peripheral blood film evaluation what lies beneath?
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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
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