Morphologic Changes in Red Blood Cells: an Illustrated Review of Clinically Important Light Microscopic Findings

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Morphologic Changes in Red Blood Cells: an Illustrated Review of Clinically Important Light Microscopic Findings Malays J Pathol 2021; 43(2): 219 – 239 REVIEW ARTICLE Morphologic changes in red blood cells: An illustrated review of clinically important light microscopic findings Laura TYRRELL1, Gary ROSE2*, Amal SHUKRI2, Samir B. KAHWASH2,3 1Division of Hematology/Oncology, Nationwide Children’s Hospital, Columbus, OH; 2Department of Pathology and Laboratory Medicine, Nationwide Children’s Hospital, Columbus, OH; 3Department of Pathology, The Ohio State University College of Medicine, Columbus, OH Abstract In this article, we provide an illustrated review that may serve as a microscope companion, as well as a reference for the diagnosis of red blood cells alterations and the interpretation of their significance. Beginners in the fields of clinical haematology and haematopathology may benefit from this manuscript’s brevity and practical points, while the more advanced will find it useful as a teaching tool. Keywords: Erythrocytes, anaemia, leukaemia, morphology INTRODUCTION anaemia, point to certain infections or toxicities, and identify signs of systemic diseases. It can Laboratory testing is an important component even be used to supplement or replace other lab of patient evaluation. The complete blood count tests in low-resource settings. (CBC) is the most common diagnostic lab test performed worldwide.1 The information gained Peripheral blood smears preparation and from a CBC can be greatly enhanced by an examination examination of a peripheral blood smear (PBS). At most academic centres, a pathologist or Proper technique in the preparation and trained technologist evaluates and reports PBS evaluation of a peripheral blood smear is findings. Clinicians (particularly haematologists) essential in properly interpreting red blood cell can benefit greatly from reviewing smears, either morphology. Ethylenediaminetetraacetic acid independently or jointly with lab personnel. (EDTA) is the preferred anticoagulant for CBC With more clinical sub-specialisation and testing and making peripheral blood smears. increased reliance on advanced testing, the Fresh blood from a capillary puncture may also practice of a clinician evaluating peripheral blood be used if the smear is immediately made on- smears has become less common. Nevertheless, it site, however, even minor delays will result in is still necessary for clinicians to have familiarity the specimen clotting. Even in anticoagulated with PBS findings and ascertain important specimens, morphological changes start to diagnostic information when a pathologist is develop one hour after collection, so prompt not available. Whether in low-resource settings processing is preferable. Smears should be without trained pathologists readily available made within 8 hours if the specimen is kept or in major medical centres after regular work at room temperature or within 24 hours if it is hours, PBS review remains a relevant skill for refrigerated at 2-8°C. clinicians. Smearing may be performed manually or by A careful review of a well-prepared PBS is a using automated devices. If available, a properly relatively simple, cost-effective way to quickly validated smearing device is generally preferable obtain important and clinically consequential due to the ability to create reproducible smears information. PBS review of red blood cell (RBC, with ample reading areas and low cellular also known as erythrocyte) morphology can destruction. Typically, peripheral blood smears narrow the differential diagnosis for unexplained are stained with variations of the Romanowsky *Address for correspondence: Gary Rose, M.D., Department of Pathology and Laboratory Medicine, Nationwide Children’s Hospital, Columbus, OH. Email: [email protected] 219 Malays J Pathol August 2021 stain, which include Giemsa, May-Grünwald- findings in erythrocytes have little significance Giemsa, Wright, and Wright-Giemsa (the latter when limited in scope. For instance, identification is used at our institution). of a single schistocyte or ovalocyte on a smear Each blood smear contains a thick end is typically clinically insignificant. However, and gradually transitions to progressively certain other red blood cell findings are always thinner regions, terminating in the somewhat significant and require investigation or clinical parabolically-shaped “feathered-edge”. correlation, no matter how rare. Examples of the Morphology is best assessed in the “reading latter include nucleated red blood cells outside of area,” between which there is no significant certain neonatal populations or finding Howell- erythrocyte overlapping (as seen in thicker Jolly bodies. regions), nor is there a linear arrangement of the Finally, good practice merits evaluating erythrocytes with large acellular gaps (as seen all cellular components of peripheral blood in thinner regions). Figure 1 shows the reading (RBCs, WBCs, and platelets) upon PBS review, area on a properly prepared and stained smear. regardless of the indications for smear review. A Selecting an optimal reading area is essential thorough review should be performed on every for proper interpretation of RBC morphology and slide. for avoiding pitfalls. For example, erythrocytes in the thinner areas of a PBS may artifactually Normal red blood cells appear to lose central pallor, resembling Normal red blood cells are biconcave disks, spherocytes.2 Additionally, artifactual rouleaux containing abundant cytoplasmic haemoglobin. may be encountered in thick areas but should This shape allows for a surface area to only be reported when seen in the reading area. haemoglobin volume ratio that is optimal for In contrast with reviewing peripheral smears function and circulation, 3 resulting in a for the presence of blasts, where even a single doughnut-like appearance on PBS. Normally, cell may require clinical justification, many red blood cells vary only slightly in shape and FIG. 1: The reading area (C) of a well-prepared PBS slide is an area where RBCs do not overlap (B – too thick) nor leave large empty spaces (D – too thin). 220 AN ILLUSTRATED REVIEW OF RBC MORPHOLOGY size. The central area (with the least amount liver disease), or genetic limitations on globin of haemoglobin) appears pale (central pallor) chain synthesis (as in thalassaemia) constitute that occupies one-third of the RBC diameter the major categories. (Fig. 2). The outer two-thirds normally exhibit a • An increase in the haemoglobin-to-RBC homogeneous distribution of haemoglobin. The volume ratio is common in RBC membrane RBC outer membrane is smooth and indistinct abnormalities. An example is hereditary from the haemoglobin-rich body of the cell. spherocytosis (HS), an RBC membrane abnormality that results in round cells tensely Factors that influence morphologic changes engorged with haemoglobin. of red blood cells Alterations in the ratio of RBC haemoglobin Alterations in the structure of haemoglobin content to RBC volume: leading to RBC deformity: • A decrease in RBC haemoglobin content to • Abnormal haemoglobin variants such as sickle volume ratio usually results in flaccid red cell haemoglobin, haemoglobin C and unstable blood cells that take the shape of a target on haemoglobin variants. PBS. Keeping in mind that haemoglobin is • Polymerisation and precipitation of normal composed of two major components, a heme haemoglobin due to deficiency of a protective molecule (consisting of a porphyrin ring with enzyme (G6PD, pyruvate kinase, etc.) iron) and globin (a tetramer of amino acid chains) can help explain the list of conditions Increased peripheral RBCs destruction or associated with increased target cells. Iron demand: deficiency (leading to lower production of • Results in the release of young, incomplete- heme), reduced production of protein (as in ly haemoglobinised RBCs (i.e. polychroma- tophilic RBCs) FIG. 2: A microscopic view from a PBS of a child who presented with iron deficiency anaemia and required transfusion. Compare the patient’s hypochromic microcytic RBCs (in red circles) with the transfused normochromic normocytic RBCs (yellow circles). Also seen are “pencil” shaped RBCs typical of iron deficiency anaemia (squares). An echinocyte (burr RBC) is seen in this field (arrow). 221 Malays J Pathol August 2021 Regulatory system dysfunction: drastically altered depending on the severity. • An example is the hypofunctional The aforementioned increase in RDW is reticuloendothelial system (as in absent, demonstrated by marked anisocytosis, or size hypo-functional spleen or even a normal variation between erythrocytes. Additionally, spleen overwhelmed by severe haemolytic relative to the severity, hypochromasia tends to anaemias) resulting in the circulation of RBCs be more pronounced in iron deficiency anaemia. with Howell-Jolly bodies. Iron deficiency anaemia should be suspected when the combination of hypochromasia and Red blood cells in hypochromic microcytic marked RBC size variability is seen. anaemias Morphologically, RBC forms known as “pencil cells”, or “cigar cells” are typically seen Microcytosis is the term used to describe red in iron deficiency anaemia. The term refers to blood cells that are inappropriately small. A hypochromic elliptocytes that may demonstrate good morphologic rule of thumb is that normal a marked increase of the long-to-short axis red blood cells should be roughly the size of ratio. While the mere presence of these cells is a non-activated lymphocyte nucleus. If most non-specific, they are relatively more frequent RBCs are smaller than a lymphocyte nucleus, in iron deficiency anaemia
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