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Int. J. Curr. Res. Med. Sci. (2017). 3(12): 44-55

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International Journal of Current Research inMedical Sciences

ISSN: 2454-5716P-ISJN: A4372-3064, E -ISJN: A4372-3061

www.ijcrims.com

Review Article Volume 3, Issue 12 -2017

Peripheral blood smears - An innovative Review

*Yadav Dinesh Prasad1, Jha Raj Kumar2, Sharma Sonia3

1Department of Biochemistry, B&C Medical College, Teaching Hospital and Research2 Department of Pathology, B&C Medical College, Teaching Hospital and Research

3Department of Microbiology, B&C Medical College, Teaching Hospital and Research*Dr. Dinesh Prasad Yadav

Associate Professor, Department of Biochemistry, B&C Medical College, Teaching Hospital andResearch, Birtamode Jhapa, NepalE-mail: [email protected]

Abstract

The peripheral blood film (PBF) is a laboratory work-up that involves cytology and somehow cell morphology ofblood cells. As basic as it is, PBF is invaluable and of great use in the characterization of various clinical diseases.This article highlights the basic science and art of importance behind the PBF. It expounds its laboratory applications,clinical indications and interpretations in the light of various clinical and sub clinical diseases. Despite advances inhematology automation and application of molecular techniques, the PBF has remained a very important diagnostictool to the hematologist and clinician. A good quality blood smear, thorough examination and proper interpretationprognosed and diagnosed with patient's clinical state should be ensured by the clinical-pathologist. Clinicians shouldbe abreast with its clinical utility and proper application of the reports in the management of patient’s state and care.

Keywords: Peripheral blood smear, Preparation, Examination, Interpretation, cells morphology

Introduction

In patient care, diagnostic formulations depend ona tripod consisting of clinical history, physicalexamination and laboratory investigations. TheLiterature reveals that as much as 70% of clinicaldecisions and diagnoses are supported bylaboratory medicine. Peripheral blood film (PBF)is a basic and a highly informative hematologicaltool at the clinician’s disposal in screening,diagnosis, prognosis and monitoring of diseaseand therapeutic outcome. An sure shot and correctunderstanding of peripheral blood interpretation is

important for a successful clinical practice. Thediagnostic relevance of a PBF is very vast. ThePBF represents the morphology of peripheralblood cells, which ensures its place in themorphologic diagnosis of various primary andsecondary blood and blood related diseases. It’sdiagnostic relevance has not been lessened byadvances in hematology automation andmolecular based techniques. [1]

DOI: http://dx.doi.org/10.22192/ijcrms.2017.03.12.007


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This article attempts to summarize the preparationand reporting of peripheral blood film, its clinicalinterpretations, manifestations and the commonperipheral blood diagnosis. This will enhance theunderstanding of PBF interpretations byClinicians.

Indications for a Peripheral Blood Film

Initiation of a PBF is often requested by theattending clinician on account of a clinicalsuspicion or less frequently initiated by thelaboratory.[2, 3] The laboratory may initiateperipheral blood film based on abnormal findingsfrom an automated count or patients clinicalinformation whose diagnosis may be justified orsupported by a peripheral blood film. The latter isguided by individual laboratory policies or localregulating guidelines.[2]

Common clinical indications for peripheral bloodfilm analysis include unexplained cytopenia:anaemia, leucopenia or thrombocytopenia;unexplained leukocytosis, lymphocytosis ormonocytosis; unexplained jaundice orhaemolysis; features of congenital haemolyticanaemias such as splenomegaly, jaundice or bonepains; suspected chronic or acutemyeloproliferative disease e.g. chronic myeloidleukaemia; suspected organ failure such as renaldisease, liver failure; features of hyperviscositysyndrome as in paraproteinaemias, leukaemichyperleucocytosis, polycythaemia; severebacterial sepsis and parasitic infections;malignancies with possible bone marrow

involvement; suspected cases of nutritionalanaemia; suspected chronic lymphoproliferativediseases such as chronic lymphocytic leukaemia;lymphoma with leukaemic spills; evaluation oftherapeutic response in haemopathies amongothers.[2, 4, 5]

Preparation of a Peripheral Blood Film slide

To ensure accurate and reliable diagnosis, pre-analytical variables that can affect the quality offilm must be controlled. These include patientpreparation and consent, blood samplingtechnique, transportation of the sample to thelaboratory and sample preservation. Bloodsampling is invasive therefore the patient/clientshould be counseled about the procedure.Commonly, blood is obtained from peripheralveins and stored in anticoagulated bottle forstipulated time according to viability of cells inparticular anticoagulant. Blood to anticoagulantratio should be in the right proportion. Rarely,capillary blood may be obtained by finger-prick.Care should be taken to ensure minimal damageto the tissues. Excess tissue fluid affects thedistribution of the cellular elements of blood.Ethylene diamine tetra-acetic Acid (EDTA) is theanticoagulant of choice. Samples should be sentto the hematology laboratory as soon as possible.Samples are best analyzed within 2 hours of bloodcollection. Delay in preparation of blood smearmay allow for degeneration of the cellularelements of blood and may result in a pseudo-thrombocytopenia (falsely reduced platelet count)due to formation of platelet aggregates.[2]

Fig: 1 Site for Peripheral Blood Smear


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Slide preparation is done by trained personnelpreferably a medical laboratory technologist, whocan ensure quality slides for microscopy.

Laboratory assistants can also be trained aboutthis technique of slide preparation.

Fig: 2 Preparation of Peripheral blood smear & prepared tongue shaped smear.

One requires slides, pipette/capillary tube andblood spreader to make PBF smear. The ‘push’(wedge) or cover-slip method is used.[6,7] Theformer is more commonly used.[7] In the wedgemethod, a drop of well mixed blood (minimum of10 gentle inversions) is placed on the base of aslide close to one end (about 1 cm from the edge)with a pipette/capillary tube. A spreader slidewith chipped edges is placed on the base slide infront of the blood and moved backwards to touchthe drop of blood which makes the blood spreadalong the base slide-width. The spreader slideshould have a smooth end to prevent the tail endof the smear from being irregular. Then, a smearis made with the spreader inclined at an angle ofabout 30 to 45 degrees to the blood. Care shouldbe taken not to apply excessive pressure on thespreader slide when smearing. This can lead toslide breaks and laboratory accidents. Smearartifacts may be caused by dirty slides, fatdroplets or poor quality slides. [8]

Laboratory safety precautions should be takenwhile working on any clinical specimen. Everyblood specimen should be treated as potentiallyhazardous. Though stains commonly used areintercalating agents that destroy microbes, they donot offer protection against HIV and HBV. Thesmear should cover two-thirds of the base slidelength and should have an oval feathered end. Asa rule, the faster and steeper the smear, the thicker

it is.[9] For instance, such smear may be adaptedfor anemic samples. The smear is properly airdried. Avoid high humidity (causes inadequatedrying) when making a smear as it commonlyaccounts for the artefactual sharp refractile borderdemarcating the area of central pallor, thusmaking hypochromia difficult to assess. Thenproceed to label the slide with pencil or crayon onthe frosted end of the slide or the head end. Thedried smear is fixed with absolute methanol orethyl alcohol and stained with a Rowmanoskystain. A properly air dried smear should be fixedwithin 4 hours of preparation but preferablywithin one hour. [6] Good fixation requires about10 to 20 minutes. Improper fixation causesartefactual burr cells (crenated red cells withrefractile borders).

Romanosky stains are mixtures of acidic dye andbasic dyes that give a differential staining of thedifferent cellular components.[10] Commonlyused stain in our environment is Leishman stainwhich is composed of polychrome methylene blue(basic component) and eosin (acidic component).May-Grunwald Giemsa or Wright-Giemsa staincan also be used.[9] The intensity of the stainingvaries with the duration of stain contact time andconcentration of the stain. It is important todetermine the adequate contact time with eachnew batch of stain made.


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Fig: 3 staining technique of Peripheral blood smear

The smear is floored with stain for about 5-10minutes, then double diluted with buffered waterand allowed for another 5–10 minutes for the cellsto pick the stain. After this, the slide is properlyrinsed under running water. Attempts should bemade to wipe the underside of the slide withcotton wool to remove excess stain. Finally, theslide is placed on a rack with the feathered endsloping upwards to dry. Stain artifact such asdebris and precipitates may be caused by over-staining (excess stain contact time) andinadequate washing under running water.Occasionally, large cells such as monocytes maybe pushed to the periphery and feathery end of thefilm and this should be noted when interpretingthe film. Infrequently, smears are made frombuffy coat layer (white area between the plasmaand red cell layer, rich in white cells and platelets)after heavy spin centrifugation especially inneutropenic specimens.

Slide preparation can be quite laborious especiallyif large numbers of specimens are to be handled.However, automated slide stainers such as adipping- style slide stainer are available.[8] Twoor more slides should be made per specimen andthe quality of the slide should be assessedimmediately. It is safer to produce a new slidethan to interpret a poor quality slide. Quality ofthe film produced depends on a proper smearingtechnique and quality of the stainingprocess.[11] For a quality differential staining tobe achieved, the stain requires an adequate

contact time to avoid over or under staining. Forquality control, the stain quality should becompared with a well made, normal, cover-slipped slide on day to day basis to detectdeterioration in stain quality.

Interpreting a Peripheral Blood Film

The hematologist may be a trained laboratoryperson but preferably a laboratory physicianespecially for slides with significant pathology areauthorized to interpretate the smears.[12, 13] Theslide is viewed at the body of the smear, usuallybeginning about one millimeter away from the tail(the monolayer part). The head of the smearshould be avoided because of high cell density.The head portion of the blood film might be ofinterest when investigating for presence ofmalaria parasites or microfilaria. The featheredend may be examined for platelet clumps andlarge cells like monocytes and blasts.

Microscopy requires a skilled systematicapproach. A quick assessment of a smear can bemade within 3 minutes but an abnormal filmwould require longer time for wider view anddifferential cell counts. Peripheral blood smearcan be used for estimation of manual blood cellcounts. With the advent of automated cellcounters which are more reliable and accurate,manual differential counts of white blood cellsusing PBF is gradually fading in routinehaematology laboratory practice. However in


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resource deprived/ poor regions where automatedcounters are not readily available, assessingdifferential cell counts from PBF a valid option.In nutshell , the value of peripheral blood smear

in assessing morphology and differential countingof blood cellular elements cannot be down-played.

Fig 4: Ideal peripheral blood smear with Head Body & Tail

Morphology of the blood cells on a PBF smear isbest discussed in line with each haemopoietic celllineage. The distribution, size, shape, color,cellular inclusions of the red blood cell (RBC)and morphology of the other major cell linesshould be carefully assessed. However, someabnormalities such as broken cells (smear orsmudge cells) may be artefacts and should betaken into consideration when reporting. Forestimating total leucocyte count, the smear cellsseen must be included in the counts to avoidspurious results.

Blood film should be interpreted alongsidepatient’s clinical details (history and physicalexamination). Results of other routine laboratorywork-ups including whole blood count,erythrocyte sedimentation rate, red cell indicesshould be part of the interpreting framework forreporting a PBF.

Red Cell morphology

The normal red cell is biconcave disc-shaped,measures about 7–8 µm in diameter, has centralpallor (approximately a third of the red celldiameter) and lacks intra-cytoplasmic inclusions.

Red cells are pink in color when stained withRowmanosky dye because the hemoglobincontent of the red cell picks upeosin.[8] Abnormal variations in cell size, shape,color, presence of intracellular inclusions andpathologic arrangement of the cells suggests apresence of abnormalities.

On microscopy, a normal sized red cell iscomparable to the size of the nucleus of a smalllymphocyte. Normally, red cells exhibit narrowvariations in size as reflected by normal red celldistribution width (RDW) of 11-15%. A widevariation in cell size is described as anisocytosis.Abnormalities of cell size can be microcyte(smaller) or macrocyte (larger RBC).Anisocytosis correlates with mean cell volume(MCV) except in combined deficiency states. Thenormal MCV range is 76–96 femtoliters. MCV<76fl suggests microcytosis while MCV >96flsuggests macrocytosis. [14] Macrocytes may beoval (ovoid) or round in shape and this hasdiagnostic implications. Oval macrocytosis isassociated with megaloblastic anaemias (folate orcobalamin deficiency), myelodysplasia and use ofdrugs like hydroxycarba-mide. Round macrocytesare seen in liver disease and alcoholism.


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Fig 5: Descreptive terms used for Red Cells

Red cell inclusions often result from defectivematuration of the erythrocytes, oxidant injury tothe cells or infections. Hypochromia reflects lowhaemoglobin content in the red cell andcommonly results from iron deficiency. Severelyhypochromic and large cells with thin border aretermed leptocytes and may also be seen in liverdiseases.

Furthermore, the arrangement of the cell maygenerate some clinical suspicion. Rouleaux

formation (stacking of the red cells like coins) inthe presence of a bluish background suggestsparaproteinaemia/plasma cell dyscrasia. Rouleauxare also seen in macroglobulinaemias.Agglutination of the red cells may be seen in coldhaemagglutinin disease (CHAD) andWaldenstroms macroglobulinaemia whileerythrophagocytosis is seen in paroxysmal coldhaemoglobinuria.


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Fig: 6 Terminology used in anaemia on the basis of Red Cells morphology

White Cell morphology

Aberrations in leukocyte morphology areassociated with a number of pathologies. A quickassessment of cell counts can be made. Normally,you see about 2 to 5 leukocytes per high powerfield (HPF). As a rule, a leucocyte/HPFapproximates about 200 and 2000 cells inperipheral blood at x10 objective and x100objective respectively. The field factor iscalculated by dividing total leucocyte counts bythe average number of leucocytes seen on tenfields.[10] Leucocytosis is suspected when WBC>5 leucocytes/HPF and leucopenia <2 cells/HPF.The more the number of cells counted, the betterthe accuracy of the cell count estimates.Therefore, cell count estimation of leucocytes will

give a better representation at low powerespecially in leucopenic specimens.

A manual review of automated counts withperipheral blood film should be performed whenflagging occurs due to excess counts. Falselyelevated leucocyte count may be generated by theautomated or manual counts due to circulatingnucleated red cells. A PBF examination can beused to correct the error. The correcting formulais given thus.[4]

Corrected WBC = [estimated WBC/(100 +Number of nucleated RBC among 100 WBC)]x 100 %.Hence, blood film remains a means of validatingabnormally high counts generated manually orfrom automated particle counters.


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In the peripheral blood, the proportion ofpolymorphonuclear (PMN) cells to mononuclearcells varies with age but in adults’ neutrophils isthe most abundant. They constitute about 40 to75% of entire leucocytes, lymphocytes about 20-45%, eosinophils 1 to 6%, monocytes 2- 10% andbasophils <1%. Reductions or increase in any ofthe white cell series may be absolute or relative.For example, relative lymphocytosis means totalwhite cell count is adequate but the lymphocytespredominate.

Mature neutrophils have segmented nucleus with2 to 5 lobes joined by a thin filament. Less matureforms include bands (stab, juvenile) forms,metamyelocyte, myelocyte, promyelocyte andmyeloblast in that order. The cytoplasm of amature neutrophil is pink or nearly colorless andpossesses moderate azurophilic and specificgranules.[19] The bands have unsegmentednuclear morphology. Neutrophilia is commonly aresponse to bacterial infections especiallypyogenic infections. Other associations ofneutrophilia include any form of acuteinflammation (such as myocardial infarction),burns, corticosteroid use (inhibits neutrophilmargination), malignancy, chronic myelogenousleukaemia.

Left shift is a term used to describe an abnormalrise in the proportion of circulating neutrophil

precursors. Normally, mature segmentedneutrophils are seen with band neutrophilpopulation less than 8% and metamyelocytes lessthan 0.5%. [9] However, an increase in theproportion of myeloid precursors is termed leftshift. Severe neutrophilia with left shift is termedleukaemoid reaction. In severe infections, toxicgranulations are seen in the neutrophils cytoplasmdue to compensatory increase in microbicidalgranules.

Right shift or neutrophil hypersegmentation is adiagnostic feature of megaloblastic anaemia. It isdefined by the presence of at least one neutrophilwith six or more nuclear segments or at least 5%of circulating neutrophils with five nuclearsegments. Neutrophil hypersegmentation may befamilial, associated with iron deficiency anaemiaor renal failure.[21]

Neoplastic lymphoid blasts when seen on bloodfilm are also large with a size comparable toactivated lymphocytes but have a high N: C ratio.Lymphoma cells are seen in leukaemic phase ofNon-Hodgkins lymphomas and usually showvarying sizes and various nuclear morphologies.Some have single deep nuclear cleavage(follicular cells), some have multiple indentationsand clefts (mantle cells).

Fig 7 : Cell differentiation and Morphology


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Relative lymphocytosis is normally seen inchildren age less than 5 years. [22] Other causesof relative lymphocytosis include acute viralinfections, connective tissue diseases,thyrotoxicosis and adrenocortical insufficiency.Causes of absolute lymphocytosis include reactive

conditions like infectious mononucleosis,hepatitis, Cytomegalovirus infections, pertussis,chronic intracellular bacterial infections (such asTuberculosis or brucellosis), chronic lymphocyticleukaemia, acute lymphoblastic leukaemia andleukaemic spills of lymphomas.

Fig 8 : various types of White Blood Cells

Eosinophils are slightly larger than polymorphsand the nucleus is usually bilobed. Their definingcharacteristic is the presence of orange-redgranules in the cytoplasm. Significanteosinophilia may be seen in allergies and parasitesinfections. However, marked eosinophilia(>1500/ml) suggest hypereosinophilic syndrome(especially with associated tissue damage) or aneoplastic entity especially when there is anassociated cellular dysplasia as in chroniceosinophilic leukaemia.

Basophils are slightly smaller than polymorphsand have large deeply basophilic (bluish) granulesthat may even totally obscure the nucleus.Basophilia is seen in hypersensitivity states andmalignant conditions like lymphomas and chronicmyeloid leukaemia.

Monocytes are the largest cells in the peripherywith blue-grey ground glass cytoplasm. Itsnucleus is large and assumes various shapes but

often horse shoe shaped. Monocytosis is seen inchronic bacterial infections such as tuberculosis,inflammatory conditions like Crohn’s disease,haematological malignancies such as chronicmyeloid leukaemia and acute myeloid leukaemia.

Platelet morphology

Platelets (thrombocytes) are approximately 2-4 x0.5 microns in dimension (which is about a thirdof a normal sized red cell) with coarsecytoplasmic granules. They are formed frombudding off of the cytoplasm of megakaryocytesin the marrow. It is expected that we seeapproximately 7– 15 platelets on x100 objective.A platelet/HPF is equivalent to approximately15,000- 20,000 platelets in circulation. Anincrease in platelet count is termedthrombocytosis while a decrease is termedthrombocytopenia. Qualitative abnormalities ofplatelets are termed thrombasthemia and requireplatelet functional studies to identify them.


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Thrombocytopenia can result from reducedproduction as in bone marrow failure syndromes,increase peripheral destruction (as in disseminatedintravascular coagulopathies and other thromboticmicroangiopathies) or increased splenic

sequestration (as in hypersplenism).Thrombocytopenia may be spurios (pseudo-thrombocytopenia) in EDTA-induced plateletaggregation or presence of clots in the bloodspecimen.

Fig 9: Platelets on PBF and abnormal forms

Causes of thrombocytosis include majorsurgeries, post splenectomy, preterm infants,haemorrhage, acute haemolysis, iron deficiency,infections, connective tissue diseases (e.g.systemic lupus erythematosus, rheumatoidarthritis), use of cytokines (thromomimetics), andcertain drugs. Thrombocytosis can be associatedwith malignant conditions especiallymyeloproliferative neoplasms (PolycythaemiaVera, myelofibrosis, essentialthrombocythaemia).

Large platelet forms may also be seen. Usually,large platelets are caused by hyperactivity ofmegakaryocytes due to increased demand.Reticulated platelets (younger larger forms) arereleased faster. Falsely elevated automatedplatelet counts may be due to red cell fragments inmicroangiopathic haemolytic anaemias, fragmentsof leukaemic cells or even fungi. Giant platelet(about the size of a normal red cell or more) isseen in inherited conditions like Bernard Souliersyndrome, May-Haggelin anomaly or WiskottAldrich syndrome and acquired states likemegaloblastic anaemia and myeloproliferativedisorders.

Reporting a Peripheral Blood Film

When laboratory results are generated, they mustbe transcribed into reports and signed by thehaematologist especially when there is asignificant PBF abnormality. The typicalreporting format begins with the patient’s bio-data, hospital number, requesting physician, dateof request, date of report and clinicalsummary/details of the patient. The body of thereport includes detailed characterization of eachof the major haemopoietic cell lines: erythrocytes,leucocytes and the platelets. This is followed by asummary of the significant findings, likelydiagnosis with differentials, other recommendedlaboratory evaluations and authorizing signatureof the laboratory physician with date.

Diagnosis from blood film must be corelated withclinical features in the patient as such; thelaboratory physician (thehaematologist/haematopathologist) holds theprivileged position of being able to marry thepatient’s symptomatology with thehaematological findings to proffer specificdiagnosis or differentials particularly in primaryhaematological disorders and other systemicdiseases with haematological manifestations.


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Reports are generated in duplicates and stored in aretrieval system (electronic or manual or both).Films/ slides should also be stored and preservedfor a minimum length of time (as stipulated bylocal guidelines) for possible retrieval or review.Slides are stored in shelves away from lightexposure.

Conclusion

Making diagnosis from a PBF requires a soundclinical database of the various possiblecytological abnormalities, their aetiologies and awealth of laboratory experience. Conclusionsfrom a PBF can be truly diagnostic for a diseasecondition such as a blood film diagnosis of sicklecell disease or chronic myeloid leukaemia. Inother cases, it is at best suggestive and requiresfurther laboratory work-ups or more advancedinvestigations such as cytochemistry, flowcytometry, cytogenetics or molecular techniquesespecially when dealing with malignancies.

Despite the major advances in genetic andmolecular techniques in diagnosis of variousdiseases the examination of blood smearmorphology remains an indispensable tool to thehaematology practice. It remains a frontlinediagnostic jigsaw in unraveling mysteries behindcryptic symptoms and signs in primary andsecondary haemopathies.

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19. Bainton DF. Morphology of Neutrophils,Eosinophils, and Basophils. WilliamsHematology. 7th Edition 2006.

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