Ann Ib Postgrad Med. 2014 Dec; 12[2]: 71–79.
PMCID: PMC4415389
PMID: 25960697
PERIPHERAL BLOOD FILM - A REVIEWAS Adewoyin1 and B. Nwogoh2
AS Adewoyin
1Dept. of Haematology & Blood Transfusion, University of Benin Teaching Hospital, Benin City, Edo State
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B. Nwogoh
2Dept. of Haematology & Blood Transfusion, University of Calabar Teaching Hospital, Calabar, Cross River State
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1Dept. of Haematology & Blood Transfusion, University of Benin Teaching Hospital, Benin City, Edo State
2Dept. of Haematology & Blood Transfusion, University of Calabar Teaching Hospital, Calabar, Cross River State
Correspondence:Dr. A.S. Adewoyin Dept. of Haematology and Blood Transfusion, University of Benin Teaching Hospital, PMB 1111, Benin City, Edo State E - Mail: moc.liamg@alomedrotcod, Phone: 07033966347
Copyright © Association of Resident Doctors, UCH, Ibadan
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Abstract
The peripheral blood film [PBF] is a laboratory work-up that involves cytology of peripheral blood cells smeared on a slide. As basic as it is, PBF is invaluable in the characterization of various clinical diseases. This article highlights the basic science and art behind the PBF. It expounds its laboratory applications, clinical indications and interpretations in the light of various clinical diseases. Despite advances in haematology automation and application of molecular techniques, the PBF has remained a very important diagnostic test to the haematologist. A good quality smear, thorough examination and proper interpretation in line with patient's clinical state should be ensured by the haemato-pathologist. Clinicians should be abreast with its clinical utility and proper application of the reports in the management of patients.
Keywords: Peripheral blood smear, Preparation, Examination, Interpretation, Reporting, Blood cells morphology.
INTRODUCTION
In patient care, diagnostic formulations rest on a tripod consisting of clinical history, physical examination and laboratory investigations. The Literature reveals that as much as 70% of clinical decisions and diagnoses are supported by laboratory medicine.1 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.
The diagnostic relevance of a PBF is enormous. The PBF exposes the morphology of peripheral blood cells, which ensures its place in the morphologic diagnosis of various primary and secondary blood and blood related diseases. It’s diagnostic relevance has not been lessened by advances in haematology automation and molecular techniques.
This article attempts to summarize the preparation and reporting of peripheral blood film, its clinical interpretations and the common peripheral blood diagnosis. This will enhance the understanding of PBF interpretations by Clinicians.
INDICATIONS FOR A PERIPHERAL BLOOD FILM
Initiation of a PBF is often a clinical request by the attending clinician on account of a clinical suspicion or less frequently initiated by the laboratory.2, 3 The laboratory may initiate peripheral blood film based on abnormal findings from an automated count or patients clinical information whose diagnosis may be supported by a peripheral blood film. The latter is guided by individual laboratory policies or local regulating guidelines.2
Common clinical indications for peripheral blood film analysis include unexplained cytopenia: anaemia, leucopenia or thrombocytopenia; unexplained leukocytosis, lymphocytosis or monocytosis; unexplained jaundice or haemolysis; features of congenital haemolytic anaemias such as splenomegaly, jaundice or bone pains; suspected chronic or acute myeloproliferative disease e.g. chronic myeloid leukaemia; suspected organ failure such as renal disease, liver failure; features of hyperviscosity syndrome as in paraproteinaemias, leukaemic hyperleucocytosis, polycythaemia; severe bacterial sepsis and parasitic infections; malignancies with possible bone marrow involvement; suspected cases of nutritional anaemia; suspected chronic lymphoproliferative diseases such as chronic lymphocytic leukaemia; lymphoma with leukaemic spills; evaluation of therapeutic response in haemopathies among others.2, 4, 5
PREPARATION OF A PERIPHERAL BLOOD FILM SLIDE
To ensure accurate and reliable results, pre-analytical variables that can affect the quality of film must be controlled. These include patient preparation and consent, blood sampling technique, transport to the laboratory and sample preservation. Blood sampling is invasive therefore the patient/client should be counselled on the procedure. Commonly, blood is obtained from peripheral veins and stored in anticoagulant bottle. Blood to anticoagulant ratio should be in the right proportion. Rarely, capillary blood may be obtained by finger-prick. Care should be taken to ensure minimal tissue damage. Excess tissue fluid affects the distribution of the cellular elements of blood. Ethylene diamine tetra-acetic Acid [EDTA] is the anticoagulant of choice. Samples should be sent to the laboratory as soon as possible. Samples are best analyzed within 2 hours of blood collection. Delay in preparation of blood smear may allow for the degeneration of the cellular elements of blood and may result in a pseudo-thrombocytopenia [falsely reduced platelet count] due to formation of platelet aggregates.2
Slide preparation is done by trained personnel preferably a medical laboratory technologist, who can ensure quality slides for microscopy. Laboratory assistants can also be trained in the art of slide preparation.
One require slides, pipette/capillary tube and blood spreader to make PBF smear. The ‘push’ [wedge] or cover-slip method is used.6, 7 The former is more commonly used.7 In the wedge method, a drop of well mixed blood [minimum of 10 gentle inversions] is placed on the base of a slide close to one end [about 1 cm from the edge] with a pipette/capillary tube. A spreader slide with chipped edges is placed on the base slide in front of the blood and moved backwards to touch the drop of blood which makes the blood spread along the base slide-width. The spreader slide should have a smooth end to prevent the tail end of the smear from being irregular. Then, a smear is made with the spreader inclined at an angle of about 30 to 45 degrees to the blood.8 Care should be taken not to apply excessive pressure on the spreader slide when smearing. This can lead to slide breaks and laboratory accidents. Smear artifacts may be caused by dirty slides, fat droplets or poor quality slides. Laboratory safety precautions should be observed when working on any clinical specimen. Every blood specimen should be treated as potentially high risk. Though stains commonly used are intercalating agents that destroy microbes, they do not offer protection against HIV and HBV. The smear should cover two-thirds of the base slide length and should have an oval feathered end. As a rule, the faster and steeper the smear, the thicker it is.9 For instance, steeper and faster smear may be adapted for anaemic samples. The smear is properly air dried. Avoid high humidity [causes inadequate drying] when making a smear as it commonly accounts for the artefactual sharp refractile border demarcating the area of central pallor, thus making hypochromia difficult to assess. Then proceed to label the slide with pencil or crayon on the frosted end of the slide or the head end. The dried smear is fixed with absolute methanol or ethyl alcohol and stained with a Rowmanosky stain. A properly air dried smear should be fixed within 4 hours of preparation but preferably within one hour. 6 Good fixation requires about 10 to 20 minutes. Improper fixation causes artefactual burr cells [crenated red cells with refractile borders].
Romanosky stains are mixtures of acidic dye and basic dyes that give a differential staining of the different cellular components.10 Commonly used stain in our environment is Leishman stain which is composed of polychrome methylene blue [basic component] and eosin [acidic component]. May-Grunwald Giemsa or Wright-Giemsa stain can also be used.9 The intensity of the staining varies with the duration of stain contact time and concentration of the stain. It is important to determine the adequate contact time with each new batch of stain made or procured.
The smear is floored with stain for about 5-10 minutes, then double diluted with buffered water and allowed for another 5–10 minutes for the cells to pick the stain. After this, the slide is properly rinsed under running water. Attempts should be made to wipe the underside of the slide with cotton wool to remove excess stain. Finally, the slide is placed on a rack with the feathered end sloping upwards to dry. Stain artifact such as debris and precipitates may be caused by over-staining [excess stain contact time] and inadequate washing under running water. Occasionally, large cells such as monocytes may be pushed to the periphery and feathery end of the film and this should be noted when interpreting the film. Infrequently, smears are made from buffy layer [white area between the plasma and red cell layer, rich in white cells and platelets] after heavy spin centrifugation especially in neutropenic specimens.
Slide preparation can be quite laborious especially if large numbers of specimens are to be handled. However, automated slide stainers such as a dipping- style slide stainer are available.8 Two or more slides should be made per specimen and the quality of the slide should be assessed immediately. Poor quality slides should be discarded and new ones produced. It is safer to produce a new slide than to interpret a poor quality slide. Quality of the film produced depends on a proper smearing technique and quality of the staining process.11 For a quality differential staining to be achieved, the stain requires an adequate contact time to avoid over or under staining. For quality control, the stain quality should be compared with a well made, normal, cover-slipped slide on day to day basis to detect deterioration in stain quality which is virtually inevitable over time with use and storage.
INTERPRETING A PERIPHERAL BLOOD FILM
The haemato-morphologist may be a trained laboratory technologist but preferably a laboratory physician especially for slides with significant pathology.12, 13 The slide is viewed at the body of the smear, usually beginning about one millimeter away from the tail [the monolayer part]. The head of the smear should be avoided as the cell density is twice that seen at the tail. The head portion of the blood film might be of interest when investigating for presence of malaria parasites or microfilaria. The feathered end may be examined for platelet clumps and large cells like monocytes and blasts.
Microscopy requires a skilled systematic approach. A quick assessment of a smear can be made within 3 minutes but an abnormal film would require longer time for wider view and differential cell counts. Peripheral blood smear can be used for estimation of manual blood counts. With the advent of automated cell counters which are more reliable and accurate, manual differential counts of white blood cells using PBF is gradually fading in routine haematology laboratory practice. However in resource deprived/ poor regions where automated counters are not readily available, assessing differential cell counts from PBF a valid option. In light of the above, the value of peripheral blood smear in assessing morphology and differential counting of blood cellular elements cannot be down-played.
Morphology of the blood cells on a PBF smear is best discussed in line with each haemopoietic cell lineage. The distribution, size, shape, color, cellular inclusions of the red blood cell [RBC] and morphology of the other major cell lines should be carefully assessed. However, some abnormalities such as broken cells [smear or smudge cells] may be artefacts and should be taken into consideration when reporting. For estimating total leucocyte count, the smear cells seen must be included in the counts to avoid spurious results.
Blood film should be interpreted alongside patient’s clinical details [history and physical examination]. Results of other routine laboratory work-ups including full blood count, erythrocyte sedimentation rate, red cell indices should be part of the interpreting framework for reporting a PBF.
RED CELL MORPHOLOGY
The normal red cell is biconcave disc-shaped, measures about 7–8 µm in diameter, has central pallor [approximately a third of the red cell diameter] and lacks intra-cytoplasmic inclusions. Red cells are pink in color when stained with Rowmanosky dye because the haemoglobin content of the red cell picks up eosin, the acidophilic components of the dye.8 Abnormal variations in cell size, shape, colour, presence of intracellular inclusions and pathologic arrangement of the cells suggests a host of abnormalities.
On microscopy, a normal sized red cell is comparable to the size of the nucleus of a small lymphocyte. Normally, red cells exhibit narrow variations in size as reflected by normal red cell distribution width [RDW] of 11-15%. A wide variation 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. The normal MCV range is 76–96 femtoliters. MCV 96fl suggests macrocytosis. 14 Macrocytes may be oval [ovoid] or round in shape and this has diagnostic implications. Oval macrocytosis is associated with megaloblastic anaemias [folate or cobalamin deficiency], myelodysplasia and use of drugs like hydroxycarba-mide. Round macrocytes are seen in liver disease and alcoholism.
Various shape abnormalities are important clues to the aetiology of anaemia and its differentials are presented in Table Table11.15-17
Table 1.
Red cell shape abnormalities and their differentials
Red Cell ShapesDifferential DiagnosisIrreversibly sickled red cells [drepanocytes]Sickle cell syndromes [SS, SC, Sβthalassemia]Target cells [codocytes, mexican hat cells]Sickle cell disease, haemoglobin C trait, haemoglobin CC disease, thalassemias, iron deficiency, Liver disease [cholestasis], asplenia,Fragmented red cells [schistocytes, helmet cells, keratocytes]Thrombotic micro-angiopathic haemolytic anaemias such as Disseminated intravascular coagulopathy [DIC], thrombotic thrombocytopenic purpura, haemolytic uraemic syndrome.Burr cells [echinocytes, crenated red cells]In-vitro artifact following prolonged storage or slow drying of the smear due to high humidity, uraemia, MalnutritionSpur cells [acanthocytes]Liver disease, Renal failure, Abetalipoproteinaemia, Spur cell anaemia, pyruvate kinase deficiencyTear drop cells [dacrocytes]Myelofibrosis, Myelophthisia [marrow infiltrations], Extramedullary haemopoiesis, Hereditary elliptocytosis, Hereditary pyropoikilocytosis, Severe iron deficiency, Megaloblastic anaemia, Thalassemias, Myelodysplastic syndromeBite cells [degmacytes]G6PD deficiency, Oxidative stress, unstable haemoglobins, congenital heinz body anaemiaPencil cellsIron deficiencyStomatocytesArtifact[ due to slow drying in humid environment], Liver disease, alcoholism, Rh-null disease, Obstructive lung diseaseElliptocytesHereditary Elliptocytosis [>25%]Basket cells [half ghost cells/Blister cells] SpherocytesOxidant damage, G6PD deficiency, Unstable haemoglobins Hereditary spherocytosis, ABO incompatibility, Autoimmune hemolytic anemia [warm antibody type], Severe burns
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Red cell inclusions often result from defective maturation of the erythrocytes, oxidant injury to the cells or infections. Howell jolly bodies are DNA remnants seen in post-splenectomy patients and in anatomical or functional asplenia due to loss of pitting action of the spleen. Basophilic stipplings or punctuate basophilia are denatured RNA fragments dispersed within the cytoplasm and are associated with haemoglobinopathies [thalassemias], lead or arsenic poisoning, unstable haemoglobins, severe infections, sideroblastic anaemia, megaloblastic anaemia and a rare inherited condition, pyrimidine 5’ nucleotidase deficiency. Siderotic granules or pappenheimer bodies appear purple on Rowmanosky stain, blue on Perl’s stain and are seen in disorders of iron utilization like sideroblastic anaemias. Intracellular parasites such as plasmodium or babesia may also be seen. Some other red cell inclusions can only be appreciated with supravital staining [reticulocyte preparations]. Heinz bodies are denatured haemoglobin [seen in oxidant injury, G6PD deficiency]. Haemoglobin H inclusions are seen in alpha-thalassemias giving rise to the characteristic ‘golf ball’ appearance of the erythrocytes. Red cells with bluish reticular fragments [ribosomal proteins and RNA] on supravital staining are reticulocytes. Reticulocytes appear as polychromatic cells on Rowmanosky stained slides. They are immature red cells newly released from the marrow sinusoids and takes about a day or two to mature in the peripheral circulation in those with intact spleen. Nucleated red cells are not normally seen in the periphery except in neonates. Their presence on blood film suggests a severe stress on the marrow forcing their premature release. Circulating nucleated red cells [erythroblasts] may be associated with increased circulating neutrophil precursors; in which case the term ‘leucoerythroblastic’ is used. 4, 15, 18 Leucoerythroblastosis occurs in the setting of marrow fibrosis, marrow stressors as seen in hypoxia, severe anaemia [haemolytic or haemorrhagic] and severe sepsis, marrow infiltrations [due to leukaemia, lymphoma, myeloma or secondary metastasis], marrow challenge with growth factors such as G-CSF and extramedullary haemopoiesis. The circulating erythroblasts may be normoblasts [normal maturation] or megaloblasts [megaloblastic changes]. The color of the red cells is reflected by its haemoglobin content. Increased haemoglobinization is termed hyperchromia. Decreased haemoglobination is hypochromia. Hyperchromic cells lack central pallor and can occur in the setting of large cell such as polychromatic cells, small cells such as microspherocytes or an abnormally shaped cell. Shape abnormalities associated with hyperchromia include irreversible sickled red cells, spherocytes and irregularly contracted cells [ICC or pyknocytes]. Spherocytes are seen in hereditary spherocytosis. Small cells termed microspherocytes [densely haemoglobinized] occur in immune haemolytic anaemia [splenic macrophages bite off portions of the membrane with the bound antibody and the cell reseals with a smaller volume]; burns and less frequently micro angiopathy. ICC lacks central pallor with irregularly [non- uniform] margins and is seen in haemoglobin SC, CC disease, oxidant injury and unstable haemoglobinopathy. Crenated red cells may be artefactual due to crenation of red cells in stored blood following delayed analysis. Hypochromia reflects low haemoglobin content in the red cell and commonly results from iron deficiency. Severely hypochromic and large cells with thin border are termed leptocytes and may also be seen in liver diseases.
Furthermore, the arrangement of the cell may generate some clinical suspicion. Rouleaux formation [stacking of the red cells like coins] in the presence of a bluish background suggests paraproteinaemia/plasma cell dyscrasia. Rouleaux are also seen in macroglobulinaemias. Agglutination of the red cells may be seen in cold haemagglutinin disease [CHAD] and Waldenstroms macroglobulinaemia while erythrophagocytosis is seen in paroxysmal cold haemoglobinuria
WHITE CELL MORPHOLOGY
Aberrations in leukocyte morphology are consistent with a number of pathologies. A quick assessment of cell counts can be made. Normally, you see about 2 to 5 leukocytes per high power field [HPF]. As a rule, a leucocyte/hpf approximates about 200 and 2000 cells in peripheral blood at x10 objective and x100 objective respectively. The field factor is calculated by dividing total leucocyte counts by the average number of leucocytes seen on ten fields.10 Leucocytosis is suspected when WBC >5 leucocytes/hpf and leucopenia