MLS-HEMATOLOGY 1

LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

Special Hematological Examinations adipocytes and primarily an unhealthy

individual this takes up around 50% of your
Bone Marrow Exam red marrow space. So, around 30 to 70 years
old this is the statistic class, it made up of
So, in performing with bone marrow as we 50% adipocytes. So, what will happen when
know this is an invasive procedure so in an individual increases by a decade? In
doing so there are a lot of things to every ten years we can expect for an
consider; individual to have an increased 10% of your
1. A proper clinical judgement- so fatty metamorphosis. So, in every decade
usually this is decided about on your that individual ages the adipocytes increase
physician and most cases the role of as well but this only happen once you aged
your medical technologist is really beyond 70 years old
mor eon handling your specimen,
create or prepare smears, in this
way we were able to create your
hematological smears wherein this
would be the basis of the physician
to identify the hematopoietic activity
in your bone marrow.
➢ Bone marrow accounts for 3.4% to One of the purposes of bone marrow
5.95 of body weight examination;
1. Hematological or nonhematologic
reason
2. Cytopenia- we can determine the
cause of an individual scoring
cytopenia
3. Metabolic- Confirmatory test for us
to be able to exclude any other
metabolism as well as infections
All of which have to go hand by hand with of
course no just you bone marrow
examination but they have to be hand by
hand with your clinical symptoms as well as
Basically, your bone marrow this is where
you PB findings. So, bone marrow should go
your hematopoietic is found so it is inside
together with the results of those two.
your spongy trabeculae which known as
your bony tissue. Surrounding that one is a
So, there are different collection sites. What
network of sinuses so; it starts with your
are the ideal collection sites for adults and
endosteum which is the vascular layer and
for children. Let’s begin with unhealthy
then it ends collecting venules. If you can
adults, usually the most favorable sides for
notice in the picture, you can see the

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

our bone marrow collection is your, your radiograph; spinous process of your
Posterior superior iliac crest for obtaining vertebrae, ribs and other marrows
your bone marrow aspirate but mind you containing holes.
this procedure can be both for bone marrow
aspirate or biopsy because once you talk So, what are the sides effects?
about your posterior superior iliac crest in - Hemorrhage- this is associated with
your biology or physiology and anatomy we platelet functions disorder or
know that in this sites there is enough thrombocytes.
marrow but aside from that it is isolated in a
sense where in this area is far from any
possible structure that could cause damages
if there are any accidental punctures. So
other sites you can consider for bone
marrow examination is your anterior
superior iliac crest so this one could be
found in the pelvis area of the patient but
this is really more ideal if the patient is in This are the devices we used to perform your
the supine position. So other sites you can bone marrow aspiration;
consider is your sternum because if you can 1. Outer puncture canola- this is you
think about it your sternum has enough use to advance your medullary cavity
marrow but only for aspiration so it cannot of your bone.
be utilized by core biopsy. Why? Because it 2. Obliterator- to prevent bone coring.
is 1 cm in thickness. What are the dangers So, what will happen when the obturator in
when we use your sternum to collect your place, the role of physician is to remove the
bone marrow? What happen is that we try to obturator and the physician is going to
prevent bursting right through the sternum insert the core biopsy needle through the
and into your heart where in you can cannula and this will be the one to replace
damage some vessels that is why sternum is the obturator inside medullary cavity so now
the least consider site for BME but also only you will have your needle inside. So, the
for aspiration. So, for our younger patient core biopsy needle is removed from the
whose age is less than 2 years old, usually punctured needle where the specimen is in
we can utilize your anterior medial surface place so what happen is the specimen now
which is of course in your tibia however expelled using your stylet
like your sternum this only for your Bone Marrow Specimen
aspiration. Now for the spinous process of
your vertebrae ribs and other red marrow ➢ Bone marrow specimen consists of
containing bones this location can be used • Aspirate: morphologic
but these are rarely used. However, this side variance, (this can be
is good use if we’re trying to study acquired through your bone
suspicious lesions that are discovered in marrow aspiration. And to

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

identify the hematologic

cells)
• Core biopsy specimen:
demonstrate bone marrow
architecture: the spatial
relationship of hematologic
cells to fat, connective tissue,
and bony stroma and used to
estimate cellularity.
• Fixatives: Zenker fixative
(potassium, mercuric Dry tap- is when the sample that you are
chloride, sodium solfate, and able to acquire fibrotic, acellular or is
packed of leukemic cells. So, what happen
glacial acetic acid), B5
is the first and second aspiration may have
fixative (aqueous mercuric, been unsuccessful. So that’s we call as your
chloride, & sodium acetate), dry tap. In this case, to confirm the results
10% neutral formalin or the sample that we really able to get by
this will allow us to retain the fibrotic cellular bacteric cells or there just
morphological conditions of the was a failure in the procedure we can do
cells. What are the core biopsy so, our core biopsy is our
confirmatory test to really confirm if it’s a
considerations ones we are trying
dry tap or it’s really the condition of our
to use fixative such as your patients. So, the core biopsy specimen
Zenker and B5? So, because they demonstrates a fibrotic marrow or an
contain mercury, they have the antimarrow (this will be our confirmatory
potential to be toxic. So towards the previous aspirates) if there is
basically, mao lang na sya ang really low cellular activity vs. an improper
sampling. So, if our core biopsy is fibrotic or
disadvantage when it comes to
empty marrow this could really be the
using your zenker. conditions of the patient however if the
biopsy shows otherwise then it could be just
Aside from that there should be an improper sampling
controlled disposal, it means dili
sya pwede basta-basta ibutang sa
sink or anywhere else. So just
like your infectious specimen
there should be a proper
management of waste when it
comes to these types of fixative.

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

Preparation of The Bone Marrow

Specimen

➢ Direct smear

➢ Anticoagulated aspirate smears

➢ crush smears

➢ concentrate (buffy coat) smears

Hematologic Exams for Diagnosis of

➢ core biopsy imprints (touch
Anemia
preparations)

Marrow Core Biopsy and Aspirate Smear Serum Iron

Stains
➢ 50 to 160 ug/dL (9-29 umol/L)
➢ wright or wright-giemsa stains ➢ Lower in iron deficiency and in
✓ preferably with longer infection and anemia of chronic
staining time disease
➢ acidic potassium ferrocyanide
✓ Prussian blue

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

Serum (Total) Iron Binding Capacity Serum Transferrin Receptor-to-Serum

Ferritin Ratio.
➢ The reference interval for adults in
250 to 400 ug/dL (45-75 umol/L) ➢ A new approach to estimate total
➢ In IDA, the serum TIBC is increased. body iron stores
➢ It is normal or decreased in the ➢ Better utilized in identifying IDA
anemia of chronic disease coexisting with ACD
Percent saturation of TIBC Reticulocyte Hb content
➢ The ratio of serum iron to TIBC is ➢ Status of erythropoiesis in the
the percent saturation of the TIBC previous 3 to 4 days may be assessed
➢ Normally, this is 20% to 55% using reticulocytes as a guide
➢ Values below 15% indicate iron-
deficient erythropoiesis Hepcidin level
➢ Valuable tool in the study of
Serum Ferritin
abnormalities of iron metabolism,
➢ In adults, the reference values are 12 hemochromatosis, anemia of
to 300 ug/L, higher values in men inflammation, and iron deficiency
than in women Serum Cobalamin Assay
➢ A good reflection of storage iron
➢ An acute-phase reactant ➢ Usual method of detecting a
➢ Less than 50 to 60 ug/L are likely to cobalamin-deficient satte
respond to iron therapy ➢ Euglena gracilis
➢ Hypothyroidism and ascorbate ➢ 200 to 900 ng/L
deficiency ➢ <100 ng/L : megaloblastic anemia
Erythrocytes Porphyrins
Methylmalonic Acid and homocysteine
➢ +zinc = zinc protoporphyrin (ZPP) Assays
➢ Increased in iron-dificient
erythropoiesis ➢ Increase in cobalamin deficiency
Serum Transferrin Receptors ➢ Normal in folate deficiency in the
absence of cobalamin deficiency
➢ Produced by shedding of membrane
TfR’s during erythrocyte maturation Deoxyuridine Suppression Test
➢ Increase in IDA and autoimmune
hemolytic anemia ➢ Measures the ability of marrow cells
➢ Lower in aplastic anemia in vitro to utilize deoxyuridine in
DNA synthesis
➢ Tritium-labeled thymidine (3H-Tdr)

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

Serum and Red Cell Folate

➢ Lactobacillus casei
➢ Red cell folate is a better test of body
folate stores and is decreased in
megaloblastic anemia due to folate
deficiency

Erythrocytes Survival studies

➢ Uses radioactive chromium (51Cr)

→ binds to β-chains of Hb sampled
every 1-2 days for 10-14 days
Autohemolysis Test
➢ Residual activity is an index of the
intravascular life span of the labeled ➢ Sterile, defibrinated blood is
red cells incubated at 37°C for 48 hours
➢ Normal blood without glucose: 0.2%
Osmotic Fragility Test to 2.0%
➢ Normal blood with glucose: 0%-
➢ Red cells are suspended in a series of 0.9%
tubes containing hypotonic solutions ➢ Increase in HS
of NaCl, varying from 0.9% to 0.0%, Sugar water test
incubated at room temperature for 30
minutes, and centrifuged.

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

Ham’s test

Hemoglobin Electrophoresis
The Double-Landsteiner Test
➢ Hb molecules in an alkaline solution
➢ For diagnosis of PCH have a net negative charge and move
➢ 1st aliquot (control): no hemolysis towrd the anode in an electrophoretic
➢ 2nd aliquot (control: hemolysis is system
present ➢ Cellulose aceatate at alhaline pH
Dithionite Tube Test • Separates the major Hb
variants S, D, G, C, and E
➢ Solubility testing from Hb A
➢ Use in screening large numbers of ➢ Fast Hbs: Hbs K, J, bart’s, N, I, and
people for the presence of Hb S or H. Hbs A2, C, E, and O arab
other sickling Hbs ➢ Slow Hb: S, D, G, and lepore
➢ Dithionite (sodium hydrosulfite)
➢ Inorganic buffer (2.3 M phosphate
buffer)

Metabisulfite slide Test

➢ Sickling test
➢ Adding sodium metabisulfite, a
reducing substance, to blood
enhances deoxygenation of Hb and ➢ Citrate agar electrophoresis at an
sickling of Hb S acidic Ph: separate Hbs that migrate
➢ +sickle cells or “holly leaf” form of together on cellulose acetate: S from
RBCs D and G, and C from E and O

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

Acid Elution Test

➢ A relatively nonpolar solvent

Acid Elution Test
weakens the internal bonds of Hb
➢ Hemoglobin other than Hb F are and decreases its stability
eluted from the red cells on an air - ➢ An unstable Hb precipitates within
dried blood film by a citric acid- 20 minutes in the nonpolar solvent
phosphate buffer (Ph 3.3). Only Hb F isopropanol, whereas a normal
remains in the fixed red cells (stain hemolysate remains clear for 30-40
pink red by eosin), and the minutes
distribution can be determined after
Ascrobic Cyanide test
staining.
➢ Blood is incubated in a solution of
sodium cyanide and sodium
ascorbate, hydrogen peroxide is
generated from the coupled oxidation
of ascorbate and oxy hemoglobin
➢ Brown color occurs rapid in G6PD
Alkali Denaturation for Hb F
deficiency
➢ Recommended assay for Hb F in the Flouroscent Spot Test
range of 2-40%
➢ Used to differentiate fetal or neonatal ➢ whole blood is added to a mixture of
blood from maternal blood found in glucose-6-phosphate (G6P), NAPD,
a newborn’s stool or vomit, or from saponin, and buffer.
maternal vaginal blood ➢ A spot of this mixture is place on
filter paper and os observed for
fluorescence with ultraviolet light

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

➢ Lack of fluorescence indicates G6PD

deficiency
➢ In testing PK deficiency:
• Loss of fluorescence of
NADH under ultraviolet
light is observed as evidence
of the presence of PK
Antiglobulin (Coombs) Test

➢ Used to detect the presence of

antibodies against circulating red
blood cells (RBC) in the body, which
then induce hemolysis
➢ DAT and IAT

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

Automation passage of each individual cell

momentarily increases the
Principle of Automation in Hematology impedance (resistance) of the
electrical path between two
The electrical Impedance principle submerged electrodes that are
located on each side of the aperture

➢ The number of pulses generated

Coultier Principle during a specific period is
➢ Detection and measurement of proportional to the number of
changes in electrical resistance particles or cells. The Amplitude
produced by cells as they transverse (magnitude) of the electrical pulse
a small aperture produced indicates the cell’s volume.

➢ Oscilloscope: display the pulses

➢ Cells suspended in an electrically thaT are generated by the cells as
conductive diluent such as saline are they interrupt the current (# pulses
pulled through an aperture (orifice in generated = # cells)
a glass tube

➢ Volume distribution histogram:

➢ As a dilute suspension of cell is allows discrimination and counting
drawn through the aperture, the of cells of specific volumes through

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

the use of threshold circuits. (size of This hydrodynamic focusing is a way to

the voltage pulse = size (volume of prevent back flow. There is a laminar flow
the cells)) and there is a heat fluid. if you notice in the
picture, the sample stream, it is being
surrounded by your sheath fluid. As the
sample passes through your central axis of
your aperture there is a sheet of fluid
➢ Volume Thresholds: separates the surrounding it. Them it’s able to create what
cell population on the histogram, and you call as your laminar flow and this allow
the count is the cells enumerated central sample stream or linear stream of
between the lower and upper set your cell and this allow you to separate and
thresholds for each population align into single file. As they pass through
the sensing zone or what you also call as
your interrogation point.
Hematocrit Determination

e.g., of hematology analyzer

Factors may affect volume measurment

➢ Cumulative RBC pulse height
1. Aperture diameter
detection
2. Protein buildup
➢ HCT = (RBC X MCV)/10
3. Coincident passage of more than one
cell at a time through the orifice
4. Orientation and deformability of
RBC
5. Recirculation of cells back into the
sensing zone

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

information on the cells’ internal

constituents
Two-dimensional distribution scatterplot

Flow cytometry

➢ Simultaneous measurement of
multiple physical characteristics of a
single cell as the cell flows in
suspension through a measuring
device
➢ As each cell passes through the
sensing zone of the flow cell, it
scatters the focused light
➢ The number of pulses generated is
directly proportional to the number
of cells passing through the sensing
zone in a specific period.
Conductivity

➢ Determined using a high-frequency

electromagnetic probe that provides

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

➢ 90° light scatter = lobularity

➢ Depolarized 90° = resolves
eosinophils because of their large
crystalline granularity
Gating

Components of a flow cytometry

automation

➢ Sheath
➢ Detectors
➢ Focused light
➢ Fluorochromes
➢ Gating
Quadrants

VCS technology
Angels of lights scatter
➢ Volume(V): using direct current
impedance, the volume of each cell
is measured
➢ Conductivity (C): radio frequency
penetrate the cell which generates the
data points of cell size and cell
internal structure.
➢ Scatter (S): mid-angle scatter
detected by a beam of laser light
which generates data about cellular
granularity and cell surface structure
➢ 0° forward angle = cell size ➢ VCS: single channel that analyzes
➢ 10° light scatter = cell structure and approximately 8,000 cells in a near-
complexity native condition.

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

This picture is multiangle polarized scatter

(MAPS)

Analysis of instrumental data output

General characteristics
➢ Histograms
➢ Symmetrical bell-shaped or gaussian
distribution
➢ Flattened curve → SD depict the
presence of subpopulations
Histogram Gaussian distribution

➢ Frequency distribution that

approximates the distribution of
many random variables and is
portrayed graphically as symmetric
bell-shaped curved

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

Mean Platelet volum in selectec disorders AG flag

Decreased MPV ➢ Abnormal curve in front of lower

➢ Aplastic anemia discriminator
➢ Megaloblastic anemia
➢ Wiskott-aldrich anemia
➢ After chemotherapy
➢ MPV, mean platelet volume
Increased MPV
➢ Idiopathic thrombocytopenic purpura
➢ After splenectomy
➢ Sickle cell anemia WU flag

➢ deviation on upper discrimination

curve

UD flag (PU flag)

➢ abnormal height at upper

discrimination
➢ this flag appears when UD exceeds
the preset height by >40%
➢ possible causes:
• PLT clumps: clotted sample
and EDTA-incobatibility (re-
collect on citrate
• Giant platelets
• Micro RBCs

Hafsah A. Samporna
 MLS-HEMATOLOGY 1
LECTURE TOPICS
JOANNE THERESE A. CARILLO, RMT
Final 2nd Semester – A.Y. 2024-2025

An electrical impedence report would • Bubbles- due to vigorous

include: shaking

➢ CBC result
➢ Scattergram
➢ Histogram
➢ flagging

Red cells and platelets

➢ isotonic
➢ 36-360 fl → RBC
➢ 2-20 fl →
Hb and WBC

➢ Hypotonic
➢ HiCN- hemoglobin
➢ 35-90- lymphocytes
➢ 90-160- monocytes
➢ 160-450- granulocytes
Hct

➢ Computed
Instrumental errors

➢ Negative: excessive lysing of RBC

➢ Positive:
• Aperture plugs- most
common problem
• Extraneous electrical pulses-
due to improper grounding

Hafsah A. Samporna