PERIPHERAL BLOOD FILM EVALUATIONWHAT LIES BENEATH?

Nawin Manachai (DVM., MSc., PhD.)

Small Animal Clinic

Department of Companion Animal and Wildlife Clinic

Faculty of Veterinary Medicine

Chiang Mai University

งานประชุมวชิาการ คณะสัตวแพทยศาสตร์มหาวิทยาลยัเชียงใหม่ 2563

Multi Systemic Disease

• ค ำถำม?

ในชว่ง 6 เดอืนทีผ่่ำนมำท่ำนดู blood smear บ่อยแคไ่หน ?

1. อย่ำงน้อย 1 คร ัง้ ตอ่สปัดำห ์2. อย่ำงน้อย 1 คร ัง้ ตอ่เดอืน3. อย่ำงน้อย 1 คร ัง้ ตอ่ 3 เดอืน4. อย่ำงน้อย 1 คร ัง้ ตอ่ 6 เดอืน5.ไม่เคยดูเลย

• Hematological disorders

-anemia

-leukopenia

-thrombocytopenia

-unexplained cytosis

-malignancies

• Non-hematological disorders

(hematologic manifestations in systemic disease)

Screening

• simply

• safe

Diagnosis

MonitoringEarly management

Peripheral blood smear (PBS)

• Peripheral blood smear (PBS)

Iron deficiencyIMHAMegaloblastic anemiaITPMyelophthisis blood pictureMAHA blood picture Hematologic malignancyBlood parasite infection

5

What is included in a complete blood count (CBC) ?

Analyzer data Blood film microscopic review

Scatter plot data

Provided by automated analyzers

Provided by automated analyzers

Peripheral blood smear (PBS)

1. EDTA-blood2. Glass slide3. Coverslip4. Fixative agent5. Staining• Wright’s stain• Diff-quick6. Light microscope7. You

Standard area…

standard areazone of morphology

feathered edge

Advantage

Verify automate analyzer results

Identify critical diagnostic features that analyzers cannot evaluate

Identify morphologic abnormalities can be present even in patients with quantitatively normal results for all hematologic parameters

stacked RBCs on

Peripheral blood film (smear)

Make blood smears soon after collection to reduce the risk of artifactsMake a good quality smear

Always start fromLOW POWER 10X

1. RBC distribution• degree of anemia• rouleaux formation• autoagglutination

2. WBC estimated number• 10-15 cell/LPF approximate to normal

3. Small number of suspected cells and large haemoparasite• nRBCs, blast• evaluate for the presence of microfilaria

4. Identify platelet clumps• might artifactually decrease platelet numbers

10X

Always start fromLOW POWER 10X

presence of microfilaria at feathered edge

presence of platelet clumpat feathered edge

Let’s proceed to …High power field with oil immersion lens x100

• Perfect in all 3 series

1. Assess to RBC2. Assess to WBC3. Assess to platelet4. Assess to others

100X

RED CELLS

• Size and staining

• Anisopoikilocytosis

• Bone marrow response

• Red cell distribution

• Others (inclusions)

Look at…

1. Size and staining

• Hypochromic microcytic -grading +1, +2, +3-mild, moderate, marked

• Normochromic normocytic

• Macrocytic

ICSH : The International Council for Standardization in Haematology

Small lymphocyte

• According to red cell size (MCV) …

Microcytic

• MCV < 60 fL (<40, cat)

• Yong animal

• Iron deficiency anemia I(D)

• Anemia of inflammation (severe)

• Lead toxicity

• Chronic blood loss

• PSS

Normocytic

• MCV 60-77 fL (40-45, cat)

Non-regenerative anemia

1.Anemia of inflammation

2. CKD

3. Sepsis

4. Hemoparasites

5. Metabolic defects

6, Hypothyroidism

7. Androgen deprivation

8.Bone marrow disease

9. Hemolytic anemia

10. Blood loss

Macrocytic

• MCV >77 fL (>45, cat)

1 Megaloblastic anemia

-Vit B12 deficiency

-Folate deficiemcy

2. Reticulocytosis

3. FeLV, MDS,

Aplastic anemia

4. Liver disease

5. Drugs

6. False due to agglutinate

RBC

14

• Iron deficiency anemia

• Microcytic hypochromic

-increased central pallor -decreased Hb-secondary to low-grade, chronic blood loss. from the GI tract (ulcers, neoplasia, parasites)

non-regenerative anemia

CKD BM diseases

AI

Endocrinopathies

• Indicates a regenerative BM response to anemia

• Blood loss

• Hemolysis

• Reflect to aggregate reticulocytes

• According to Red cell staining (MCH and MCHC)…

Hypochromic RBC

Normohromic RBC

Polychromasia RBC

• Hypochromic microcytic anemia in dog

VET MED CMU

Peripheral Blood Film…Dog 3 yoPCV = 30%Hb = 10 g/dLMCV = 57 fLChronic diarrhea withbloody content for several weeks

• Iron deficiency (ID)• Chronic blood loss

2. Anisocytosis Variation in RBC size on a blood smear, normally correlate to Red Cell Distribution Width (RDW) measure by automates

- Normal finding in cat- Frequently found with poikilocytosis- Assess to others morphologic changes

A) Normal distribution of RDW(11-14% in dog)(14-20% in cat)

B) Increase distribution of RDW- Anisocytosis

VET MED CMU

Important to assess RBC morphology…. (HPF100X)

• To progress understanding of the type of anemia present and its possible causes

• Poikilocytes may be removed premature from circulation

• Some abnormalities have specific significance relating to a particular

• Disease while others may be non-specific

• Spleen and liver an important role

• Aniso-poikilocytosis

Aniso-poikilocytosis

1.Schistocyte

(fragmented cell)

2.Dacrocyte

(tear drop cell)

BM diseases: myelofibrosis

3.Acanthocyte (spur cell)

- Lack of central pallor

4.Echinocyte (bur cell)

5.Eliptotocyte (ovalocyte)

6.Codocyte/Leptocyte

(target cell)

7.Stomatocyte

8.Spherotocyte

Micro-spherocyte

9. Keratocyte (bite cell)

(Basket/blister cell) (oxidative injuries)

Red cell fragmentation (fragmented RBC) caused by mechanical injury to RBC in vasculature (shearing force)Common in dogs with DIC, vasculitis, hemangiosarcoma, TTP, HUS

• Schistocytes.. (fragmented RBCs)

• Microangiopathic hemolytic anemia (MAHA)

• MAHA blood picture (1-2 / HPF)

Presentation Title

Feline, 1 yo M anorexia, depress and presence of skin and mucosal hemorrhage

PE:Pale mmPetechial andEcchymosisHemorrhage

Peripheral blood film

-Acanthocytes +3-Schistocytes +2

Coagulogram

ThrombocytopeniaPT and aPTTprolongation

Underlying disease?

• MAHA blood picture

• Acanthocytes (spur cells)...

Pathologic: Abnormal lipid composition of RBC membraneIncrease free cholesterol in plasma e.g. liver failure, hepatic lipidosis in catSevere liver disease : Spur cell anemiaFragile RBCFragmentation injury: DIC ; co-finding with schistocyte or keratocyte (MAHA blood picture)

-No central pallor-Irregular spine

• Echinocyte (crenation, burr cell)…

Normal or artifacts: Drying artifacts in smear preparation, prolonged storage in EDTA

Pathologic: Electrolyte abnormality, uremia, rattlesnake envenomation glomerulonephritis

Type I Type III

Presence of central pallorRegular spiculeChange in membrane lipidor pH or oxidative injury

• Echinocyte and Acanthocyte

VET MED CMU

Peripheral Blood Film…Dog 12 yo, malePCV = 32%Hb = 10 g/dLMCV = 60 fLAbdominal effusionALT = 400 U/L HALP = 362 U/L HAlbumin = 2 g/dL LLiver US : multiple hyperechoic mass

• Echinocytosis from hematotoxin suspected snake venom

VET MED CMUVET MED CMU

Parameter Day 1 Day2

Hct (%) 54 27

Hb (g/dl) 19 9.7

Plt (cell/ul) 82,000 36,000

WBC (cell/ul) 15,100 49,650

Band(cell/ul)

- 5,165

Creat.(mg/dl) 1.6 2.83Identify morphologic abnormalities can be present in patients with quantitatively normal results for all hematologic parameters

• Stomatocyte… (mouth-like)

• Pathologic: anemia (non-specific): Hereditary stomatocytosis in Alaskan Malamute

cirrhosis, obstructive liver disease

• Leptocyte… (target cells)

• Fold in the membrane a central bar of Hb Artifacts: Drying artifacts in smear preparation and excessive EDTA

• Pathologic (dog): anemia (non-specific) Hepatic diseases cirrhosis esp. cholestatic diseasePost-splenectomy Renal disease (nephrotic syndrome), Regenerative anemia Anemia of inflammation

Increase ratio of surface area to volume

Decreased ratio of Hb(relative or absolute)

27

• Leptocyte… (target cells) : Chlolestatic disease

VET MED CMU

Peripheral Blood Film…Dog 8 yo, malePCV = 28%Hb = 9.1 g/dLMCV = 58 fLAbdominal enlargementALT = 350 U/L HALP = 1,240 U/L HCholesterol= 250 mg/dL HLipemia serum

• Spherocytes (micro-spherocyte)...

Suggestive of immune-mediated hemolytic anemia (IMHA) (only spherocyte found) or RBC fragmentation (a few spherocyte + schistocyte, acanthocyte etc.)Only observed in dogs due to prominent central pallor

Remove a portion of a damaged RBCs cell membraneResult from partial phagocytic removal of antibody-coated membrane Spherocytes are prematurely removed from circulation by splenic macrophages because of their reduced ability to deform

• No central pallor• Smaller than RBCs

• Spherocytosis... with other IMHA blood picture

VET MED CMU VET MED CMU

-Spherocyte-nRBC-Polychromasia-Autoagglutination

• Keratocytes (helmet cells)…..

Red cell fragmentation caused by a ruptured vesicle or oxidative injury

Often result from oxidative damage to RBC membraneDIC, hepatic lipidosis in cats

3. Bone marrow response…

Minor evidence of regeneration of BM response

Increased RBC size (MCV)

- macrocytosis

Increased Howell-Jolly bodies

-nuclear remnant

-splenic diseases or splenectomized dogs

-small number in healthy cats

Polychromasia(with anisocytosis)

Nucleated red blood cells (nRBC)

Aggregate reticulocyte(NMB)

• Blood loss• RBC destruction (hemolysis)• Myelophthisis (BM disease)

3. Bone marrow response…

VET MED CMU VET MED CMU

PolychromasiaNucleated red blood cells (nRBC)

CLL with hemolytic anemia in cat with FeLV

Small lymphocyte nRBC

• Don’t be confuse!! between small lymphocyte and nucleated red cell

Small lymphocyte Nucleated Red Cell

VET MED CMU VET MED CMU

4. Red cell distribution…

Rouleaux formation

• Normal in healthy horses and cats • Excessive rouleaux formation indicates

hyperglobulinemia and /or hyperfibrinogenemia-Inflammatory disease: FIP, Reactive lymphadenopathy-Neoplasia: B-cell neoplasm : MM, CLL

Autoagglutination

• Due to the binding of Ab to RBC• Immune-mediated hemolytic anemia• Suggesting for Saline dilution test to confirm

• Rouleaux formation -stacks formation of RBCs

• Autoagglutination-grape-like formation of RBCs

• Saline dilution testClues: Rouleaux formation and Agglutination

Rouleaux formation

Agglutination

• Rouleaux formation

VET MED CMU VET MED CMU

Feline Female 3 m ADSH abdominal enlargement

Parameter value

Hct (%) L

Hb (g/dl) L

Plt (cell/ul)

WBC (cell/ul) 35,103 H

Band(cell/ul)

399 H

Lymphocyte 3,989

VET MED CMU

VET MED CMU

TP = 9.5 g/dAlbumin = 2.5 g/dlGlobulin = 5.5 g/dlA to G = 0.4

VET MED CMU

VET MED CMU

FNA:VET MED CMU

PBS: VET MED CMU

• Rouleaux formation

TP = 8.4 g/dL HAlbumin=3.1 g/dLGlobulin = 5.3 g/dL HA/G ratio = 0.58 L

• Reactive lymphadenopathy

VET MED CMU

TP = 7.2 g/dL Albumin = 3 g/dLGlobulin = 4.2 g/dL A/G ratio = 0.71

2 weeks

• Autoagglutination

VET MED CMU VET MED CMU

C Female 5 yo poodlenegative all type of blood parasiteby SNAP and PCR

Diagnosis: Primary IMHA

Basophilic stippling

Howell-Jolly body

HaemoparasiteBabesia spp.

Mycoplasma spp

5. Others (RBC inclusion)…

VET MED CMU

VET MED CMUVET MED CMU

Arise from variety sorces• metabolic • physiologic• pathogenic conditions

WHITE CELLS

• number and predominate cells

• toxic granulation, vacuolation

• left shift of maturation, blast

• WBC inclusions

• atypical lymphocyte (reactive or blasts or variants)

• Others

Look at…

• Number and predominate cells…

VET MED CMU VET MED CMU VET MED CMU

Lymphocytes predominate Neutrophil predominate

• 10-15 cell/LPF approximate to normal

• Left-shifts (with maturation)

• Increased concentration of immature neutrophils in blood band, metamyelocytes or precursor forms

(with maturation)

• Indicates more consumption of the neutrophils

• Left shift often occurs along with toxic change, which indicates the release of granulocyte precursors due to an intense demand for inflammatory cells in peripheral tissues. Band (neutrophil) cell

metamyelocyte

Severe inflammatory diseases

indicates enhanced neutrophil turnover and reduced

maturation time, tissue demanded

intense stimulation of granulopoiesis

• Morphologic criteria

Döhle bodies: blue inclusion in the cytoplasm

Basophilia cytoplasm

Cytoplasmic vacuolation

Toxic granulation

Giant neutrophils (cats) refer to metamyelocyte

• Mild to occasionally toxic change - secondary

to any strong marrow stimulation (necrosis,

hemolysis and IMHA

• Moderate to mark toxic change - sepsis/endotoxemia

Döhle bodies Basophilia cytoplasm

Giant neutrophils Cytoplasmic vacuolation

• Toxic change of neutrophils

• Toxic change of neutrophils : canine pyometra

History and physical examination

C, Female 12 yo, breed:YorshirFever, PU/PD depress and anorexia 2-3 daysweakness PE.: Pale mm 7% dehydration

Parameter Result

HCT L 30

Hb L 11.1

RBC L 4.7

MCV 64.2

MCH 23.6

MCHC H 36.8

WBC H 20.430

Band H 9.602

• Left-shifts with toxic changeToxic neutrophils

• Hypersegmented neutrophil

• Prolonged corticosteroid administration• Hyperadrenocoticism• Chronic inflammation• Megaloblastic anemia

5 lobes more than 5 %6 lobes, only 1 cells

VET MED CMUVET MED CMU

• Morphologic alterations in lymphocytes (atypical lymphocyte)

• Reactive lymphocyte

• Large granular lymphocyte (LGL)(variants)

• Lymphoblast (neoplastic lymphocyte)

Mild to moderate increase amount of cytoplasm more deeply basophilic stainingNuclear chromatin less condensed

• immune (antigenic)-stimulated lymphocytes withupregulated synthesis of inflammatory mediators, and/or immunoglobulins (antibodies), or both

• suggest >> active systemic antigenic stimulation

• Plasmacytoid reactive lymphocyte • Monocytoid reactive lymphocyte

• Reactive lymphocytes

48

• Reactive lymphocytes in FIP

VET MED CMU

F Female 3 m ADSH feverWBC = 39,890 HBand = 399 HNeutrophil = 35,103 HLymphocyte = 3,989Mono = 300

Viral infection

Variants of lymphocyteGranular Lymphocytes or Natural Killer Cells Granular

infrequently in blood smearsa few distinct azurophilic granules Increased numbers may occur with chronic immunestimulation (especially chronic ehrlichiosis in dogs)

Large granular lymphoidleukemia (LGL) from dogs

• Large granular lymphocyte (LGL)

• Important to differentiate reactive lymphocytes from neoplastic lymphocytes (leukemia or disseminated form of lymphoma)

• In general, reactive lymphocytes occurring secondary to

systemic immune stimulation with small number

Neoplastic lymphocyte from CLL

Monotonous populationof small lymphoblastwith coarse chromatin

• Neoplastic lymphocytes (lymphoblast)

VET MED CMU

• Others

Mast cells (Mastocytemia) Smudge cell in feline CLL

VET MED CMU

• Others : Acute myeloid leukemia : M2 (suspected) • Myeloblast with maturation

VET MED CMUVET MED CMU

VET MED CMU

C Female 8 yo Mixed WBC = 89,320 vomit depress and feverPCV =26% Hb=9 g/dL Plt = 71,000 cell/uL

PLATELETS

number

morphology and staining

clumping

Look at…

• Platelet assessment

Verify analyzer results of thrombocytopenia

Before estimating platelet counts from a blood smear….Examination the feathered edge of the smearplatelet clumps

-difficult venipuncture or excessively turbulent blood flow-collected from small peripheral veins, collapse -in cats: reactive platelets, which clump readily on sample collection

false thrombocytopeniaFeathered edge large platelet clumps

• False thrombocytopenia

VET MED CMU VET MED CMU

• Platelets number estimation

• 8–15 platelets per high power field (×100)

>>> considered to normal

• Report

-increased

-adequate

-decrease

.

• Approximate number…(semi-quantitative)

• Macro-platelet (giant platelet)

-mostly no clinical significance-minor criteria for reactive thrombopoiesis

• Activated platelet --> shape change- Disk to sphere- Develop projection or filipods

VET MED CMU

• Morphology…

• Immune-mediated thrombocytopenia; (IMT or ITP)

Platelet 19,000 cell/uLMacro plateletsNormal leukogram• PT = 5.4 sec N• aPTT = 10.7 sec N

Platelet 270,000 Monocytosis

Platelet 118,000Left shift

Taper-downPrednisolone 0.5 mg/kgFor 14 days

Platelet 307,000Normal leukogram

Platelet 214,000

• C poodle F 10 yo hematuria marked thrombocytopenia (isolated)

complete response by prednisolone Tx for 2 m.-no clinical bleeding and others-Plt return to normal

Prednisolone 1 mg/kgFor 1 m.

Taper-down qodPrednisolone 0.5 mg/kgFor 14 days

VET MED CMU

• Peripheral blood film report

• Red cellAnisocytosis +3Microcytic hypochromic +1Spherocyte+3nRBC (10/100WBC)autoagglutination• WBCIncrease neutrophil and bands predominate• Plateletmicro-platelet adequate

VET MED CMU

Peripheral blood smear (PBS)apart of CBCCritical diagnostic value- RBC-WBC-Platelet-Other

• Summary

History and Physical Exam

CBC

MCV+RDWReticulocyte count

Blood smear

Other Lab

61

• Special Thx

Lab. of Hematology Unit StaffSmall Animal Teaching HospitalFaculty of Veterinary MedicineChiang Mai University