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.

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.

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 rightproportion.

• Samples are best analyzed within 2 hours of blood collectionas delay in preparation of blood film may allow for thedegeneration of cellular elements of blood and may result ina pseudo-thrombocytopenia (Bain, 2005).

• Commonly used stain in our facility is Leishman stain whichis composed of polychrome methylene blue and eosin. Whilemethylene blue stains the acidic content of the cell (nucleus),the eosin which is acidic stains the cytoplasm of the cell(Ochei and Kolhatkar, 2007).

•

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 thecell density is twice that seen at the tail.

• The head portion of the film might be of interest wheninvestigating for the presence of malaria parasite ormicrofilaria.

The fathered end may be examined for platelet clumpsand large cells like monocytes and blasts (Adewoyin andNwogoh, 2014).

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

WHITE BLOOD CELLS (LEUCOCYTES)

GRANULOCYTES

•Neutrophils

•Eosinophils

• Basophils.

AGRANULOCYTES

•Lymphocytes

•monocytes

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

A stained blood film showing neutrophils (Slomianka, 2009)

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).

A stained blood film showing eosinophil (Slomaianka, 2009)

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).

A stained blood film showing basophils (Slomianka, 2009)

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. Largelymphocytes are sometimes indented.

• Cytoplasm: small lymphocytes has little (small) cyplasmic space thatstains dark blue, while large lymphocytes has abundant cytoplasmicspace.

• Main function: Both effect the immune defense system (inform of Tand 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).

A stained blood film showing lymphocyte (Slomianka, 2009)

MONOCYTES

• Size: measures 15-20 microns in diameter.

• Nucleus: Kidney shaped, stains unevenly with astringy appearance.

• Cytoplasm: Abundant and stains greyish blue andvacuole may be seen.

• Main function: Phagocytic in function and capableof 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 bacterialinfections. (Adewoyin and Nwogoh, 2014)

A stained blood film showing monocyte (Slomianka, 2009)

DIFFERENTIAL WHITE BLOOD CELL COUNT: WHAT YOU SHOULD KNOW

• Differential white blood cell (diff. WBC) count isperformed to determine the relative number of each typeof white cell present (Cheesbrough, 2010).

• The white blood cell (WBC) count on its own is not veryinformative in evaluating the state of health of anindividual.

• The presence of a normal WBC does not mean that all iswell with a patient (Munster, 2012). In view of this, it iscommon practice to provide a so-called WBC differentialcount.

• The standard WBC differential divides the white bloodcells into the 5 major sub-population which includelymphocytes, monocytes, neutrophils, eosinophils, andbasophils (Munster, 2013).

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).

METHODS OF DIFFERENTIAL COUNT

• Battement method

• Longitudinal method

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).

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 findenough WBCs to perform a 100-cell differential. In this situation, adifferential is usually performed by counting 50 cells. A notation onthe 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 maybe performed to increase the accuracy of the differential. Theresults are then divided by 2 and a note made on the report that 200leucocytes were counted.

• Calculate the absolute number of each white cell type by multiplyingthe number of each cell counted in % by the total WBC count.

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

• 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

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.

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).

RBCs Abnormal MorphologyNOMENCLATURE 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

• 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)

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

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).

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).

ANOMALOUS CHANGES• Pelger-Huet anomaly

• Muy-Hegglin anomaly

• Alder-Reilly anomaly

(Ochei and Kolhatkar, 2007).

PLATELETS• Platelets (Thrombocytes) are approximately 2-4 by 0.5

microns in dimension (which is about a third of anormal size red cell) with coarse cytoplasmic granules(Adewoyin and Nwogoh, 2014).

• They are formed from budding off of the cytoplasmicof megakaryocytes in the marrow.It is expected that wesee approximately 7-15 platelets on x100 objective(Bain, 2005).

• A decrease in platelet count is termed thrombocytopenia.

• Qualitative abnormalities of platelet are termedthrombasthenia and require platelet functional studies toidentify them (Hoffbrand, 2011).

A stained blood film showing platelets (Slomianka, 2009)

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).

SYSMEX SP-1000I™ AUTOMATED

HEMATOLOGY SLIDE PREPARATION UNIT

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.

• 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)

The Sysmex HemoSlider

The RAL Stainer

Mythic 22 Full blood count Autoanalyzer.

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.

PRINCIPLE OF OPERATION

USES OF HAEMATOLOGY AUTO- ANALYZER

• Full Blood Count (FBC)

• Estimation of Red Cell Indices

(MCV, MCH, MCHC)

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

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.

THANKS

FOR

YOUR

AUDIENCE

REFERENCESAdewoyin 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 ofMedicine, 353: 498-507.

Basu, S. (2005). Blood cell and bone marrow morphology. The Science ofLaboratory Diagnosis, second edition.

Cheesbrough, M. (2010). Blood Films. District Laboratory Practice inTropical Countries, second edition, Cambridge University Press, pp.319-329.

Constantino, B. T. and Cogionis, B. (2000). Nucleated RBCs – significancein 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.

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 themodern haematology. SEED haematology.Sysmex. Available athttp://www.sysmex-europe.com/...SEED/sysmex-pdf.Accessed December 12,2013.

Ochei, J. and Kolhatkar, A. (2007). Examination of peripheral bloodsmear. Medical laboratory science: Theory and Practical. TataMcGraw-Hill publishing company limited. New Delhi, pp. 288-302.

Slomianka, L. (2009). Blue histology – blood. School of Anatomyand Human Biology. University of Western Australia.

Tefferi, A. and Hanson, C. A. (2005). How to interpret and pursuean abnormal complete blood cell count in adults. Mayo ClinicalProcedure, 80(7):923-936.