DIFFERENTIAL COUNTING ▪ Basophil

o Takes up the basic component of the stain

▪ Also known as “Diff count” in the laboratory o Nucleus = lobulated
▪ “Leukocyte Type Number Fraction” o Granules = blue-black which obscures the nucleus
▪ Expresses in percent (%) the relative number of the different types of WBC ▪ Lymphocyte
present in the peripheral blood o Nucleus = dark violet
▪ Counting 100 WBCs in a stained blood smear o Robin’s egg blue cytoplasm
▪ Counting = the counting of 100 WBCs ▪ Monocytes
▪ Differential = when we count 100 WBCs in a stained peripheral blood smear, we o Striking feature = vacuoles
differentiate the WBC according to its specific type o Nucleus = monocytoid
▪ Example: 68 neutrophils in 100 WBCs (68 neutrophils in relative/in relation to o Cytoplasm = ground-glass appearance
100 WBCs)
▪ 4 general steps involved: ▪ 4 other procedures are needed to be known when performing blood smear:
o Prepare blood smear – before staining, it should be air dried completely o Be vigilant or alert of the presence of immature cells
(air drying is dependent on the thickness of the smear) o Presence of abnormal red cell morphology
o Stain the smear – Wright stain o Take not the number of platelets
➢ Basic component – methylene blue (stains the NUCLEUS) o Presence of blood parasites (microfilariae and malarial parasites)
➢ Acid/eosinophilic component – eosin (stains the ▪ Leukocyte Estimate Count
CYTOPLASM) o Purpose: to know if the WBC count in the PBS is sufficient
➢ Mixture of the basic and acid component (stains the o How to perform:
NEUTROPHILIC COMPONENT) ➢ Scan the entire area of the Zone of Morphology using 5 LPO
➢ Neutrophil = takes up the NEUTRAL part of the stain ➢ Count the WBCs in the entire 5 LPO
o Count the cells ➢ Whatever the total WBCs counted in 5 LPO, divide it by 5 and
o Report the result (both in absolute and relative reporting) multiply it with 0.2 X 10^9/L (constant WBC estimation factor)
▪ Check smear for quality ➢ The result should be within the normal reference range: 4,000-
o Perform leukocyte estimate count 11,000 cells per cubic millimeter
o Well-made blood smear: Bullet shaped (gradual transition from the thick ➢ If the result is less than the normal range, diff count cannot be
portion of the head to the thin portion of the tail) performed because there is leukopenia
o Zone of morphology = where morphology of red and white cells are
intact; an area before the tail portion; where morphology and diff count PERFORMING THE COUNT
are performed
▪ Count 100 WBCs and at the same time, differentiate the WBCs according to their
109 𝑇𝑜𝑡𝑎𝑙 # 𝑜𝑓 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 type
𝑊𝐵𝐶 𝑥 = 𝑥 0.2 𝑥 109 /𝐿 ▪ Methods of scanning the smear
𝐿 5
o Purpose: To ensure that the cells are counted only once/to prevent
▪ Neutrophil repetition
o Takes up the neutral part of the stain
o Nucleus = dark violet o METHODS:
o Granules = lilac to pink ➢ Cross-sectional/Crenellation Method – routinely used method
for differential counting; counting the WBCs from side to side
▪ Eosinophil starting from the zone of morphology towards the head portion
o Takes up the eosin or acidic component of the stain • It is unlikely for the counting to reach the head portion,
o Nucleus = characteristically bi-lobed but not most of the time; dark blue because if the estimated WBC count is within the
or dark purple normal range, the 100 WBCs are localized in ZOM
o Granules = red orange • Up, down, down, up in a serpentine manner
 ➢ Longitudinal/Streak Method – counting WBCs from the ZOM o Report the result
towards the head portion; not routinely used ➢ Relative Count – not an informative count
➢ Battlement Track Method – scanning method for cell counts
➢ Meander Method # 𝒐𝒇 𝒔𝒑𝒆𝒄𝒊𝒇𝒊𝒄 𝑾𝑩𝑪
➢ Four-field Meander Method 𝑿 𝟏𝟎𝟎
𝟏𝟎𝟎

o Identify the cells and categorize each cell to its specific type Example: 68 neutrophils in 100 WBCs. 68% is the relative
o If there are more than 5 metarubricytes or nucleated RBCs, compute for count of neutrophils.
the corrected WBC count
Method of reporting: (in order)
𝑢𝑛𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 𝑥 100 WBC count – 10,000 cells per cubic millimeter
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 = Neutrophils – 25
# 𝑜𝑓 𝑁𝑅𝐵𝐶 𝑝𝑒𝑟 100 𝑊𝐵𝐶 + 100
Report as: corrected WBC count Lymphocytes – 68
Monocytes – 02
o Do not progress to far into the thick area (thick portion = rouleaux Eosinophils – 05
formation) Basophils – 00
➢ The thick portion obscures the WBC morphology 100%
o Do not use the very thin area (thin portion = RBCs appear to be fully
hemoglobinized meaning the the RBCs have no central area of pallor) Absolute count of neutrophils: 25/100 X 10,000 = 2,500
o When the count is <1 X 10^9/L = LEUKOPENIA neutrophils per cubic millimeter
➢ Count 50 WBCs and indicate in the notation result Absolute count of lymphocytes: 68/100 X 10,000 = 6,800
➢ Buffy coat smear in a severely leukopenic patient lymphocytes per cubic millimeter
• Centrifuge the specimen for 5 mins
• Aspirate the buffy coat using Pasteur pipet INTERPRETATION: Relative neutropenia only (in the relative
• Place a drop of the buffy coat on a slide and make a count, the neutrophil is only decreased). Relative and absolute
smear lymphocytosis (increased in both relative and absolute counts)
• Air dry and stain using Wright Stain
➢ Absolute Count – gives the actual number of WBC type per
• Count 100 WBCs for differential counting
microliter/cubic millimeter/millimeter
o If it shows abnormal distribution
• Only done by identifying first which WBC count is
➢ Perform 200 cell count, average (by dividing it by 2) & indicate
in result showing an abnormal relative count (increased or
decreased)
o Reference range for Differential count
• Those that have abnormal relative counts are the only
CELL RELATIVE types computed for the absolute count
ABSOLUTE COUNT
TYPE COUNT
Relative Count x WBC count
Conventional SI
Neutrophil 35-71 1,500-7,400 1.5-7.4
o BAND can be counted under neutrophils
Band 0-6 0-700 0.0-0.7
Lymphocyte 24-44 1,000-4,400 1.0-4.4
Monocyte 1-10 100-1,000 0.1-1.0
Eosinophil 0-4 0-400 0.0-0.4
Basophil 0-2 0-200 0.0-0.2
 o Neutrophil 2. Schilling’s Classification
➢ Increase – neutrophilia ▪ According to their granulations specifically for the appearance of the secondary
➢ Decrease – neutropenia granules
o Lymphocyte ▪ Groupings of cells
➢ Increase – lymphocytosis Myeloblasts & promyelocytes 0 (not normally seen in PBS because
➢ Decrease – lymphopenia they do not contain secondary
o Monocyte granules)
➢ Increase – monocytosis Myelocytes 0 (not normally seen because
➢ Decrease – monocytopenia primary granules are still seen)
o Eosinophil Metamyelocytes/Juvenile (earliest cell 0-1%
➢ Increase – eosinophilia seen in PBS with secondary granules)
➢ Decrease – eosinopenia Stab 3-5%
o Basophil Segmented neutrophil (more 51-67%
➢ Increase – basophilia commonly encountered with secondary
➢ Decrease – basopenia granules)

▪ Shift to the left → increase in young forms

METHODS IN CLASSIFYING AND COUNTING NEUTROPHILS o Regenerative = accompanied by high WBC count
o Degenerative = accompanied by low or normal WBC count
1. Arneth’s Classification ▪ Shift to the right → increase in mature forms
▪ Classifies neutrophils according to their age by taking note of the number of lobes
▪ Number of lobes

Class I 1 round or indented nucleus (blast) 5%

Class II 2 lobes 35%
Class III 3 lobes (more common) 41%
Class IV 4 lobes 17%
Class V 5 or more lobes (hypersegmented) 2%
100%

▪ Shift to the left → increase in Class I & II (increase in immature forms)

▪ Shift to the right → increase in Class IV & V (increase in the hypersegmented
forms)
▪ Normal Arneth’s index → 60%
o Add the percentages of Class I and II, plus the 50% of Class III