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HAEMATOLOGY - A Core Curriculum© Imperial College Presshttp://www.worldscibooks.com/medsci/p688.html

b1015 Haematology: A Core Curriculum

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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 possibleclinical significance

� The meaning of ‘normal range’ and ‘reference range’� The approximate normal ranges for the white cell count, haemo-

globin concentration, mean cell volume and platelet count inhealthy adults

� How to interpret a blood count and develop a differential diagnosis� The meaning and clinical significance of an ‘erythrocyte sedi-

mentation rate’

The Full Blood Count

The term ‘full blood count’ (FBC) refers to a group of tests performedsimultaneously on a blood sample to assess whether there is any haema-tological abnormality. The tests that are almost always included in an FBCare shown in Table 2.1. In modern haematological practice these tests areperformed on large automated analysers capable of processing many hun-dreds of samples in a day. Further tests are sometimes also included. The

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The Blood Count and Film 21

blood count is performed on a blood specimen that has been anticoagu-lated by being mixed with ethylene diaminetetra-acetic acid (EDTA), achelating agent that prevents clotting by binding calcium. If you areobtaining a blood specimen from a patient for an FBC be sure to use atube containing EDTA, add the correct volume of blood and mix the bloodsample 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 glasscounting chamber. Nowadays it is counted by an automated instrumentthat detects individual cells flowing in a stream through a sensor.Recognition is either because the cell interrupts a beam of light or becauseit alters the electrical current flowing between two electrodes. A WBC isperformed on a sample in which the mature red cells have been lysed bysolutions to which they are exposed within the instrument so that the onlycells that will be counted are the white cells and any nucleated red bloodcells that might be present.

Table 2.1. The full blood count.

Normal range Normal range Test Abbreviation Units in men* in women*

White blood cell count WBC ×109/l 3.7–9.5 3.9–11.1Red blood cell count RBC ×1012/l 4.32–5.66 3.88–4.99Haemoglobin Hb g/dl 13.3–16.7 11.8–14.8

concentrationHaematocrit Hct l/l 0.39–0.50 0.36–0.44Mean cell volume MCV fl 82–98Mean cell MCH pg 27.3–32.6

haemoglobinMean cell MCHC g/dl 31.6–34.9

haemoglobinconcentration

Platelet count ×109/l 168–411† 188–445†

* From Bain, B.J. (2006). Blood Cells. Fourth Edition. Blackwell Publishing, Oxford.† Reported ranges from different instruments vary.

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The red blood cell count

The red blood cell count (RBC, also known as the red cell count) was ini-tially determined with a counting chamber. As for the WBC, it is nowdetermined by an instrument that counts red cells flowing through a sen-sor. However, this time the red cells are not lysed. White cells will actuallyalso be included in the count but because they are infrequent in relation tothe red cells this does not usually introduce much error.

The haemoglobin concentration

The haemoglobin concentration (Hb) is determined by lysing red cells andmeasuring light transmitted through a diluted sample of the blood at aspecific wavelength after conversion of the haemoglobin to a stable form.

The haematocrit

It is possible to measure the proportion of red cells in an anticoagulatedblood sample by centrifuging a tube of the blood and comparing theheight 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 performedbecause it is not suitable for dealing with large numbers of blood speci-mens. The modern equivalent is called a haematocrit (Hct). It has thesame significance as a PCV but instruments calculate it rather than meas-uring 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). Thiswas a very laborious technique so it was not done very often. Moderninstruments estimate the MCV from the height of the electrical impulsegenerated by interruption of a light beam or an electrical current. Thesame electrical impulse is therefore used both to count the cells and to sizethem.





The mean cell haemoglobin

The mean cell haemoglobin (MCH) is the average amount of haemoglo-bin in an individual red cell. Instruments calculate it by dividing thehaemoglobin in a given volume of blood by the number of red cells in thesame volume.

The mean cell haemoglobin concentration

The mean cell haemoglobin concentration (MCHC) is the average con-centration of haemoglobin, rather than the absolute amount, in anindividual red cell. Fig. 2.1 is a visual representation of the differencebetween the MCH and the MCHC. Instruments calculate it by dividing thehaemoglobin in a given volume of blood by the proportion of the wholeblood sample that is occupied by red cells.

The red cell distribution width

The red cell distribution width (RDW) is a measurement of the amount ofvariation in the size of the red cells.

Fig. 2.1. A diagram to illustrate the difference between the mean cell haemoglobin(MCH) and the mean cell haemoglobin concentration (MCHC).




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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 latterthree values are:

MCV (fl) = PCV (l/l) ×⋅ 1000RBC (cells/l) × 10−12

MCH (pg) = Hb (g/dl) × 10RBC (cells/l) × 10−12

MCHC (g/dl) = Hb (g/dl)PCV (l/l)

The red cell indices are very useful for indicating the likely cause ofanaemia.

The reticulocyte count

A reticulocyte count is a supplement to an FBC, rather than a normal partof it. It can be performed using a microscope, counting the percentage oferythrocytes that have developed a ‘reticulum’ or network of precipitateddye after incubation of the blood sample with a dye such as methyleneblue (Fig. 2.2). This is a method for identifying ribonucleic acid (RNA)within red cells, thus demonstrating that these are cells newly releasedfrom the bone marrow (1–3 days old). Reticulocyte counts can also beperformed by automated instruments, using either the same principle asthe reticulocyte count with a microscope or alternatively, using a fluores-cent dye that binds to RNA.

The Differential White Cell Count

The different types of white cell can be distinguished from each other byexamining a stained blood film. If a hundred cells are counted and the





percentages are multiplied by the WBC, the absolute number of cells ofeach type can be calculated. Calculating the percentage or absolute num-ber of each cell type is referred to as a differential count. It is much moreuseful to calculate the absolute count than to try to make deductions fromthe percentages. For example, if a patient had 5% neutrophils and 95%lymphocytes this could indicate either a severe reduction of neutrophils ora marked increase in lymphocytes.

Many automated instruments can recognise the five normal types ofwhite cell and can detect the presence of nucleated red blood cells(NRBC) and other cells that are not normally present in the circulatingblood. If only normal cells are present they are capable of performing adifferential count. If abnormal cells are present it is usually still necessaryto perform a differential count on a blood film. However, someinstruments are also capable of counting NRBC. Normal ranges for thedifferential white cell count are shown in Table 2.2.

Haematological Terminology

Haematologists use precise terms to refer to abnormalities in the bloodcount and film. Since they use these terms to report abnormalities to

Fig. 2.2. A reticulocyte preparation, showing a reticulum of precipitated dye followingincubation of blood with methylene blue.




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clinical staff it is necessary for all doctors to understand them. The termsused to describe quantitative abnormalities are shown in Table 2.3; unfor-tunately these terms are not necessarily logical or consistent so you justhave to learn how they are used. The terms used to describe erythrocytesand 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 forcomparison. A reference range is derived from a carefully defined popula-tion, the reference population: the definition will include the age andgender of the subjects and sometimes the ethnic origin or other relevantdetails. If a requirement to be in good health is part of the definition then a‘reference range’ becomes a ‘normal range’. Conventionally, referenceranges and normal ranges include the central 95% of results from thereference population. Ideally, tests would give a clear separation of healthysubjects (the normal range) and an unhealthy population, as shown inFig. 2.3a. More often, the situation will be as shown in Fig. 2.3c, with asmall amount of overlap between sick and well people. Sometimes a testgives poor separation of sick and well (Fig. 2.3b). Either the laboratory orthe 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 willalso be included; the test becomes sensitive but at the cost of a high rate offalse positives. If the threshold is moved down so that no healthy person is

Table 2.2. The differential white cell count.

Normal range Normal rangeTest Units in men* in women*

Neutrophil count ×109/l 1.7–6.1 1.7–7.5Lymphocyte count ×109/l 1.0–3.2Monocyte count ×109/l 0.2–0.6Eosinophil count ×109/l 0.03–0.46Basophil count ×109/l 0.09–0.29

*From Bain, B.J. (2006). Blood Cells. Fourth Edition. Blackwell Publishing, Oxford. Figures are foran automated instrument and will differ slightly between instruments; figures for manual differentialcounts will have broader limits.





Table 2.3. Terminology used for describing quantitative abnormalitiesin blood counts.

Term Meaning

Anaemia Reduced HbPolycythaemia or Either term can be used to indicate an increase

erythrocytosis in the RBC, Hb and HctLeucocytosis Increased WBCLeucopenia Reduced WBCThrombocytosis Increased platelet countThrombocytopenia Reduced platelet countNeutrophilia Increased neutrophil countNeutropenia Reduced neutrophil countLymphocytosis Increased lymphocyte countLymphopenia* or Reduced lymphocyte count

lymphocytopeniaMonocytosis Increased monocyte countEosinophilia Increased eosinophil countBasophilia† Increased basophil countReticulocytosis Increased reticulocyte countReticulocytopenia Reduced reticulocyte count

*The term ‘lymphopenia’ is particularly illogical because it is used to mean a reducedlymphocyte count not a reduced amount of lymph.† Basophilia has a quite different alternative meaning; it also means an increaseduptake of basic dyes by the cytoplasm of a cell so that when examined with a micro-scope it appears blue.




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Table 2.4. Terminology used for describing erythrocytes and their abnormalities.

Term Significance Picture

Normocytic Of normal sizeNormochromic With normal staining

characteristics

Anisocytosis Increased variation in size

Poikilocyte An erythrocyte of abnormalshape

Poikilocytosis Increased variation in shape

Microcyte Smaller than normalMicrocytosis The presence of microcytes

(Continued)





Table 2.4. (Continued )


Macrocyte Larger than normalMacrocytosis The presence of macrocytes

Hypochromic Paler than normal (more thana third of the diameter ofthe cell is pale)

Hypochromia The presence of hypochromiccells

Polychromasia Having a blue tinge

Elliptocyte An erythrocyte that is ellipticalin shape

(Continued)




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Ovalocyte An erythrocyte that is oval inshape

Macro-ovalocyte An oval macrocyte

Spherocyte An erythrocyte that is sphericalin shape and is thereforelacking central pallor

Irregularly An erythrocyte that lackscontracted cell central pallor and has an

irregular outline

Teardrop cell An erythrocyte that is shaped(dacrocyte) like a tear

(Continued)







Target cell An erythrocyte that hashaemoglobin concentratedat the periphery of the celland also as a dot in the centre

Sickle cell An erythrocyte with a crescentor sickle shape

Stomatocyte An erythrocyte with a slit-like‘stoma’

Schistocyte A small piece of a red cellor fragment

(Continued)




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Acanthocyte An erythrocyte that has a smallnumber of spicules ofirregular length

Echinocyte or An erythrocyte that hascrenated cell a large number of

short regular spicules

Rouleaux Red cells stacked up like aflattened pile of pennies

Agglutination Red cells forming irregularclumps

(Continued)





Table 2.4. (Continued ).


Howell–Jolly body A fragment of the nucleusremaining in a maturered cell (stains purple)

Pappenheimer An iron-containing granulebody (stains navy blue)

Basophilic Fine or coarse darkstippling blue-staining dots

scattered throughthe cytoplasm

falsely classified as sick, there are no false positives but the test becomesvery insensitive (a lot of false negative results). Fortunately, the overlapbetween 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 normalrange that represents 95% of the apparently healthy population. For exam-ple, if the upper 20% of results for serum cholesterol in a typical Westernpopulation indicated an increased risk of myocardial infarction, an indi-vidual and his physician might aim to alter his life style and medicationsso that his results fell in the bottom 80% of the reference range rather thanthe central 95%. This bottom 80% would be the health-related range.




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Fig. 2.3. Paired histograms showing the possible results of applying a test in sick andhealthy individuals: (a) an ideal test; (b) a non-ideal test that will necessarily result in eitherfalse positive or false negative results, depending on the threshold chosen; (c) a test that showssufficient discrimination between sick and healthy individuals to be useful in clinical practice.

When laboratories collect data to establish a normal range they per-form the specified test on healthy volunteers using exactly the sameinstruments and methods that they are going to use for analysing patientsamples. They will also analyse the data using appropriate statisticalmethods. If the data have a Gaussian distribution, e.g. the Hb (Fig. 2.4),then the arithmetic mean ± two standard deviations (or, more strictly, 1.95standard deviations) can be used. The WBC, however, has a logarithmicdistribution (Fig. 2.5) and the data have to be converted to logarithmsbefore they can be analysed. Other non-Gaussian distributions have to bedealt with by other statistical techniques.

It is important when interpreting laboratory tests to take account of thefollowing: (i) the normal range may not be appropriate; (ii) the test resultmay be within the normal range but abnormal for that patient; and (iii) thetest result might be outside the reference range but actually normal.

Laboratories usually have only two normal ranges, for adult men and foradult women. Normal ranges for children often differ from those for adults,normal ranges for pregnant women differ form those of non-pregnantwomen (e.g. the Hb is lower and the WBC and MCV are higher) and normalranges for individuals of African ancestry differ from those of NorthernEuropeans (the FBC, neutrophil count and platelet count are lower).





Fig. 2.4. A histogram of Hb results in healthy subjects showing a Gaussian distribution.

Fig. 2.5. A histogram of WBC results in healthy subjects showing a skewed distributionthat will become Gaussian on logarithmic transformation.




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A test result for an individual patient may still be within the normalrange but nevertheless be abnormal for that individual. For example, a mancould have a major gastrointestinal haemorrhage and next day, as a result ofhaemodilution, 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 subjectsthere 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 itis very probable that at least one will be abnormal. For example, if theeight tests that usually form part of the FBC are performed it is likely thata 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, youshould ask yourself the following questions: (i) is this result abnormal forthis patient?; (ii) if it is abnormal, is it a trivial abnormality that should beignored or might it be important?; (iii) if it might be important, is it soabnormal 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 toconsider the clinical history, any medications the patient is taking, anyabnormalities found on physical examination and the results of any othertests that have already been done. You may then need to develop a differ-ential diagnosis and do extra tests to find out the cause.

The differential diagnosis indicated by various blood count abnor-malities will be dealt with in the chapters that follow but will be outlinedhere, 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 dif-ferent components of the differential count to see which parts areabnormal. The automated instrument that produces the WBC is likely to





have produced an automated differential count. If the abnormality ispurely quantitative, e.g. an increased neutrophil count, then the clinicalhistory may reveal the cause (e.g. known bacterial infection) so that nofurther haematological tests are indicated. On other occasions there maybe immature cells present or abnormalities of mature cells so that theautomated instrument either does not produce a differential count or‘flags’ the result, indicating an abnormality. In this instance a blood filmis usually needed.

The neutrophil count

In deciding if a neutrophil count is elevated or reduced it is important tomake a comparison with a relevant reference range (e.g. counts are higherin 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 thebone marrow; (ii) inability of the bone marrow to respond sufficiently toincreased need, e.g. in infected neonates; (iii) peripheral destruction, e.g.drug-induced immune destruction; or (iv) abnormal pooling, e.g. in thespleen in hypersplenism.

The lymphocyte count

A high lymphocyte count can be due to increased production of lympho-cytes or to altered distribution within the body. Increased production canbe reactive, e.g. to viral infection, or be the result of leukaemia. A highlymphocyte count due to mobilisation of lymphocytes from tissues occursas a transient acute response to stress, e.g. following severe trauma or amyocardial infarction. Lymphocytosis due to redistribution of lympho-cytes within the body also occurs following splenectomy.

A low lymphocyte count can be due to inherited and acquired immunedeficiency, e.g. HIV infection, or can be a stress response to illness, surgeryor trauma, in that case being mediated by corticosteroids. A stress-inducedlymphocytosis is followed by a stress-induced lymphopenia.




The monocyte count

An increased monocyte count is usually reactive, as the result of chronicinfection, inflammation or malignancy and is the result of increased pro-duction by the bone marrow. Less often it represents leukaemia.

A reduced monocyte count is usually due to inadequate production bythe bone marrow. It is uncommon but when it does occur it renders thepatient 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 dueto increased bone marrow production.

A low eosinophil count occurs as a stress reaction, mediated bycorticosteroids.

The basophil count

An increased basophil count is often a feature of a haematological neo-plasm and results from increased bone marrow production. It isdiagnostically useful since reactive basophilia (e.g. due to hypothyroidismor ulcerative colitis) is uncommon.

A reduced basophil count is rarely noted and is even more rarely ofdiagnostic 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 thecells or by trying to work out the mechanism of the anaemia. A classifi-cation of anaemia based on cell size is shown in Table 2.5. This approachis very useful and often indicates a likely diagnosis and the tests that areneeded to confirm it. If consideration of clinical features and erythrocytesize does not suggest a diagnosis it can be useful to seek evidence of amechanism of the anaemia, as shown in Table 2.6.

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Polycythaemia

Polycythaemia refers to an increase of RBC, Hb and Hct. These labora-tory abnormalities can be the result of a true polycythaemia, in which thetotal volume of red cells circulating in the bloodstream is increased. Thiscan also be the result of an acute or chronic reduction of plasma volume,referred to as pseudpolycythaemia. The cause of an acute reduction inplasma volume, e.g. shock, burns or dehydration, will be apparent fromthe clinical history but a chronic pseudopolycythaemia requires laboratorytests to distinguish it from true polycythaemia. The differential diagnosisof polycythaemia will be discussed in Chapter 9.

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 chronicdisease (common) (vitamin B12 or folate disease

Thalassaemia deficiency or exposure Blood loss(common in some to certain drugs) Renal failureethnic groups) Myelodysplastic syndromes Bone marrow

Hypothyroidism suppression (e.g. byAplastic anaemia chemotherapy)Haemolysis

Table 2.6. Classification of anaemia according to mechanism.

Mechanism Possible supporting evidence

Failure of bone marrow production Low reticulocyte count, lack of polychromasiaBlood loss Clinical evidence and later increased

reticulocyte countIncreased destruction (i.e. Increased reticulocyte count, polychromasia,

haemolysis) increased bilirubin concentration, increasedlactate dehydrogenase, cells of abnormalshape (spherocytes, elliptocytes, fragments)

Red cell pooling in the spleen plus Clinical evidence — presence of splenomegalyincreased plasma volume




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Thrombocytosis

A high platelet count is almost always due to increased production by thebone marrow. The exception is following splenectomy, when it is due toredistribution. Thrombocytosis is usually reactive, due to infection, inflam-mation, blood loss or malignancy. Less often it is the result of a chronichaematological neoplasm known as a myeloproliferative neoplasm.

Thrombocytopenia can be the result of: (i) failure of bone marrowproduction; (ii) increased destruction by antibodies; (iii) increased con-sumption during coagulation; (iii) blood loss with failure to replaceplatelets that are lost; or (iv) pooling in an enlarged spleen. The causes ofthrombocytopenia will be discussed in more detail in Chapter 11.

The Erythrocyte Sedimentation Rate

This test involves mixing blood with the correct amount of citrate antico-agulant, allowing it to sediment in a tube of precise dimensions andmeasuring the number of mm the red cells have sedimented at the end ofone 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 isincreased by an increase in large plasma proteins (such as fibrinogen, α2macroglobulin and immunoglobulins, particularly immunoglobulin M)and by a reduction in albumin concentration. The ESR is increased bypregnancy and by infection, inflammation, tissue infarction and malig-nancy. Although this test is very non-specific, it remains useful formonitoring 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 famil-iar with the terminology and the abbreviations that are usually used. Youalso need to know the approximate normal range for common measure-ments. By the time you get to the end of this book, or your haematologycourse, you should also be able to interpret a blood count and film, developa differential diagnosis and explain what tests you would do next.


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