Haematology – Making the most of it in practice.

Karen Jackson BVSc, MANZCVSc, Dipl. ACVPIDEXX Laboratories – Sydney

Unit 20, 38-46 South St, Rydalmere, NSW, 2116

Haematology is the study of blood and blood forming tissues with blood being a mixture of formed elements (e.g. WBCs, RBCs, platelets) within plasma. The complete blood count (CBC) is the most commonly used test to assess these formed elements. CBCs contain large amounts of information about each element (WBCs, RBCs, and platelets) and can be quite daunting when first examined; however, with time and hopefully this lecture, the interpretation and evaluation of CBCs will become easier and relied upon as an essential component of the diagnostic evaluation of your patients.

CBCs are indicated for screening to assess general health, as an adjunct to the diagnostic evaluation of an ill patient, as a monitoring tool in response to previous haematologic disease, and to screen for immunodeficiency and coagulation status. The CBC includes quantitative and qualitative analysis of WBC, RBC, and platelets. It can also variably include plasma protein, plasma characteristics, and reticulocyte count.

Quantitative analysis includes machine or manual counting methods while the qualitative analysis includes machine dot plots/scattergrams and blood smear evaluation. Both quantitative and qualitative portions are of equal importance. The focus of this session will be on the benefits of performing and evaluating blood smears, how best to do this in-house, and what you should be looking for.

Specimen collection and handlingTo study formed elements of blood it must be anticoagulated.Tips: Fill vacutainers/tubes to the proper level to assure correct anticoagulant:blood Rock gently Process immediately or refrigerate up to 24h Atraumatic venipuncture avoids haemolysis and/or initiation of coagulation

cascade. Ideally, blood smears should be prepared with freshly drawn blood. Do not store blood smears in refrigerator; condensation will cause water artifact.

Anticoagulants:1. EDTA (lavender or purple top):

Potassium EDTA binds calcium, which is essential to clotting. EDTA can cause clumping of platelets and spurious thrombocytopenia (low)Occasionally plasma is drawn off for biochemical analysis, but this EDTA

plasma cannot be used for calcium concentration (since EDTA binds Ca to cause a spurious decrease), or potassium (adds K+ to cause a spurious increase).

2. Heparin (green top) This is the anticoagulant of choice for avian and reptilian species.

3. Citrate (blue top)For coagulation studies; +/- platelet counts if EDTA causing constant clumping

4. No anticoagulant (red top or clot tube)For serum chemistries; NOT used for the CBC.

The blood clots and serum (plasma minus clotting factors) is extracted for biochemical analysis.

Making a blood smearDraw up a sample of blood using a microhaematocrit tube (do not plug the tube).

Place a small drop of blood on one end of the slide.

Use a clean slide (designate as spreader) to drag this drop to make a smear. The spreader slide is backed up into the drop of blood and then shoved forward, dragging a film of blood with it, as the direction arrow shows in the figure below.

The slide with the drop of blood on it should be held firmly on the table during the spreading. The spreader slide should be shoved forward in a firm manner to avoid chatter. Increasing the angle of the spreader slide will result in thicker, shorter smears. Decreasing the angle will result in thinner, longer smears. The following techniques are incorrect.

A correctly made smear will have a colorful sheen when held up to the light, reflective of a monolayer of cells. This sheen is visible in the unstained smear, but not after staining.

Staining a blood smearUsing ' diff-quick': IT IS DIFFICULT TO OVERSTAIN BUT UNDERSTAINING IS COMMON so err on the long time rather than the short.

1st: light blue solution (fixative); dip at least 10 times, or until solution runs off smoothly (the slide can be left virtually indefinitely in the fixative so consider 20s-1min standard)2nd: eosin solution; dip at least 10 times, or until the solution runs off smoothly 3rd: dark blue solution; dip at least 10 times, or until the smear is a uniform color 4th: rinse in tap water 5th: stand on edge to drain off water and let air-dry

Blood smear evaluation

1. Assess quality: check your smear using the microscope to see if WBC are spread throughout relatively evenly or if they are all bunched up at the feathered edge. If most of the WBC are found at the feathered edge, then you need to prepare your smears slightly faster. Also, check to see that there is a zone just short of the feathered edge where RBC are evenly spread without crowding (i.e. a monolayer).

2. Once adequate quality is determined: Determine the leukocyte differential and then calculate absolute values Evaluate neutrophil morphology: toxic change, cytoplasmic inclusions Evaluate platelet frequency and estimate a count Evaluate RBC morphology: anisocytosis, polychromasia, poikilocytosis Examine CBC quantitative information and add qualitative information (i.e.

blood smear/morphology and/or dot plot information) to evaluate the full CBC.

Systematic Approach to Examination of the Blood SmearA. Low magnification (4x or 10x)

1. Determine smear adequacy/quality (Staining? Cell morphology? Cell distribution?)

2. Examine red cell distribution – Agglutination? Rouleaux?3. Note the presence of platelet clumps (often feathered edge but also body)4. Note the presence of large/atypical cells or parasites (feathered edge common)5. Locate monolayer (2-3 10x fields in from the feathered edge on a well prepared

smear – where red cells only rarely present in doublets and evenly spread). Estimate white cell number (normal ~ 18-51 per 10x field or the avg. of 10 fields at 20x ~ WCC)

6. Also within monolayer estimate RBC density. If anaemic (i.e. decreased density) initiate evaluation for features of regeneration, poikilocytosis, or haemic parasites which might aid in determining the cause.

B. Intermediate magnification (40x dry or 50x oil)1. Within the monolayer perform a 100-cell WBC differential. Count nRBCs as in

excess of the 100 WBCs. If >5% of the differential is nRBCs consider correcting the WBC if using impedence technology. Count 200 cells if you estimate the WBC increased.

2. Evaluate basic morphologic features of RBCs: estimate polychromasia, anisocytosis, and poikilocytosis (e.g. spherocytes, schistocytes, acanthocytes). Be sure to remain in the monolayer for accuracy.

C. High magnification (100x oil)1. Complete the evaluation of RBC morphology: identify any inclusions/parasites

(e.g. Mycoplasma haemofelis, Babesia spp.), Heinz bodies, and better characterization of any poikilocytosis previously seen

2. White cell morphology: identify toxic changes (e.g. cytoplasmic vacuolation, cytoplasmic basophilia, Dohle bodies), inclusions, and cells of questionable origin

3. Estimate platelet count (1 platelet ~15-20 x 10E9/L if platelet clumping not identified – therefore 8-29 platelets per 100X field suggests “normal range” platelet number in the dog). Note presence of large platelets – particularly in Cavaliers. Check for Anaplasma platys (North Qld, NT).

Red cell arrangements: important abnormalities

1. RouleauxGrouping of RBCs in “coin stack” appearance.Mild-moderate normal in cats, moderate to marked

normal in horses, any in dogs or excessive in cats/horses associated with inflammation (increased globulins)

2. AgglutinationClumping of RBCs secondary to

autoantibody crosslinkingSeen almost exclusively with IMHA (can also

happen in transfusion mismatch)Note affects: RBC and therefore MCV and

MCHC. Also calculated HCT but not PCV. Hb measurement not affected.

Red cell parasites

1. Mycoplasma spp.Formerly known as Haemobartonella spp. and/or

Eperythrozoon spp.Gram-negative, non-acid fast bacteria, small (05-

1um), cocci/rods/rings, can present in chains and are predominantly on external surface of RBC

Different species affect different species of animal: haemofelis, haemominutum, turicensis – cats; haemocanis, haemominutum, haematoparvum – dogs, suis – pigs, ovis – sheep/goats, wenyoni, haemobos – cattle, haemolamae – alpacas/llama

PCR most sensitive method of detection and only method of speciation

Often dissociate from RBCs when in EDTAVariable clinical significance. Haemofelis most clinically significant in small

animal medicine as can cause significant, often regenerative, anaemia in cats.

2. Babesia spp. Protozoal organism, variable size (1-5um), often

teardrop or round in shape, often in pairs, with a distinct nucleus (red compared to granular blue of the remainder of the organism) and within RBCs

Different species affect different species of animals: canis canis, canis vogeli, canis rossi, large unnamed (North Carolina), gibsoni, conradae, microti-like – dogs; felis – cats; bigemina, bovis – cattle; caballi, equi – horses

PCR most sensitive method of detection and best method of speciation

Variable clinical significance

RBC Morphology: Important abnormalities

1. AcanthocyteUnevenly spiculated cells Liver disease, abnormal lipid balance, neoplasia (esp.

haemangiosarcoma), post-splenectomy

2. Basophilic stipplingRibosomes and polyribosomes aggregating together and staining blueMarkedly regenerative anaemias (particularly cattle but also rarely other species), without regeneration consider lead toxicity

3. EccentrocyteHaemoglobin eccentrically pushed to one side with opposing RBC membranes adheredOxidative injury: endogenous (e.g. DKA, lymphoma, hyperthyroidism, inflammatory disease) and exogenous (e.g. zinc, paracetamol, onion/garlic, prolonged propofol, vitamin K)

4. EcchinocyteCrenated cell, EVEN spiculesMost often drying artifact. Rarely electrolyte abnormalities (horses predominantly)

5. Heinz bodiesAggregates of oxidized, precipitated haemoglobin attached to internal surfaces of RBC membranesOxidative injury (see Eccentrocytes) in most speciesFew small Heinz bodies (<5%) normal in catsStain blue with New Methylene Blue stain (easy to enumerate)

6. KeratocyteBlister appearance, single projection (“apple-stem” cell), or two

projections (“helmet” or “basket” cell)Fragmentation: iron deficiency, thrombosis, DICLiver disorders

7. SpherocytePerfectly round cell, smaller diameter, without central pallor,

only assessable in the dog (cat/horse no central pallor in health). Not biconcave disc anymore.

Many = Immune-mediated haemolysis, snake biteFew/rare = nonspecific with bee sting, DIC, inflammatory disease, oxidant injury

8. SchistocyteRBC fragments with pointed extremitiesDIC, thrombosis, vascular neoplasia (dogs), severe iron deficiency, myelofibrosis, glomerulonephritis, heartworm

White cell distribution/estimation

Even distribution throughout monolayer and feathered edge

Monolayer estimationAt 10x: 18-51/field ~ normalAt 20x: avg. WBC/field (confirm over 10fields) ~WCC x 10E9/L.

Differential: At 40x/50x or 100x perform 100-200 cell differentialNeutrophilsLymphocytesMonocytesEosinophilsBasophilsnRBCsOthers

For interpretation use this differential to calculate the absolute values (e.g. 89% neutrophils with 10 x 10E9/L 8.9 x 10E9/L)

White cell morphology: important abnormalities

1. Neutrophil toxic changeCharacteristics are cytoplasmic and include

cytoplasmic basophilia, cytoplasmic vacuolation, Dohle bodies, and rarely giant neutrophils and neutrophil granulation

Seen with inflammation

2. Neutrophil left shiftingCharacteristics are nuclear and include lack of

segmentation, C-shaped (bands), kidney bean shaped (metamyelocytes), and round (myelocytes) nuclei.

Seen with inflammation and rarely Pelger-Huet anomaly and chronic myeloid leukaemia

3. Reactive lymphocytesCharacteristics include deeply basophilic cytoplasm,

slightly increased amounts of cytoplasm, slightly larger nuclei, occasional paranuclear clearing (Golgi)

Seen with antigenic stimulation

4. Atypical cellsClassifiable atypical cells: mast cells, macrophages, lymphoblasts,

rubricytes/rubriblastsUnclassifiable atypical cells: unclassed blasts

Platelet estimation/distribution/morphology

At 4x or 10x scan feathered edge and body for platelet clumpsIn monolayer at 100x estimate platelets/field 15-20 x this avg ~ plt estimationAssess for large platelets

ReferencesCornell University College of Veterinary Medicine. EClinPath: The on-line textbook. https://ahdc.vet.cornell.edu/clinpath/modules/INDEX.HTM

Harvey (2012) Veterinary Hematology: A diagnostic guide and color atlas St Louis, Missouri: Elsevier

IDEXX Laboratories. IDEXX Learning Center. https://www.idexxlearningcenter.com/idexx/default.aspx Stockham, S; Scott, M (2008) Fundamentals of Veterinary Clinical Pathology 2nd ed. Ames: Wiley-Blackwell.

Valenciano, A; Cowell, R (2014) Diagnostic Cytology and Hematology of the dog and cat 4th ed St Louis, Missouri: Elsevier