Quality Samples_Haemotology

Haematology is the study of blood disease and diseases of the blood forming organs. Many people say that haematology is more of an art than a science and this is particularly appropriate when one considers the morphological assessment of red cells, white cells and platelets which is a critical part of a haematological assessment. Although cell numbers are obviously important, a complete cell count (CBC) or full blood count (FBC) on its own is simply not enough . At CTDS every haematology submission has an automated cell count performed and also has a white cell differential and cell morphology examination made on on a prepared blood film. All blood smears are examined in the first instance by a qualified haematologist and then, if certain criteria are met, also by a veterinary pathologist. For routine haematology we recommend the submission of whole blood taken into EDTA anticoagulant (or Lithium heparin for exotics or small samples that also require biochemistry) and a freshly prepared blood film made at the time of phlebotomy. The cell morphology of a freshly prepared smear is generally superior to that of a sample taken and stored in EDTA and assists our evaluation greatly (glass microscopy slides and slide holders are available from CTDS on request). For coagulation profiles, PT and or APTT submissions please submit a sample taken into sodium citrate also.

Quality samples for haematology

It is especially important for haematology that the blood sample is not clotted -the presence of fibrin clots in haematology samples give results that are erroneous. At CTDS we check every sample for the presence of fibrin clots before analysis and examine the smear for evidence of platelet clumping.

Anticoagulants in haematology
Anticoagulant	Tube Top Colour				Test required
EDTA Whole Blood (1ml)		or			FBC, CBC, Coagulation profile, all screens and profiles.
Citrate (1ml)	!	or			PT, APTT and Coagulation profile
Lithium Heparin whole blood (1ml)		or	!		Avian and reptilian haematology -also GshPX and Lead
! check label - green tops maybe heparin or citrate (coagulation)

Good sampling techniques:

• Wherever possible, sample from larger vessels e.g. jugular

• Immediately transfer blood from the syringe into anticoagulated tubes first ensuring the tip of the syringe does not touch the side of the tube (fill serum tubes last)

• As soon as blood is transferred into the anticoagulated tube, mix the sample by gentle inversion 20 times, or by rolling the tube on a flat surface for 30 seconds

• Ensure anticoagulant tubes (particularly EDTA) are filled to the recommended level and do not overfill

• Ensure all tubes containing anticoagulant are within the expiry date shown on the tube label

N.B. Citrate tubes have a very short shelf life – order just before use. Citrate tubes should be stored in the refrigerator

Blood smear preparation

Using either fresh EDTA whole blood or a drop of fresh blood from the tip of a syringe:

• Place a small drop of blood onto a clean, grease free glass slide.

• Take another clean glass slide as a spreader, preferably one with a bevelled edge, and draw this back into the blood drop

• Allowing the blood to flow across the spreader surface

• Holding the spreader at an angle of approximately 30 degrees, move the spreader "gently" forward in a single smooth action

• Spread to the end of the slide - do not lift the spreader from the slide

• Label the smear and allow to air dry (do not heat)

• All fresh blood films are invaluable but a "perfect" blood smear should have a smooth rounded tail, approximately one cell thick at its tail, and cover approximately 2/3rds of the length of the slide

• The cells should be distributed evenly in a layer one cell thick over the tail of the smear

Common problems

• If the blood film "falls off" the end of the slide try either using less blood and/or moving the spreader faster

• If the blood film is too short or too thin, try using a little more blood and/or spreading more slowly

• The prescence of "smudged" white cells, although occasionally clinically significant, is often associated with "hard" spreading

• Red cells that overlap at the tail - too much blood and or spread too slowly

• Absence of white cells in the body and many white cells at the tail - spread too slowly or lifting the spreader before smear completion N.B. it is normal to have more white cells at the tail but an absence of white cells in the body is not normal

Common haematology terms and abnormalities

Anisocytosis	Anisocytosis means that the red cells are of unequal size. It is a feature of many anaemias, and other blood conditions, and does not have much diagnostic value. The 'red cell distribution width' (RDW) is a quantitative measure of the degree of anisocytosis. The RDW is useful in the differential diagnosis of microcytic anaemia
Acanthocytes	Acanthocytes (also known as "spur cells") may be described as red cells with finger-like projections - typically 5-10 irregular, blunt projections (which vary in width, length and surface distribution and should not be confused with echinocytes). These cells have a decreased survival time and may be observed in liver disorders, increased blood cholesterol content or from the presence of abnormal plasma lipoprotein composition .
Dohle bodies	Dohle bodies appear as single or multiple light blue or grey staining areas in the cytoplasm of a neutrophil. They are rough endoplasmic reticulum containing ribonucleic acid (RNA) and may represent localised failure of the cytoplasm to mature. Dohle bodies are found in infections, poisoning, burns, and following chemotherapy.
Echinocytes	Echinocytes (also called "crenated cells") are morphologically altered red blood cells that appear to have numerous, fine, uniform spicules throughout the cell membrane. Echinocytes are often overlooked as an artifact of preparation e.g. due to storage or slow drying bloodsmears, however several disease processes (e.g. lymphosarcoma (partially as a result of chemotherapy), pk deficiency, uremia) and toxins have been found to alter the red blood cell membrane which leads to the formation of echinocytes.
Haemobartonellosis Feline infectious anaemia (FIA) also known as Mycoplasma felis	The most common red cell parasite in the UK is Haemobartonella felis which is a gram negative epicellular parasite found in feline erythrocytes. Red blood cell destruction is due primarily to immune-mediated events and direct injury to red blood cells induced by the organism is minimal. The attachment of the organism to erythrocytes commonly leads to the development of antibodies against the organism as well as to erythrocyte antigens so positive Coomb's tests are common. Clinically haemobartonellosis and primary immune haemolytic anaemia are difficult to differentiate. For the diagnosis of both these conditions an EDTA sample and fresh air dried blood film are required.
Howell Jolly Bodies	Howell-Jolly bodies are round, purple staining nuclear fragments of DNA in the red blood cell. They are usually observed singly in haemolytic anaemia, following splenectomy, and in cases of splenic atrophy. Multiple Howell-Jolly bodies may be observed in cases of megaloblastic anaemia.
Macrocytes	Macrocytes are red cells with an increased size, 9-12µm in diameter. They may be found in liver disease and megaloblastic anaemia, when associated with vitamin B12 or folic acid deficiency, the macrocytes may appear slightly oval in shape.
Normochromic	Normochromic describes the red cells as being of normal colour i.e. indication of haemoglobin content, for the species
Normocytic	Normocytic describes the red cells as being of normal size i.e. diameter for the species.
Poikilocytosis	Poikilocytosis is a term which indicates that red cells of abnormal shape are present on the blood film. Of itself it is fairly non-specific. Some particular types of poikilocyte are very informative, however. The 'tear-drop' poikilocyte is a characteristic feature of marrow fibrosis, but it can also be seen in other conditions.
Schistocytes	Schistocytes are red blood cell fragments that result from membrane damage encountered during passage through vessels. They occur in microangiopathic haemolytic anaemia, severe burns, uremia, and haemolytic anemias cause by physical agents, as in disseminated intravascular coagulation (DIC). They are sometimes referred to as "bite cells".
Spherocytes	Spherocytes are red cells which are almost spherical in shape. They are not biconcave like a normal red blood cell and do not have the central area of pallor which a normal red cell shows. These cells are associated with haemolytic anaemia

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