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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
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*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
MCV=
Hct
( )
RBC count
x 10
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
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 =
CORRECTED RETICULOCYTE
COUNT
CRC =
Correction Factor
(maturation days)
1
1.5
2
2.5
3
ERYTHROCYTE SEDIMENTATION
RATE (ESR)
PLASMA FACTORS
Elevated
Cholesterol
Lowered
Albumin & Lecithin
Defribination
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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
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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1)
2)
3)
4)
5)
4)
5)
6)
7)
8)
MCV ( fL ) 10-15L
MCH (pg) 1012g
RBC Distribution Width (%)
Morphology
Reticulocyte
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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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.
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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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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
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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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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