HEMATOLOGY (Lecture)

LABORATORY EVALUATION OF RBC

Hemoglobin Determination
Cyanmethemoglobin/ Hemoglobin
cyanide
Reference method approved by CLSI
Alkaline Drabkin solution
Potassium Ferricyanide
Potassium Cyanide
Sodium Bicarbonate- incubation
time is 10 mins
Dihydrogen Potassium
Phosphate- 3 mins
Surfactant stabilizes the
solution, avoid precipitation with
other proteins

Hemoglobin is oxidized to
methemoglobin then to
cyanmethemoglobin
Absorbed at 540nm, g/dL
SOURCES OF ERROR
1) Drabkins solution should be stored
in brown bottle in a dark place.
2) Carboxyhemoglobin converts to
cyanmethemoglobin for 1 hour.
3) Hemoconcentration
4) Calibration errors: pipettes,
instruments, cuvettes. Reduce error
20%
5) Increased absorbance
False
with high leukocyte
count (>30 x 109/L)
False
platelet count (>700 x
9
10 /L)
Hyperlipemia

Turbidity caused by Hb S and

Hb C resist hemolysis
Precipitation of abnormal
globulins
CLINICAL SIGNIFICANCE
Total red cell mass = 600gHb carry
800ml of O2
O2 tension in the lungs: 100mmHg
where 95-98% of Hb is bound to O2
O2 tension in the tissues:
<200mmHg where <30% of O2
remain in the Hb
Cyanosis
5g/dL of Hb
1.5 g/dL of methemoglobin
0.5 g.dL of SHb
Methemoglobinemia (Hereditary
type)
10-25%- no apparent
symptoms
35-50%- external dyspnea &
headaches
>70% - lethal
Carboxysulfhemoglobinemia
SulfHb binds with carbon
monoxide
Sulfhemoglobinemia
Clostridium perfringens
bacteremia
Carboxyhemoglobinemia
Symptom manifestation 20-30% HbCo
Hemolytic Anemia endogeneous Co
Smokers- elevated Hct,
polycthemia

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KLEIHAUER- BETKE METHOD /

Cellulose Acetate at pH 8.6

ACID DILUTION TEST

Hb F in fetal cells resists acid

elution
Dense staining Hb F cells vs
Hb A ghost cells to obtain
percentage
Detect presence of Hb F cells
in maternal circulation
Differentiates hereditary
persistence of Hb F from
other condition associated
with high Hb F levels.
Resists acid elution

Citrate Agar at pH 6.2

*Lightly stained- Hb A
* Darkly stained- Hb F
*Electrophoresis
- determines HbF
HEMOGLOBIN S SOLUBILITY TEST
Hb S is insoluble when
combined with reducing agent,
sodium dithionite
Crystallize & give turbid
appearance.
Follow up positive results with
Hb electrophoresis
HEMOGLOBIN ELECTROPHORESIS
Identification of normal and
abnormal Hb.
Net negative charges & buffer
pH
Hb migrate from negative
region toward positive region
1. Cellulose Acetate at pH 8.6
2. Citrate Agar at pH 6.2

MICROHEMATOCRIT
Volume of packed RBCs that
occupies a given volume of
whole blood
Packed cell volume, % or l/L
CAPILLARY Hct TUBE
7 cm long
Uniform bore about 1mm
Filled at least 5cm
Sealed at empty end
Sealed end away from the
center
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Gross Examination
PLASMA
Orange or green : Bilirubin
Pink or red :
Hemoglobinemia
Cloudy : 1 or 2 hrs after a
fatty-rich meal, nephrosis,
hyperglobinemia
SOURCES OF ERROR
1) Improper sealing of capillary
tubes
2) Increased concentration of
anticoagulant
3) Not properly mixed specimen
4) Time & speed of centrifugation
5) Buffy coat reading
6) Reading and measurement
7) Trapped plasma
8) Blood loss
9) Fluid loss in dehydration
10) Improper specimen collection
RULE OF THREE
Used for quick, visual check of
results for Hct and Hb

Hct is 3x the value of Hb 3

Normocytic, normochromic
RBC INDICES
MEAN CELL VOLUME (MCV)
Average volume of RBCs
Femtoliters (fL) or 10-15 L
80-96 fL ( 80.0 96.1)

MCV=

Hct

( )
RBC count

x 10

MEAN CELL HEMOGLOBIN (MCH)

Average weight of Hb in a RBC
Picograms (pg) or 1012g
28-33pg (27.5 33.2)

MCH=

Hb(g/dL)
RBC count

x 10

MEAN CELL Hb
CONCENTRATION (MCHC)
Concentration of Hb in each
RBC
g/ dL
33-36 g/ dL ( 33.4-35.5)
MCH=

Hb(g /dL)
Hct ( )

x 100

RBC COUNT
TOTAL RBC COUNT =
cells counted x dilution factor
area x depth
RETICULOCYTE COUNT
Assess erythropoeitic activity of the
bone marrow
Number of reticulocytes

MILLER DISC

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 2 squares :
smaller

Specimen with low Hct; low retic

falsely elevated.
Hct relate to mature RBC and
not retic.
Correction factor of 45%
1
9

the area of the larger

square
112 cells in small square
1008 cells in large square

1)
2)
3)
4)

RETICULOCYTE COUNT
RNA + NMB or BCB = dye
ribonucleoprotein complex
Dark blue network (reticulum or
strand)
At least 2 dark blue granules
SOURCES OF ERROR:
Very anemic or polycythemic patient.
Retic count = retic (infants)
Mixing of blood & stain 1:1
Moisture in the air, poor drying of
slide
Confusion with other RBC inclusions
(Howell-Jolly, Heinz)
ARC =

Reticulocyte ( ) x RBC count

100

CORRECTED RETICULOCYTE
COUNT

CRC =

Reticul ocyte ( ) x Hct ( )

45

RETICULOCYTE PRODUCTION INDEX

Shift reticulocytes
>2-4 days to lose reticula
Polychromasia in morphology
Correction is made for evaluation of
erythropoiesis.
RPI = Reticulocyte (%) x (Hct (%) 45)
Maturation time

Patients Hct Value

(%)
40-45
35-39
25-34
15-24
<15

Correction Factor
(maturation days)
1
1.5
2
2.5
3

ERYTHROCYTE SEDIMENTATION
RATE (ESR)
PLASMA FACTORS
Elevated

Fibrinogen and other globulins

(decrease zeta potential)

Cholesterol
Lowered
Albumin & Lecithin
Defribination
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RED CELL FACTORS

1) Anemia
2) Directly proportional to cell weight
3) Inversely proportional to surface
area
4) Poikilocytosis
SOURCES OF ERROR
1) Increased anticoagulant
concentration
2) Anticoagulants- sodium or
potassium oxalate
3) Tilting of tube
4) Temperature
5) Prolonged standing
6) Patients with severe anemia
7) Disorders preventing rouleaux
formation
8) Bubbles in the tube
9) Room temperature
10) Filled up to zero mark
11) Clotted specimen

1)
2)
3)

Stages of ESR
10 mins : little sedimentation
40 mins : constant rate of settling
10 mins : slow sedimentation, cell
packing
MODIFIED WESTERGREN
Taller column allows detection
of highly eleveated ESRs
4 parts blood
1 part 3.8% sodium citrate or 0.85%
sodium chloride
mm/hr
WINTROBE

Shorter column allows

increased sensitivity in
detection of mildly elevated
ESRs
Oxalate anticoagulated
blood
EDTA or citrated blood
TUBE SPECIFICATION
WESTERGREN
300mm length
200mm
graduation/
markings
5.5 0.5 mm
external bore
2.65 0.15 mm
internal bore

WINTROBE
115 mm length
100mm graduation/
markings
3mm internal bore
Top 0 to bottom 100;
ESR, red markings,
left side
Top 100 to 0 bottom,
Hct, white
markings, right side

ESR TUBE
It has anticoagulant inside
Plastic tube has stopper

INCREASED ESR
1) RA
2) Pregnancy
3) Bacterial infection
4) Malignancy
5) Tissue damage
6) MM
7) Waldestrom macroglobinemia
8) Severe anemia
DECREASED ESR
1) Polycythemia
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2) Sickle cell anemia

3) Spherocytosis
4) Poikilocytosis

1)
2)
3)
4)
5)

FALSELY INCREASED ESR

Tilted column
Hemolysis
Increase room temperature
Vibration
Refrigerated sample not returned to
room temperature
FALSELY DECREASED ESR
1) Clotted sample
2) Excess anticoagulant
3) Old blood
4) Delay testing
5) Bubbles
6) Low room temperature
7) Narrow ESR column diameter

RBC DISTRIBUTION WIDTH

(Variation in size)
Presence or degree of
anistocytosis
Significanr increased values
CV or SD
RR: 11.6% - 14.6%
**HISTOGRAM
a scatter blot to determine the
variation
RBC PARAMETER
1) RBC concentration (x 1012/L)
2) Hemoglobin (g/dL)
3) Hematocrit (% or l/L)

4)
5)
6)
7)
8)

MCV ( fL ) 10-15L
MCH (pg) 1012g
RBC Distribution Width (%)
Morphology
Reticulocyte

RBC PARAMETER INTERPRETATION

1) Hb conc: anemia
2) MCV: cell size
3) MCHC: cell Hb conc
4) RDW: anistocytosis
5) Morphologic description & correlate
with numerical values and
reticulocyte response
6) Review remaining information
(patients history)
RBC MORPHOLOGY
ANISOCYTOSIS
Variations in size of RBC
Detected by reviewing MCV

Normocyte
Biconcave disc/ discoyte
7-8m dia ; 2.5 cm thick
Clear area of 1/3 of the cell
Microcyte
<80 (fl) ; <6m
Iron deficiency anemia
Thalassemia
Other microcyte anemia

Hb = small RBC
Macrocyte

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 >8.5 9.0 m with an MCV of

>100fl
Vitamin B12 & Folate
deficiencies
Alcoholism, chemotherapy,
reticulocytosis, leukemia, etc

Appears larger than mature

cells.
Hemolytic anemia, newborns,
recovery from acute hemorrhage

Megalocyte
Largest
Red cells, 12- 21 m

VARIATION IN HEMOGLOBIN
CONTENT
Normochromic
Hb concentration in the red cell
is within reference range
Hyperchromic
Increased concentration of Hb in
red cell
Smaller central pallor
Hypochromic
Decreased concentration of
hemoglobin in the red cell
Erythrocytes have increased area
of central pallor
IDA, Thalassemia, Anemias
association with definitive
haemoglobin production.
Polychromasia
Young red cells, containing
residual RNA
Stain pinkish gray to pinkish
blue color on Wrights stained
blood smear.

Qualitative Grading of Hypochromia

Slight
Central pallor is >1/2 but 2/3
of RBC, pale outer rim of Hb
MODERATE
Central pallor is 2/3 of the cell, pale
outer rim of the cell.
MARKED
Only thin rim of pink cytoplasm
remains

ABNORMAL RBC ARRANGEMENTS

1) Rouleaux
RBC arranged in stacks of
coins or flat plates
Increased or abnormal plasma
proteins
Can be dispersed by mixing
with saline
Increased ESR
2) Agglutination
Irregular clumps / RBC from Ag-Ab
reactions
Cold agglutinins
POIKILOCYTES
Variation in shape of RBC
1) Acanthocyte
Spur cell / Thorn cell
Unevenly spaced pointed
projections

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Small spherical with irregular

thornlike
Lack central pallor
Excessive cholesterol in the
membrane
Alcoholic cirrhosis
Post splenectomy
Abetalipoprotenemia
2) Blister cell
Pyknocyte
Single/ multiple vacuoles/ markedly
thin areas at the periphery results
trauma to red cells.
-Heinz bodies
-HA
-MAHA
3) Keratocyte
Helmet cells / Bite cells
One/ several notches with projections
that looks like horns
Result from RBC on fibrin stand
4) Shistocyte
Schizocyte / Degmacyte / Fragmented
cell
Ripped to shread
Fragments of red cells with varying
shape
Resulting membrane damage

5) Echinocyte / Burr cell

Spiculated red cells with short equally
spaced
Evenly distributed projections near the
entire surface
Seen in old anticoagulated blood
6) Elliptocyte
Pencil cell / Cigar cell / Camell cell

Elongated cells with central area of

pallor and Hb at both ends
7) Knizocyte
Pinched cell
With more than 2 concavities with dark
stick of Hb in the central with pale
area on either end.
8) Leptocyte
Thin cells
Thin flat cells with Hb at the periphery
Thalassemia
9) Drepanocyte
Sickle cell / Meniscoyte
Thin, elongated cells with pointed ends
and may be curved, straight or S, V, or
L shaped
Polymerized Hb S showing crescent or
boat shape
10) Stomatocyte
Mouth cell / Cup form /
Mushroom cup
Uniconcave red cells with an
oval or slit like central pallor

11) Codocyte
Targets cells/ Mexican Hat /
Bulls eye / Platycyte / Greek
Helmet cell
Thin, bell-shaped with bulls
eye at the center
12) Dacrocyte
Teardrops
Round cells with single pointed
extremity
Pear-shaped
13) Speherocyte

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 Rounded red cells with dense Hb

content
Lack central pallor and increase staining
intensity
Smaller than a normal cell, associated by
increased with OFT
INCLUSION BODIES
1) Basophilic stippling
Punctuate basophilia, blueberry bagel
2) Cabot ring
Figure of inclusion
Blue-thread-like or reddish violet
3) Hb CC Crystal
Bar of Gold
Hexagonal crystals with
blunt ends and darkly
stained, angular crystals
4) Hb SC Crystals
Straight with parallel sides
and one blunt pointed
protruding end.

Single large dense dot near

the cell membrane

8) Pappenheimer Bodies
Iron containing bodies near
the periphery of erythrocyte
Dark staining granules in
Wrights stain

5) Hb H
Golf ball cell
Small greenish blue
inclusions of precipitated Hb
6) Howell- Jolly Bodies
Small, round bodies
composed of DNA usually
located eccentrically in the
red cells
7) Heinz Bodies
Denatured / Ppt Hb

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ANEMIA
Refers to a decrease in the normal
concentration of Hb or RBC count
Below the lower limit of the
95% reference interval for the
individual (age, sex, %
geographic location.)
*ANEMIA is NOT a disease of an
underlying disorder.
SYMPTOMS OF ANEMIA
General Signs & Symptoms of an
Anemic Individual

1) Due to decrease in O2 transport

Fatigue
Syncope
Dyspnea
Angina pectoris
Systemic organ failure
2) Due to decrease in blood volume
Pallor
Postural Hypertension
3) Due to increased cardiac output
Palpitation
Congestive heart failure

PHYSIOLOGIC RESPONSES TO
ANEMIA
1) Shift to the right & increased 2,3 DPG
2) Selective redistribution of blood flow
3) Increased cardiac output
4) Increased RBC production

MECHANISM OF ANEMIA
Effective Erythropoiesis
Red cells that are produced in the
bone marrow that reaches the
circulation and its lifespan.
Production of functional RBC
Supply peripheral circulation
with adequate number of cells
Ineffective Erythropoiesis
Red cells that are produced in
the bone marrow does not the
circulation and its lifespan.
Production of defective
erythroid precursors
Low blood hemoglobin
Decreased normal circulating
RBCs
Insufficient Erythropoeisis
Decreased erythroid precursor
in BM
Decreased RBC production
Iron deficiency
Deficiency of EPO
Loss of erythroid precursors
Suppression of erythroid
precursors
RELATIVE
Normal RBC mass
Increased plasma volume
Conditions that result to
hemodilution
*Pregnancy & volume overload
ABSOLUTE
Decreased RBC mass
Normal plasma volume

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Mechanisms involved:
Decreased delivery of RBC into
circulation
Impaired or defective
production
Failure of BM to response
(reticulocytopenia)
Increased loss of RBCs from the
circulation
*Acute bleeding, accelerated
destruction
*Respondent BM
(reticulocytosis)
ERYTHRON
Rate of production = Rate of
destruction
DISTURBANCES ON THE
ERYTHRON
1) Normal red cell destruction but
red cell production
2)
red cell destruction but
normal cell production
3)
Red cell destruction &
red cell production
4) Normal red cell destruction &
normal cell production
CLASSIFICATION OF ANEMIA
1) Decreased production of RBC
a. Marrow damage /
Marrow infiltration
b. Decreased EPO
c. Iron Deficiency
2) Nuclear Maturation

3) Cytoplasmic Naturation
4) Hemolytic
a. Acute
b. Chronic
c. Congenital
- Red cell
- Hemoglobinopathies
- Acquired
MORPHOLOGIC
CLASSIFICATION
1) Macrocytic, Normochromic
2) Microcytic, Hypochromic
3) Macrocytic, Hyperchromic
4) Microcytic, Normochromic
PHYSIOLOGIC CLASSIFICATION
1) RPI is <2.0 inches : ineffective
erythropoiesis
-Anemia is due to hypoproliferate
anemia & maturation disorder
2) RPI is >3.0 : effective
erythropoiesis
- Anemia is due to
hemolysis & blood loss
anemia
LABORATORY DIAGNOSIS OF
ANEMIA
1) Efficient use and interpretation
of laboratory measurements
2) Careful history and physical
examination
3) Determine the Hb, Hct or
erythrocyte count lies below :
- The reference intervals
for age, sex
- Patients previous values

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4) Underlying cause or mechanism

for the anemia

LABORATORY FINDINGS
Increased numbers of polychromatic
macrocytes
Increased erythropoiesis due to
hemorrhage hemolysis
History (blood loss)
Physical examination
(jaundice, splenomegaly)
Finding suggestive of hemolysis
>Poikilocytes:
Sickle cells
Target cells
Hemoglobinopathies
Hb C, Hb D, Hb E and
thalassenia
Fine basophilic stippling
Oval macrocytes and
hypersegmented
Macrocytic Anemia

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