ST.
ALEXIUS COLLEGE - MEDICAL TECHNOLOGY DEPARTMENT
DIFFERENT BLOOD CELL COUNT
A. RETICULOCYTE COUNT
B. ERYTHOCYTE SEDIMENTATION COUNT
C. MANUAL CELL COUNT
D. DIFFERENTIAL CELL COUNT
1. RETICULOCYTE COUNT
GENERAL INFORMATION
RETICULOCYTE COUNT
used to assess the erythrocyte activity of the bone marrow
more precise method for assessment is through flow cytometry
Differentiation between reticulocytes and other red cell inclusions
PAPPENHEIMER BODIES stain darker shade of blue than
reticulofilamentous material of reticulocytes
HEMOGLOBIN H undergoes denaturation in the presence of new
methylene blue, resulting in round inclusion bodies that stain
greenish blue
HEINZ BODIES are also stained supravitally but they stain a lighter
shade of blue than reiculocytes and stain well with methyl violet
RETICULOCYTE COUNT
REAGENTS, EQUIPMENTS AND PROCEDURES
SAMPLE - whole blood EDTA
STAIN - SUPRAVITAL STAIN; to detect presence of RNA, the RBC must
be stained while they are still living
a. NEW METHYLENE BLUE (produces more reliable results
than BCB)
b. BRILLIANT CRESYL BLUE
CALCULATION:
RETICULOCYTE COUNT
METHOD:
a. MILLER DISC
composed of two squares with the area of smaller square
measuring 1/9 of the large square
RBCs are counted in the smaller square and reticulocytes are
counted in the large square
minimum of 112 cells should be counted in the small
square
Calculation:
RETICULOCYTE COUNT
SOURCES OF ERROR
Blood and stain are not mixed before the smear is made
Moisture in air and or poor drying
Presence of RBC inclusions (eg. Howell Jolly Bodies, Heinz Bodies etc.)
ABSOLUTE RETICULOCYTE COUNT (ARC)
actual number of reticulocytes in 1L of whole blood
CORRECTED RETICULOCYTE COUNT (CRC)
RETICULOCYTE COUNT
RETICULOCYTE PRODUCTION INDEX (RPI)
correction done considering the presence of marrow reticulocytes
(shift reticulocytes) in the peripheral blood; seen in patients with
extremely low hematocrit.
PATIENT HEMATOCRIT (%) MATURATION TIME (Days)
40-45 1
35-39 1.5
25-34 2
15-24 2.5
<15 3
2. ERYTHROCYTE
SEDIMENTATION RATE
GENERAL INFORMATION
ERYTHROCYTE
SEDIMENTATION RATE
to detect and monitor an inflammatory response
a specific amount of blood is allowed to sit in a vertical position for a
period (1 hour)
the distance that red cell fall is reported as mm/hr
STAGES OF ESR:
1. INITIAL ROULEAUX FORMATION/LAG PHASE (10 minutes)
sedimentation rate is slight
2. PERIOD OF FAST SETTING/DECANTATION PHASE (40 minutes)
rapid and constant rate of sedimentation
3. FINAL PACKING (10 minutes) - slow rate
FACTORS AFFECTING ESR
1. ERYTHROCYTE FACTOR
a. SIZE AND MASS - the larger the particle, the faster the rate of fall
b. RBC agglutination - increases ESR
c. Altered RBC Shape - failure to form rouleaux
d. Severely decreased RBC Count - aggregation & rouleaux formation
are increased
2. PLASMA COMPOSITION
i. RBCs are negatively charged so they repel each other
a. Fibrinogen and acute phase proteins - increase ESR
b. ALBUMIN - decreases ESR
c. CHOLESTEROL - increases ESR
FACTORS AFFECTING ESR
3. MECHANICAL FACTORS
a tilt of 3 degrees can cause an increase up to 30%
increase temperature = increase ESR
length & diameter of ESR tube; ESR tube with a narrower bore =
decrease ESR
4. OTHERS
If anticoagulant concentration is increased, ESR is low
Anticoagulants sodium & potassium oxalate and heparin cause the
RBCs to shrink (increases ESR)
Bubbles in the column invalidate the result.
METHODS FOR ESR
a. WINTROBE METHOD b. MODIFIED WESTERGREN METHOD
BORE SIZE: 3mm in diameter BORE SIZE: 2.5 mm in diameter
GRADUATION: up to 100 mm GRADUATION: up to 200 mm
Anticoagulant: Double Oxalate Anticoagulant: EDTA or 0.109M Trisodium Citrate
Dilution: No Dilution Dilution:
4 parts Whole Blood, 1 part citrate
4 parts EDTA Whole Blood, 1 PART 0.85% NaCl
c. AUTOMATED ESR
NOTES ABOUT ESR
ESR should be set up within 2 hours of blood collection; if EDTA is used,
test may be set up within 6 hours
Westergren Method is the standard method
Stratified Sedimentation = poor separation of RBC and plasma layer due
to increased reticulocyte count
Sources of Error
a. Excess EDTA = falsely low ESR
b. Temperature = should be done at 20-25 degree celsius
c. more than 60 minutes = false increase
3. MANUAL CELL COUNT
GENERAL INFORMATION
MANUAL CELL COUNT
done to check on the validity of electronic methods for calibration purposes
done when counts exceed the linearity of the machine
done as a back -up method especially when instrument is non-functional
REAGENTS, EQUIPMENTS & PROCEDURES
a. HEMOCYTOMETER
measurements:
1 ruled area = 9mm2
1 WBC square (composed of 16 smaller squares) = 1mm2
1 square inside a WBC square = 0.0625 mm2
Central Square: (composed of 25 smaller squares)
1 small square inside the central square = 0.04 mm2
1 smallest square = 0.0025 mm2
MANUAL CELL COUNT
MANUAL CELL COUNT
MANUAL CELL COUNT
DIFFERENTIATION BETWEEN RBC AND WBC FOR COUNTING
RBC COUNT
DILUTING FLUIDS: HAYEM'S, GOWER'S, TOISSON'S, BETHEL'S, NORMAL
SALINE SOLUTION, 3.8% SODIUM CITRATE, FORMOL CITRATE
CHARACTERISTICS OF THE DILUTING FLUID: ISOTONIC SOLUTION
PIPETTE PROPERTIES
a. SIZE OF BULB : LARGER
b. COLOR OF THE BEAD: RED
c. VOLUME OF THE BULB: 100
d. SIZE OF THE BORE: SMALLER
e. CALIBRATION: 0.5; 1; 101
f. DILUTION: 1:200
g. DILUTION FACTOR: 200
MANUAL CELL COUNT
DIFFERENTIATION BETWEEN RBC AND WBC FOR COUNTING
WBC COUNT
DILUTING FLUIDS:
2-3% GLACIAL ACETIC ACID
1% HYDROCHLORIC ACID
TURK'S SOLUTION
CHARACTERISTICS OF THE DILUTING FLUID: ISOTONIC SOLUTION
PIPETTE PROPERTIES
a. SIZE OF BULB : SMALLER
b. COLOR OF THE BEAD: WHITE
c. VOLUME OF THE BULB: 10
d. SIZE OF THE BORE: LARGER
e. CALIBRATION: 0.5; 1; 11
f. DILUTION: 1:20
g. DILUTION FACTOR: 20
NOTES ABOUT MANUAL CELL
LEUKOCYTOSIS
COUNT
WBC COUNT >30 x 10'9/L = dilute to 1:100
WBC COUNT >300 x 10'9/L = dilute to 1:200
LEUKOPENIA
<3.0 x 10'9/L = dilute to 1:10
POLYCYTHEMIA = dilute to 1:301 (20ul WB + 6mL RBC Diluting Fluid)
SEVERE ANEMIA = dilute to 1:101 ((20ul WB + 2mL RBC Diluting Fluid)
In WBC Count, the difference between two rule areas should agree within 10%.
RODAK: Count the two ruled areas and average the number of cells before computing for the cell
count;
BROWN: Count and calculate the cell counts on each of the ruled area and average for final result
RANGE OF ERROR: WBC = 15% ; RBC = 10-20%
SOURCES OF ERROR:
a. Dust and fingerprints
b. Uneven flow of diluted blood into the chamber
Underfilled/Overfilled chamber
NOTES ABOUT MANUAL CELL
COUNT
CORRECTED WBC COUNT
done when WBC COUNT is too high due to the presence of circulating
nRBC (nucleated red blood cell)
when there are >10 nRBCs/100 WBC counted
4. DIFFERENTIAL CELL
COUNT
GENERAL INFORMATION
DIFFERENTIAL CELL COUNT
done to determine the count of each type of white blood cell present in the blood
reported as:
i. RELATIVE COUNT (%) - not very informative; denotes number of specific cell
type per 100 WBC
Relative Count = number of specific leukocyte x 100
100
ii. ABSOLUTE COUNT (x10'9/L) - very informative; denotes number of specific cell
type per liter of blood
Absolute Count = Relative count x WBC Count (x10'9/L)
DIFFENTIAL CELL COUNT
Increased ABSOLUTE COUNT occur in:
NEUTROPHIL - inflammation, bacterial infection, acute myelogenous leukemia
EOSINOPHIL - allergic reaction, scarlet fever, parasitic infection, eosinophilic
leukemia
LYMPHOCYTES - viral infections, whooping cough, infectious mononucleosis,
lymphocytic leukemia
MONOCYTES - brucellosis, tuberculosis
BASOPHIL - allergic reaction, basophilic leukemia
DIFFENTIAL CELL COUNT
AUTOMATED DIFFERENTIAL CELL COUNT
A. CELLS ARE CLASSIFIED BY SELECTIVE CYTOCHEMICAL STAIN
Example:
Technicon D/90 System - a photo-optical analysis of light scatter and absorption
determines the differential count. Used to differentiate most cell types.
PEROXIDASE Stain: stains eosinophils most intensely followed by neutrophils,
lymphocyte, blast cells, nRBCs, platelets and cellular debris are stained poorly
MONOCYTES - determined by alpha-naphthylbutyrate esterase activity
BASOPHILS - identifies by ALCIAN BLUE STAINING
B. DIGITAL IMAGE PROCESSING SYSTEM
this system uses computers to identify specific WBC types in stained smear
Example: Corning LARC (Leukocyte Automatic Recognition Computer)
DIFFENTIAL CELL COUNT
C. UNSTAINED CELLS ARE CLASSIFIED BY PHASE MICROSCOPY ON THE BASIS
OF SIZE AND REFRACTIVE INDEX
D. FLOW THRU SYSTEM
this system identifies specific WBC type in solution
Example: Coulter Counter, Hemalog D, Cytofluorograph
NEUTROPHIL COUNT
ARNETH'S COUNT - BASED ON AGE OR LOBULATION
NORMAL ARNETH'S INDEX - 60% (I + II + 1/2 III)
MATURITY CLASS NUCLEAR PERCENTAGE
CLASS I
CHARACTERISTIC
YOUNG One round or indented nucleus (blast) 5%
CLASS II Two Lobes
35%
MATURE CLASS III Three Lobes 41%
CLASS IV Four Lobes 17%
OLD
CLASS V Five or more lobes 2%
NEUTROPHIL COUNT
SHIFT TO THE LEFT - Increased in Class I and Class II
increase of IMMATURE CELLS; decrease in segmentation
Hemorrhage, Acute Infection, Leukemia, Polycythemia Vera, Pyogenic
Infection & TB
SHIFT TO THE RIGHT - Increased in Class IV and Class V
increase of MATURE CELLS, increase in segmentation
Congenital Hypersegmentation, Liver Disease, Sprue
NEUTROPHIL COUNT
SCHILLING'S COUNT - ACCORDING TO GRANULATION
CELLS % IN BLOOD SMEAR
MYELOBLAST & PROMYELOCYTE 0%
MYELOCYTE 0-1%
METAMYELOCYTE 0-1%
STAB 3-5%
SEGMENTER 51-67%
EOSINOPHIL COUNT
METHODS: uses diluting fluid containing:
Pilot's a. PROPYLENE GLYCOL & DISTILLED WATER - lyse all RBC
Randolph's b. SODIUM CARBONATE - to accelerate staining of eosinophils
Friedmann's c. ACETONE - to prevent lysis of eosinophils
d. PHLOXINE - stains eosinophil granules
Dunger's
Mannose
uses same procedure as in WBC Count except for all
Discombe's
of the 9 primary squares counted
a. Depth factor = 10
b. Dilution factor = 10
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