DAVAO DOCTORS COLLEGE

MEDICAL LABORATORY SCIENCE DEPARTMENT

STUDENT NOTES: HEMA 1
LBORATORY EVALUATION OF RED BLOOD CELLS

When to perform manual counting? squares. The total number of smaller

squares in the central secondary square
1) Counts exceed the linearity of an instrument
is 400.
2) Instrument is nonfunctional (no backup)
f. As a rule, RBCs are counted on the five
3) Remote laboratories (3rd world countries)
of the tertiary squares (designated as R1,
4) Disaster situation.
R2, R3, R4, and R5) totaling to 80
I. Manual RBC/WBC Count (Hemocytometer)
smaller squares.

1) Parameters of Improved Neubauer Chamber

(Hemocytometer)
a. When viewed from the top, the
hemocytometer has two raised platforms
surrounded by depressions on three
sides which are sometimes called
“moats”.
b. The chamber depth is exactly 0.1 mm
lower than the lateral platforms. Each
of the halves of the central platform
consists of a primary square measuring
3x3 mm (9mm² ) and is subdivided into
nine secondary squares each measuring
1 x 1mm.
c. The four-corner secondary squares
(designated as W1, W2, W3, and W4) 2) Manual RBC Count
are used for WBC count and are a. RBC Pipette
subdivided into 16 tertiary squares. i. Red bead
d. The central secondary square is divided ii. Stem is the portion from 0.0 to
into 25 tertiary squares, each measuring 1.0
0.2 mm. iii. Mixing chamber is the area
e. Furthermore, each of these tertiary from 1 to 101
squares is subdivided into 16 smaller
 is directly proportional to the
hemoglobin concentration.
b. Anticoagulant: EDTA or Heparin
c. Dilution: 1:251
d. Blood Volume: 20ul
e. Reagent Volume: 5 ml
f. Wavelength: 540 nm
g. Measures: % Transmittance
b. Diluting fluid: Isotonic Saline
c. Dilution: 1:100 / 1:200
III. Microhematocrit Determination
d. Objective: 40x
e. Area Counted: 0.2 mm2 (5 small
squares of center square
 Volume of packed red blood cells that occupies
f. Formula: a given volume of whole blood
i.  Often referred to as the packed cell volume
(PCV)
 Reported either as a percentage (e.g., 36%) or in
3) Manual WBC Count liters per liter (0.36 L/L).
a. WBC Pipette a. Volume: 3 quarters full plain capillary
i. White bead tube
ii. Stem is the portion from 0.0 to b. Length of plug: 4 mm long
1.0 c. Centrifuge speed: 10,000 – 15,000 g
d. Sealant: Nonabsorbent clay
iii. Mixing chamber is the area
from 1 to 11
i. Rule of Three Method
a. Quick visual check of the results of the
hemoglobin and hematocrit.
b. Apply only to samples that have
normocytic, normochromic RBCs.
c. Hemoglobin to Hematocrit ratio of 3:1
+/- 3
d. Value discrepant indicates abnormal red
blood cells or error.
b. Diluting fluid: Hypotonic Saline
c. Dilution: 1:10 / 1:20
d. Objective: 40x IV. Red Blood Cell Indices
e. Area Counted: 4 Corners of Large
Squares
f. Formula: b. Initial classification for anemia.
i. c. To determine the average volume and
hemoglobin content and concentration of the
red blood cells in the sample.
d. Quality control check.
II. Hemoglobin Determination e. Composed of MCV, MCH, and MCHC.

i. Mean Cell Volume (MCV)

1) Cyanmethemoglobin Method b. Average volume of the red blood
a. Principle cell
i. Hemoglobin (Fe2) + K3Fe c. Expressed in femtoliters (fL), or
(CN)6  10^15L
Methemoglobin (Fe3) + KCN d. Formula:
 Cyanmethemoglobin
i.
ii. Absorbance of the
cyanmethemoglobin at 540 nm e. Reference Interval:
i. Normal = 80 to 100 fL
 ii. Microcytic = <80 fL b. RBCs are counted in the smaller square,
iii. Macrocytic = >100 fL and reticulocytes are counted in the
2) Mean Cell Hemoglobin (MCH) larger square.
a. Average weight of hemoglobin in a red
blood cell
b. Expressed in picograms (pg), or 10^12 g
c. Formula:
i.
d. Reference Interval:
i. Normal = 26 to 32 pg
3) Mean Cell Hemoglobin Concentration (MCHC)
a. Average concentration of hemoglobin in
each individual red blood cell
b. Expressed as grams per deciliter
(formerly given as a percentage):
c. Formula: c. A minimum of 112 cells should be
counted in the small square, because this
i.
is equivalent to 1008 red cells in the
d. Reference Interval: large square
i. Normal = 32 to 36 g/dL d. Formula:
i.

V. Reticulocyte Count
e. Reference Interval
i. Adult = 0.5% to 1.5%
 Assess the erythropoietic activity of the bone ii. Neonates = 2.5% to 6.5%
marrow
 Principle:
VI. Absolute Reticulocyte Count
a. Supravital stains bind, neutralize, and
cross-link RNA. These stains cause the
ribosomal and residual RNA to
coprecipitate with the few remaining a. Actual number of reticulocytes in 1 liter (L) or 1
mitochondria and ferritin masses in microliter (mL) of blood
living young erythrocytes to form b. Formula:
microscopically visible dark-blue i.
clusters and filaments (reticulum).
c. Reference Interval:
i. 20 to 115 x10^9/L
1) Reticulocyte count without Miller disc
a. Count 1000 RBCs under the oil
immersion objective lens (1000x total VII. Corrected Reticulocyte Count
magnification).
b. Formula:
i. a. Few RBCs  low hematocrit  falsely elevated
reticulocyte
b. Used of correction factor as 45%
c. Reference Interval: c. Formula:
i. Adult = 0.5% to 1.5% i.
ii. Neonates = 2.5% to 6.5%
2) Reticulocyte count with Miller disc d. Reference Interval:
a. Reduce the labor-intensive process. i. HCT of 35% = 2 to 3% reticulocyte
b. Disc is composed of two squares, with ii. HCT of <25% = 3 to 5 % reticulocyte
the area of the smaller square measuring
1/9 the area of the larger square. VIII. Reticulocyte Production Index
a. Disc is inserted into the eyepiece of the
microscope
 a. Anemia  shift reticulocytes  falsely h. Difference of the Tubes/Methods:
increased.
b. Patient’s hematocrit is used to determine the
appropriate correction factor (reticulocyte
maturation time in days):

X. Sugar Water Screening Test

c. Formula:
i. a. Principle
i. Whole blood is incubated at 37 degrees
d. Reference Interval: Celsius in a low ionic strength sugar
i. RPI >3 = adequate bone marrow water solution, which promotes binding
response of complement components, particularly
ii. RPI <2 = inadequate bone marrow C3 to the red cell surface. Normal RBCs
response don’t hemolyze, but with PNH, which is
very sensitive to complement mediated
lysis, do hemolyze.
IX. Erythrocyte Sedimentation Rate
b. Specimen:
i. Oxalate or citrated whole blood
c. Reagent:
a. Detect and monitor the course of inflammatory i. Sucrose solution
conditions such as rheumatoid arthritis, d. Interpretation;
infections, or certain malignancies. i. Positive = If patient’s supernatant
b. Nonspecific for inflammatory diseases. contains hemolysis (PNH, other
c. Increase in plasma cell myeloma, pregnancy and hemolytic states)
anemia.
d. Low sensitivity and specificity.
e. Principle: XI. Acidified Serum Lysis Test: Ham Method
i. When anticoagulated blood is allowed to
stand at room temperature undisturbed
for a period of time, the red blood cells a. Principle:
settle toward the bottom of the tube. i. Erythrocytes are incubated with fresh
ii. The ESR is the distance in millimeters and heated serum to test for hemolysis.
that the red blood cells fall in 1 hour. Weak acid is used in specific serum cell
f. Stages of ESR: mixtures to maximize hemolytic
i. Lag / Rouleaux formation – 10 min activity.
ii. Sedimentation/settling – 40 min ii. The presence of hemolysis, depending
iii. Slow sedimentation / Packing – 10 min on the test conditions, may be observed
g. Reference Interval in cases of antibody-sensitized coated
erythrocytes, spherocytes, or
paroxysmal nocturnal hemoglobinuria
(PNH).
b. Interpretation:
i. Positive = PNH, Aplastic anemia,
HEMPAS, Leukemia, Myelogenous
Leukemia
XII. Bone Marrow Examination persons.
e. Interpretation:
i. Fluorescence = Normal G6PD
a. Performed by a physician to examine the cellular ii. Partial = Mild deficiency
activities of the marrow. iii. Little or No fluorescence = G6PD
b. Presents the early developmental events that deficiency
produce the blood picture seen in peripheral
blood or evidence of an underlying systemic III. Ascorbate Cynaide Test
disease.
i. Wright-Giemsa stain usually used.
ii. Prussian Blue as special stain
iii. Cytochemical stains for enzymes. a. Principle
iv. H & E stain for histological i. Measures the ability of the cell to
examination. detoxify hydrogen peroxide when
incubated with ascorbate. Cyanide in
inhibits RBC catalase to detoxify
I. Donath-Landsteiner Screening Test hydrogen peroxide and inhibits
gluthathione peroxidase system. In
result, red cell with G6PD deficiency
a. Principle: will be oxidized and will develop
i. Used to demonstrate the presence of this methemoglobin  reaction mixture of
extremely potent hemolysin. This brown color.
antibody requires cold incubation to b. Interpretation:
exhibit hemolysis in the patient’s serum. i. Positive = brown color within 1-2 hours
A positive result is diagnostic of (EDTA) or brown color within 2-4
paroxysmal cold hemoglobinuria (PCH), hours (heparin & ACD)
the rarest form of autoimmune ii. Negative = remains/looks the same
hemolytic anemia.
b. Procedure
IV. Hemoglobin Electrophoresis: Cellulose Acetate
i. Patient’s serum is incubated with normal
group O, P-positive cells at 4 degrees Method and
Celsius.
ii. Mixture is warmed to 37 degrees
Celsius (temperature which complement 1) Cellulose Acetate Method:
is bound) and hemolysis occurs if the D- a. Simple method for detection
L antibody is present. b. Preliminary identification of both
normal and abnormal hemoglobins.
c. After, hemoglobin must be quantitated
II. Glucose-6-Phosphate Dehydrogenase Activity in by other methods. Requires
Erythrocytes: Visual Fluorescent confirmation by other methods.
d. Principle
i. A small quantity of red cell
a. Principle hemolysate is placed on the
i. The enzyme glucose-6-phosphate cellulose acetate membrane
dehydrogenase (G6PD) catalyzes the between the center and the
following reaction: cathode (negative pole) of an
ii. (NF) G6PD (F at 340 to 370nm) G6P + electrophoretic chamber. An
NADP  6-phosphogluconate + electric field is created in the
NADPH chamber through the use of a
b. The observed rate of the appearance of bright power supply and generation of
fluorescence is proportional to the blood G6PD current through a buffer at
activity. alkaline pH (8.0-8.6).
c. G6PD deficiency is one of the most prevalent ii. Hemoglobin molecules have a
hereditary erythrocyte enzyme deficiencies. net negative charge at alkaline
d. A deficiency of this enzyme can produce drug- pH and migrate on the
or stress-induced hemolytic anemia in afflicted membrane toward the anode
 (positive pole). Owing to a. Screening test for fetal hemoglobin (F)
variations in the amino acid b. Provides accurate and precise quantitation of the
content, the net charge of percentage of Hb F in blood.
different hemoglobin type c. Recommended method for quantitation in level
varies, and this determines each range of 2-40%.
hemoglobin’s rate of mobility in d. Principle
the electric field. i. Hb F resists denaturation exhibited by
e. Stain: the other hemoglobins at alkaline pH.
i. Ponceau S After a specified period, denaturation is
f. Interpretation: stopped by addition of sat. ammonium
i. Based on known migration sulfate, which lowers the pH and
patterns of various hemoglobins precipitate any denatured hemoglobin.
and by comparison of controls. The test solution is filtered, leaving Hb
2) Citrate Agar Method F that can be quantitated.
a. Routinely performed at acid pH. e. Formula:
b. Differentiates some hemoglobin variants i.
that migrate together on cellulose
acetate. f. Reference Interval:
c. Detects small amounts of Hb A or F in i. Adult = <1%
the presence of large amount of the
others VII. Isopropanol Precipitation Test
d. Principle:
i. Same as with cellulose acetate
but in different medium and pH.
a. Principle
i. Nonpolar solvents in a hemoglobin
V. Dithionite Tube Test / Solubility Test solution cause the internal bonding
forces of the hemoglobin molecule to
weaken and the molecule’s stability to
a. Screening test for the detection of sickling decrease. Thus, in 17% isopropanol
hemoglobin solution incubated at 37 degrees Celsius,
b. Inexpensive, high degree of accuracy stability of normal hemoglobin is
c. Non-specific for hemoglobin S borderline and it begins to precipitate
d. Positive also in Hb Barts, Hb C Harlem after 40 minutes. Presence of unstable
e. Principle: hemoglobin results in lesser and rapid
i. Red cells are lysed by saponin, allowing precipitation.
hemoglobin to escape. Sodium b. Interpretation:
dithionite binds and removes oxygen i. Normal = precipitate after 40 minutes
from the test environment. Hb S ii. Positive = precipitation within 5 to 20
polymerizes in the resulting minutes.
deoxygenated state and forms a
precipitate in a high-molarity phosphate VIII. Heat Denaturation Test
buffer solution. The precipitate is
consist of tactoids (liquid crystals that
refract, deflect light and make solution
turbid). a. Principle
f. Interpretation: i. Washed RBCs are hemolyzed with
i. Presence of sickling hemoglobin = water, a phosphate buffer is added, and
Turbidity the mixture is allowed to incubate at 50
ii. Normal = Clear and lines (background) degrees Celsius for 3 hours. Many
are visible unstable hemoglobin are heat sensitive
and their partial denaturation causes the
partial appearance of a flocculent
VI. Alkali Denaturation Test precipitate within 1 hour, where as
normal blood shows little.
IX. Osmotic Fragility Test iii. PK deficiency = with add ATP 0-0.8%
hemolysis

a. Principle XI. General Principles of Hematology

i. OFT reflects the shape of RBCs.
Instrumentation
Because of the decrease surface area-to-
volume ratio, spherocytes have a limited
capacity to expand in hypotonic
solutions and therefore lyse at higher 1. Electronic Impedance
concentrations of saline than normal a. The impedance principle of cell
biconcave red cells. In contrast, cells counting is based on the detection and
that are hypochromic or flatter has
measurement of changes in electrical
bigger capacity to expand thus lyse
much as higher concentration. resistance produced by cells as they
b. Procedure traverse a small aperture.
i. Suspend the red cells in a series of b. Cells suspended in an electrically
saline (NACl) solutions ranging from conductive diluent such as saline are
0.85 to 0.1% and incubating for 30 pulled through an aperture (orifice) in a
minutes at room temperature. glass tube.
Absorbance is measured at 540 nm.
c. Calculation: c. In the counting chamber, or transducer
assembly, low-frequency electrical
i.
current is applied between an external
d. Reference Interval:
electrode (suspended in the cell dilution)
i. Normal hemolysis begins at 0.45% and
completely at 0.35% and 0.30% NaCl. and an internal electrode (housed inside
the aperture tube).
d. Electrical resistance between the two
X. Autohemolysis Test electrodes, or impedance in the current,
occurs as the cells pass through the
sensing aperture, causing voltage pulses
a. To screen for PK deficiency and G6PD that are measurable.
deficiency.
2. Radiofrequency
b. Also to screen for hereditary spherocytosis
c. Not sensitive nor specific a. Low-voltage Direct Current impedance,
d. Principle as described previously, may be used
i. Measures the spontaneous lysis of red in conjunction with RF resistance, or
cells incubated at 37 degrees Celsius for resistance to a highvoltage
48 hours. During this time, depletion of electromagnetic current flowing
glucose and ATP results in membrane between both electrodes simultaneously.
loss and spherocyte formation. Glucose
b. Although the total volume of the cell is
added to the blood may protect against
autohemolysis partially or completely. proportional to the change in DC, the
e. Three patterns of hemolysis: cell interior density (e.g., nuclear
i. Type 1 (G6PD deficiency): Slightly to volume) is proportional to pulse size or
moderate increased but is partially change in the RF signal.
corrected with glucose. c. Conductivity, as measured by this high-
ii. Type 2 (PK deficiency): greatly
frequency electromagnetic probe, is
increased and glucose has no effect,
ATP corrects the hemolysis. attenuated by nucleus-to-cytoplasm
iii. Type 3 (HS): Greatly increased but can ratio, nuclear density, and cytoplasmic
corrected by either glucose or ATP. granulation.
f. Reference Interval: d. DC and RF voltage changes may be
i. Normal = 0.2-2.0% hemolysis detected simultaneously and separated
ii. G6PD deficiency = with add glucose by two different pulse processing
0.0-0.9% hemolysis
circuits.
 3. Optical Scatter detection of interference in the laser
a. Optical scatter may be used as the beam and enable enumeration and
primary methodology or in combination differentiation of cell types. Optical
with other methods. scatter may be used to study RBCs,
b. In optical scatter systems (flow WBCs, and platelets.
cytometers), a hydrodynamically e. As the cells pass through the sensing
focused sample stream is directed zone and interrupt the beam, light is
through a quartz flow cell past a scattered in all directions.
focused light source. f. Light scatter results from the interaction
c. The light source is generally a between the processes of absorption
tungstenhalogen lamp or a helium-neon diffraction (bending around corners or
laser (light amplification by stimulated the surface of a cell), refraction (bending
emission of radiation). Laser light, because of a change in speed), and
termed monochromatic light because it reflection (backward scatter of rays
is emitted at a single wavelength, caused by an obstruction).
differs from brightfield light in its
intensity, its coherence and its low
divergence or spread.
d. These characteristics allow for the

References:
Lotspeich-Steininger, C., Steine-Martin, E. A., Koepke,
J., Clinical Hematology, Principles, Procedures and
Correlations, JB Lippincott Company, East Washington
Square, Philadelphia, Pennsylvania, USA, 1992, pp 59-
106
Rodak, B., Fritsma, G., Keohane, L., Hematology,
Clinical Principles and Applications, 4th Edition, 2012,
Elsevier Saunders, Missouri, USA, pp 87-100
Denila, H., Hematology for Medical Laboratory Science,
Philippines, pp 20-26