Lesson 3: LABORATORY EVALUATION OF RBCS – PART 1

Topic: Hematocrit
Hemoglobin
Blood Dilution and RBC Count
Red Blood Cell Indices
Reticulocyte Count
Erythrocyte Sedimentation Rate
Osmotic Fragility Index

Learning Outcomes: At the end of this module, you are expected to:

1. State the clinical significance of the reticulocyte count, corrected reticulocyte count, and
the reticulocyte production index.
2. State the clinical significance of the erythrocyte count and the reason for the difference
in the RBC counts of children, male adults and female adults.
3. Discuss the importance of hematocrit, red blood cell indices, and erythrocyte
sedimentation rate in the clinical diagnosis of certain diseases and the various factors
that affect the results of these tests.
4. Compute and interpret for the Red Blood Cell Indices (Mean Corpuscular Volume, Mean
Corpuscular Hemoglobin, Mean Corpuscular Hemoglobin Concentration)
5. Discuss the Red Cell Distribution Width (RDW)
6. Correlate the results of Hct, ESR & blood indices to diseases affecting man.
7. Discuss the proper procedures, sources of error in the determination, and clinical uses.
8. Differentiate the methods of ESR, Hct, and OFT.

LEARNING CONTENT

Introduction:

A Medical Technologist / Medical laboratory scientist could determine the

rate of production of red blood cells in the peripheral circulation by performing
reticulocyte count.

This module includes Reticulocytes; Erythrocytes; Hemoglobin; Hematocrit; RBC

Indices; Erythrocyte Sedimentation Rate; and Osmotic Fragility Test. This module is
designed to provide students with theoretical background about the laboratory methods
of RBC studies and their clinical significance.
Lesson Proper:

Reticulocyte Count

Basic Information

 Assess erythropoeitic activity of the Bone marrow

 Anticoagulant of choice: EDTA
 Normal Reticulocyte Count:
Adult: 0.5 to 1.5%

Newborn: 2 to 6%

 Principle: Whole blood is stained with supravital stain. Any

non-nucleated red cell that contains 2 or more particles of
blue stained granufilamentous material is a reticulocyte.

Formula

1. For Routine Microscopy

# 𝑜𝑓 𝑟𝑒𝑡𝑖𝑐𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 (%) = 𝑥 100
1000 (𝑅𝐵𝐶)

2. Using Miller Disc

 inserted into the eyepiece of the microscope and the grid is
seen
 composed of two squares; RBCs are counted in the smaller
square (B), and reticulocytes are counted in the larger square
(A).
 Formula:

𝑇𝑜𝑡𝑎𝑙 𝑅𝑒𝑡𝑖𝑐𝑠 𝑖𝑛 𝑆𝑞𝑢𝑎𝑟𝑒 𝐴

𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 (%) = 𝑥 100
𝑇𝑜𝑡𝑎𝑙 𝑅𝐵𝐶 𝑖𝑛 𝑆𝑞𝑢𝑎𝑟𝑒 𝐵 𝑥 9

3. Absolute Reticulocyte Count

 is the actual number of reticulocytes in 1 liter (L) or 1 microliter (mL) of blood
 Formula:
𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 (%) 𝑥 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (𝑥 1012 ⁄𝐿)
𝐴𝑅𝐶 (𝑥 109 ⁄𝐿) = 𝑥 1000
100

 Reference Range: 20 X 109/L - 115 X 109/L

4. Corrected Reticulocyte Count

  is also known as Reticulocyte Index or Hematocrit Correction
 used to correct the reticulocyte counts in patients with anemia
 Formula:
𝑃𝑎𝑡𝑖𝑒𝑛𝑡 𝐻𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡 (% 𝑜𝑟 𝐿/𝐿)
𝐶𝑅𝐶 =
45% (0.45 𝐿/𝐿)

 Reference Range:
o Hct of 35% (0.35L/L) expected to have 2% - 3%
o Hct of 25% (0.25L/L) expected to have 3% - 5%
o NV: same w/ the stated NV of Retics count – 0.5% - 2%

5. Corrected Reticulocyte Count

 also known as Shift Correction
 a general indicator of the rate of effective erythropoiesis in patients w/ anemia
 Formula:
𝐶𝑅𝐶
𝑅𝑃𝐼 =
𝑀𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑇𝑖𝑚𝑒

Maturation Time Correction Factor:

Patient’s Hematocrit Correction Factor
Value (%) (Maturation Time, Days)
40–45 1
35–39 1.5
25–34 2
15–24 2.5
<15 3

 Reference Range:
o >3 – indicates adequate bone marrow
o <2 – indicates inadequate erythropoietic response
 Clinical Significance:
Elevated RPI Decreased RPI
1. Chronic Hemolysis 1. BM failure
2. Recent Hemorrhage - Aplastic Anemia
3. Response to 2. Ineffective
Therapy Erythropoiesis
- Megaloblastic
Anemia
*Vit. B
12
*Folate
Deficiency

Methods of Reticulocyte Count

 1. Wet Method (Water)
a. NEW Methylene Blue Method (NMB)
b. Cook, Meyer, Tureen Method (BCB)
c. Seiverd’sMethod (MB or BCB)
2. Dry Method (Alcoholic)
a. Schilling’s Rapid Method (BCB)
b. Sabin’s Method (NR – Neutral Red & JG – Janus Green)
c. Seiverd’s (MB or BCB)
d. Osgood- Wilhelm (MB)

Note: NMB and BCB

 bind, neutralize & cross link RNA

 these stains cause the ribosomal & residual RNA to coprecipitate with
the few remaining mitochondria & ferritin in young erythrocytes to
form reticulum (filaments) & microscopically visible dark – blue clusters

Clinical Significance of Reticulocyte Count

Increased Reticulocyte Count Decreased Reticulocyte Count

Indicates Hemolysis aplastic anemia,
in conditions in which bone marrow is not
Physiologic Increase at birth, menstruation & producing red blood cells (decreased
pregnancy erythropoiesis),
acute benzol poisoning,
hemolytic anemias, pernicious anemia &
individuals with IDA receiving iron therapy, chemotherapeutic radiation – induced
thalassemia, hypoproliferation (Azathioprine, Chloramphenicol,
sideroblastic anemia, Dactinomycin, Methotrexate & other
high elevation, meds like LEVODOPA, malarial chemotherapy meds
meds, CORTICOTROPIN, &
fever-reducing meds and in
acute and chronic blood loss

Sources of Error in Reticulocyte Count

1. Refractile appearance ( bodies) of RBCs are confused as retics

2. RBC inclusions are mistaken as retics

Red Blood Cell Count

 Transports Oxygen and removed waste products from the Body’s tissue
 Total number of red blood cells/L of whole blood
 Factors affecting RBC count:
o Posture – Low in recumbent position
o Exercise or Excitement – Higher than those under basal conditions
 o Dehydration – Higher in severe hemoconcentration
o Age-Higher in newborn infants
o Sex- Lower in women
o Altitude – Higher in high altitude than those at sea level
 Hemocytometry
o estimation of the total number of blood cells in a given volume
o numerical evaluation of formed elements of blood
o consists of counting chamber or hemocytometer, pipettes & diluting fluid
o microscopic method
o Instruments used in hemocytometry
1. Diluting Fluid
 used mainly to disperse blood cells to facilitate counting of cells
 Characteristic of an ideal diluting fluid:
o ISOTONIC (RBC); HYPOTONIC (WBC)
o Has a high specific gravity
o Easy to prepare
o Good preservative
o Has a buffer action
o Does not initiate growth of molds
o Stable
o Non allergenic
o Non -corrosive
 RBC Diluting Fluids
o Dacie’s Fluid – Formol citrate fluid; BEST DILUTING FLUID for
RBCs; can be kept for a long period of time; cell morphology not
altered
o NSS (0.85%) – for emergency use; ideal to use in case of
excessive rouleax formation & autoagglutination
o 3.8% Na Citrate
o Hayem’s
o Gower’s
o Toissons’s
o Bethel’s
 WBC Diluting Fluids - lyze red blood cells except nucleated red blood
cells
o 2-3% Glacial Acetic Acid
o 1% HCL
o Tuerk’s solution: Glacial acetic acid & Methyl violet
 Platelet Diluting Fluids
o Rees & Ecker d.f. – light microscopy
o 1% Ammonium oxalate – phase contrast microscopy
2. Pipets/Pipettes
 Thoma Pipette
o Consist of graduated capillary tube, mixing bulb with glass bead
and aspirating tube
o Parts: stem, bulb, rubber tube
RBC Thoma Pipette

WBC Thoma Pipette

Thoma Pipette RBC WBC

Color of bead RED WHITE
Markings 0.5, 1.0, 101 0.5, 1.0, 11
Volume of the bulb 100 10
Dilution Range 1:100 to 1:1000 1:10 to 1:100
Size of bore Smaller Larger

o Dilution Factor Formula:

𝑉𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝑡ℎ𝑒 𝑏𝑢𝑙𝑏 (𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡)
𝐷𝐹 =
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑏𝑙𝑜𝑜𝑑 𝑢𝑠𝑒𝑑 (𝑣𝑎𝑟𝑖𝑎𝑏𝑙𝑒)

Example:
 100
𝑅𝐵𝐶 = = 200
0.5
10
𝑊𝐵𝐶 = = 20
0.5
o Manual Method Dilution:
 1:200 (RBC)
 1:20 (WBC)
o Coulter Counter Dilution:
 1:50,000 (RBC)
 1:500 (WBC)
3. Hemocytometer
 an instrument used to count the blood cells
According to TYPE: According to RULINGS
Open type (Spencer, Burker, Levy, Levy- Thoma
Hausser
Closed type (Thoma – Zeiss) Tuerk
Addis Fuchs – Rosenthal
Exton Neubauer
Petroff Improved Neubauer
Bass – Jones

 Open type – Spencer,

Improved Neubauer =
most commonly used
counting chamber
 invented by Louis-Charles
Malassez
 consists of a thick glass
microscope slide.
 Depth = 0.1 mm
 Grids/Ruled Areas:
o RBC use 5 small
squares in the
center large square
o WBC use 4 corner large squares
o Unopette for WBC use all 9 large squares
o Platelet use 25 small squares in the center large square

 Neubauer Counting Chamber

o Total Area – 9 mm2
 o Depth – 0.1 mm
o Total volume – 0.9 mm3
o Area of the large square – 1 mm2
o Area of the smaller central square – 0.04 mm2
o Area of the smallest square – 0.0025 mm2
o Dimensions – 3 x 3 x 0.1mm
o also used for counting sperm cells

 Speirs - Levy Counting Chamber:

o Total Area – 10 mm2
o Depth – 0.2 mm
o Total volume – 2.0 mm3
o Dimensions – 2 x 5 x 0.2mm
o Use: for rapid, direct counts
of eosinophil cells ( & basophils) in peripheral blood for the
assay of adrenal cortical hormone level in clinical experiments
and for use in the administration of
ACTH for the treatment of arthritis

 Fuchs – Rosenthal Counting Chamber:

o Total Area – 16 mm2
o Depth – 0.2 mm
o Total volume – 3.2 mm3
o Dimensions – 4 x 4 x 0.2 mm
o Use: gold standard for quantification of
leukocytes (eosinophils, basophils) in cerebrospinal fluid (CSF).

 Rule in Counting in Hemocytometer

o Count cells: Top or left triple boundary
lines
o Do not count cells touching: Bottom or
right boundary lines

 Procedure
1. Mix blood
2. Aspirate to 0.5 mark
3. Wipe excess blood
4. Suck RBC diluting fluid up to 101 mark
5. Mix blood and diluting fluid
6. Discard first few drops
7. Charge
8. Stand for 3 minutes
9. Count!
  General Formula
o RBC Count (RBC in millions / cu.mm)
= Number of red blood cells counted X area correction
factor X Depth factor X Dilution
= Number of red blood cell s counted X 5 X 10 X 200
or
= Number of red blood cell s counted X 10,000
 To convert values to SI unit, multiply answer by 0.000001
o SI unit RBC count = X 10 12/ L
 Reference Range
o Male: 4.6 – 6.0 M / cu.mm (5.5 – 6.5)
4.6 – 6.0 x 1012/L
o Female: 4.0 – 5.4 M / cu.mm (4.5 – 5.5)
4.0 – 5.4 x 1012/L
o At birth: 5.0 – 6.5 M/cu.mm
1.0 – 6.5 x 1012/L
 Clinical Significance
Low Value High Values
 Blood loss (Hemorrhage)  High altitude
 BM failure  Congenital heart disease
 Iron, Folate, Vitamin  Polycythemia vera
deficiencies  Dehydration
 Hemolysis
 Certain cancers
 Anemia
 After age 50
 In recumbency
 After meals (as much as 10%
lower)

**END of LESSON**

Hemoglobinopathies Laboratory Diagnosis

a. Hemoglobin Electrophoresis
 Alkaline Electrophoresis
o Cellulose Acetate Electrophoresis
o uses medias such as: paper, starch blocks,
cellulose
o with buffer
o at alkaline pH 8.6
 o this technique separates the hemoglobin fractions S, F, A, C,
and A2
 Citrate Agar Electrophoresis
o takes place at acid pH 6.2
o uses citrate agar with buffer ions
o Hb S, D, G, C, E, and O = Hb fractions that citrate agar at acid
pH separates

th
Hb S Glutamine is replaced by VALINE; 6 amino acid of beta chain
Hb C Glutamine is replaced by LYSINE; 6th amino acid of beta chain
st
Hb D Glutamine is replaced by GLYCINE; 121 amino acid of beta chain
Hb E Glutamine is replaced by LYSINE; 26th amino acid of beta chain
Hb O Glutamine is replaced by LYSINE; 121st amino acid of beta chain

b. Denaturation Procedures - tests for Hb F quantitation and determine

the amount of fetal blood that has mixed with maternal blood
following delivery
 Alkaline Denaturation
o Principle: Hb F are alkali resistant
o Methods: Kleihauer Betke (NaOH); Singer Method (KOH)
o Specimen: RBC hemosylate from EDTA WB
o Reagent: Cyanmethemoglobin reagent;mSodium /Potassium
hydroxide
 Acid Elution Test
o Principle: Hb F are resistant to acid
elution
o Specimen: Blood smears from EDTA
WB or Capillary blood
o Reagent: Erythrosin; Erlich’s /
Mayer’s hematoxylin
o Results: Deep pink RBCs – increase
Hb F
Pale ghost cells – decrease Hb F

oElevated Hb F : can be seen in beta-thalassemia and

paroxysmal nocturnal hemoglobinuria (PNH).
c. Chromatography
 Colorimetric method
 Quantitation of Hb A1 (Glycosylated Hb)
 accomplished by cation exchange
minicolumn chromatography
 includes highpressure liquid
chromatography (HPLC)
d. Hb Solubility Test
 most common screening test for Hb S
 EDTA, Na Citrate or Heparinized WB
 Reagent: Na dithionate
  Positive result: Turbidity
Laboratory Evaluations

I. Colorimetric Method:
o Direct Visual Colorimetric Method
i. Tallquist Method
o uses absorbent pads with lithographed color scale
o 50% error
ii. Dare’s Hemoglobinometer
o 2 glass plates with a rotating disc of tinted glass
o 30% error
iii. Acid Hematin Method
o uses 0.1N Hcl to convert Hb into acid
hematin (a brown solution) which is diluted with distilled water
drop by drop until the color matches that of the standard
o Method: Sahli-Hellige Method
o Materials: Sahli pipette volume: 20
ul/0.02ml
Sahli type hemoglobinometer
o Disadvantage:
a. Operator’s personal
perception in matching colors
b. Addition of DW drop by drop
is time consuming
c. Abnormal Hbs are not measured
iv. Alkaline Hematin Method
o uses 0.1N NaOH alkali to convert Hb into alkaline hematin
o Methods: Wu; Clegg & King
o Disadvantage: Hb F is not measured since it is resistant to
denaturation by alkali
o Photoelectric Colorimetric Method - Spectrophotometric Method (540 nm)
i. Oxyhemoglobin Method (HbO2)
o 0.007 N NH4OH or 0.1% Na2CO3 will convert Hb into
oxyhemoglobin
ii. Cyanmethemoglobin Method (HiCN)
o also known as hemiglobin cyanide; ferrihemoglobin cyanide
o measures all forms of hemoglobin except sulfhemoglobin
o Reagent: Detergent – modified Drabkin’s Reagent = with pH 7.0 –
7.4
a. Dihydrogen K phosphate: shortens
incubation/conversion time for 10 – 15mins into 3 mins
b. K ferricyanide: converts Hb into Methemoglobin
c. K cyanide: converts methemoglobin into
cyanmethemoglobin
d. Non-ionic detergent (such as Sterox or Triton X):
promotes RBC lysis
o Sources of Error and Corrective Measures
 Sources of Errors Corrective Measures
1. Lipemia Can be corrected using a patient blank
2. Increased WBCs Can be corrected by centrifuging test mixture & testing Hb
on the supernatant fluid
3. HbS & HbC Dilute Hb with DW

4. Increased globulins Use Dihydrogen K phosphate

5. Overanticoagulation Causes no effect on Hb determination

II. Specific Gravity or Gravimetric Method:

o the specific gravity of the blood is the ratio of the
o weight of the same volume of water
o Method: CuSO4 (at 1.055)
o Specific gravity of blood: 1.055
o If the drop of blood:
Sinks – high specific gravity
Floats – low specific gravity

III. Gasometric Method:

o indirect method of Hb determination based on the O2 contents of the blood
o based on: 1gm Hb can carry 1.34 ml O2
o abnormal Hb are not measured having no affinity to O2
o Method: Van Slyke’s
o Formula
𝑂𝑥𝑦𝑔𝑒𝑛 (𝑚𝑚𝐻𝑔)
𝐻𝑏 =
1.34

IV. Chemical Method:

o indirect method of Hb determination based on the Iron contents of the blood
o based on: 1gm Hb contains 3.47 mg iron
o Methods:
i. Wong’s – conc. H2SO4 & potassium persulfate
ii. Kennedy’s
o Formula
𝐼𝑟𝑜𝑛 (𝑚𝑔)
𝐻𝑏 =
3.47

Note:

1. To convert
a. g Hb to % Hb
𝑔 𝐻𝑏
% 𝐻𝑏 = 𝑥 100
𝑆𝑡𝑑 𝐻𝑏 (16)

b. % Hb to g Hb
% 𝐻𝑏
𝑔 𝐻𝑏 = 𝑥 𝑆𝑡𝑑 𝐻𝑏 (16)
100
 2. Reference Range
a. Male: 13.5 – 18 g/dl
b. Female: 12 – 16 g /dl
c. Newborn: 14 – 26 g / dl

3. Clinical Significance
Low Hb High Hb
 Anemia  Sickle cell anemia
 Blood loss  Thalassemia
 Iron, folate and Vitamin B6, B12  Transfusion reaction
deficiencies  Hemolysis
 Dehydration
 Polycythemia vera
 High altitude

Laboratory Methods:

1. Adam’s Microhematocrit
o Tube: Capillary tube
Lenght: 70 - 75mm
Bore size: 1- 1.2mm
Band color: Red – Heparinized
Blue – Non-heparinized
*Capillary tubes should be at least – 2/3 or
5cm (50mm) full
Sealing/plug: end of the tube with the colored ring
: at least 4 mm long (4-6 mm)
o Centrifugation:

Speed: 10,000 – 15,000 g

Time: 5 minutes
2. Wintrobe’s Microhematocrit
o Tube: Wintrobe tube
Length: 115 mm / 11.5 cm
Bore: 3.0 mm
Right Graduation lines: 100 (10) - 0
o Centrifugation:
Speed: 2,000 – 2,300 rpm
Time: 30 minutes
 Case 1: Case 2: Case 3:

HGB = 12 g/dL HGB = 9 g/dL HGB = 15 g/dL

HCT = 36% (0.36 L/L) HCT = 32% HCT = 36%

According to the rule of According to the rule of According to the rule of

three, three, three,

HGB (12) X 3 = HCT (36) HGB (9.0) X 3 = HCT (27 HGB (15) X 3 = HCT (45
versus actual value of versus obtained value of
32) 36)

An acceptable range for An acceptable range for An acceptable range for

the hematocrit would be
hematocrit would be hematocrit would be
33% to 39%. 24% to 30%, 42% to 48%,

These values conform to so these values do not so these values do not

the rule of three. conform to the rule of conform to the rule of
three. three.

Laboratory Evaluation

 Rule of Three
o a quick visual check of the results of the hemoglobin and hematocrit
o value of the hematocrit should be three times the value of the hemoglobin plus
or minus 3:
HGB X 3 = HCT + 3 (0.03 L/L)
o for NORMOCYTIC NORMOCHROMIC red cells
o a value discrepant with this rule may indicate abnormal red blood cells, or it
may be the first indication of error.

 Procedure (Microhematocrit)
o Fill capillary tube approximately three – quarters full with anticoagulated blood
o Seal the end of the tube with the colored ring with clay and wax
o Centrifuge for 10,000 G for 5 minutes
 o Determine hct using microhematocrit reader
o Reference Range:
Male 40 – 54%
Female: 35 – 49%

Neonates: 48 – 68%

 Sources of Error
False Increased
 Insufficient centrifugation
 Inclusion of buffy coat
 Disorders such as sickle cell anemia,,
macrocytic anemias, hypochromic
anemias
 Dehydration
False Decreased
 Improper sealing of capillet
 Increased conc. of anticoagulants
 Prolonged centrifugation
 Acute blood loss

 Clinical Significance
High Hct Low Hct
 Dehydration  Anemia
 Polycythemia vera  Blood loss
 High altitude  BM failure
 Hemolysis

Red Blood Indices:

 1. Mean Cell Volume 2. Mean Cell Hemoglobin 3. Mean Cell Hemoglobin
(MCV) (MCH) Concentration (MCHC)

Definition - the average volume of - average weight / content - the average

the red blood cell of hemoglobin in a red concentration of
- Indicator of the red cell blood cell hemoglobin in each
size individual red blood cell

S.I. unit : femtoliters (fL), or picograms (pg), or grams per deciliter (g/dl)
-15 -12
10 L 10 g
3
Conventional Unit: um Uug %

Formula: Hct Hb Hb
MCV = ------- x 10 MCV = ------- x 10 MCV = ------- x 100
RBC RBC Hct

Reference Range: 80 – 100 fl 28 – 32 pg 32 – 36 g/dl

4. Red Cell Distribution Width (RDW)

o Numerical expression of variation in red cell size
Variation in red cell size – Anisocytosis

Variation in red cell shape – Poikilocytosis

o Estimate RBC anisocytosis
o the higher the RDW the more varied the size of the red cells
o calculated through automated machines
o RDW = CV (Coefficient of Variation) of RBC Histograms
* CV = (SD / MCV) x 100

o Reference range:11.6% - 14.6%

5. Color Index
o average volume of Hb in each erythrocyte as compared w/ the average amt in a
normal erythrocyte
% Hb

C.I. = ----------- Reference Range: 0.90 – 1.10

 % RBC

Hb

% Hb = ----------------------- x 100

Std. Hb. (16)

RBC count

% RBC = ---------------------------- x 100

Std. RBC count (5)

6. Volume Index
o average size of RBC as compared w/ the average size of a normal RBC

% Hct

V.I. = ----------- Reference Range: 0.90 – 1.10

% RBC

Hct

% Hct = ----------------------- x 100

Std. Hct (47)

RBC count

% RBC = ---------------------------- x 100

Std. RBC count (5)

 7. Saturation Index
o average amount of Hb per unit volume of cell in relation to the normal

Color Index % Hb

S.I. = ------------------ or ------------

Volume Index % Hct

Reference Range: 0.80 – 1.20

8. Mean Corpuscular Diameter (MCD)

o mean or average diameter of the RBC
o Method of measurement : Price –Jones Technic
uses a camera lucida wherein the stained smear is projected onto a paper

o Price-Jones Curve : distribution curve of the measured diameters of red blood

cells

9. Mean Corpuscular Average Thickness (MCAT)

o Formula is: the volume is equal to the area multiplied by the height
o Data needed: MCV & MCD

MCV

MCAT in microns = -----------

(MCD)2
2
Reference Range: 1.7 – 3.5 micra

Erythrocyte Sedimentation Rate (ESR)

Basic Information

 rate of settling of RBCs from their plasma after the addition of anticoagulant
 measure the degree of settling of erythrocytes in plasma for 60 minutes
 depends on stacking of RBCs – Rouleaux formation
 Importance:
o nonspecific indicator of inflammation
o good index for the presence of hidden but active diseases (TB &
carcinoma)
 o measures the suspension stability of RBCs determines the abnormal
concentration of fibrinogen & serum globulin
 Values: women – increase ESR
man – decrease ESR (due to the conc. of androgenic steroids in males

Eunuchs (castrated male) – increase ESR

 stages of ESR: (60 mins)

a. First 10 mins – initial rouleaux formation
b. Next 40 mins – period of rapid RBC settling
c. Last 10 mins – period of final settling or packing

 Clinical correlation of ESR:

 Factors affecting RBC Sedimentation in ESR

Increased ESR Normal ESR
Pregnancy (after3rd mo.) TB, Menstruation PV
Acute & chronic infections Multiple myeloma Congestive heart failure
Rheumatic fever Waldenstrom’s macroglobulinemia Hypofibrinogemia
Rheumatoid arthritis Subacute bacterial endocarditis Poikilocytes
Myocardial infarction Cryoglobulinemia
Nephrosis Hypothyroidism
Acute hepatitis Hyperthyroidism
 Laboratory Evaluation

 Methods:
Method Anticoagulant Reference Range Others
1. Wintrobe Ammonium- M: 0-9 mm/hr
Potassium oxalate F: 0-20 mm/hr
(known as double C: 0 – 13 mm/hr
oxalate, balanced
oxalate, Paul-
Heller’s solution,
2. Westergren 3.8% Na citrate M: 3-5 mm/1 hr Reference or recommended
7-15mm/2hrs method
F: 4-7mm/1hr
12-17mm/2hrs
3. Graphic-Cutler 3.8% Na citrate M: 0-8 mm/hr Cutler tube:
F: 0-10 mm/hr 0-50 mm graduation
Menstruation:
12mm/hr
4. Linzenmeier 3.8% Na citrate M: 350-600 mins Linzenmeier tube:
F: 300-600 mins 1ml – mark 18mm
Menstruation:600 mins
5. Micromethods 5% Na citrate M: 1-5 mm/hr MicroLandauTube
a. Micro Landau F: 1-8 mm/hr -2 marks: 12.5 & 62.5
(modification of mm above the tip
Linzenmeier-Raunert)
b. Smith Micro
c. Crista or Hellige-
Vollmer
6. Roarke - Ernstene Heparin 0.35 – 0.5 mm/min

7. Bray’s (modified 1.3% Na oxalate M: 47% Bray’s tube

Cutler Method) F: 42% -both sides similar to
Winthrobe tube ; - ESR & Hct

 Comparison between Wintrobe Method & Westergren Method

Difference WINTROBE WESTERGREN

Length 115 mm / 11.5 cm 300 mm

2.5 mm
Bore 3 mm - Internal: 2.65 mm
- External: 5.5 mm
L: 0 -100 (10) Up to 200 mm
for ESR
Graduation
R: 100 (10) – 0
for Hct
Original W. M.:
- 3.8% Na Citrate
Anticoagulant Double oxalate
Modified W. M.:
- EDTA

Amt of blood 1 ml 2.4 ml

Twice
Reading Once - after 1 hr & after 2
Hrs

 Zeta Sedimentation Rate

o a micromethod; most sophisticated of the improved methods
o does not require correction of anemia (unaffected by anemia)
o Materials:
a. Special capillary tube – 75mm long; 2.0mm inner
diameter, 2.3 mm outer diameter
b. Zetafuge – special centrifuge
o Procedure:
 *the special capillary tube is filled (3/4 full) w/ well mixed EDTA-
anticoagulated WB (approximately 0.2 ml)
* the capillary tube is closed at one end w/ a sealing clay to a depth of
approximately 5mm
* spinned in the zetafuge at 4 spin cycles at 400
rpm/45 secs. to det. Zetacrit (ZHct)
o Formula
𝐻𝑐𝑡 (%)
𝑍𝑆𝑅 (%) = 𝑥 100
𝑍𝐻𝑐𝑡 (%)
o takes approximately 4 mins to perform
o Zetacrit (%) is read at the “knee” of the curve
o Reference Range:
Male & Female – 40 – 51%
Borderline normal – 51 – 54%
Mildly elevated – 55 – 59%
Moderately elevated – 60 – 64%
Markedly elevated - >65%

 Automated ESR Systems

o Ves-Matic system
*determine ESR by use of an optoelectronic sensor,
which measures the change in opacity of a column
of blood as sedimentation of blood progresses
*20 minutes
o Sedimat 15
*uses the principle of infrared measurement
*15 minutes
o ESR STAT PLUS system
*based on centrifugation
*suitable for pediatric patient
*smaller required sample volume and shorter testing
time

 Disposable Kits
o uses Dispettes
* disposable plastic tubes w/ safety caps for the columns that allow the
blood to fill precisely to the zero mark.
*safety cap makes the column a closed system and eliminates the error
involved in manually setting the blood to the zero mark.

 Sources of Error
o Errors inherent to the SAMPLE
RBCs: Rouleaux formation – increase ESR
 Poikilocytes causes – decrease ESR

Plasma components:

Acute phase reactants (alpha-1, alpha-2,

fibrinogen often – increase ESR

Albumin & Lecithin – retards ESR

Cholesterol – increase ESR

o Technical Errors
*ESRs should be performed within – 4 hrs of bld collection

- prior to testing:
may be stored at 4oC up to 24 hrs – must be
rewarmed by holding the specimen at ambient
room temperature for at least 15 minutes
prior to testing

*Excess/over- anticoagulation of blood causes false – decreaseNa/K

oxalate & heparin anticoagulants causes the red blood cells to shrink -
increase

*a tilt of 3o can cause errors up to – 30% increase

*Effect of Temperature: 18 – 25oC ideal temp
Increase temp. causes false – increase

Decrease temp. causes false – decrease

Osmotic Fragility Test

Basic Information

 Measure the ability of the red blood cells to take up fluid without lysing
 used to measure erythrocyte resistance to hemolysis while being exposed to varying
levels of dilution of a saline solution
 Primary factor : SHAPE OF THE RBC
 Specimen: Heparinized blood or defibrinated blood
 follows the Law of Osmosis:
The transfer of solution from lower to higher concentration wherein
RBC + hypotonic solution will result to swelling of RBC
RBC + hypertonic solution will result to shrinking of
RBC
 Specimen: WB (EDTA)
 Laboratory Methods

1. Sanford Method
o Different conc. of hypotonic solution
o Initial solution used: 0.5% NaCl
o Interpretation:
No hemolysis – tubes w/ compact sediment & clear solution
Initial Hemolysis – 1st tube from the left w/ not
so compact sediment and w/ faint pink solution
Complete hemolysis – 1st tube from the left without
sediment and with dark red solution
o Reference Range:
IH – tube 22
CH – tube 17
Tube # 25 24 23 22 21 20 19 18 17 16 15 14

Drops of 25 24 23 22 21 20 19 18 17 16 15 14
0.5% NaCl
Drops of D.W. 0 1 2 3 4 5 6 7 8 9 10 11

Drop of blood 1 1 1 1 1 1 1 1 1 1 1 1

% NaCl .50 .48 .46 .44 .42 .40 .38 .36 .34 .32 .30 .28

o Procedure:

 12 tubes

 Gently shake the tubes & let stand for 2 hours

 Centrifuge
 Interpret degree of hemolysis
 - % NaCl concentration = Tube # x 0.02
Example: Tube 25 x 0.02 = 0.50% NaCl

2. Modified Sanford Method

o Reduced volumes (in terms of mL)
3. Giffin and Sanford Method
4. Dacies’s Method
o Hemolysis is determined with the use of Spectrophotometer

Precaution in EOFT
1. The blood sample should be obtained w/a minimum of stasis & trauma
2. Test procedure should be set up ASAP
3. The capillary pipet must be held in approximately the same angle so as to ensure
uniform size of drops
 4. Blood should directly fall on the saline solution & not on the dry sides of the tubes

Clinical Significance:

Increased EOFT: Decreased EOFT:

(Decreased Surface Area – Volume Ratio) (Increased Surface Area – Volume Ratio)

SPHEROCYTES Sickle Cells (Drepanocytes)

- Hereditary Spherocytosis Target Cells (Codocytes/Leptocytes)
- Autoimme Spherocytosis Hypochromic Cells
- HDN (ABO) Reticulocytes
AIHA IDA
HTR Thalassemias
Senile /Old Cells
Poisoning
Burns