The Blood Count and Film

Transcription

1 2 The Blood Count and Film What Do You Have to Know? The constituent parts of a blood count The meaning and clinical significance of a reticulocyte count The meaning of the words that are frequently used to describe abnormalities in the blood count and blood film and their possible clinical significance The meaning of normal range and reference range The approximate normal ranges for the white cell count, haemoglobin concentration, mean cell volume and platelet count in healthy adults How to interpret a blood count and develop a differential diagnosis The meaning and clinical significance of an erythrocyte sedimentation rate The Full Blood Count The term full blood count (FBC) refers to a group of tests performed simultaneously on a blood sample to assess whether there is any haematological abnormality. The tests that are almost always included in an FBC are shown in Table 2.1. In modern haematological practice these tests are performed on large automated analysers capable of processing many hundreds of samples in a day. Further tests are sometimes also included. The 20

2 The Blood Count and Film 21 Table 2.1. The full blood count. Normal range Normal range Test Abbreviation Units in men* in women* White blood cell count WBC 10 9 /l Red blood cell count RBC /l Haemoglobin Hb g/dl concentration Haematocrit Hct l/l Mean cell volume MCV fl Mean cell MCH pg haemoglobin Mean cell MCHC g/dl haemoglobin concentration Platelet count 10 9 /l * From Bain, B.J. (2006). Blood Cells. Fourth Edition. Blackwell Publishing, Oxford. Reported ranges from different instruments vary. blood count is performed on a blood specimen that has been anticoagulated by being mixed with ethylene diaminetetra-acetic acid (EDTA), a chelating agent that prevents clotting by binding calcium. If you are obtaining a blood specimen from a patient for an FBC be sure to use a tube containing EDTA, add the correct volume of blood and mix the blood sample with the anticoagulant. The white blood cell count The white blood cell count (WBC, also known as the white cell count) was initially determined by counting cells using a microscope and a glass counting chamber. Nowadays it is counted by an automated instrument that detects individual cells flowing in a stream through a sensor. Recognition is either because the cell interrupts a beam of light or because it alters the electrical current flowing between two electrodes. A WBC is performed on a sample in which the mature red cells have been lysed by solutions to which they are exposed within the instrument so that the only cells that will be counted are the white cells and any nucleated red blood cells that might be present.

3 22 Chapter 2 The red blood cell count The red blood cell count (RBC, also known as the red cell count) was initially determined with a counting chamber. As for the WBC, it is now determined by an instrument that counts red cells flowing through a sensor. However, this time the red cells are not lysed. White cells will actually also be included in the count but because they are infrequent in relation to the red cells this does not usually introduce much error. The haemoglobin concentration The haemoglobin concentration (Hb) is determined by lysing red cells and measuring light transmitted through a diluted sample of the blood at a specific wavelength after conversion of the haemoglobin to a stable form. The haematocrit It is possible to measure the proportion of red cells in an anticoagulated blood sample by centrifuging a tube of the blood and comparing the height of the column of red cells with the total height of the blood sample. This test is called a packed cell volume (PCV). It is no longer performed because it is not suitable for dealing with large numbers of blood specimens. The modern equivalent is called a haematocrit (Hct). It has the same significance as a PCV but instruments calculate it rather than measuring it directly. This is done by multiplying the average size of a red cell, the mean cell volume (MCV), by the number of red cells in a litre of blood (the RBC). The mean cell volume The mean cell volume (MCV) was once estimated by dividing the PCV (obtained by centrifugation) by the RBC (from a counting chamber). This was a very laborious technique so it was not done very often. Modern instruments estimate the MCV from the height of the electrical impulse generated by interruption of a light beam or an electrical current. The same electrical impulse is therefore used both to count the cells and to size them.

4 The Blood Count and Film 23 The mean cell haemoglobin The mean cell haemoglobin (MCH) is the average amount of haemoglobin in an individual red cell. Instruments calculate it by dividing the haemoglobin in a given volume of blood by the number of red cells in the same volume. The mean cell haemoglobin concentration The mean cell haemoglobin concentration (MCHC) is the average concentration of haemoglobin, rather than the absolute amount, in an individual red cell. Fig. 2.1 is a visual representation of the difference between the MCH and the MCHC. Instruments calculate it by dividing the haemoglobin in a given volume of blood by the proportion of the whole blood sample that is occupied by red cells. The red cell distribution width The red cell distribution width (RDW) is a measurement of the amount of variation in the size of the red cells. Fig A diagram to illustrate the difference between the mean cell haemoglobin (MCH) and the mean cell haemoglobin concentration (MCHC).

5 24 Chapter 2 The platelet count Platelets are counted by the same principles as red cells and white cells. They can be distinguished from red and white cells by their smaller size. The red cell indices The term red cells indices refers to the RBC, MCV, MCH and MCHC. The formulae (which you do not need to know) for calculating the latter three values are: MCV (fl) = MCH (pg) = MCHC (g/dl) = PCV (l/l) 1000 RBC (cells/l) Hb (g/dl) 10 RBC (cells/l) Hb (g/dl) PCV (l/l) The red cell indices are very useful for indicating the likely cause of anaemia. The reticulocyte count A reticulocyte count is a supplement to an FBC, rather than a normal part of it. It can be performed using a microscope, counting the percentage of erythrocytes that have developed a reticulum or network of precipitated dye after incubation of the blood sample with a dye such as methylene blue (Fig. 2.2). This is a method for identifying ribonucleic acid (RNA) within red cells, thus demonstrating that these are cells newly released from the bone marrow (1 3 days old). Reticulocyte counts can also be performed by automated instruments, using either the same principle as the reticulocyte count with a microscope or alternatively, using a fluorescent dye that binds to RNA. The Differential White Cell Count The different types of white cell can be distinguished from each other by examining a stained blood film. If a hundred cells are counted and the

6 The Blood Count and Film 25 Fig A reticulocyte preparation, showing a reticulum of precipitated dye following incubation of blood with methylene blue. percentages are multiplied by the WBC, the absolute number of cells of each type can be calculated. Calculating the percentage or absolute number of each cell type is referred to as a differential count. It is much more useful to calculate the absolute count than to try to make deductions from the percentages. For example, if a patient had 5% neutrophils and 95% lymphocytes this could indicate either a severe reduction of neutrophils or a marked increase in lymphocytes. Many automated instruments can recognise the five normal types of white cell and can detect the presence of nucleated red blood cells (NRBC) and other cells that are not normally present in the circulating blood. If only normal cells are present they are capable of performing a differential count. If abnormal cells are present it is usually still necessary to perform a differential count on a blood film. However, some instruments are also capable of counting NRBC. Normal ranges for the differential white cell count are shown in Table 2.2. Haematological Terminology Haematologists use precise terms to refer to abnormalities in the blood count and film. Since they use these terms to report abnormalities to

7 26 Chapter 2 Table 2.2. The differential white cell count. Normal range Normal range Test Units in men* in women* Neutrophil count 10 9 /l Lymphocyte count 10 9 /l Monocyte count 10 9 /l Eosinophil count 10 9 /l Basophil count 10 9 /l *From Bain, B.J. (2006). Blood Cells. Fourth Edition. Blackwell Publishing, Oxford. Figures are for an automated instrument and will differ slightly between instruments; figures for manual differential counts will have broader limits. clinical staff it is necessary for all doctors to understand them. The terms used to describe quantitative abnormalities are shown in Table 2.3; unfortunately these terms are not necessarily logical or consistent so you just have to learn how they are used. The terms used to describe erythrocytes and their abnormalities are shown and illustrated in Table 2.4. Normal Ranges and Reference Ranges Once we have got a laboratory result how do we know if it is normal? Laboratories issue test results with a reference range or normal range for comparison. A reference range is derived from a carefully defined population, the reference population: the definition will include the age and gender of the subjects and sometimes the ethnic origin or other relevant details. If a requirement to be in good health is part of the definition then a reference range becomes a normal range. Conventionally, reference ranges and normal ranges include the central 95% of results from the reference population. Ideally, tests would give a clear separation of healthy subjects (the normal range) and an unhealthy population, as shown in Fig. 2.3a. More often, the situation will be as shown in Fig. 2.3c, with a small amount of overlap between sick and well people. Sometimes a test gives poor separation of sick and well (Fig. 2.3b). Either the laboratory or the clinician looking after the patient has to decide the threshold to accept. If the figure chosen includes all sick people then a lot of normal people will also be included; the test becomes sensitive but at the cost of a high rate of false positives. If the threshold is moved down so that no healthy person is

8 The Blood Count and Film 27 Table 2.3. Terminology used for describing quantitative abnormalities in blood counts. Term Anaemia Polycythaemia or erythrocytosis Leucocytosis Leucopenia Thrombocytosis Thrombocytopenia Neutrophilia Neutropenia Lymphocytosis Lymphopenia* or lymphocytopenia Monocytosis Eosinophilia Basophilia Reticulocytosis Reticulocytopenia Meaning Reduced Hb Either term can be used to indicate an increase in the RBC, Hb and Hct Increased WBC Reduced WBC Increased platelet count Reduced platelet count Increased neutrophil count Reduced neutrophil count Increased lymphocyte count Reduced lymphocyte count Increased monocyte count Increased eosinophil count Increased basophil count Increased reticulocyte count Reduced reticulocyte count *The term lymphopenia is particularly illogical because it is used to mean a reduced lymphocyte count not a reduced amount of lymph. Basophilia has a quite different alternative meaning; it also means an increased uptake of basic dyes by the cytoplasm of a cell so that when examined with a microscope it appears blue.

9 28 Chapter 2 Table 2.4. Terminology used for describing erythrocytes and their abnormalities. Term Significance Picture Normocytic Normochromic Of normal size With normal staining characteristics Anisocytosis Increased variation in size Poikilocyte Poikilocytosis An erythrocyte of abnormal shape Increased variation in shape Microcyte Microcytosis Smaller than normal The presence of microcytes (Continued)

10 The Blood Count and Film 29 Table 2.4. (Continued ) Term Significance Picture Macrocyte Macrocytosis Larger than normal The presence of macrocytes Hypochromic Hypochromia Paler than normal (more than a third of the diameter of the cell is pale) The presence of hypochromic cells Polychromasia Having a blue tinge Elliptocyte An erythrocyte that is elliptical in shape (Continued)

11 30 Chapter 2 Table 2.4. (Continued ) Term Significance Picture Ovalocyte An erythrocyte that is oval in shape Macro-ovalocyte An oval macrocyte Spherocyte An erythrocyte that is spherical in shape and is therefore lacking central pallor Irregularly contracted cell An erythrocyte that lacks central pallor and has an irregular outline Teardrop cell (dacrocyte) An erythrocyte that is shaped like a tear (Continued)

12 The Blood Count and Film 31 Table 2.4. (Continued ) Term Significance Picture Target cell An erythrocyte that has haemoglobin concentrated at the periphery of the cell and also as a dot in the centre Sickle cell An erythrocyte with a crescent or sickle shape Stomatocyte An erythrocyte with a slit-like stoma Schistocyte or fragment A small piece of a red cell (Continued)

13 32 Chapter 2 Table 2.4. (Continued ) Term Significance Picture Acanthocyte An erythrocyte that has a small number of spicules of irregular length Echinocyte or crenated cell An erythrocyte that has a large number of short regular spicules Rouleaux Red cells stacked up like a flattened pile of pennies Agglutination Red cells forming irregular clumps (Continued)

14 The Blood Count and Film 33 Table 2.4. (Continued ). Term Significance Picture Howell Jolly body A fragment of the nucleus remaining in a mature red cell (stains purple) Pappenheimer body An iron-containing granule (stains navy blue) Basophilic stippling Fine or coarse dark blue-staining dots scattered through the cytoplasm falsely classified as sick, there are no false positives but the test becomes very insensitive (a lot of false negative results). Fortunately, the overlap between healthy and sick is not often as extreme as shown in Fig. 2.3b. It can be useful to have a health-related range, rather than a normal range that represents 95% of the apparently healthy population. For example, if the upper 20% of results for serum cholesterol in a typical Western population indicated an increased risk of myocardial infarction, an individual and his physician might aim to alter his life style and medications so that his results fell in the bottom 80% of the reference range rather than the central 95%. This bottom 80% would be the health-related range.

15 34 Chapter 2 Fig Paired histograms showing the possible results of applying a test in sick and healthy individuals: (a) an ideal test; (b) a non-ideal test that will necessarily result in either false positive or false negative results, depending on the threshold chosen; (c) a test that shows sufficient discrimination between sick and healthy individuals to be useful in clinical practice. When laboratories collect data to establish a normal range they perform the specified test on healthy volunteers using exactly the same instruments and methods that they are going to use for analysing patient samples. They will also analyse the data using appropriate statistical methods. If the data have a Gaussian distribution, e.g. the Hb (Fig. 2.4), then the arithmetic mean ± two standard deviations (or, more strictly, 1.95 standard deviations) can be used. The WBC, however, has a logarithmic distribution (Fig. 2.5) and the data have to be converted to logarithms before they can be analysed. Other non-gaussian distributions have to be dealt with by other statistical techniques. It is important when interpreting laboratory tests to take account of the following: (i) the normal range may not be appropriate; (ii) the test result may be within the normal range but abnormal for that patient; and (iii) the test result might be outside the reference range but actually normal. Laboratories usually have only two normal ranges, for adult men and for adult women. Normal ranges for children often differ from those for adults, normal ranges for pregnant women differ form those of non-pregnant women (e.g. the Hb is lower and the WBC and MCV are higher) and normal ranges for individuals of African ancestry differ from those of Northern Europeans (the FBC, neutrophil count and platelet count are lower).

16 The Blood Count and Film 35 Fig A histogram of Hb results in healthy subjects showing a Gaussian distribution. Fig A histogram of WBC results in healthy subjects showing a skewed distribution that will become Gaussian on logarithmic transformation.

17 36 Chapter 2 A test result for an individual patient may still be within the normal range but nevertheless be abnormal for that individual. For example, a man could have a major gastrointestinal haemorrhage and next day, as a result of haemodilution, his Hb has fallen from his usual level of 16.5 g/dl to 14 g/dl. This is still within the normal range but is very abnormal for him. If the normal range represents results from 95% of healthy subjects there will always be normal results that fall outside the normal range. This will be so for 5% of results for each test. If a lot of tests are done it is very probable that at least one will be abnormal. For example, if the eight tests that usually form part of the FBC are performed it is likely that a third of patients will have an abnormal result. How to Interpret a Blood Count and Develop a Differential Diagnosis When interpreting a blood count, or any other laboratory result, you should ask yourself the following questions: (i) is this result abnormal for this patient?; (ii) if it is abnormal, is it a trivial abnormality that should be ignored or might it be important?; (iii) if it might be important, is it so abnormal that there is a clinical urgency in dealing with the result?; and (iv) what is the likely cause of the abnormality? To determine the likely cause of an abnormality you might need to consider the clinical history, any medications the patient is taking, any abnormalities found on physical examination and the results of any other tests that have already been done. You may then need to develop a differential diagnosis and do extra tests to find out the cause. The differential diagnosis indicated by various blood count abnormalities will be dealt with in the chapters that follow but will be outlined here, according to which test is abnormal. Abnormal white cell counts If the WBC is abnormal it is necessary to go further and look at the different components of the differential count to see which parts are abnormal. The automated instrument that produces the WBC is likely to

18 The Blood Count and Film 37 have produced an automated differential count. If the abnormality is purely quantitative, e.g. an increased neutrophil count, then the clinical history may reveal the cause (e.g. known bacterial infection) so that no further haematological tests are indicated. On other occasions there may be immature cells present or abnormalities of mature cells so that the automated instrument either does not produce a differential count or flags the result, indicating an abnormality. In this instance a blood film is usually needed. The neutrophil count In deciding if a neutrophil count is elevated or reduced it is important to make a comparison with a relevant reference range (e.g. counts are higher in pregnancy and lower in some individuals of African ancestry). A high neutrophil count is usually reactive to infection, inflammation, trauma or surgery, and is due to increased production by the bone marrow. Rarely, it is due to leukaemia. A low neutrophil count can be due to: (i) inadequate production by the bone marrow; (ii) inability of the bone marrow to respond sufficiently to increased need, e.g. in infected neonates; (iii) peripheral destruction, e.g. drug-induced immune destruction; or (iv) abnormal pooling, e.g. in the spleen in hypersplenism. The lymphocyte count A high lymphocyte count can be due to increased production of lymphocytes or to altered distribution within the body. Increased production can be reactive, e.g. to viral infection, or be the result of leukaemia. A high lymphocyte count due to mobilisation of lymphocytes from tissues occurs as a transient acute response to stress, e.g. following severe trauma or a myocardial infarction. Lymphocytosis due to redistribution of lymphocytes within the body also occurs following splenectomy. A low lymphocyte count can be due to inherited and acquired immune deficiency, e.g. HIV infection, or can be a stress response to illness, surgery or trauma, in that case being mediated by corticosteroids. A stress-induced lymphocytosis is followed by a stress-induced lymphopenia.

19 38 Chapter 2 The monocyte count An increased monocyte count is usually reactive, as the result of chronic infection, inflammation or malignancy and is the result of increased production by the bone marrow. Less often it represents leukaemia. A reduced monocyte count is usually due to inadequate production by the bone marrow. It is uncommon but when it does occur it renders the patient susceptible to infection. The eosinophil count An increased eosinophil count is usually reactive, as a result of allergy (including some adverse drug reactions) or parasitic infection, and is due to increased bone marrow production. A low eosinophil count occurs as a stress reaction, mediated by corticosteroids. The basophil count An increased basophil count is often a feature of a haematological neoplasm and results from increased bone marrow production. It is diagnostically useful since reactive basophilia (e.g. due to hypothyroidism or ulcerative colitis) is uncommon. A reduced basophil count is rarely noted and is even more rarely of diagnostic importance. Anaemia The cause of the anaemia may be apparent from the clinical history. If not, a differential diagnosis can be developed by considering the size of the cells or by trying to work out the mechanism of the anaemia. A classification of anaemia based on cell size is shown in Table 2.5. This approach is very useful and often indicates a likely diagnosis and the tests that are needed to confirm it. If consideration of clinical features and erythrocyte size does not suggest a diagnosis it can be useful to seek evidence of a mechanism of the anaemia, as shown in Table 2.6.

20 The Blood Count and Film 39 Table 2.5. Classification of anaemias according to cell size. Cell size Microcytic Macrocytic Normocytic Causes Iron deficiency Liver disease Early stages or iron (common) Alcohol excess deficiency and Anaemia of chronic Megaloblastic anaemia anaemia of chronic disease (common) (vitamin B 12 or folate disease Thalassaemia deficiency or exposure Blood loss (common in some to certain drugs) Renal failure ethnic groups) Myelodysplastic syndromes Bone marrow Hypothyroidism suppression (e.g. by Aplastic anaemia chemotherapy) Haemolysis Mechanism Table 2.6. Classification of anaemia according to mechanism. Possible supporting evidence Failure of bone marrow production Blood loss Increased destruction (i.e. haemolysis) Red cell pooling in the spleen plus increased plasma volume Low reticulocyte count, lack of polychromasia Clinical evidence and later increased reticulocyte count Increased reticulocyte count, polychromasia, increased bilirubin concentration, increased lactate dehydrogenase, cells of abnormal shape (spherocytes, elliptocytes, fragments) Clinical evidence presence of splenomegaly Polycythaemia Polycythaemia refers to an increase of RBC, Hb and Hct. These laboratory abnormalities can be the result of a true polycythaemia, in which the total volume of red cells circulating in the bloodstream is increased. This can also be the result of an acute or chronic reduction of plasma volume, referred to as pseudpolycythaemia. The cause of an acute reduction in plasma volume, e.g. shock, burns or dehydration, will be apparent from the clinical history but a chronic pseudopolycythaemia requires laboratory tests to distinguish it from true polycythaemia. The differential diagnosis of polycythaemia will be discussed in Chapter 9.

21 40 Chapter 2 Thrombocytosis A high platelet count is almost always due to increased production by the bone marrow. The exception is following splenectomy, when it is due to redistribution. Thrombocytosis is usually reactive, due to infection, inflammation, blood loss or malignancy. Less often it is the result of a chronic haematological neoplasm known as a myeloproliferative neoplasm. Thrombocytopenia can be the result of: (i) failure of bone marrow production; (ii) increased destruction by antibodies; (iii) increased consumption during coagulation; (iii) blood loss with failure to replace platelets that are lost; or (iv) pooling in an enlarged spleen. The causes of thrombocytopenia will be discussed in more detail in Chapter 11. The Erythrocyte Sedimentation Rate This test involves mixing blood with the correct amount of citrate anticoagulant, allowing it to sediment in a tube of precise dimensions and measuring the number of mm the red cells have sedimented at the end of one hour. The normal range is 1 10 mm in an hour for a man and 0 20 mm in an hour for a woman. The erythrocyte sedimentation rate (ESR) is increased by anaemia and decreased by polycythaemia. It is increased by an increase in large plasma proteins (such as fibrinogen, α2 macroglobulin and immunoglobulins, particularly immunoglobulin M) and by a reduction in albumin concentration. The ESR is increased by pregnancy and by infection, inflammation, tissue infarction and malignancy. Although this test is very non-specific, it remains useful for monitoring chronic inflammatory conditions, such as rheumatoid arthritis, and in the follow up of Hodgkin lymphoma. Conclusions In order to interpret a full blood count and blood film you need to be familiar with the terminology and the abbreviations that are usually used. You also need to know the approximate normal range for common measurements. By the time you get to the end of this book, or your haematology course, you should also be able to interpret a blood count and film, develop a differential diagnosis and explain what tests you would do next.