Reporting and grading of abnormal red blood cell morphology

Transcription

1 International Journal of Laboratory Hematology REVIEW The Official journal of the International Society for Laboratory Hematology INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY Reporting and grading of abnormal red blood cell morphology B. T. CONSTANTINO Mississauga, ON, Canada Correspondence: Benie T. Constantino, 5591 Haddon Hall Road, Mississauga, ON L5M 5G4, Canada. com doi: /ijlh SUMMARY In spite of the continual standardization of test result formats, the improvements of laboratory technologies, publications of reference guidelines, and the advancements in hematology analyzers, the methods of reporting or grading abnormal red blood cell morphology still vary among laboratories everywhere. This article describes the methods or systems of reporting abnormal red cell morphology and the conditions associated with the abnormalities. Received 3 December 2013; accepted for publication 24 February 2014 Keywords Red blood cell morphology, grading system, standardization INTRODUCTION More than four decades ago, equipped with the earliest automated hematology Coulter Counter Model S to count blood cells and to determine the size (MCV) and hypochromia (MCH) of the red cells, an attempt was made to promote uniformity (standardization) in grading hematologic abnormalities [1]. In spite of this, and the continual standardization of test result formats, publications of reference guidelines, the improvements of laboratory (lab) technologies, and the advancements in hematology instrumentations [1 7], the methods of reporting or grading abnormal red blood cell morphology (RBC-M) still vary among laboratories everywhere [8, 9]. This is probably because some reference textbooks and laboratories have different grading systems and grading levels of reporting results [1 4, 10]. The grading system represents the method for reporting results, whereas the grading level indicates the relative percentage (%) or fraction of abnormal cells in the peripheral blood film (PBF). For example, one reference textbook [11] grades elliptocytes/ovalocytes as slight (2 5%), moderate (6 15%), and marked (>16%) compared to other reference [4] which rates the same abnormality as 1+ (6 10%), 2+ (10 25%), 3+ (25 50%), and 4+ (>50%). Moreover, other laboratory [9] reports the same red cell changes as + (1 5%), ++ (5 25%), and +++ (>25%). Undoubtedly, the lack of a uniform grading system can lead to inconsistent and confusing results as reflected in these examples. The grading system and grading level vary; three types of grading systems can be observed. The grading level shows two varying formats: one with three levels and the other, four grade levels format. The grading level differences, however, are statistically significant, although they may not be clinically. Currently, there are two systems or methods of grading RBC-M [2 4, 11]. While some laboratories report or grade the degree of morphologic abnormalities 1

2 2 B. T. CONSTANTINO REPORTING AND GRADING OF ABNORMAL RBC MORPHOLOGY numerically (plus) from 1+ to 4+, others used descriptive terms, such as slight (few), moderate, or marked, and/or rare or occasional. At times, others just report the abnormal morphologic findings as present. Because there is no evidence that either system is better than the other, the author emphasizes that maintaining consistency within a chosen system is a good laboratory practice and is also recommended by laboratory accrediting services. For the grading system (numerical or descriptive) to be clinically meaningful, it should be clearly defined by their corresponding grading levels and be understood by the technologists and clinicians. Tables 1 and 2 show the condensed reference guide for grading abnormal RBC-M and the conditions associated with different grade levels, respectively. These tables are handy and can be easily used as a reference guide when performing microscopy. This article describes the grading systems or methods of reporting RBC-M and the conditions associated with the abnormalities, and explains the reasons for standardizing the method of reporting RBC-M nationally or internationally. ANALYSIS AND INTERPRETATIONS OF RESULTS The reporting or grading of abnormal RBC-M is an essential component of hematology process and can serve as an invaluable aid in the diagnosis of a variety of disorders (Tables 1 and 2). The grading of RBC-M represents the magnitude or degree of morphological changes brought about by intrinsic or extrinsic factors involved in the pathogenesis of a specific disease () [21]. Some specific red cell aberrations including spherocytes, target cells, elliptocytes, macrocytes, and hypochromic microcytes are indicative of a particular disorder. They appear in varying percentages or fractions of cells at certain stages of many s (disorders), thus they may not be regarded as distinguishing features of a single disease. However, they are significant for they reflect common pathways of red cell destruction, altered function and structure, or abnormal production and metabolism. For example, spherocytes (decreased surface membrane redundancy) may be seen in varying percentages in patients with autoimmune hemolytic, microangiopathic hemolytic, hemoglobinopathies, or after thermal injury. Since different types of abnormal red cells arise by different etiologic processes, the diagnosis of a disease can be made by assessing PBF in conjunction with clinical and other automated laboratory data such as numerical and graphical data [22, 23]. The interpretation of the reference guide (Table 1) shown for illustration of the grading system of RBC-M is self-explanatory. This simplified reference guide is presented in a two-system or three-graded format: 1+ or slight, 2+ or moderate, and 3+ or marked. Notice there is no 4+, rare, or occasional comment. The clinical interpretation or significance of grade 3+ and 4+ is almost the same markedly increased, thus it is practical to merge them into one grade and simply reports 3+. The rare or occasional comment is meaningless or redundant. Although the grading of abnormal results looks simple, the grading itself is complicated as it requires experience and expertise to identify and distinguish many abnormal cells. Specific details on some morphological disorders such as burr cells, Howell-Jolly bodies, stomatocytes, teardrops, agglutination, microcytes, sickle cells and others and how to examine and grade abnormal RBC- M and their mechanisms of formation are discussed elsewhere [see Refs. 4, 8, 13 18]. As a rough guide, the size of the nucleus of normal lymphocyte and normal red cell is almost the same. Any deviation in size, shape, and staining properties represents abnormal red cells. Schistocytes/fragments are small pieces (fragments) of red cells with irregular outline characterized by triangular, crescentic, and half-moon-shaped cells. They are seen especially in syndromes of microangiopathic hemolytic such as thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. The presence of <1% fragments in the PBF is considered normal. However, the finding of >1% schistocytes with accompanying low platelets and absence of additional severe RBC morphological abnormalities may signify an underlying pathological disorder and should never be ignored. Put simply, for the schistocyte count to be considered clinically meaningful, it should represent the main morphological alteration in the PBF [24]. Some hematology analyzers are capable of detecting and producing a red cell fragments count, the presence of which may be reflected as a high takeoff on the left baseline side of the histogram curve [23]. The count, however, must be correlated with

3 B. T. CONSTANTINO REPORTING AND GRADING OF ABNORMAL RBC MORPHOLOGY 3 Table 1. Reference guide for grading red blood cell morphology [1 4, 9 12] Red blood cells (cell type) Normal (nonspecific) 1+ (%) (Slight/few) 2+ (%) (Moderate) 3+ (%) (Marked) Hypochromasia >40 (MCH pg) pg <18 Polychromasia >20 Microcytes (MCV fl) fl <60 Macrocytes (MCV fl) fl >125 Schistocytes (Fragments) >15 Elliptocytes/Ovalocytes >50 Rouleaux >50 Spherocytes >20 Target cells >25 Acanthocytes >30 Burr cells 30% Report if present Irregularly contracted red cells (Bite cells) 4% Stomatocytes 30% Teardrop cells* 4% Agglutination Dimorphic red cells Dual population Howell-Jolly bodies Oval macrocytes Pappenheimer bodies Parasites Sickle cells pg, picogram; fl, femtoliter. *Teardrop cells accompanied by NRBCs can be reported even <4%. the morphologic grading level. Although identification of RBC fragments apparently seems straightforward, there is no precise consensus definition of schistocytes, hence the variability in the morphological interpretation and reporting between laboratories and observers [25]. Elliptocytes/ovalocytes are cells with oval or elliptical shapes. Often times, these terms are interchangeable. In normal subjects, <15% of red cells may be slightly oval or elliptical, whereas in patients with hereditary elliptocytosis (or sometimes in thalassemia trait), >25 75% of red cells are elliptical [26]. Although elliptical cells of around 15% have been considered as normal, recent studies indicate no higher than 5% is normal [27]. Elliptical cells are also present in patients with dyserythropoiesis, such as megaloblastic and severe iron deficiency. Infiltrative disorders of the bone marrow such as in metastatic carcinoma are associated with formation of elliptocytes and teardrop cells. Rouleaux are red cell aggregates resembling a stack of coins. They are caused by an increase of asymmetric macromolecules, such as globulin and fibrinogen. Associated clinical conditions include multiple myeloma, acute infection/inflammation, and macroglobulinemia. These alterations will result in an increased ESR and a moderate-to-marked rouleaux in the PBF [28]. Slight rouleaux formation is of normal occurrence especially in patients with low red cell counts because of the relative fibrinogenemia; so in this case, their slight presence be reported as nonspecific. Spherocytes are small dense spheroidal red cells with normal volume enclosed within greatly diminished surface area, hence the decreased surface volume ratio and the decreased deformability and filterability. They are usually absent in the blood films of healthy individuals. It is useful to distinguish red cell variations between hereditary spherocytosis (HS) and acquired one. In HS, large numbers of almost the same size of

4 4 B. T. CONSTANTINO REPORTING AND GRADING OF ABNORMAL RBC MORPHOLOGY Table 2. Conditions associated with abnormal RBC morphology based on their grading [4, 8, 13 20] Cell type Slight* (1+) to Moderate* (2+) Marked 3+ Schistocytes (Fragments) Elliptocytes/ Ovalocytes Rouleaux Spherocytes Target cells Acanthocytes Hypersplenism Myeloid metaplasia Megaloblastic Iron deficiency Cancer cytotoxic chemotherapy Enzymes deficiencies Premature infants Renal graft rejection Infection Severe sepsis Myelofibrosis Megaloblastic Severe iron deficiency Sickle cell Hypersplenic state Metastatic carcinoma Sideroblastic state Thalassemia trait Post splenectomy Liver disease Hemoglobinopathies Older population of transfused red cells Heart valve prosthesis Heinz body hemolytic Premature infants Myelofibrosis Newborn/Premature infants Thalassemia minor Postsplenectomy Severe iron deficiency Newborn/Old age Severe burns Sideroblastic Enzymes deficiencies Anorexia nervosa/ starvation Vitamin E deficiency Hypothyroidism Thalassemia major Severe burns Mechanical hemolytic (prosthetic heart valve) Hereditary pyropoikilocytosis Metastatic carcinoma Chronic renal failure Unstable hemoglobin Malignant hypertension Hereditary pyropoikilocytosis Myelofibrosis Hemoglobin C trait South East Asian ovalocytosis Hyperfibrinogenemia Hyperglobulinemia Chronic inflammatory Disorders Microangiopathic hemolytic Hereditary pyropoikilocytosis Severe burns Hypersplenism Clostridium perfringens A-C and A-S trait Thalassemia major Sickle cell Sickle-thalassemia Renal disease Postsplenectomy Neonatal or acquired hepatitis Uremia McLeod phenotype Myxedema Microangiopathic hemolytic Disseminated intravascular coagulation Vasculitis syndromes Hereditary elliptocytes Multiple myeloma Waldenstrom s Macroglobulinemia Hereditary spherocytosis Autoimmune hemolytic Hemolytic transfusion reaction ABO incompatibility C-C disease S-C disease Hb. E disease Liver disease Abetalipoproteinemia Alcoholic liver disease *Note the grading level of some conditions may vary from slight to moderate and even to marked depending on the severity, intensity, and duration of the disorders. spherocytes are present without other abnormal cells other than the signs of erythropoietic regeneration. The automated MCV is within normal range, but the MCHC may be high normal or increased [29], whereas, in acquired one, small to large numbers of spherocytes in varying sizes are present together with other red cell

5 B. T. CONSTANTINO REPORTING AND GRADING OF ABNORMAL RBC MORPHOLOGY 5 abnormalities as seen in pyropoikilocytosis, immune hemolytic, or hemoglobinopathies. The MCHC is usually normal. Target cells (TC) are the morphologic expression of increased surface-to-volume ratio which signify either excessive surface membrane formation or excessive loss of volume or hemoglobin (Hb) content of the red cells. The red cells may look like a bull s-eye, hence the name target. TC may be microcytic, normocytic, or macrocytic. Major causes of microcytic TC or decreased Hb content include severe iron deficiency, thalassemia trait, and certain hemoglobinopathies such as Hb C and Hb E. Some causes of normal TC include sickle cell and sickle cell thalassemia. Macrocytic TC are uncommon, but may be observed in patients with liver disease, postsplenectomy, and in premature infants. It must be emphasized, however, that TC may be an artifact of blood film preparation due to slow air drying or over anticoagulation of blood sample. Acanthocytes are spheroidal dense cells with multiple unevenly distributed spikes of varying length. The presence of few acanthocytes is significant, particularly when it is associated with target cells and Howell-Jolly bodies, as it may indicate asplenia [12]. In some asplenic cases, an unusual increased of lymphocyte counts resembling chronic lymphocytic leukemia may be observed. In all these examples, the grading level of red cell abnormalities depends on the relative fraction or percentage of red blood cells with abnormality and/or the relative degree of abnormality in individual cells seen in the PBF [4]. Their degree or rate of variability, however, is influenced by the severity, intensity, and duration of the illness, rapidity and extent or stage of the (hemolytic) process, and/or whether the abnormality is due to intracellular or extracellular causes such in the RES or in the spleen. When reporting hematologic abnormalities, the total morphologic picture must be presented in conjunction with automated numerical and graphical pictures so that the physicians can relate this picture to the clinical picture of the patient for better diagnosis and health care. COMMENTS Despite the modern hematology instrumentation and technical advancement in the clinical laboratory, review of patient s complete blood count and peripheral blood film (PBF) remains the mainstay of hematologic diagnosis. In other words, the automated findings of microcytic or fragmented red cells have yet to be verified microscopically. Note, however, that because of the tremendous improvements in both the precision and accuracy as well as flagging of many abnormal red cells by modern hematology analyzers, automated results including slightly hypochromic microcytic red cells and slightly reduced platelets can be autoreleased. Depending on laboratory protocol, a qualifying comment can be affixed to the results, such as microcytosis, consider iron deficiency and/or thalassemia. This modification allows technologist to focus more on significant abnormalities, resulting in reduce overall workload and operating costs, decrease turnaround time, and increase productivity in the laboratory. The ability to microscopically verify or grade other abnormal RBC-M and use technology to help sort through that process is inherently subjective and thus may be prone to variation as it is influenced by the skill and experience of the technologists. A simpler and more consistent reporting/grading format may not only be a cost-effective way but also a more systematic and efficient laboratory process that improves the level of reproducibility between observers in the laboratory and thus may lessen or preclude variability in reporting results. The purpose of a red cell morphology report is to convey to the physician the abnormal findings in a legible, comprehensible, and concise manner that will enable judgment on the clinical significance of the abnormalities. Thus, the reporting or grading of abnormal RBC-M should be based on clinical significance and age of the patients. Some morphologic alterations, including sickle cells, fragments, oval macrocytes, Howell-Jolly bodies, acanthocytes, and spherocytes, are quite specific and significant even when they occur in small numbers. In small numbers, other morphologic variations, including target cells, ovalocytes/elliptocytes, round macrocytes, teardrop cells, polychromasia, stomatocytes, and burr cells, are ambiguous and of little diagnostic significance. They become significant only when they appear in considerable numbers (Table 2). Moreover, there are some other red cell abnormalities, such as dimorphic red cells, dual population, or agglutination, which do not need to be quantified and should be reported merely as present. This quantitative descriptor present can be used only if the abnormal red cells in questions meet the

6 6 B. T. CONSTANTINO REPORTING AND GRADING OF ABNORMAL RBC MORPHOLOGY necessary numbers required (Table 2) or as per laboratory protocol so as to avoid misinterpretation of results by the clinicians. Be aware that the morphological characteristics of the newborn s erythrocytes differ considerably from their counterpart in older infants, children, and adults [30]. Children and adult may show up to 1% red cell fragments, whereas full-term infants may show 3% fragments, some polychromasia, spherocytes, teardrops, acanthocytes, target cells, nucleated red blood cells, or Howell-Jolly bodies. Premature infants may even show increased red cell abnormalities. Because of this, the grading of abnormal RBC-M should take into consideration these distinctions to ensure an accurate interpretation of results and their use in patient care. Ideally, standardized definitions and reporting format should be employed within all laboratories. Although standardization would take much time and energy to conform laboratories throughout the world to this system, it would be more pragmatic and beneficial if all laboratories in one country in particular and the whole world used a uniform or standardized method of reporting abnormal RBC-M. The benefits of standardization are enormous including reducing costs (cost-productive), increasing efficiency, protecting the health of the patients, improving the quality of health care, and promoting the global harmonization of medical reporting of results so you can be sure your clinical laboratory result interpretations will be same everywhere. Everywhere in the world you will find that the same morphological alterations in size, shape, color, and staining properties of the red cells, or even the pathophysiologic mechanisms in their formations can be observed there is no racial barrier. Simply put, the hypochromic microcytic red cells of iron deficiency or the fragmented red cells of hemolytic can have the same morphological alterations seen virtually in all patients whether in the Philippines, in Canada, or in other countries. It is undeniable that the clinical laboratory is experiencing globalization by virtue of ever faster travel, improve laboratory technologies and communications and thus the need to harmonize and standardize clinical laboratory reporting of results has increased in importance. The clinical implications of such globalization include the comparability of results so that a patient and/or his physician may compare results obtained in one region or country to another results obtained in a different location. For example in reporting spherocytes, one laboratory location may grade result of 9% spherocytic cells as 1+ or slight [10] while others report or grade the same result of 9% as ++ or moderate [9] and 3+ or marked [4]. Without standardization, the differences between two results may be confusing which may lead to inconsistent diagnostic interpretation, thereby affecting treatment and clinical outcome. This lack of standardization and the virtual similarities of abnormal RBC morphological findings everywhere as well as the benefits of standardization further reinforce the importance of standardization of reporting abnormal RBC-M results in all laboratories. In view of the foregoing statements, therefore, the standardization of reporting abnormal RBC-M is essential and can be accomplished not only within a laboratory, but also in laboratories within a country and internationally. In addition, with standardization and increased sophistication of hematology instrumentation, the evaluation of red cell morphology is becoming more systematic and consistent among laboratory professionals. REFERENCES 1. Walton JR. Uniform grading of hematologic abnormalities. Am J Med Technol 1973;39: Connors DM, Wilson MK. A new approach to the reporting of red cell morphology. J Med Technol 1986;3: Napoli VM, Nichols CW, Cleck S. A semiquantitative estimate method of reporting abnormal RBC morphology. Lab Med 1980;11: Gulati G. Blood Cell Morphology Grading Guide, 3rd edn. Chicago, IL: American Society for Clinical Pathology Press; 2009: Hookey L, Dexter D, Lee DH. The use and interpretation of quantitative terminology in reporting of red blood cell morphology. Lab Hematol 2001;7: Briggs CJ, Linssen J, Longair I, Machin SJ. Improved flagging rates on the Sysmex XE compared with the XE-2100 reduce the number of manual film reviews and increase laboratory productivity. Am J Clin Pathol 2011;136: Carr J, Gaesaman S, Czader M. Performance evaluation of the new Unicel DxH 800 Coulter cellular analysis system in a large hospital setting. Lab Med 2012;43:

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