Introduction to haematological techniques.ppt

Hematology

1. Introduction to basic
haematological
techniques
Dr. Shafini Bt Mohd Yusoff

2. Content

Blood collection and handling

Basic haematological techniques

3. LIST OF TESTS
Routine Tests

ROUTINE HEMATOLOGY (FBC,
ESR, FBC/DC, FBP)

ROUTINE COAGULATION
(APTT/PT, INR, D-Dimer, Fib, DIC
SCREENING)
Specialized tests
1.SPECIAL COAGULATION ( Lupus
Anticoagulant, Factor Assay, Platelet
Aggregation, Thrombophilia Workup,
VW Workup, Anti Xa screening, DIC
screening)
2.HEMOLYSIS - G6PD screening
3.HB Electrophoresis (Hb Analysis)
4.Molecular Study (α & β-thalassemia,
BCR-ABL, JAK2, AML)
5.Flowcytometry (PNH, Leukemia,
Lymphoma)
6.Bone Marrow Aspiration
7.Serum Ferritin

4. Aim:

To produce reliable and accurate
results to all patients & clinical
personnel

6. Collection and handling of
blood

7. Causes of misleading results
Precollection
Food or water intake within 2 hrs
Smoking
Physical activity
Stress
Drugs or dietary supplement
During collection
Diff times (diurnai variance)
Posture
Haemoconcentration fr prolonged tourniquet
Excessive positive pressure when drawing blood into syringe
Incorrect tube
Capillary vs venous blood
Handling of specimen
Insufficient or excess anticoagulant
Inadequate mixing of blood
Error in patient/specimen identification
Storage condition
Delay in transit to lab

8. Differences between venous
& capillary blood

PCV, RBC count and Hb of capillary
blood is slightly greater than the
venous blood.

leucocyte and neutrophil counts are
higher by 8%, and monocytes by 12%
in capillary blood.

The platelet count is higher in venous
blood than in the capillary blood by
about 9%.

9. Anticoagulants
EDTA

acts by removing the calcium

Suitable for routine hematological work
Not suitable for coagulation problems
Trisodium citrate

The anticoagulant of choice in
coagulation studies
in a ratio of 9:1 is used
(blood:anticoagulant)

Used for ESR in a ratio of 4:1

10. Anticoagulants (cont)

Heparin

This might be used at a conc. of 10-20 iu/ml of
blood

Should not be used for making blood films as it
gives faint blue colouration to the background

It is the best anticoagulant for osmotic fragility
test and immunophenotyping.

Not suitable for blood counts because it often
induces platelet & leucocytes clumping

11. Effects of storage on blood
cell morphology (Qualitative)

If blood is allowed to stand in the lab before films
are made, degenerative changes occur.

By 3 hours changes are evident

By 12-18 hours changes become striking

Best to count leucocytes & platelet within 2 hrs

Changes: separated nuclear lobes, ragged
cytoplasmic margin/ less well defined, vacuoles in
the cytoplasm, lobulation of the nucleus,
crenation & sphering of rbc

NRBC disappear in blood within 1-2 days at room
temperature

12. Quantitative effects of
storage on blood

The red cells start to swell with the result of
increase in PCV, MCV and osmotic fragility.

PT slowly increases

ESR decreases

WBCs and platelets gradually fall

Reticulocyte count begins to fall within 6 h.

Hb remains unchanged for days

Inappropriate handling of blood during
transfer to the lab may cause hemolysis,
partial coagulation & cell disintegration

13. What are the common tests?
1.Quantitative measurements: FBC/CBC
RBC count, Hb estimation, PCV/Hct, Red
cell indices; MCV, MCH, MCHC,RDW and
reticulocytes

Total WBC count and differential
Platelet count.
2. Blood film:
preparation and interpretation.

14. Manual

Low cost

Labor intensive

Less precise

As standard
Automated

High capital

Rapid performance and less labour

Precise

Needs calibration and maintenance.

15. Hb estimation
Manual method using the
cyanmethaemoglobin
method.
Principle of the test is
dilution of blood in a
solution containing
potassium cyanide and
potassium ferricyanide.
 Hb will be converted
cyanmethaemoglobin
(HiCN).
The absorbance of the
solution is then measured
by spectrophotometry

16. Hb estimation

Automated method modification of
the manual HiCN method.

Non cyanide reagents are used.

17. Packed cell volume/Hct

The ratio of volume
occupied by the
packed red cells to the
volume of the whole
blood.

Modes of
measurements

Manual (PCV)

Automated :
generation of
electrical pulses (Hct)

18. RBC count
Manual : laborious,
obsolete and inaccurate.
Counting of RBC using a
special counting chamber.
Automated method: The
RBCs are counted
automatically by two
methods

Aperture impedance:
where cells are counted
as they pass in a stream
through an aperture.

Or by light scattering
technology

19. Histograms showing
the size
distribution of
white cells, red
cells and platelets.

Sizing is based on
impedance
technology.

20. Red cell indices

MCV= PCV/RBC count (fl)

MCH= Hb/RBC count (pg)

MCHC= Hb/PCV (g/l)

RDW

21. Hematology analyzer

three main physical technologies
used
1.electrical impedance
2.flow cytometry
3.fluorescent flow cytometry

22. Electrical impedance

Principle of cell
counting: based on
the detection and
measurement of
changes in electrical
resistance produced
by cells as they
traverse a small
aperture.

23. Electrical impedance

It is used in almost every hematology analyzer.

Whole blood is passed between two electrodes through an
aperture so narrow that only one cell can pass through at a
time.

The impedance changes as a cell passes through.

The change in impedance is proportional to cell volume,
resulting in a cell count and measure of volume.

Impedance analysis able to produce CBCs and three-part
WBC differentials (granulocytes, lymphocytes, and
monocytes) but cannot distinguish between the similarly
sized granular leukocytes: eosinophils, basophils, and
neutrophils.

24. Flow cytometry

A single-cell stream passes
through a laser beam.

the scattered light is measured
at multiple angles to determine
the cell’s granularity, diameter,
and inner complexity.

Give detailed information about
the morphology of blood cells

It is an excellent method for
determining five-part WBC
differentials.

Laser flow cytometry is more
expensive than impedance
analysis

25. Fluorescent flowcytometry

Adding fluorescent reagents 

extends the use of flow cytometry to
measure specific cell populations.

Cellular fluorescence is used to measure 
RNA (reticulocytes), DNA (nucleated red
cells), and cell surface antigens.

useful for the analysis of platelets,
nucleated RBCs, and reticulocytes.

27. Blood film

28. Preparation and staining of
blood films

Spreading technique

Labelling blood films

Fixing blood films

staining

29. Technique for making a
blood film

Correct technique for
making a wedge-spread
blood film.

The film of blood should be
narrower than the glass slide
and should not extend to the
end of the slide.

It should be narrower than
the cover slip that will be
applied.

30. Spreading technique

Place a small drop
of blood in the
centre line of a
slide about 1 cm
from one end.

Without a delay,
place a spreader in
front of the drop at
an angle of about
30
o
to the slide.

31. 
Move the spreader
back to make
contact with the
drop.

The drop should
spread out quickly
along the line of
contact.

32. Spreading technique

With a steady
movement of the
hand, spread the
drop of blood
along the slide.

33. Spreading technique

The spreader must
not be lifted off
until the last trace
of blood has been
spread out.

Automated
methods for blood
film spreading can
also be used.

34. Preparation and staining of
blood films
Labelling blood films
The film should be labelled
immediately after spreading by
writing either the lab reference
number or the name of the patient
and the date in pencil.

35. Preparation and staining of
blood films
Fixing blood films
To preserve the morphology of the
cells, blood films should be fixed
without delay.
Methyl alcohol (methanol) is the
fixative of choice

36. Staining of blood films

Romanowsky stains are routinely used, they
depend one two components:

Azure B/methylene blue: basic cationic dye
stains acidic cellular components such as
nucleic acid and basophilic granules in varying
shades of blue

Eosin Y: acidic anionic dye stains the basic
components such as hemoglobin and
eosinophilic granules an orange to pink color.

The neutral components of the cells are stained
by both components of the dye, producing
variable colors.

37. 
Amongst the Romanowsky stains used are:

Jenner is the simplest

Giemsa is the most complex

Leishman, it is intermediate, widely used in
routine.

Wright stain, used widely in North America.

39. Good blood films
A satisfactory blood film should not extend
to the edges of the glass slide.
 It should be evenly spread and should not
be thicker at the tail.
It should not have transverse or
longitudinal ridges or streaks.
 For adequate examination it should be
covered with a cover slip which is wider
than the film of blood so that the edges of
the blood film can be examined.

40. Good and bad blood films
Blood films should first be
examined macroscopically
to make sure that they are
of satisfactory quality for
examination.
Films a-e are
unsatisfactory. Only film f
is satisfactory. Film a is
unevenly spread and too
thick at the tail.
 Films b and c are too long
and b is also too broad.
 Film d is too short and
thick and film e is too close
to one edge of the glass
slide.
√
Х Х Х
Х Х

41. ERRORS MAY OCCUR DURING
STAINING

42. 1-Colour of nuclei blue to black

Eosin concentration too low

Staining time too short

Smear too thick

Inadequate time incubating in buffer solution

43. 2.Staining is too pink in colour

Incorrect proportion of azure B = eosin Y

pH of buffer too low

Excessive washing in buffer solution

44. 3.Pale Staining

The use of old staining solution

45. 4.Blue background
 Inadequate fixation
Prolonged storage before fixation
The use of heparinized blood

46. Method of examination of
blood films
Should examined macroscopically then
examined microscopically, using a low
power objective such as X 20 or X 40 or X
50.
The edges and the tail of the film should be
examined for large abnormal cells, platelet
aggregates or fibrin strands.
Fibrin strands are very pale
blue,sometimes almost colourless, and
deform the red cells through which they
pass. When they are present the platelet
count is often falsely low.

47. Method of exam of PBF
(cont)
Examination of a blood film should be
systematic.
 Having examined the edges and the tail
and excluded artefacts that make the film
unsuitable for assessment, cells of various
lineages should be assessed.
 The ideal part of the film to examine for
red cell morphology is the area where the
red cells are touching but without much
overlap.

48. 
Examining red cells in the tail of the
film will give a false impression of
spherocytosis and examining a part
of the film that is too thick will make
recognition of white cells unreliable.

49. Thin area
Thick area
Area of ideal
thickness

50. Most film examination should be done with a low
to medium power objective, e.g. X 20, X 40 or X
50.High power (X 100) should be used only for
assessment of fine cellular details.
 Get into the habit of systematically assessing
erythrocytes, leucocytes and platelets with regard
to both cell numbers and cytological features.
 Examination of the blood film should include an
assessment as to whether the automated blood
count is correct.

51. 
Assessment of the film should be
done in the light of the patient's age,
gender and clinical details.

When clinically indicated, a
differential count should be
performed, the results being
reported as absolute counts.

52. Manual differential
leucocyte count
Differential WBC counts are usually
performed by visual exam. of blood
films which are prepared on slides.
Even in the well-spread films, the
distribution of the various cell types is
not totally random.
Neutrophils and monocytes
predominate at the margins and the
tail; lymphocytes in the middle of the
film.

53. bodyHead
Lymphocytes+
Polymorphs++
Tail

54. Manual differential
leucocyte count

55. Reporting the differential
leucocyte count
The differential count, expressed as
the percentage of each type of cell,
should be related to the total
leucocyte count and the result
reported in absolute numbers
(x10
9
/l).
Myelocytes and metamyelocytes, if
present, are recorded separately from
neutrophils.
Band cells either recorded separately
or counted with the neutrophils.

56. Correcting the WBC count
for nucleated red blood cells

When NRBC are present, they will be
included in the total WBC count (by
automation).

So for correction of the count; the
NRBC should be counted with the
total WBC (total 100 cells)

Then the corrected WBC count will be
equal to total WBC count-(total count
x NRBC/100)

57. FBP

Reporting

Clinical information
(History + Physical
Finding)

Haemogram
(FBC + Histogram)

Peripheral Blood Film
(stained + mounted)

58. FBP Reporting

Rbc: Hb level + morphology + others

Wbc: counts + morphology + others

Platelet:counts + morphology

Impression:

Suggestion:

59. Normal blood film

60. Normal blood - dense area
- 20X

61. Normal blood- thin area

62. Normal blood-slide edge

63. Special Tests

64. 
Reticulocytosis is a
feature of
increased red cell
production.

New methylene
blue is used to
stain the
reticulocytes.

65. Haemoglobin H inclusions

haemoglobin H disease
have a significant
proportion of cells
containing haemoglobin H
inclusions [blue arrow].

These are small pale blue
inclusions distributed
evenly through a red cell,
giving an appearance
which has been compared
to a golf ball.

Increased numbers of
reticulocytes [red arrows]
are also apparent.

66. ESR estimation

Erythrocyte sedimentation rate

Measured by the use of Westergren
Tube

Old open method, closed method
and automation

67. Mechanism of erythrocyte
sedimentation

The rate of fall of the red cells is
influenced by a number of factors.

Basically it depends upon the
differences in specific gravity
between red cells and plasma.

68. 
Rouleaux formation and red cell
clumping make them easier to
sediment.

Rouleaux formation and clumping
are controlled by the fibrinogen and
other acute phase proteins.

Rouleaux is also enhanced by
immunoglobulins.

69. 
Anaemia, by altering the ratio of red
cells to plasma, encourages rouleaux
formation and sedimentation.

70. Significance of ESR
measurement

Clinically useful in disorders associated
with increased production of acute-phase
proteins.

Screening test

Most of acute or chronic infections and
most neoplastic and degenerative
diseases are associated with changes in
the plasma proteins which lead to
acceleration of the sedimentation.

71. Kleihauer test
Kleihauer test showing a
single positive
(haemoglobin F-
containing) cell.
 A Kleihauer test is used to
detect fetal cells in the
maternal circulation,
particularly in order to
quantitate fetomaternal
haemorrhage in Rhesus-
negative women carrying a
Rhesus-positive fetus.
 The test can also be used
to detect adult cells with a
high content of
haemoglobin F.

72. Thank you