Peripheral blood smear

examination
Rakhee Kar
Additional Professor
JIPMER, Puducherry
 Blood film examination may still help to..

• Diagnose a disease
• Monitor response
• Detect pre-analytical or
analytical error in output
of automated analyzer
• Correlate instrument flag
• Additional information
 Layout
Examination of a peripheral smear-general

RBC morphology

WBC morphology

Platelet morphology

Hemoparasites

Artifacts and trouble shooting

Window to the bone marrow

Examination of a peripheral smear-general

 A: Good look at the slide-
Quality of smear and staining
B: At scanner/low power-Choose optimal
assessment area and technique
1.RBCs are uniformly and
singly distributed

2.Few RBCs are touching or

overlapping
3.Normal biconcave RBC
appearance
4. 200 to 250 RBC per 100x
 Optimal Assessment Area

Thin area Thick area

"Spheroidocytes" or flattened
Rouleaux is normal in
red cells. True spherocytes will
be found in other (good) areas such areas. Confirmed
of smear. in thin areas.
 A word of caution:
• Beware of:

Tailing

Low WBC counts

• You are likely to find suspects in the tail end

and edges of smear
Importance of low power (10x)

Platelet distribution/
clumps

Fibrin strands
 C: At high power (40x), observe

• RBC -Morphology

• WBC-Estimate of TLC, DC
• Choose a portion of the PS where there is only slight
overlapping of the RBCs.

• Count 10 fields, take the average number of WBC/ hpf.

• Multiply the average number of white cells by 2000.

 D: In oil immersion (100x)
• RBC, WBC, Platelet morphology

• RBC inclusions

• Hemoparasites

• Estimate of platelet count

1. Estimate the number of platelets per oil immersion field.

2. Take an average of at least 5-6 fields.

3. Multiply the average by 15,000.

 Platelet count
Alaska Med. 2004 Oct-Dec;46(4):92-5.
Platelet count assessment from peripheral blood smear (PBS).
Webb DI1, Parker L, Webb K.

Abstract

OBJECTIVE:
To visually count platelets in a peripheral blood smear and compare with an automated machine platelet count.
METHODS:
Thirty-five peripheral blood smears were made from blood specimens counted on an automated blood cell machine: twenty-three
thrombocytopenic specimens, 1 with high platelet count and 11 with normal counts. Ten and 25 high-power fields were microscopically
averaged and then multiplied by 15,000 and 20,000 to arrive at a platelet count in 1,000 per microliter. Comparisons between visual and
machine counts were drawn.
RESULTS:
There was fair concordance in 27 specimens. In three specimens underestimation was found, overestimation in five. A 15,000 multiplier gave
slightly better results than 20,000. Average in 10 high-power fields was as good as 25. Abnormal counts could be assessed as well as normal.

CONCLUSION:

Average in 10 high-power field on a blood film microscopically and

multiplying by 15,000 gives a platelet count reasonably close to automated

machine counts in thousands per microliter

RBC Morphology
 Assessment of RBCs
1. Size and shape and their variation
(Anisocytosis,Poikilocytosis)
2. Relative hemoglobin content.
3. Polychromatophilia.
4. Rouleaux formation or agglutination
5. Inclusions.
 RBC size
MCV

Microcytic Normocytic Macrocytic

Hypochromic Normochromic
Morphologic classification of anemia

Microcytic Normocytic Macrocytic

hypochromic normochromic normochromic
• Iron Deficiency • Diseases of kidney, • Megaloblastic
(IDA) liver, endocrine anemia
• Chronic organs • Liver
Infections • Early IDA disease/alcoholism
• Thalassemias • Marrow infiltration • Hypothyroidism
• Hemoglobino- • Marrow hypoplasia • MDS
pathies • Haemolytic anemias • Aplastic anaemia
• Sideroblastic • Myelopthisic
Anemia anaemias
Microcytic hypochromic Anemias
Hemoglobin

Heme Globin
Thal/ hemoglobinopathies

Porphyrin Iron
Sideroblastic
anemia

Deficiency Defective utilization

IDA ACD
 D/D of microcytic hypochromic
anemia
Iron deficiency anemia Thalassemia trait
Moderate anisopoikilocytosis, Mild Anisocytosis, target cells
pencil cells, elongated cells
thrombocytosis+/-
Homozygous thalassemia
(Thal Major, intermedia)
 D/D of microcytic hypochromic
anemia
Sideroblastic anemia- Lead poisoning
Dimorphic anemia basophilic stippling
 Dimorphic anemia

• Two different population of RBC

– Macrocytes with normocytes

– Microcytes with normocytes

– Macrocytes with microcytes

• MCV – low, normal or high

 Dimorphic anemia

Causes
• Response to iron or vitamin therapy

• Blood transfusion in a pt. with

microcytic/macrocytic anemia

• Myelodysplastic syndromes

• Sideroblastic anemias
Macrocytic Anemias
Megaloblastic Non-megaloblastic
• MCV > 110 fl • Liver disease/alcoholism
• Folate or B12 deficiency • Hypothyroidism
• MDS
• Aplastic anaemia
• Myelopthisic anaemias
• Hemolytic anemias with
florid retic response
• Neonates
Megaloblastic Anemia
Pancytopenia,
macrovalocytes,
HSP, cabot rings,
nRBCs with
megaloblastic
maturation
 Other macrocytic anemias
Anemia associated with Liver disease Retic response (Polychromatophils )

Aplastic Anemia
 Normocytic normochromic anemia

• Diverse group

• Both hypo- and hyper-

proliferative anemias

• Retic count important

to delineate the type

 Clues to a hemolytic process
• Polychromasia
• n-RBC
• Specific morphologic
abnormalities
Clues to inherited hemolytic anemias
and other stories that RBCs tell..
Membranopathies
Enzymopathies
Hemoglobinopathies
Spherocytic anemia

Coombs test
History
 Spherocytes on PS
Causes Clues
• Hereditary Sherocytosis • Uniform-sized spherocytes

• Auto Immune Hemolytic • Variable spheros, nRBCs,

Anemia Agglutination +/-

• Hemolytic Ds of Newborn • Many nRBCs

• Transfused RBC • Spheroidocytes

• MAHA • Microspherocytes, schistocytes

• Burns • Spheros, smaller RBC fragments

 Clues to other membranopathies..
Elliptocytes Stomatocytes
 Clues to G6PD Deficiency
Blister cells Bite cells

Dapsone-induced hemolysis
Pyrimidine 5’ nucleotidase deficiency
 All enzymopathies do not have
characteristic morphology
• PK deficiency
Thalassemias and hemoglobinopathies

Sickle cell anemia

Sickle beta thalassemia

Thalassemias and hemoglobinopathies

Homozygous Hb C disease
 Target cells

Microcytic Normocytic Macrocytic

• Severe Iron • Sickle cell anemia • Liver disease

Deficiency (IDA) • Sickle thalassemia • Post splenectomy
• Thalassemia trait • Preterm infants
• Hb E disease
• Hb C disease
Clues to acquired hemolytic anemias
and other stories that RBCs tell..
 Agglutination

Addl clues:

Cold Tight clusters

Polychromatophils
agglutinin
Spherocytes
disease n-RBCs
 Rouleaux formation

Addl clues:
Proteinaceous
background
Plasmacytoid cells
Schistocytes
Microangiopathic hemolytic anemia
 Thrombotic thrombocytopenic purpura (TTP)

 Hemolytic uremic syndrome (HUS)

Schistocytes
 HUS/TTP-related disorders •Well hemoglobinised
Disseminated carcinoma broken cells
Chemotherapy/drugs
•Predominant finding in
Transplant-associated microangiopathy

 Pregnancy and postpartum period absence of other red cell

HELLP syndrome abnormality
TTP/HUS
•Look for low platelets,
 Malignant hypertension
and other signs of
 Disseminated intravascular coagulation
hemolysis
KERATOCYTE

HELMET CELL

TRIANGLE
MICROSPHEROCYTES
SCHISTOCYTE
Some more poikilocytes..
 Spur cell (Acanthocyte)

Longer and lesser

number of spikes
 Burr cell (Echinocyte)

Anemia of renal
disease

Shorter and more

number of spikes
Tear drop cell (Dacryocyte)

MA
RBC inclusions on Romanowsky
stains
Basophilic stippling
Howell Jolly bodies
Pappenheimer bodies
Howell Jolly bodies
Int. Jnl. Lab. Hem. 2015, 37, 1–7
WBC Morphology
 n-RBCs give spurious WBC count
• Look at the counts (TLC).
• Does it need any correction?

Eg: TLC-15000
nRBC: 50/100 WBC
Hemolytic anemia, LEB picture etc
Corrected TLC??
 Distribution (DLC)

• Look at the distribution (DLC).

• Any penias or philias? (look at absolute count)

• Any morphologic abnormality?

• Any abnormal cells ?

Morphologic abnormalities of
neutrophils
Nucleus
Pelger-Huet anormaly

Hereditary
Myelodysplastic syndrome

Hypersegmentation

Megaloblastic anemia
 Neutrophil-morphology
Cytoplasm
Hypogranulation
Myelodysplastic syndrome

Toxic granule

Bacterial infection

Vacuolization
Bacterial infection
Pseudo Pelger- Huet and hypogranulated
neutrophils

MDS
 Toxic Granulation
• Increased basophilic
granules

in neutrophils.

• Seen in severe infections,

burns, malignancies, and
pregnancy.

• Distinguish from
basophils.
 Dohle Bodies
• Sky blue inclusions in
cytoplasm of
neutrophils.
• Seen in infections,
burns, myleproliferative
disorders, and
pregnancy.
• Composed of RER and
glycogen granules.
Chediak Higashi syndrome
 Leukocyte vacuolation

Chanarin–Dorfman syndrome Lipid Storage Disease

Any left shift?
Neutrophils & Precursors
Leukemoid reaction CML
Grade 0

Grade 4
LAP Score
Normal Lymphocyte
morphology
Transformed lymphocyte
(atypical lymphocyte)

Dengue
IM
 Abnormal
Lymphoid cell Mantle cell
lymphoma
morphology Blastoid
variant
 Eosinophils

Nuclear hypersegmentation of neutrophils,

eosinophils, and basophils due to hydroxyurea
 Time for a blast!

Lineage is often made out on PS morphology.

Myeloblast Lymphoblast
 What else?

Subtype is often made out on PS morphology.

AML M2 AML M5
What is the emergency?

APL- variant with DIC

APL- variant
 But we may not be lucky always…
Blasts/ abnormal cells may not stare at you; you
might have to look for them
 Acute promyelocytic leukemia-classical
 Sub-leukemic leukemia
 Hypoplastic AML
 MDS (RAEB 1/2)
 Associated myelofibrosis/ myelonecrosis (LEB)
Platelet morphology
 What does the PS tell?

• Estimate of count.

(normal, thrombocytopenia, thrombocytosis)

• Does it tally with counter value.

• Any spurious low or high count.

• Any morphologic abnormalities

 Spurious low counts
(counts low, appear adequate on smear and
normal in morphology)
1. EDTA induced
pseudothrombocytopenia 2. Platelet satellitism
1. Exposure of cryptic Ag on • EDTA
IIb/IIIa by EDTA • Uncertain mechanism
 Spurious low counts
3. Non-EDTA 4. Partial clotting of sample
• Procedural
Low counts/ Spurious low counts
(platelets large in size)

• Large platelets
– ITP
–Bernard Soulier
Syndrome
– May Hegglin anomaly
– Immature reactive
platelets
• ITP on steroids
• Post chemotherapy
recovery
 Large platelets

D: Giant platelets in primary myelofibrosis, cellular phase.

E: Bizarre plateletsin essential thrombocythemia.
F: Giant platelets and a megakaryoblast in acute megakaryoblastic
leukemia.
G: Stripped megakaryocytic nucleus.
H: Bernard-Soulier syndrome.
I: May-Hegglin anomaly. Large Döhle-like inclusions (arrow) and giant platelets
 Spurious high counts

MAHA

Cryoglobulins

Burns patient
Bacterial overgrowth in stored sample
 Spurious high counts
Rarer than pseudothrombocytopenia

• Microcytic red cells – Hb H

• Fragmented red cells – TTP, DIC
• Leukemic fragments, apoptotic cells - TLS
• Cryoglobulinemia
• Hyperlipidemia
• Bacteria, fungi, parasitised red cells
• Inadvertant heating of sample
Hemoparasites
 HEMOPARASITE
Any infectious organism
spending a part of its life cycle
in blood with a demonstrable
life form in any of the blood
cells or their marrow
precursors
 Peripheral smear in Malaria
THICK SMEARS THIN SMEARS
• 2-3 drops of blood • 1 drop of blood

• Increased sensitivity • Decreased sensitivity

• Decreased specificity • Increased specificity

• All parasites • Intracellular forms also

extracellular well made out.
 Plasmodium – ring forms

Pl. vivax

Pl. falciparum Pl. knowlesi

Pl. ovale Pl. malariae

Plasmodium – trophozoite form

Pl. falciparum
Pl. vivax

Pl. knowlesi

Pl. ovale Pl. malariae

 Plasmodium- schizont
Pl. falciparum
Pl. vivax

Pl. knowlesi
Pl. malariae

Pl. ovale
 Plasmodium - gametocytes
Pl. vivax

Pl. falciparum

Pl. knowlesi

Pl. malariae
Pl. ovale
 Other diagnostic clues

• Monocytosis

• Pigments in monocytes, neutrophils either in

peripheral smear or marrow

• Sometimes features of hemolysis

Malarial pigment
 Wuchereria bancrofti - Microfilaria

• PERIPHERAL BLOOD :
 Direct wet mount with no staining
 Giemsa or Leishman stained thick blood smear
 Post DEC provocation test
• Examine the extreme tail end of a smear under scanner view
to screen
Artefacts and trouble shooting
 Features of a well-stained PBS
• Macroscopically: color should be pink to purple
• Microscopically:
RBCs: Orange to salmon pink
WBC: Nuclei is purple to blue
Cytoplasm is pink to tan
Granules are lilac to violet
Eosinophil: Granules orange
Basophil: Granules dark blue to black
 Pale staining
 Too Acidic Stain:
RBC pale, WBC barely visible
1. Insufficient staining time
2. Prolonged buffering or washing
3. Old stain
 Correction:
1) Lengthen staining time
2) Check stain and buffer pH
3) Shorten buffering or wash time
 Dark staining
 Too Alkaline Stain:
RBC gray, WBC too dark, Eo granules gray
1. Thick blood smear
2. Prolonged staining
3. Insufficient washing
4. Alkaline pH of stain components
5. Heparinized sample
 Correction :
1. Check pH
2. Shorten stain time
3. Prolong buffering time
 Water Artifact
• Moth eaten RBC,
• Heavily demarcated central pallor
• Crenation
• Refractory shiny blotches on the RBC
What contributes to the problem:
1. Humidity in the air during air drying
2. Water absorbed from the humid air into the alcohol based
stain
Solution:
1. Drying the slide as quickly as possible.
2. Fix with pure anhydrous methanol before staining.
3. Use of 20% v/v methanol
Summary
 A drop of blood tells a story
• A simple tool; yet exceedingly important.
• Can tell some things that no automated
hematology analyzer can.
– Type/ cause of anemia
– Likely nature of leukemia / lymphoma
– Cause of thrombocytopenia
– Bugs in the scene
• Guide to do or not to do a BM examination
Thank you