White Blood Cell Differential Count DR. Yasir M. Khaleel M.B.Ch.B, M.Sc.

Definition The relative percentage of each type of white blood cells in peripheral blood. White Blood Cells (leucocytes) Total 4.0 – 11.0 × 10 9 /L Absolute Relative Percentage Average Neutrophils 2.5 – 7.5 × 10 9 /L 50-70% 65% Lymphocytes 1.5 – 3.5 × 10 9 /L 25-35% 30% Monocytes 0.2 – 0.8 × 10 9 /L 2-10% 6% Eosinophils 0.04 – 0.44 × 10 9 /L 1-5% 3% Basophils 0.01 – 0.1 × 10 9 /L 0.4-1% 0.5%

Microscopic exam 10× (low fold): overall smear quality, rouleaux, agglutination or parasites 100× (oil Len): WBC Diff, RBC morphology

tail body head

Counting Area: Observe one field and record the number of WBC according to the different type then turn to another field in the snake-liked direction *avoid repeat or miss some cells

Normal peripheral blood smear

White blood cells There are three types of granulocyte named according to their staining characteristics in blood films. They are neutrophils,eosinophils and basophils. Mononuclear cells are divided into lymphocytes and monocytes.

Stab (band) neutrophil Diameter:12-16 Cytoplasm : pink Granules : primary & secondary Nucleus : dark purple blue dense chromatin

Band form Neutrophils There are smaller numbers of cells of neutrophil lineage with non- segmented nuclei. They are referred to as neutrophil band cells or band forms. They are less mature than segmented neutrophils. An increased number of band cells is referred to as a left shift'.

Segmented neutrophil Diameter: Cytoplasm: very pale blue Granules: primary secondary lilac-staining (purple) Nucleus: dark purple blue dense heterogeneous chromatin 2- 5 lobes

Neutrophils The neutrophils in the circulating blood are mainly mature segmented neutrophils. neutrophilic because they owe their color to uptake of both the acidic and the basic components of the stain

Eosinophil Diameter: Cytoplasm : full of granules Granules: large refractile orange-red Nucleus: blue dense chromatin 2 lobes like a pair of glass

Basophil Diameter: Cytoplasm: pink Granules: dark blue – black obscure nucleus Nucleus: blue

Lymphocyte Diameter: small 7-9 large Cytoplasm : rim, clear, pale blue Granules: small (a granular) large (a variable number of pinkish-purple granules) Nucleus: dark blue / round dense homogeneous chromatin

Lymphocytes divided into B(20%) &T(70%) both of them divided into small & large

Normal lymphocytes Lymphocytes are the smallest WBC. They have large condensed nucleus, with a scanty pale blue cytoplasm.

Monocyte Diameter: 14-20(are the largest normal blood cells) Cytoplasm: grey blue Granules: dust-like lilac color granules(purple) Nucleus: blue large irregularly shaped and folded

The red blood cells here are normal, happy RBC's. They have a zone of central pallor about 1/3 the size of the RBC. The RBC's demonstrate minimal variation in size (anisocytosis) and shape (poikilocytosis). A few small fuzzy blue platelets are seen. In the center of the field are a band neutrophil on the left and a segmented neutrophil on the right. Samples of Blood films: 1

A normal mature lymphocyte is seen on the left compared to a segmented PMN on the right. An RBC is seen to be about 2/3 the size of a normal lymphocyte Samples of Blood films: 2

Here is a monocyte. It is slightly larger than a lymphocyte and has a folded nucleus. Monocytes can migrate out of the bloodstream and become tissue macrophages under the influence of cytokines. Note the many small smudgy blue platelets between the RBC's. Samples of Blood films: 3

In the center of the field is an eosinophil with a bilobed nucleus and numerous reddish granules in the cytoplasm. Just underneath it is a small lymphocyte. Eosinophils can increase with allergic reactions and with parasitic infestations. Samples of Blood films: 4

There is a basophil in the center of the field which has a lobed nucleus (like PMN's) and numerous coarse, dark blue granules in the cytoplasm. They are infrequent in a normal peripheral blood smear, and their significance is uncertain. A band neutrophil is seen on the left, and a large, activated lymphocyte on the right. Samples of Blood films: 5

Terms Leu c ocytosis Vs Leu c openia k/c Neutrophilia Vs Neutropenia Lymphocytosis Vs Lymphocytopenia Monocytosis Vs Monocytopenia Eosinophilia Basophilia Granulocytosis Agranulocytosis

Erythrocytes Sedimentation Rate ESR DR. Yasir M. Khaleel M.B.Ch.B, M.Sc.

ESR Erythrocyte Sedimentation Rate: Def: The distance that erythrocytes have fallen after 1 hour in a vertical column of anti-coagulated blood under the influence of gravity. The most satisfactory method was provided by Westergren 1921

ESR is a non-specific test for inflammation. It is easy to perform, widely available and inexpensive making it a widely used screening test Normal ESR not exclude disease High ESR not confirm the disease unless proved by other tests. It is also used a monitoring test for response to treatment in conditions in which it is raised (tuberculosis, autoimmune diseases etc(

Basics: The ESR test is performed in the laboratory by placing anti-coagulated blood in an upright tube (Westegren's most often). At the end of an hour of this, the rate of the RBC sedimentation is measured

Factors affecting the ESR A- Plasma related factors: Increased in the concentration of fibrinogen and Immunoglobulins due to inflammation and tissue injury will increase rouleaux formation and hence the rate of sedimentation. Plasma proteins especially fibrinogen, adhere to the red cell membranes and neutralize the surface negative charges, promoting cell adherence and rouleaux formation. Any condition associated with increase in plasma fibrinogen will lead to elevated ESR ( infection, inflammation, malignancy) Plasma albumin retards sedimentation of RBCs.

The RBC's here have stacked together in long chains. This is known as "rouleaux formation" and it happens with increased serum proteins, particularly fibrinogen and globulins. Such long chains of RBC's sediment more readily. This is the mechanism for the sedimentation rate, which increases non-specifically with inflammation and increased "acute phase" serum proteins.

B-The RBC related factors: 1-The size or mass of the RBC: macrocytes have faster rate of fall so read higher ESR with macrocytic anaemia. 2- Shape of RBCs that show alterations in their bioconcavity, like spherocytes and sickle cells (these RBCs can’t form rouleaux), usually do not exhibit increase rate, unless there is severe anemia. 3- RBC count :Increase red cell mass will retard the sedimentation rate e.g. polycythemia, Vs anaemia

C-Technical factors: 1-Perpendicularity of sedimentation tube, slight deviations from the absolute vertical can increase the result. ( False high) 2-Temperature (RT C°) higher temperature cause false high results due to reduction in plasma viscosity (e.g. direct sun light) 3-Vibration cans reduce the ESR. 4- Time ( 1 hour ) 5-Air bubble ( not allowed) 6- Excess or Less anti-coagulant 7- Clotted sample ( not allowed)

Reference Range Adults < 50 years: Females 0-20 mm/hr ; Males 0-15 mm/hr Adults > 50 years: Females 0-30 mm/hr ; Males 0-20 mm/hr For older ages (Females : (Age +10)/2; Males: Age/2) Children(<10) 0-10 mm/hr

Physiological variations  Sex: females have higher ESR than males.  Age: old age specially after 60 have higher ESR. Children have lower ESR reading.  Pregnancy and Menstruation: higher ESR  Weight: Obesity: slightly higher ESR reading.  Drugs: Aspirin and other NSAIDs, and cortisone may be associated with slightly lower ESR reading, while OCP, vitamin A and theophylline are associated with high ESR reading  Recent meal before the test (might) be associated with slightly high ESR reading.

 Slightly Elevated ESR, simply repeat the test after 1-2 months.  ESR ≥100 mm/hr is strongly associated with serious underlying disorders like: * Connective tissue disease, * Infections ( specially chronic infections like TB) * Malignancies Clinical Evaluation of High ESR Reading

ESR is helpful in diagnosing two specific inflammatory diseases, temporal arteritis and polymyalgia rheumatica. Some conditions with very high ESR >100 mm/hr - Multiple myeloma - Connective tissue disorders - SLE, RA and other autoimmune diseases - Tuberculosis - Malignancies - Severe anemia

IMPORTANT Erythrocyte sedimentation rate is a non-specific test and is not diagnostic of any particular disease. It has a high sensitivity but low specificity. Never base a diagnosis solely on an ESR value, either normal or high. Interpretation of the result should always be along with the patient's clinical history, examination findings and results of other tests done. If high ESR is encountered without any obvious reasons, patient should be reassured and the test repeated after a reasonable time (1-2 months). There is no need to extensively search for an occult disease without repeating it again.

Procedure - Patient should have NO meal in the last 4 hours. -The blood sample must be mixed with anticoagulant agent in this test. Put 0.4 ml sodium citrate ml blood. OR Put 0.2 ml sodium citrate ml blood. OR Put 0.5 ml sodium citrate+ 2 ml blood. 1. Mix gently with out shaking for at least 2-3 minutes then put in the graded tube and leave it stand vertically on the stand for 1 hour. 2. Read the amount of plasma that appeared without moving it. The Result: ESR= ( ? ) mm/hr.

Thank You Dr. Yasir M. Khaleel