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o Excluded in counting:
o BOTTOM:
Note: W (4 corner squares) is for WBC counting. R (5 central
o Cells touching
squares) is for RBC counting. the middle and
o Primary square = 9 mm2
outermost
o Secondary square = 1 mm2 border (don’t
o Tertiary square = 0.04 mm2 count L2 and
o Quaternary square = 0.0025 mm2 L3)
Note: Tertiary (5 x 5 squares) and quaternary squares (five Rs) are o RIGHT:
for RBC counting
o Cells touching
the middle and
outermost
border (don’t
count L2 and
L3)
Formulas:
Manual RBC count
# 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝐷𝐹 𝑥 𝑑𝑓 𝑥 𝐴𝐹
• Whereas:
DF = dilution factor
Enlarged tertiary square (one R central square)
L1=innermost L2=middle L3=outermost ❖ RBC Dilution Factor
101−1
➢ DF =
• Improved Neubauer’s ruling 𝑝𝑜𝑖𝑛𝑡 𝑜𝑓 𝑎𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛
11−1
DF = of this?
0.5
Answer: loss of body fluids
DF = 1:20
Note: Dilution factor may vary ▪ Technical factors
depending on the blood cell
concentration.
o Over anticoagulation
▪ Result: diluted
blood
df = depth factor
➢ Space between cover slip and
▪ False ↓ count (RBC,
hemocytometer WBC, platelet)
➢ 10mm (constant)
o Clotted specimen
AF = area factor
25
▪ False ↓ count (all)
• AF = 𝑡𝑒𝑟𝑡𝑖𝑎𝑟𝑦 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑢𝑠𝑒𝑑
o Hemolyzed sample
Note: Area factor is not constant
▪ RBCs are destroyed
• Routine area factor ▪ False ↓ count (RBC,
AF =
25 Hct)
5 ▪ Hemoglobin is not
AF = 5
affected
Note: Routine RBC count uses 5
▪ False ↑ count
tertiary squares. Therefore: Routine (platelet)
area factor is 5
Example: HEMATOCRIT
# of cells counted (average) = 481 Principle:
Dilution factor = 1:200
depth factor = 10mm ▪ Measure packed cell volume
Area factor = 5mm2 o Mechanical centrifugation
= (481) 𝑥 (200) 𝑥 (10𝑚𝑚) 𝑥 (5𝑚𝑚2 ) 2 Hct Procedures
Answer:
I. Macrohematocrit
= 4.81 x 10 6
/mm3 or /μL (Conventional unit) ➢ Uses Wintrobe tube
Note: 1mm3 = 1 μL ➢ Not done anymore
= 4.81 𝑥 1012 /L (SI unit)
Disadvantages:
➢ Needs large volume of blood
Factors that affect manual RBC Count: ➢ Longer reading time
▪ 30 mins.
▪ Physiologic factors
➢ More trapped plasma in between
o Dehydration
packed cells
▪ Result:
▪ False ↑ count (Hct)
Hemoconcentration
▪ False ↑ count (Hct)
II. Microhematocrit
▪ Vomiting
➢ Done in the lab
▪ Diarrhea
➢ Uses capillary tube
▪ Burn injuries
1. Blue tube
▪ No anti-
coagulant
Case study: (If asked)
2. Red tube
A patient has vomited for 3-4 days. CBC ▪ Heparinized
result: ↑RBC ↑Hgb ↑Hct. What is the cause
Hem a tol ogy 1 WH GB |4
Note: The only hemoglobin that can’t be This is consistent with what?
converted to cyanmethemoglobin is Thalassemia minor
sulfhemoglobin
c) Anemia of chronic disease (ACD)
▪ Anemia of chronic
inflammation
Types of Drabkin’s solution d) Sideroblastic anemia
Hem a tol ogy 1 WH GB |6
❖ Types of Hemoglobin S
Example:
1. Hemoglobin SS
➢ Sickle cell anemia 3x1012/L x 3 = 9
➢ Completely abnormal
genes 9 ± 1.5 = 7.5 – 10.5 g/dL (Hgb range)
o Mutation in both β- 13 g/dL x 3 = 39
globin chains
2. Hemoglobin AS 39 ± 3% = 36 – 42% (Hct range)
➢ Sickle cell trait
➢ Only one gene is abnormal
o Mutation in only one Case study:
β-globin chain
3. Hemoglobin SD An automated machine released a result
of:
4. Hemoglobin SG
5. Hemoglobin SE RBC ct = 3 x 1012/L
Note: SD, SG, and SE are sickle cell diseases Hgb = 18 g/dL
Hct = 29%
Factors that affect RBC sickling:
Hem a tol ogy 1 WH GB |7
o Reticulocytosis
▪ Elevated <2 RPI
reticulocyte count
➢ Bone marrow is no longer effective
▪ Bone marrow is still
in correcting the anemia
effective in
➢ Excessive hemolytic anemia
producing RBCs
o Anemia
Case study: o Destruction of RBCs before
120 days
A patient is suspected with anemia.
Lab results: Erythrocyte Sedimentation Rate
low Hgb, low Hct, high reticulocyte count Clinical Significance: For inflammatory
process determination
➢ Bone marrow is not the cause of
anemia Inflammation = ↑ESR
low Hgb, low Hct, low reticulocyte count Principle: Place anticoagulated blood for 1
hour (undisturbed)
➢ Bone marrow is the cause of
anemia Phases (1hr)
o Aplastic anemia
1st – Roleaux formation (10 mins)
2nd – Fast settling of RBCs (40 mins)
2. Absolute reticulocyte count (ARC)
3rd – Final packing of RBCs (10 mins)
𝑟𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑜𝑢𝑛𝑡 𝑥 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 𝑥10
2 ESR Methods
3. Corrected reticulocyte count (CRC)
𝐻𝑐𝑡 a. Wintrobe
𝑟𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑜𝑢𝑛𝑡 𝑥
0.45
Hem a tol ogy 1 WH GB |9
➢ Oxalate citrate
(anticoagulant)
➢ 110-115 mm overall tube
length
➢ 0 – 100 mm ruled area
b. Westergren
➢ EDTA citrate (anticoagulant)
➢ 300 mm overall tube length
➢ 0-200 ruled area