PERIPHERAL BLOOD SMEAR Lymphocytosis Methylene Blue

increased lymphocytes viral stains acidic (nucleic acid) property of the cell
normal: 10-47% Eosin Y
Method Advantage Disadvantage
a. Absolute/True Lymphocytosis acidic
Coverslip Bone Marrow preparation Difficult to master and to label stains basic (cytoplasm) property of the cell
b. Relative/False Lymphocytosis
Good for WBC To thin which can break easily stains the protein part, hemoglobin,
false increase
even cell distribution Slip has to mount to the side lymphocytes is about 50% eosinophilic granules
Wedge Easy to label Larger cells/Artifacts go to large cells are pushed at the edge of the smear
Immature WBC (abnormal the edge of the smear which Staining Process
cells) can be easily can contribute to relative 1. Fixation
observed at the edges of lymphocytosis Different Poikilocytes: 10% volumes/volume Methanol
the smear Pushing can introduce trauma Smudge cells prevent water contamination
to cells (Basket cells, Smudge 40% can attract humidity in the air
cells) Nuclear remnants/fragments of lymphocyte
looks like a finger
2. Addition of buffer solution
Glass slide & Coverslip pH 6.4-6.8 aged distilled water
Beacons Method Basket cells pH 6.4 0.05 M NaPO4
Cytoplasmic remnants/fragments of granulocyte pH 7.2 observe Malarian pigments/stiplings
Remember: Wedge Method/Double slide/Slide spreader looks like a net 3. Staining
Methylene Blue
used to transfer blood: Eosin Y
Echinocyte
capillary tube 4. Washing remove excess stain
no pathologic relevance
applicator sticks 5. Drying
diagnosis: equidistant, regular-sized pointed spicules
apply blood: 1 cm from the edge of the slide
Ideal Smear
drop of blood: 2-3 mm diameter
1.2 mm diameter of capillary tube
spread immediately Macroscopic:
angle: 30-45 (best angle: 30) ideal: purple
ideal spreading: smooth blue: slides stained after 1 week or longer
ideal EDTA concentration: 1.25-1.75 nm/ml Cells mistaken as Echinocytes:
K2EDTA best used to prevent crenation Burr Cells
terminate smear: half-inch at the end has pathologic relevance
observe all cells at the thin area diagnosis: irregularly spaced, irregular-sized blunt spicules Microscopic:
routine pH: 6.8 caused by: increase blood urea nitrogen Neutrophil granules pinkish-tan; neutral color & pH
due to Kidney problem/Renal pick up staining characteristics of both
Types of Wedge Method: insufficiency stain
Eosinophil granules striking bright red-orange
a. Push-type
Acanthocyte attracts more Eosin Y
b. Pull-type
has pathologic relevance Basophil granules blue-black
diagnosis: irregularly spaced, irregular-sized pointed graying-blue = improper stain
Criteria for good smear:
spicules
Monocyte has fine azuredast
blurry appearance in cytoplasm
- 2/3 of the slide
Stains: red-blue-grayish with ground glass
tongue shape appearance
occupy the whole width Leishman
May Grunwald Lymphocyte blue
feather edge
Jenner-Giemsa has scanty cytoplasm
lateral edges are visible
film is smooth Wright-Giemsa Platelets lilac; pinkish-tan; purple
no holes or spaces crispier granules RBC red (Wrights Giemsa)
transition from thick (near the heel)-thin-feathered most used fade central pallor
rainbow appearance near the feather edge under light Romanowsky sharp central pallor = hypochromic
modified Wright-Giemsa
polychrome stain
Too Blue Too Red 100x OIO Grading Chart of central pallor
Thick smear Morphology is evaluated +1
Alkaline stain WBC is generally performed +2 2/3
Inadequate washing Thin smear 100 WBC is counted & classified to obtain percentages of +3
Heparinized blood: heparin + Acid stain types +4 Thin ring
Wright stain Excessive washing Segmenters can be distinguished from Bands
Hyperproteinemic patients Inclusions are seen o Distribution
RBC: Howell-Jolly not overlapping because of the zeta potential that repels the
protein stains blue
WBC: Dohle bodies RBC from each other
Reactive or Abnormal cells are seen

Examination: Differential Count o Polychromasia

RBC Morphology representing Reticulocyte
Macroscopic Size
o blue-red color
bluer overall than normal discoid with central pallor stains to see inclusions
due to hyperproteinemia Anicocytosis New Methylene Blue
Roleux Formation (Pseudoagglutination) reference: Lymphocytes Brilliant Cresil Blue (BSB)
holes all over the film (lipids)
check hemoglobin o Shape Grading chart
grainy appearance Poikilocytosis Slight 1%
agglutinated/clump RBC (destroyed) depends on severity of abnormal shape +1 3%
caused by antibodies against RBC antigen can be reported using grading chart
+2 5%
accumulation of nucleated cells Skistocyte
false elevated: leukocytes +3 10%
Target cell/Codocyte
blue specks out at the feathered edge Sickle Cell +4 >11%
increase WBC & platelet
Grading Chart o Inclusion
Normal 5% no inclusions
Microscopic not graded, reported as negative and positive
Slight 5-10%
must be examined at the heel Sickle Cell
+1 10-25%
Popenheimer bodies Sideroblastic
10x objectives +2 25-50% Howell-jolly
overall film quality, color, distribution +3 50-75% Basophilic stiplings
feather edge should be checked quickly for WBC distribution +4 >75%
presence of Rouleaux formation Distribution
scanned quickly for any large abnormal cells (+4) Nucleated RBC o Roleaux formation
presence of fibrin strands only in bone marrow high protein (except Albumin)
specimen must be rejected anemic patients high ESR reading
snowplow effect excreted immediately
presence of more than four times the number of cells per due to hypoxia +1 3-4/hp
field at the edges / feather compared with the monolayer orthochromatophilic stage +2 5-10
area of the film mistaken from lymphocytes
+3 >10
scanty cytoplasm
40x High-dry or 50x OIO false elevated coun
efficient for validating/verifying instrument values when a total o Agglutinated RBC
microscopic assessment of the film is not needed o Hemoglobin concentration discoid shape is disrupted
WBC estimate Central Pallor: 1/3 of total diameter grainy appearance of the smear
Hypochromic has sharp edge antibodies against RBC antigen
select an area in which the RBCs are separated from one
Hyperchromic Auto Anti-I
another with minimal overlapping
Normochromic Mycoplasma pneumoniae
average WBC per field x2000 (x40 magnification)
Auto Anti-i
average WBC per field x3000 (x50 magnification)
Epstein-Barr: infectious mononucleosis
 Auto Anti-P Diseases: Normocytic
Paroxysmal cold hemoglobinuria viral infection Leukemia
Incompatible blood Poikilocytosis Myelofibrosis
HDN Myelogenous Leukmia Diamond Blackfan
increased Basket cells Fanconi Anemia
Grading Chart especially in patients undergoing chemotherapy other bone marrow problem
+1 3-4/hpf all cells (especially leukemic cells) are fragile
+2 5-10/hpf Macrocytic, Hyperchromic
Chronic Lymphoblastic Leukemia (CLL) Megaloblastic Anemia
+3 >10/hpf
increased Smudge cells hypersegmented neutrophil
H. Spherocytosis

Pyruvate-Kinase Deficiency Microcytic, Hypochromic

+1 +2 +3 presence of Burr Cells Anemia of chronic disease
Burr cells Thalassemia
Acanthocytosis Thalassemia Minor male with Fe deficiency
Ovalocyte Abetalipoproteinemia Iron deficiency
Target cell presence of Acanthocytes
1-5/hpf 6-10/hpf >10/hpf Sideroblastic (dimorphism)
Stomatocyte absence of B-Lipoprotein
Bizzare RBC important for lipid transportation Reticulocytosis
Poikilocytes Hemolytic Anemia
Dacrocytes (tear McLeod Phenotype
drop cell)
low Kell Antigen
Acanthocytes affects RBC shape
3-10/hpf 11-20/hpf >20/hpf
Schistocytes causes Neuroacanthocytosis
Spherocytes
Polychromatophilia
Hyperproteinemia
Multiple Myeloma
plasma defect
Platelet Macroglobulinemia (Waldens Strongs)
average platelet per field x20000
average platelet per field xRBC count/200 RBC with Agglutination (Antibody against RBC Antigen)
Auto-immune Hemolytic Anemia
WBC Hemolytic Transfusion Reaction
Hemolytic Disease of the Fetus & the Newborn
50x or 100x magnification
Cold Heme Agglutinin Disease
increased Neutrophil bacterial
increased Lymphocyte viral
increased Eosinophil allery/parasitic

shift to the left shift to the right

immature WBC Hypersegmented Neutrophil
1 neutro w/ 6 lobes
5 neutro w/ 5 lobes
100 neutro (lobe count)