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Normal platelets, variability and artefact
Platelet size: Circulating platelets are bi-convex discs of around 2–3 µm diameter (these form less than a quarter the area of normal red cells when spread on a glass slide). Platelet size can vary in response to illness - for example, platelets responding to increased need (such as immune thrombocytopenia) may be larger. Large platelets are arbitrarily defined as being more than one quarter the size of a red cell, and some platelets of this size will be found in the blood of many normal individuals (although infrequent in number generally accepted as <5%). The ‘giant platelet’ is different: a single platelet that has a size equal to or greater than the size of a red cell is ‘giant’ – such cells are rare in normal blood, and may indicate underlying illness. In essence, and film with frequent large platelets, or with more than the occasional giant platelet should be checked carefully for underlying disease.
IMAGE: Low power with size range a23 (do we also do a normal?)
Platelet number: In normal health platelet counts lie between 150-400 x109/l. This means that platelets are significantly less frequent than red blood cells (approximately 1 platelet to 10-20 red cells). This ratio (1:15) can be helpful in assessing platelet number, but take care since the actual ratio may vary according to the area of the blood film examined and the number of red cells present. Also, since the most frequent reason to count platelets manually is when they are clumped, a visual determination can provide only an approximate answer (however this can still be very helpful to clinicians where a spurious elevation or reduction in count is present). Elevated or reduced numbers of platelets can occur in a wide range of conditions, and altered number is not specific to single disease states. This section focuses mainly on morphological aspects, but the more frequent or important causes are outlined in the tables.
Table of causes via link Platelet appearance: Electron microscopy describes four zones within platelets: (i) the peripheral membrane zone rich in adhesive molecules; (ii) the submembrane area of tubules and filaments involved that determine shape; (iii) the granular zone with alpha and dense platelet granules; (iv) a tubular system that promotes shape change. However, detailed structure cannot be determined on blood smears, where the most common appearance is a densely-stained round or irregular cell fragments. However, platelets do have a morphology that can be seen on well-stained smears. Furthermore, immediately following spreading the interaction between platelet and glass slide may cause some activation with features of partial activation apparent, and structural detail may be seen - particularly in larger forms. Images (1): platelets may appear uniformly granular, but partial activation may cause short membrane projections, and a spread form with a central granular and peripheral membrane zone (fried egg) may be seen (a1, a2, a3). Images (2): Occasionally platelets may overlie red cells or abut against them; in such cases the platelet cytoplasm which is otherwise not easily seen can cause a characteristic ‘halo’ that displaces haemoglobin (a4) or indents the red cell membrane (a5).
Distinguishing platelets from other elements A number of features may be mistaken for platelets; and it is important to distinguish these morphologically. The key is to know how a platelet should appear – the stained shade, density and shape all contribute - if something appears unusual then examine it closely. Some examples are shown in the images below:
IMAGES Stain debris are the most frequently encountered problem, and may superficially resemble platelets and may occasionally be a source of confusion when platelet counts are very low, but is generally distinguished based on colour, density, or shape. Debris (a6, a7) Other elements that can cause issues (including to automated counters), these include infecting organisms, cryoglobulin, or red cell fragments. Often recognizing the presence of these elements is easy if you think about them, but you need to think about them! Infections (a24) Cryoglobulin (a25) Microspherocytes (a26) Fragments (need)
Artefacts affecting platelets Artefacts affecting platelets can be recognised morphologically. The most frequent platelet aggregation or clumping. This phenomenon induced only in the anticoagulated samples and is caused by non-specific antibody associated particularly with EDTA anticoagulant and has no clinical significance, but may cause difficulty in interpreting platelet counts. Platelet clumps will usually be seen throughout the film, but are most obvious at the end of the blood smear. A related, but distinctive, phenomena is ‘satellitism’ where the platelets form a rosette around neutrophils and again has no clinical significance. Finally, in samples damaged by heat there may be extensive platelet clumping and degranulation with amorphous clumps that are difficult to recognise. Aggregation (a8) Burns/old (a9) Satellitism (a10)
TABLE: Causes of high platelet counts
Haematological neoplasms Key features: although not always the case, platelet counts may be very high (>1000x109/l), there may be morphological abnormalities affecting the platelets, or characteristic changes affecting other lineages Look for marked variability and enlargement of platelets; where more marked morphological abnormalities arise this may suggest transformation. Where part of an MDS/MPD or acute leukaemia, the features affecting other cell types will be more conspicuous. o essential thrombocythaemia o prefibrotic stage of primary myelofibrosis o polycythaemia vera (there may also be iron deficiency) o chronic myeloid leukaemia (platelet count is rarely affected alone) o myelodysplastic/myeloproliferative neoplasm: refractory anaemia with ring sideroblasts and thrombocytosis o rare cases of acute myeloid leukaemia (t(3;3) or inv(3))
Reactive and other causes Key features: The platelet count is usually <1,000 x 109/l with relatively normal platelet appearances. The cause may be obvious to the clinical team, or other features of the blood film such as iron deficiency, signs of inflammatory response or splenic hypofunction Some more frequent causes are shown in the table. • Chronic inflammation: including rheumatoid arthritis, inflammatory bowel disease, tuberculosis • Acute blood loss: haemolysis or haemorrhage • Iron deficiency • Cancer • Splenectomy or hyposplenism TABLE: Causes of low platelet counts
Decreased Platelet Production Key features: This group of causes will generally have additional morphological clues within other cell lineages, there may be changes of form affecting the platelets themselves The most commonly encountered are: • Bone marrow infiltration: as part of haematological (or less frequently non-haematological) • Bone marrow failure: aplastic anaemia or chemotherapy (rarely poisons) • Ineffective production: MDS or nutritional deficiency • Inherited states: Bernard‒Soulier syndrome MYH9-related disorders grey platelet syndrome
Increased Platelet Destruction Key features: A compensatory increase in platelet number may cause the platelets to be larger; it is important to look for features of cause (these may be absent in immune causes). • Viral illness including mononucleosis, hepatitis and HIV • Malarial infection • MAHA including DIC and sepsis, HUS, TTP or physical destruction • Pregnancy gestational 100-150 but consider ITP, pre-eclampsia or HELLP • Immune ITP or autoimmune, drug induced
ABNORMAL PLATELET MORPHOLOGY Large or giant platelets with mainly normal appearance Definition: large platelets are arbitrarily defined as having a diameter of 4um or greater (equivalent to about a quarter the size of a normal red cell (a12) Large platelets can occur in normal blood, but when frequent they are associated with particular pathological states: • High counts: Myeloproliferative disorders (typically in early disease larger forms will be found, but appearances will be normal) • Low counts: Accelerated platelet production: immune thrombocytopenic purpura or other conditions (mature in blood) Congenital large platelets o Bernard‒Soulier syndrome o Glanzmann thrombasthenia; o MYH9-related disorders (a27)
Small platelets with normal appearance Definition: Seen in Wiscott-Aldridge: the MPV measured by automated counters is <5fl compared with 7.5-12 for normal platelets, converting this to a flattened area on a slide is difficult, but expect the average area to be around half of the normal size. Associated with low platelets • Wiskott‒Aldrich syndrome (images)
Platelets with abnormal or absent granules a15 Associated with…. • Myelodysplastic Syndrome • Myelofibrosis • Grey and pseudo-grey platelets (a13, a14)
Profoundly abnormal platelet forms Associated with….. • megakaryocyte fragments (a17, a18) • micromegakaryocytes (a16, a19) • circulating megakaryocyte nuclei (a20) • megakaryoblasts (a21, a22)