In patient’s care, diagnostic formulations rest on a tripod consisting of clinical history, physical examination and laboratory investigation. The literature reveals that as much as 70% of clinical decisions and diagnosis are supported by laboratory medicine (WHO, 2015; Adewoyin & Nwogoh, 2014). Peripheral blood film (PBF) is a basic and a highly informative haematological tool at the clinician’s disposal in screening, diagnosis and monitoring of disease progression and therapeutic response. An adept understanding of peripheral blood interpretation is important for a successful clinical practice (Adewoyin & Nwogoh, 2014). Therefore, the ability to prepare, stain and report correct findings of a peripheral blood film is a skill that every Medical Laboratory Scientist should desire and study extensively to get expertise in. This is important as its role in the investigations and diagnosis of diseases mostly anaemia and most other haematological disorders cannot be over emphasized. To me, it is the hallmark of haematology.

1. BLOOD FILM EXAMINATION: ITS RECENT
INVESTIGATIVE METHODOLOGY IN THE DIAGNOSIS
OF DISEASE
A SEMINAR PRESENTED TO
THE DEPARTMENT OF HAEMATOLOGY
FEDERAL TEACHING HOSPITAL,
ABAKALIKI
BY
SCT. NWUDELE CHIBUEZE
SCT. IWUCHUKWU CALISTUS KANAYO.
DATE: 4TH NOVEMBER, 2015.

2. INTRODUCTION
• In patient’s care, diagnostic formulations rest on a tripod consisting of clinical
history, physical examination and laboratory investigation.
• The literature reveals that as much as 70% of clinical decisions and diagnosis are
supported by laboratory medicine (WHO, 2015; Adewoyin & Nwogoh, 2014).
• Peripheral blood film (PBF) is a basic and a highly informative haematological
tool at the clinician’s disposal in screening, diagnosis and monitoring of disease
progression and therapeutic response.
• An adept understanding of peripheral blood interpretation is important for a
successful clinical practice (Adewoyin & Nwogoh, 2014).
• Therefore, the ability to prepare, stain and report correct findings of a
peripheral blood film is a skill that every Medical Laboratory Scientist should
desire and study extensively to get expertise in.
• This is important as its role in the investigations and diagnosis of diseases
mostly anaemia and most other haematological disorders cannot be over
emphasized. To me, it is the hallmark of haematology.

3. HINTS ON BLOOD FILM QUALITY
• To ensure accurate and reliable results, pre-analytical
variables that affect the quality of film must be controlled
(Adewoyin and Nwogoh, 2014).
• Blood to anticoagulant ratio should be in the right
proportion.
• Samples are best analyzed within 2 hours of blood collection
as delay in preparation of blood film may allow for the
degeneration of cellular elements of blood and may result in
a pseudo-thrombocytopenia (Bain, 2005).
• Commonly used stain in our facility is Leishman stain which
is composed of polychrome methylene blue and eosin. While
methylene blue stains the acidic content of the cell (nucleus),
the eosin which is acidic stains the cytoplasm of the cell
(Ochei and Kolhatkar, 2007).
•

4. WHICH PART OF THE SLIDE SHOULD
BE EXAMINED FOR WHAT?
• The slide is viewed at the body of the film, usually
beginning about one millimeter away from the tail
(the monolayer part).The head should be avoided as the
cell density is twice that seen at the tail.
• The head portion of the film might be of interest when
investigating for the presence of malaria parasite or
microfilaria.
The fathered end may be examined for platelet clumps
and large cells like monocytes and blasts (Adewoyin and
Nwogoh, 2014).

5. smear showing a viewable side in a slide (Slomianka, 2009)

6. WHITE BLOOD CELLS (LEUCOCYTES)
GRANULOCYTES
• Neutrophils
• Eosinophils
• Basophils.
AGRANULOCYTES
•Lymphocytes
•monocytes

7. NEUTROPHILS
• Size: measures 10-14 microns
• Nucleus: are lobulated, connected by thin strands of
chromatin and stain deep reddish purple.
• Cytoplasm: are abundant and stains light pink.
• Main function: capture and destroy invading
organisms and other foreign toxic materials.
• Reference range: Absolute Number
Adults: 40-75% 1.5-7.5 x 109/L
Children: 20-45% 1.5-6.5 x 109/L
Increase is Neutrophilia as in bacteria infections
Decrease is Neutropenia as viral infections

8. A stained blood film showing neutrophils (Slomianka, 2009)

9. EOSINOPHILS
• Size: measures 12-17 microns
• Nucleus: lobulated (mostly 2 and three occasionally) and
contain nucleoli.
• Cytoplasm: not clearly visible because it contains large
round orange-red granules and occasionally vacuoles.
• Main function: associated with 1gE antigen-antibody
reactions.
• Reference range: Absolute number
• 1-6% 0.02-0.6 x 109/L
• Increase is eosinophilia as seen in helminth infections
(Hoffbrand, 2011).

10. A stained blood film showing eosinophil (Slomaianka, 2009)

11. BASOPHILS
• Size: measures approximately 10 microns (smaller than
neutrophils and eosinophils)
• Nucleus: usually bilobed, and stain deep purple-blue, but
obscured with cytoplasmic granules.
• Cytoplasm: slightly basophilic and contains large granules
which stain purple or black.
• Main function: Phagocytes (contains heparin, histamines and
serotonin).
• Reference range: Absolute number
• 0-1% 0.01-0.1x 109/L
• Increase is basophilia as seen in myeloproliferative
disorders
(Ochei and Kolhatkar, 2007).

12. A stained blood film showing basophils (Slomianka, 2009)

13. LYMPHOCYTES
• Size: The small lymphocytes is approximately size of red blood cells (10
microns) while large lymphocytes measures 12-16 microns.
• Nucleus: Round or irregular and dark mauve staining. Large
lymphocytes are sometimes indented.
• Cytoplasm: small lymphocytes has little (small) cyplasmic space that
stains dark blue, while large lymphocytes has abundant cytoplasmic
space.
• Main function: Both effect the immune defense system (inform of T
and B-lymphocytes).
Reference range : Absolute number
 In adults: 21-40% 1.2-4.0 x 109/L
 In children: 45-70% 6.0-8.5 x 109/L
 Increase is lymphocytosis as seen in protozoa infections
 Decrease is lymphocytopenia as seen in HIV/AIDS.
(Cheesbrough, 2010).

14. A stained blood film showing lymphocyte (Slomianka, 2009)

15. MONOCYTES
• Size: measures 15-20 microns in diameter.
• Nucleus: Kidney shaped, stains unevenly with a
stringy appearance.
• Cytoplasm: Abundant and stains greyish blue and
vacuole may be seen.
• Main function: Phagocytic in function and capable
of ingesting a large number of bacteria.
• Reference range: Absolute number
2-8% 0.2-1.0 x 109/L
Increase is monocytosis as seen in chronic bacterial
infections. (Adewoyin and Nwogoh, 2014)

16. A stained blood film showing monocyte (Slomianka, 2009)

17. DIFFERENTIAL WHITE BLOOD CELL COUNT:
WHAT YOU SHOULD KNOW
• Differential white blood cell (diff. WBC) count is
performed to determine the relative number of each type
of white cell present (Cheesbrough, 2010).
• The white blood cell (WBC) count on its own is not very
informative in evaluating the state of health of an
individual.
• The presence of a normal WBC does not mean that all is
well with a patient (Munster, 2012). In view of this, it is
common practice to provide a so-called WBC differential
count.
• The standard WBC differential divides the white blood
cells into the 5 major sub-population which include
lymphocytes, monocytes, neutrophils, eosinophils, and
basophils (Munster, 2013).

18. OBSERVING AND RECORDING NUCLEATED
RED BLOOD CELLS (nRBCs)
• If nRBCs are observed while performing the
differential they need to be reported.
• Correct the WBC count if the nRBC count is
greater than 10nRBCs/100.
• Use the following to calculate corrected WBC
(cWBC)
• nRBC = WBC x 100/(nRBC +100)
(Constantino et al., 2000; Adewoyin and Nwogoh, 2014).

19. METHODS OF DIFFERENTIAL COUNT
• Battement method
• Longitudinal method

20. PROCEDURES
• Place a drop of immersion oil on the lower third of
the blood film and cover with a down cover glass
(Cheesbrough, 2010).
• Examine the blood film under the low power (x10)
objective.
– To evaluate the quality of the blood film.
– To estimate roughly the red cells and white cells count
(Ochei and Kolkatkar, 2007).

21. EXAMINATION OF THE BLOOD FILM UNDER
OIL IMMERSION (X100) OBJECTIVE
• Take the differential count leucocytes of 100-cell and record your
result using manual chart format or automated cell counter.
• When the WBC is very low (below 1000/uL), it is difficult to find
enough WBCs to perform a 100-cell differential. In this situation, a
differential is usually performed by counting 50 cells. A notation on
the report must be made that only 50 white cells were counted.
Multiply each percentage by 2.
• When the WBC is very high (>50,000/uL), 200-cell differential may
be performed to increase the accuracy of the differential. The
results are then divided by 2 and a note made on the report that 200
leucocytes were counted.
• Calculate the absolute number of each white cell type by multiplying
the number of each cell counted in % by the total WBC count.

23. A stained blood film showing different white blood cells (Gillet, 2009)

24. • Observe and report the morphological
abnormalities in red cells.
• Observe and report the abnormalities in leucocytes.
• Evaluate platelet count and morphology .
• Observe for the presence of blood parasites.
• Observe inclusion bodies or other abnormalities
(Ochei and Kolhatkar, 2007).
BLOOD FILM REPORT

25. RED BLOOD CELL MORPHOLOGY
• Scan area using x100 (oil immersion).
• Observe 10 fields.
• Red cells are observed for size, colour,
haemoglobin content or pallor, shape, presence
or absence of inclusion bodies
NORMAL MORPHOLOGY
• Normocytic: normal cell size and shape.
• Normochromic: normal haemoglobin content
and colour.

26. ABNORMAL MORPHOLOGY
• Red cell morphology must be scanned in
a good counting area. Two questions
should be asked:
oIs the morphology seen in every field?
oIs the morphology pathologic or
artificially induced?
(Adewoyin and Nwogoh, 2014).

27. RBCs Abnormal Morphology
NOMENCLATURE OF RED CELL SHAPES
NEW TERMINOLOGY OLD TERMS
• Discocyte Biconcave disc
• Echinocyte (I-III) Burr cell, crenated cell, berry cell
• Acanthocyte Spur cell, acanthoid cell
• Stomatocyte Mouth cell, cup form, mushroom cap.
• Spherocyte Spherocyte, microspherocytes
• Schizocyte Schistocyte, helmet cell, fragmented cell
ELLIPTOCYTE AND OVALOCYTE
• Drepanocyte Sickle cell
• Codocyte Target cell
• Dacryoctye Teardrop cell, tennis racket cell

28. • QUALITATIVE GRADING OF RBC MORPHOLOGY
Grade Degree of Abnormalities
• Marked1 to 5 cells/10 fields Slight
• 6 to 15 cells/10 fields Moderate
• > 15 cells/10 fields
Grading Inclusions
• Rare 0 to 1/hpf
• Few 1 to 2/hpf
• Moderate 2 to 4 /hpf
• Many > 5/hpf
• hpf, high-power field (Gillet, 2009)

29. A stained blood film showing red blood cells with platelets (Slomianka, 2009)

30. WHITE BLOOD CELL MORPHOLOGY
• Most alteration in leucocytes morphology can
be classified into three categories:
• Toxic or reactive changes
• Anomalous changes
• Malignant changes
(Bain, 2005).

31. TOXIC OR REACTIVE CHANGES
• Hyper-segmentation of nucleus
• Cytoplasmic vacoulation
• Toxic granulation
• Double bodies
• Basket cells or smudge cells
• Turk cells
• Reactive lymphocytes
• Barr bodies
• Auer Bodies/Auer rods
(Bain, 2005).

32. ANOMALOUS CHANGES
• Pelger-Huet anomaly
• Muy-Hegglin anomaly
• Alder-Reilly anomaly
(Ochei and Kolhatkar, 2007).

33. PLATELETS
• Platelets (Thrombocytes) are approximately 2-4 by 0.5
microns in dimension (which is about a third of a
normal size red cell) with coarse cytoplasmic granules
(Adewoyin and Nwogoh, 2014).
• They are formed from budding off of the cytoplasmic
of megakaryocytes in the marrow.It is expected that we
see approximately 7-15 platelets on x100 objective
(Bain, 2005).
• A decrease in platelet count is termed thrombocytopenia.
• Qualitative abnormalities of platelet are termed
thrombasthenia and require platelet functional studies to
identify them (Hoffbrand, 2011).

34. A stained blood film showing platelets (Slomianka, 2009)

35. AUTOMATION IN BLOOD FILM EXAMINATION
 The laboratory practice of haematology has
evolved tremendously over the past few decades
with automated analyser generated complete blood
counts (CBC) having fully replaced the original
manual individual parameter assay methods
(Seed, 2013).
In line with the principles of good laboratory
practice, standardised slide making and staining
procedures will guarantee good quality peripheral
blood smears. The best form of standardisation is
automation (Seed, 2013).

36. SYSMEX SP-1000I™ AUTOMATED
HEMATOLOGY SLIDE PREPARATION UNIT

37. FEATURES AND ADVANTAGES OF SYSMEX SP-1000I™
• Improves and standardizes smear turnaround time.
• Rapid smear preparation with first-in, first-out slide
preparation and staining.
• Reflexive slide preparation: applies laboratory defined criteria
to prepare smears.
• Low sample volume requirements: onboard micro-sample
mode aspirates 60 μL of sample volume to prepare and stain
quality smears.

38. • Uses a combination of unique slide cassette and bathless
staining process.
• Automatically adjusts the angle, speed and blood volume
based on HCT value of sample.
• Flexible operation and Consistently produces quality smears.
• Has ability to stain pre-made smears (e.g. body fluid, bone
marrow samples) and can produce multiple smears
automatically.
• In routine operations, the SP-1000i provides rapid, automated
preparation of peripheral blood smears to help laboratories
meet and standardize smear review turnaround times
(Seed, 2013)

39. The Sysmex HemoSlider

40. The RAL Stainer

41. Mythic 22 Full blood count Autoanalyzer.

42. HAEMATOLOGY AUTO- ANALYZER
• An automated system for Complete blood count (CBC).
•
PRINCIPLE OF HAEMATOLOGY AUTO-
ANALYZER
• The principle is based on measurement of cells in a
fluidic system (Flow cytometry) with a complex of
optical systems. Cells are counted based on their sizes,
granularity and volume.
• Exists in different Models, Example Mythic 18 auto-
analyzer, Mythic 22 auto-analyzer, Sysmex autoanalyzer
and mindray autoanalyzers.

43. PRINCIPLE OF OPERATION

44. USES OF HAEMATOLOGY AUTO- ANALYZER
• Full Blood Count (FBC)
• Estimation of Red Cell Indices
(MCV, MCH, MCHC)

45. CARE AND MAINTENANCE
• Read carefully the manufacturer’s manual and then
prepare SOP for use, care and maintenance.
• The equipment should be operated under suitable
temperature.
• Protect from dust by covering with its protective
covering.
• Regular and periodic checks should be done on the
working reagents.
• Maintenance should be done by a well trained
personel

46. CONCLUSION
• In order to ensure that the microscopic review will
provide a report that can be trusted for clinical
judgement, the quality of the smear and stain must be
optimal. The best way to achieve this is by means of
automation of both slide-making and staining.
• However manual knowledge of film making, staining
and report cannot be overemphasised since the
efficiency of automation depend on the knowledge of
the user.

47. THANKS
FOR
YOUR
AUDIENCE

48. REFERENCES
Adewoyin A. S. and Nwogoh, B. (2014). Peripheral Blood Film – A review.
Annals of Ibadan Postgraduate Medicine, 12(2): 71-79.
Bain, B. J.(2005). Diagnosis from the Blood Smear. England Journal of
Medicine, 353: 498-507.
Basu, S. (2005). Blood cell and bone marrow morphology. The Science of
Laboratory Diagnosis, second edition.
Cheesbrough, M. (2010). Blood Films. District Laboratory Practice in
Tropical Countries, second edition, Cambridge University Press, pp.
319-329.
Constantino, B. T. and Cogionis, B. (2000). Nucleated RBCs – significance
in the peripheral blood films. Laboratory Medicine, 31(4): 223-229.
Gillet, P. (2009). Haematology White Blood Cell, Anaemia Classification.
Tropical Laboratory Medicine Unit, pp. 1-52.

49. Hoffbrand, A. V. (2011). Megaloblastic anaemia. In: A. V.
Hoffbrand, D Catovsky E. G. Tuddenham, A. R. Green (eds).
Postgraduate Haematology. 6th ed. Wiley Blackwell.
Munster, M. (2013). The role of peripheral blood smear in the
modern haematology. SEED haematology.Sysmex. Available at
http://www.sysmex-europe.com/...SEED/sysmex-pdf.
Accessed December 12,2013.
Ochei, J. and Kolhatkar, A. (2007). Examination of peripheral blood
smear. Medical laboratory science: Theory and Practical. Tata
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Slomianka, L. (2009). Blue histology – blood. School of Anatomy
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