- blockage, wet pipette.
Hb tube – dirty tube.
Hemoglobinmeter- Defective or improper
comparator.

59. 1. Not taking exact amount of blood
2. Not cleaning the outer surface of Hb-tube.
3. Not giving the enough time or giving more than
required time to react with HCl.
4. Visual error
5. Adding more amount of distilled water.
6. During reading keeping stirrer in the Hb tube.

60.  Cyanmethaemoglobin Method
 Tallqvist Method
 Oxyhaemoglobin method
Alkaline – Haematin method
Chemical method

61. This is most accurate & most wildly used method of
estimation of Hb.
It is internationally recommended method for best
quality control.

62. BLOOD + POTASSIUM CYANIDE, ferricynide
= Methaemoglobin & carboxyhaemoglobin
Then , cyanmethaemoglobin
Absorbance of solution is measured in photoelectric
calorimeter at wavelength of 540 nm.

63. Reagent
1. Drabkin’s Cyanide- ferricynide solution.
Composition
Pot. ferricyanide – 200mg
Pot. Cyanide -- 50 mg
Sod bicarbonate – 1gm
Distilled water -- 1000ml

64. Cyanide is poisonous
Highly recommended to use the rubber teat .
Turbidity may cause faulty results.

65. Take 0.02 ml of blood
Mix with 4ml of 0.04% liquor ammonia
The formed solution is matched in a colorimeter at
540nm

66. • Common sources of error in measuring hemoglobin
include anything that will cause turbidity and
interfere with a Spectrophotometric method.
• Examples are a very high WBC or platelet count and
hemoglobin's that are resistant to lysis.

67. Iron content of Hb is 0.347 %
Total iron content of blood is estimated chemically &
Hb content is found out by dividing result by 3.47.

68.  A drop of blood collected finger is absorbed on a
piece of blotting paper
Dried for a few seconds
Compared with the references standard of color
paper made by manufacturer
Used in survey purpose.

69. Visual error
Subjective variability
Results are not reliable
If figure is squeezed then false results

70. Can be measured by Two methods which includes
 Counting RBC’s by manual method
 Electronic cell counter

71. Male 5.0 +/- 0.5 x 1012/l
4.5 to 5.5 million / cb. mm
Female 4.3+/- 0.5 x 1012/l
3.8 to 4.8 million / cb. mm

72. Principle
The blood is diluted 200 times in the red pipette and
the cell are counted in the counting chamber,
knowing the dilution employed, their number in
undiluted blood can easily be calculated.

73. RBC pipette
Neubauer chamber

74. 1. NaCl - 0.05gm- Maintain tonicity
2. Na2SO4- 2.05gm -Anticoagulant
3. HgCl2- 0.25g – antifungal
4. Distilled water 100ml- medium for solution.

75.  Position the Neubauer chamber over microscope.
 Finger prick
 0.5ml of blood sample
 Hayem’s fluid up to 101mark
 Shake the RBC pipette
 Discard 2-3 drops
 Load on Neubauer chamber.
 Wait for 2-3min
 Counting of RBCs
 That is multiplied by the 10,000

76. 1mm
1mm
Area= 1X1=1mm, Total nos of squre 400. area of each squre= 1/400mm.

77. Area= 1X1=1mm,
Total nos of squre 400.
Area of each squre=
1/400mm.

78. Area of smallest square= 1/400 sq mm
Depth= 0.1mm
Vol of smallest square= 1/4000cb mm
Nos of RBCs counted are in 80 square
Hence 80 X 1/4000= 1/50.
Hence nos of RBCs in 1cb mm vol is
= 50 X 200 X nos conted.
i.e. 10,000X nos counted.

79. Very high WBC
High concentration of very large platelets
Agglutinated RBC’s,
Roulex formation
Cell fragments or other debris

80. Inaccurate amount of blood taken.
Inaccurate amount of fluid
Mannual error during taking fluid
Improper mixing
Improper loading – improper distribution
Counting errors
Calculation error
Improper instruments

81. Clinical Significance:
1. Increase in Total leukocyte count of more than
10,000/cu mm is known as leukocytosis .
2. Decrease or less than 4,000/cu mm as leucopenia.

82. - Adults : 4,000-10,000/cu mm
- At Birth : 10,000-25,000/cu mm
- 1 to 3 years : 6,000-18,000/cu mm
- 4 to 7 years : 6,000-15,000/cu mm
- 8 to 12 years : 4,500-13,500/cu mm

83. The Glacial acetic acid lyses the red cells
The gentian violet slightly stains the nuclei of the
leukocytes.
The blood specimen is diluted 1:20 in a W.B.C
pipette with the diluting fluid.
The cells are counted under low power of the
microscope by using a counting chamber.
The number of cells in a undiluted blood are
reported per cu mm of whole blood

84. 1) Double oxalated or EDTA blood
2) Capillary blood.

85. 1. Glacial acitic acid (hemolysis of RBCs without
affecting WBCs)
2. Gention violet (stains the nuclei of leucocytes)
3. Distilled water act as medium.

86. Position the Neubauer chamber over microscope.
Finger prick
0.5ml of blood sample
Turks (WBC) fluid up to 11mark
Shake the WBC pipette
Discard 2-3 drops
Load on Neubauer chamber.
Wait for 2-3min
Counting of WBC in (X)
That is multiplied by the 50

87. Inaccurate amount of blood taken.
Inaccurate amount of fluid
Improper mixing
Improper loading – improper distribution
Counting errors
Calculation error
Improper instruments

88.  Unusual RBC abnormalities that resist lysis
 Nucleated RBC
 Fragmented WBCs
 Unlysed particle
 Specimen containing fibrin, cell fragmnets or other
debris