Hem a tol ogy 1 WH GB |1

Routine RBC Tests Relative polycythemia – false

Midterms polycythemia
Mr. Normel Adarve
PIPETTES
o RBC Count
1. RBC pipette
o Hematocrit (Hct)
➢ 101 mark
o Hemoglobin (Hgb)
➢ 0.5 mark
o Red blood cell indices
o Reticulocyte count ▪ Point of aspiration for routine
o Erythrocyte Sedimentation Rate testing only
❖ Aspirate below 0.5 mark
RBC COUNT ➢ For patients with
suspected polycythemia
Principle:
➢ More dilution is needed
• Dilution of blood
o Diluting fluid ❖ Aspirate above 0.5 mark
o Thoma pipette ➢ For patients with
• Counting of blood cells suspected anemia
o Hemocytometer ➢ Less dilution is needed

Clinical Significance: Check if there is 2. WBC pipette

enough RBC production ➢ 11 mark
➢ 0.5 mark
❖ Anemia
▪ Point of aspiration for routine
➢ Less than the reference value
testing only
❖ Polycythemia
➢ Greater than the reference value DILUTING FLUIDS (isotonic)
➢ Lyse erythrocytes
Assay Units Reference
(adults) Intervals 1. Dacie’s
➢ Most ideal
RBC, male x106/μL 4.20–6.00
➢ Formalin and citrate
(x1012/L)
➢ Prevents clumping of RBCs
RBC, female x106/μL 3.80–5.20 2. Hayem’s
(x1012/L)
HGB, male g/dL (g/L) 13.5–18.0
➢ Allows clumping of RBCs
(135–180) ▪ Caused by hyperproteinemia
HGB, female g/dL (g/L) 12.0–15.0 3. Bethel’s
(120–150) 4. Gower’s
HCT, male % (L/L) 40–54
(0.40–0.54) 5. Toisson’s
HCT, female % (L/L) 35–49 6. Normal saline solution (NSS)
(0.35–0.49)
Source: Rodak’s Hematology 5th edition

Note: Do not use RBC count, Hemoglobin (Hgb) count, • Hemocytometer

and Hematocrit (Hct) count to confirm
anemia/polycythemia. o Counting chamber

Absolute anemia – true anemia

Relative anemia – false anemia
Absolute polycythemia – true
polycythemia
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(count L1, L2,

and L3)
o BOTTOM:
o Cells touching
the innermost
border (count L1
only)
o RIGHT:
o Cells touching
the innermost
border (count L1
only)

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

o Included in counting: Routine RBC count

o TOP: 101−1
DF = 0.5
o Cells touching
DF = 1:200
the 3 borders
(count L1, L2, Note: Dilution factor may vary
depending on the blood cell
and L3) concentration.

❖ WBC Dilution Factor

11−1
o LEFT: ➢ DF = 𝑝𝑜𝑖𝑛𝑡 𝑜𝑓 𝑎𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛
o Cells touching
the 3 borders Routine WBC count
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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
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▪ w/ anti- ▪ Physiologic factors

coagulant ▪ Technical factors
o Intravenous (IV) line
▪ Result: diluted
Note:
Use red tube if blood is from fingerstick procedure. blood
Use blue tube if blood is transferred from EDTA ▪ False ↓ count
tube. (Hct)
Note: (Board exam question) o Hemolyzed sample
Filling of tube and sealing of tube are done on the ▪ False ↓ count
side of red ring. (Hct)
o Prolonged tourniquet
application
▪ Hemocontration
▪ False ↑ count
(Hct)
o Inclusion of buffy coat
in Hct reading
▪ False ↑ count
(Hct)

Buffy coat composition:

1. WBCs
2. Nucleated RBCs
3. Platelets
4. Immature cells
Diseases that increase buffy coat
❖ Capillary tube = 70 mm level
▪ Fill tube with blood
o 50 mm 1. Leukemia
▪ Tube w/o blood ➢ Cancer
o 20-25 mm 2. Leukemoid reaction
▪ Clay and parafilm ➢ Bacterial/viral infection
o 4-6 mm ➢ Bone marrow reaction
o Sealant
❖ Centrifugation o Overcentrifugation
o RCF = 10,000 – 15,000 ▪ No effect on Hct
gravitational force
o RPM = 10,000 – 12,000 ▪ Maximum
packing time
Packed cell volume % was already met
o PCV% < Reference value o
▪ Anemia o Undercentrifugation
▪ False ↑ count
Note: To confirm what type of (Hct)
anemia, do a peripheral blood o Improper sealing of
smear exam tube
o PCV% > Reference value ▪ False ↓ count
▪ Polycythemia (Hct)
▪ Washing out
▪ Physiologic factors
Factors that affect Hct result:
o Poikilocytes
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▪ Abnormal shape of 1. Original

the RBC ➢ Sodium bicarbonate
▪ False ↑ and False ↓ ▪ Buffer solution
count (Hct) 2. Modified
➢ Dihydrogen potassium
Poikilocytes that (↑) increase phosphate
Hct ▪ Buffer solution
a) Macrocytosis Advantages of Modified Drabkin’s
b) Sickle-cell
c) Thalassemia major ▪ Shorter incubation time
▪ Not affected by abnormal
Reason: There are more trapped precipitating proteins
plasma in between packed cells.
Note: In measuring cyanmethemoglobin,
Poikilocytes that (↓) decrease
use a spectrophotometer.
Hct (540 nm wavelength)
a) Crenated RBCs
b) Microcytosis
Anemias w/ microcytic-hypochromic blood
o Dehydration picture: (ITAS)
▪ False ↑ count (Hct)
➢ Microcytic
HEMOGLOBIN o Small RBCs
➢ Hypochromic
2 Hgb Procedures
o No Hgb content
1. Acid Hematin
➢ Principle: Hgb is converted a) Iron deficiency anemia
into acid hematin ▪ Common in females
➢ Uses 0.2 N HCl o Due to menstruation
➢ Dark-brown color b) Thalassemia
2. Cyanmethemoglobin ▪ Common in males
➢ Principle: Hgb is converted
Two types:
to methemoglobin then into
cyanmethemoglobin • Major – Hgb and Hct
➢ Uses Drabkin’s solution are below the reference
value
Active components of Drabkin’s solution:
• Minor – Hgb or Hct are
▪ Potassium cyanide within the reference
• KCN value
• Methemoglobin to
Case Study:
cyanmethemoglobin
▪ Potassium ferricyanide A male patient with microcytic-
• Hemoglobin to hypochromic blood picture. CBC result:
methemoglobin Hgb is normal.

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
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Factors that affect cyanmethemoglobin • Dehydration

or Hgb results: (LIHA) • Blood acidosis
• Low blood O2 level
▪ Lipemia
o False ↑ count (Hgb) Note:
o Milky appearance of To measure hemoglobin, RBCs must be lyzed.
plasma
▪ Turbid RBCs that resist lyzing are called non-lyzing
▪ Icteric RBCs
o False ↑ count (Hgb) ➢ False ↓ count (Hgb)
o Dark-brown/orange plasma
▪ Turbid Examples of non-lyzing RBCs
▪ High WBC count
• Sickle cell
o False ↑ count (Hgb)
• Target cell
▪ Abnormal plasma proteins
• Severly hypochromic RBCs
o False ↑ count (Hgb)
• Nucleated RBCs
Note: These factors express turbidity.
Turbidity increases light absorbance.

Beer’s Law ↑ turbidity = ↑ RULE OF 3

concentration
• RBC count x 3 = Hgb x 3 = Hct
▪ Abnormal hemoglobin • Range computation
o False ↓ count (Hgb) o add/subtract 1.5 for Hgb
o Hemoglobin S o add/subtract 3% for Hct
▪ A point mutation on • BLOOD PICTURE MUST BE
the 6th amino acid of o Normocytic
the β-globin chain o Normochromic
▪ Glutamine mutated
into Valine
Note: A single hemoglobin is composed of 2 α-
globin and 2 β-globin chains

❖ 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

Note: Using the rule of three, ➢ Iron deficiency

Hgb range must be: 7.5-10.5 g/dL ▪ Prolonged coughing
Hct range must be: 51-57% ➢ Anemia of
chronic disease
➢ Hgb doesn’t coincide with RBC
▪ Inflammation/arthritis
count Hct using Rule of 3 ➢ Anemia of
➢ Don’t report yet chronic
➢ Falsely elevated Hgb count inflammation
o Check specimen for
➢ Macrocytic & Hyperchromic
presence of
o Megaloblastic anemia
▪ (LIHA)
➢ Normocytic (w/ low Hct and low
▪ elevated platelet
Hgb)
count
o Aplastic anemia
RED BLOOD CELL INDICES ▪ Bone marrow is
involved
➢ Perform if ↓ in both Hct and Hgb
❖ Mean cell volume Note: If either Hgb or Hct is normal, there
o Volume or size of RBC may not be a presence of anemia
o Average (80-100 fL)
• Mean cell hemoglobin
▪ Femtoliter
concentration
𝐻𝑐𝑡
𝑥 10 o % of hemoglobin in one
𝑅𝐵𝐶 𝑐𝑡
specimen
Blood picture: o Measures entire specimen
o Average (32-36%)
Normocytic - MCV is within reference 𝐻𝑔𝐵
value 𝑥 100
𝐻𝑐𝑡
Microcytic – MCV < 80 fL
Macrocytic – MCV > 100 fL
❖ Mean cell hemoglobin
o Weight of hemoglobin in
RBC
o Average (26-32 pg)
Assay Units Reference
▪ Picogram Intervals
𝐻𝑔𝑏
𝑥 10
𝑅𝐵𝐶 𝑐𝑡 MCV fL 80–100
Blood picture: MCH pg 26–34
Normochromic – MCH is within reference MCHC g/dL 32–36
value
Hypochromic – MCH < 26 pg RDW % 11.5–14.5
Hyperchromic – MCH > 32 pg RETIC x 103/μL 20–115
(x 109/L)
RETIC % 0.5–2.5

Case study: (Blood pictures) Source: Rodak’s Hematology 5th edition

➢ Microcytic & Hypochromic RETICULOCYTE COUNT

o ITAS
• Supravital stains
▪ Male
o RBCs turn into blue
➢ Thalassemia
o To see inclusions
▪ Female
 Hem a tol ogy 1 WH GB |8

1) New Methylene Blue (NMB) 4. Reticulocyte production index

2) Brilliant Cresyl Blue (BCB)
𝐶𝑅𝐶
Other stainable inclusions: # 𝑜𝑓 𝑑𝑎𝑦𝑠
• Howell-Jolly bodies Note: (# of days the reticulocytes mature
o Megaloblastic anemia in the circulation)
o Thalassemia
• Heinz bodies • Normal no. of days (maturation)
o Unstable Hgb o 1 day
o G6PD deficiency Hematocrit level Maturation days
• Hemoglobin H 45 ±5 1 day
o α-Thalassemia major 35 ±5 1.5 days
• Pappenheimer bodies 25±5 2 days
o Sideroblastic anemia 15 ±5 2.5 days
<10 3 days
Formulas:
1. Relative reticulocyte count
Evaluation of results: (RPI)
# 𝑜𝑓 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 𝑥 100
>3 RPI
1000 𝑅𝐵𝐶 𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑
Note: You can observe 1000 RBCs on 10 fields (oil ➢ Bone marrow is still effective in
immersion objective). correcting the anemia.

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

Assay Unit Reference

Intervals
Male 0-15 mm/hr <50 yrs
0-20 mm/hr >50 yrs
Female 0-20 mm/hr <50 yrs
0-30 mm/hr >50 yrs
Children 0-10 mm/hr