Hematology 1
Hematology 1
Hematology 1
Page 1 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
MANUAL METHOD OF CBC
Reports will include:
Hematocrit THREE (3) MAJOR PHASES OF HEMATOPOIESIS
s
o Decreased in both red cells and platelets due to Hemoglobin Compounds Globin Chain Composition
overcrowding of precursors as seen in the bone in Early Life Development
marrow. Embryonic Forms
Gower I 2 Zeta and 2 Epsilon
Portland 2 Zeta and 2 Gamma
Gower II 2 Alpha and 2 Epsilon
Fetal Hemoglobin 2 Alpha and 2 Gamma
Page 2 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
Adult Hemoglobin Delta chain can be temporarily acting as a
Hb A1 2 Alpha and 2 Beta substitute for the missing globin chain, most
Hb A2 2 Alpha and 2 Delta esp. for adults.
Produced before birth Consistently
produced but undetectable
CELLULARITY
s
Epsilon and Zeta Chains All cells will be concentrated in the blood islands of the
o Have the same phenomenon yolk sac for the first month AOG, which will be decreasing
o They are synthesized in the highest level at zero subsequently before the 3rd month AOG.
month, and subsequently decline on the 3rd month Cellularity peaks on the hepatic period before on the
AOG. second AOG (care of the liver).
Liver
Synthesized at zero month declines at 3rd month
o It can produce red cells even after 2 weeks after birth.
o It takes over blood cell production once yolk sac
Alpha Chain
declines.
o Starts initially to reproduce during the mesoblastic
Lymph Nodes
phase at zero month.
Bone Marrow
o Consistently increases, plateaus, but never declines.
o Before the 5th month AOG
Reason why it is detectable in adults.
Increases Plateaus Never declines Different Bones
o They are capable of storing red cells after months.
o The capacity of Tibia and Femur will be diminished
Gamma Chain
o Produced before 3rd month AOG. before age 25.
o Consistently increases, peaks, plateaus, and
declines.
o Lowest level during 6th months post-partum.
Hence, the fetal hemoglobin shouldn’t be
detected when the baby grows.
Increases Peaks Plateaus Declines
Beta Chain
o Has a dramatic presentation.
o Produced before 3rd month AOG, but not detectable.
o Has a slow progression.
o Peaks before birth, consistently increasing joining
alpha chain in its plateau; hence, in adult hgb ang
dominant is alpha 2 and beta 2.
Peaks before birth Consistently increasing
Joins alpha chain in its plateau
Delta Chain
o Produced before birth
o It is consistently produced but not in detectable levels. Before Age 1, all of the bones of the body are
haematopoietically active.
o It is there but not significant.
o However, because of retrogression at age 4 to 7,
o In Thalassemia, we see the disease according to the
there is the beginning of fat deposition in the bone
globin chain that is not present.
marrow.
Page 3 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
Ideal site for bone marrow collection
o Adult: Superior Ileac Crest (most ideal site)
o In the medulla of long bones, the majority is yellow
marrow.
Core Biopsy Trephine Jamshidi needle Cytokine Source Target Cell Effect
(a small fraction of the biopsy needle EPO Peritubular BFU-E and Stimulates
bone is obtained) Interstitial cells CFU-E proliferation of
Aspiration Aspiration
University of erythroid
(extracting red marrow needle
Illinois sternal progenitors and
from the patient) needle prevents apoptosis
BM collection is one of the most bothersome and of CFU-E.
aggressive collection. G-CSF Endothelial Neutrophil Stimulates
BM studies are done to: cells, precursors, granulocyte
1. Check the M:E ratio. placenta, fibroblasts, colonies,
2. Check for cellularity. monocytes, leukemic differentiation of
macrophages myeloblasts progenitors toward
RATIONALE – M:E Ratio Observation neutrophil lineage
Normal Infection Leukemia and maturation
2:1 to 4:1 6:1 25:1 GM-CSF T cells, Bone marrow Promotes antigen
(Average: 3:1) macrophages, progenitor presentation, T cell
M is higher than E (even if they come from same endothelial cells, dendritic homeostasis,
common progenitor), WHY? cells, cells, hematopoietic cell
o Remember: These cells come from the common fibroblasts, macrophages, growth factor
mast cells NKT cells
myeloid progenitor (CFU-GEMM) - source.
IL-2 CD4+ T cells, T cells, NK Cell
o Mas mabilis ang turnover ng other blood cells
NK cells, B cells, B cells, growth/activation of
compared to the red cell.
cells Monocytes CD4+ and CD8+ T
o The red cell has BFU-E (Burst-forming Unit)
cells, suppress
Hence, it is guaranteed that even if you have Treg responses,
individual cell precursors, they can undergo mediator of
multiple mitotic divisions to give out the need for immune tolerance.
red cells.
IL-3 Activated T Hematopoietic Proliferation of
In infections, the M cells increases because the body cells, NK cells stem cell and hematopoietic
needs it. After the infection, the ratio will return back to progenitors progenitors
normal values. IL-6 T cells, T cells Costimulation with
In Leukemia, there is a high M ratio due to unregulated macrophages, B cells other cytokines,
production. fibroblasts Liver cell
growth/activation of
NORMAL MARROW CELLS
s
T cells and B cells,
All developing hematopoietic cells megakaryocyte
Macrophages maturation, neural
Mast cells differentiation,
Osteoblasts acute phase
(commonly confused as plasma cells) reactant
Osteoclasts IL-10 CD4+, Th2 T T cells Inhibits cytokine
(commonly confused as megakaryocytes) cells, CD8+ T Macrophages production, inhibits
Mast Cells cells, macrophages
o Cells in the bone marrow, they are not blood cells. monocytes,
macrophages
o They only make use of the blood for transport medium
IL-12 Macrophages T cells T cell, Th1
to go to the tissues.
differentiation
IL-15 Activated CD4+ T cells CD4+/CD8+ T cell
CD4+ T cells CD8+ T cells proliferation
NK Cells CD8+/NK cell
cytotoxicity
IFN-α Dendritic cells, Macrophages, Antiviral, Enhances
NK cells, T NK cells MHC expression
and B cells,
-- INTENTIONALLY LEFT BLANK -- macrophages,
fibroblasts,
endothelial
cells,
osteoblasts
Page 4 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
REMEMBER:
The specific growth factor that can induce growth for such cell.
IL-3, IL-5, IL-6, IL-11, and IFN-α – board exam essentials
G-CSF – specific for associated term (granulocyte)
IL-3 – affect almost all cells
IL-15 – specific for affecting T cell maturation and development.
ERYTHROCYTES
PRODUCTION
PRECURSOR VS PROGENITOR
PRECUROSOR PROGENITOR
from CFU-GEMM, non-committed
pronormoblast is it could develop to any
developed and is now cell
the precursor cell ex. Common myeloid
committed to become a progenitor- can give out
red cell granulocyte, erythrocyte,
megakaryocyte or monocyte
CFU-GEMM
progenitor cell for all types of blood cell except for
(1st column) general rule of maturation:
lymphocyte As cells mature- decrease in size
CFU-L As the cells mature- loss of basophilia
will give out/ source of lymphocyte
Page 5 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
o Cell will initially start with a blue color. Because of Composed of DNA
maturation the blue color will change into PINK Seen in normal red cells but they are
removed by the spleen.
(2nd column) As cells mature the N:C ratio decreases So, when there are Howell-jolly bodies
Size of the nucleus decreases. in the initial cells produced, they will
There will be disappearance of the nucleus that will be circulate in the spleen and after they will
applicable for red cells only because for other cells there is be eaten by the spleen.
imposition of the unique nuclear characteristics There will be a lot of Howell-jolly bodies
seen in patient with removed spleen.
(3rd column) As the cell matures the chromatin pattern Stays in the BM for 24 hrs. before it goes to the peripheral
becomes more coarse, dense, and clumped. circulation for another day and will fully become a mature
Open/ loose chromatin appearance- euchromatin cell
Euchromatin will become heterochromatin as it goes down
1. PRONORMOBLAST/ PROERYTHROBLAST
Rubriblast- equivalent to the rubriblastic nomenclature
Earliest recognizable precursor of the red cell
Intensely blue in color
BFU-E
In basic microscopy: they appear are very dark circular cells
o The light can no longer penetrate it burst forming unit erythroid
Capable of mitotic cell division stays for 1 week
Stays in the bone marrow in that stage for approx. 24 hours.
o Pronormoblast will gather materials for globin synthesis CFU-E
1 week
2. BASOPHILIC NORMOBLAST/ PRORUBRICYTE
Another cell exclusively seen in the bone marrow for about
24 hrs.- undergoes mitotic division Pronormoblast
They are needed to be highlighted because they are the 1st 7 days
stage to begin hemoglobin synthesis
There will be a decrease in the size of the nucleus but a
Mature red cells
deeper staining/ intensity of the blue color in the cytoplasm
o What causes the very blue color? 1 pronormoblast= 8-32 red cells depending on the #
the organelles are highly active in protein of mitotic divisions
synthesis= stain more blue
Is it possible to reduce 18-21 days? Yes, especially in the
case of hypoxia
3. POLYCHROMATOPHILIC NORMOBLAST HYPOXIA
Equivalent to rubricyte Low levels of oxygen in the tissue and the body needs to act on
Polychromatic because there is an increase in hemoglobin this
synthesis that will affect the blue color of cytoplasm 3 basic methods in which the body will address the lack of
o Polychromia= increases hgb synthesis oxygen delivery:
Seen in the bone marrow for about 24 hrs. 1. Chemical response:
Last stage capable of mitotic cell division Increase in 2,3-DPG
o Shift to the right of the oxyhemoglobin
4. ORTHOCHROMIC NORMOBLAST/ METARUBRICYTE
Not capable of mitosis dissociation curve
Possesses a single color because hemoglobin synthesis is Release of oxygen content
peaking 2. Physical response
Very high hemoglobin= pink in color Selective redistribution of blood
Stage wherein it extrudes the nucleus o Priority of blood supply: vital organs (brain)
Nucleated red blood cell (ex. namumutla)
3. Hematologic response
5. POLYCHROMATOPHILIC ERYTHROCYTE
Slow but sure that it will address the lack of oxygen
Despite the pink color, there are remnants of RNA and
ribosomes that are seen in the cytoplasm
Roles of RNA and ribosomes: For the completion of HYPOXIA AND EPO
hemoglobin synthesis
Stain RNA and ribosomes supravitally= reticulocyte / a. Early release of reticulocytes
reticulum Tries to affect the receptor that are found in the
When the nucleus is extruded, will there be remaining DNA
endothelium of the trilaminar sinus of the bone
in the cell? YES
o they are known to be the HOWELL-JOLLY marrow so that they will allow the passage of
BODIES reticulocyte
Page 6 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
Reason why we see shift reticulocytes EXTRAVASCULAR HEMOLYSIS
o Shift reticulocyte- reticulocytes that did not stay in
the bone marrow for 1 week
o They should stay for 1 week in the bone marrow
before going to the peripheral circulation, but the
body needs red cell to work on oxygen delivery
METABOLIC PATHWAYS
RBC HEMOLYSIS
MICROHEMATOCRIT METHOD
A small amount of whole blood is
centrifuged to determine maximum
Page 7 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
Principle packing of erythrocytes, expressed as Limitation: Only for normocytic and normochromic RBC.
PCV or Hct
Anticoagulated Whole Blood (EDTA) LABORATORY METHODS: BLOOD CELL INDICES
Capillary blood collected in
Specimen Heparinized Capillary Tubes MCV: Mean cell volume
Requirement Specimens should be stored at room
s temperature and be processed within
6 hours after blood collection.
Note: more than 6 hours causes RC to be
macrocytic when exposed to anticoagulant for a
long period of time. = False ↑
Capillary hematocrit tubes (with red
band or plain) In automated determinations, MCV makes use of
Reagents and Non-absorbent sealing clay electronic impedance / Coulter principle.
Equipment Microhematocrit reader device With range = Normocytic
Microfuge with an RCF of 10,000 to <80: Microcytic
15,000 RPM >100: Macrocytic
Fill at least two capillary tubes MCH: Mean corpuscular hemoglobin
approximately 2/3 full. (2 HCT prep)
Seal the unfilled end.
Procedure Centrifuge.
Determine microhematocrit value
using the microhematocrit reader.
Results should agree within 0.02L/L
for duplicates. In manual determinations, Hgb is identified with the used
E.g.: 0.45 in the first HCT reading then 0.47 in of Cyanmethemoglobin principle.
the 2nd HCT reading, it is acceptable because
Within range = Normochromic
0.02 is the difference.
<26: Hypochromic
TECHNICAL ERRORS
>32: ask yourself if it is a macrocyte or spherocyte =
Excess anticoagulant (red cell shrinks) False ↓
cause increase MCH & MCHC
Insufficient mixing of blood prior to obtaining Hct
MCHC: Mean corpuscular hemoglobin concentration
sample may increase or decrease Hct False ↑
Improper sealing of capillary tube
Inadequate centrifugation or allowing tubes to stand too
long after the centrifugation. False ↑
Including a large buffy coat in the reading False ↑
Improper use of the microhematocrit reader False ↑ / ↓ g/dL
Heat sealing of capillary tubes may damage the blood
sample (bursting of RC) False ↓ Useful for the evaluation of iron therapy, anemia.
PHYSIOLOGIC ERRORS However, the more significant indices for Red cell
Trapped plasma may cause the Hct to be falsely evaluation are: MCV and MCH
increased by as much as 0.02 L/L. RDW: red cell distribution width
Note: trapped plasma causes: spherocytes, single cells, target
cells,
Certain abnormal rbc shapes (spherocytes and sickle
cells interfere with rbc packing)
During the first few hours of acute blood loss.
Dehydration increases Hct Elaborated with the use of histograms.
Note: Water comprises the majority of your plasma, wherein Ratio that determines red cell population of homogeneity
dehydration would cause increase RCs. (uniformity in size)
Specimen collection errors may alter the results. False ↑ High RDW: Anisocytosis
NORMAL VALUES
NEWBORN 0.50-0.58 OTHER INDICES
INFANTS 0.35-0.40
s
CHILDREN 0.38-0.44 VI
WOMEN 0.37-0.43 Same as MCV
MEN 0.40-0.50 Hct x 2.3
Formula: M
LABORATORY METHODS: RULE OF THREE RBC ∈ x 20
mm 3
Normal Values: 0.9 – 1.1
SI
Same as MCHC
Page 8 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
C.I . o Exiting blood vessels
Formula: o Uses integrin and selectin
V .I o Perform phagocytosis
Normal Values: 0.80 – 1.20
MCD
Prince – Jones Method (Direct Micrometry)
Normal Values: 6 – 9 u
A result >9 is considered macrocytic
MCAT
Hemoglobin content
MCV
( )
2
Formula: MCD
π
2
Normal Values: 1.7 – 2.5 u
GRANULOCYTES
NEUTROPHILS
STAGES OF NEUTROPHILS
1. Myeloblast
o Azurophilic/primary
o Quadrangle nucleus
2. Promyeloblast
o 15 – 20 azurophilic granules
3. Myelocyte
o Start of secondary/specific granule synthesis
o “Dawn of Neutrophilia” – 1st stage that shows
different color of granules
o D-shaped/circular nucleus
o Last stage capable of cell division
4. Metamyelocyte
o Juvenile cells
o Presence of indented nucleus
o Tertiary/gelatinase granules are produced
5. Band/Stab
o Increase constriction of nucleus (C-shaped)
o Synthesis of secretory vesicle
6. Segmenter/Neutrophil
o 3 - 5 lobes
o Hyposegmentation: Pelger-Huet Anomaly
NEUTROPHIL FUNCTION
o Hypersegmentation: Megaloblastic anemia Attachment
o 1 neutrophil: 16 myeloblast Includes killing and ingestion
NEUTROPHIL GRANULES
Primary Secondary Tertiary Secretory
(Azurophilic (Specific Granules Granules
Granules Granules) (Vesicles)
Myeloperoxidase B2- Gelatinase CD11b/CD18
Microglobulin
Acid B- Collagenase Lysozyme ALP
glycerophosphat
ase
Cathepsin Gelatinase Acetyltrans Vesicle-
ferase Associated
membrane 2
Defensin Lactoferrin B2- CD10, CD13,
Diapedesis Microglobu CD14, CD16
Page 9 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
lin Colorless cytoplasm
Elastase Neutrophil - Cytochrome Do NOT contain hydrolytic enzymes
Gelatinase- D558
Associated BASOPHIL FUNCTION
Protein Responds to adrenal corticosteroids
Proteinase-3 Others - C1q receptor Immediate hypersensitivity
Others - - Complement o IgE + basophil: degranulation
receptor 1 Delayed hypersensitivity reaction
o Cutaneous basophil hypersensitivity
Other Functions:
o For IgE synthesis
o Angiogenesis: repair blood vessel due to production
of mitogenic factor
o Control helminth
EOSINOPHILS
Myelocyte
o Stage first recognized
Large granules
o Blue
o Has crystalloid “Charcot Leyden crystals” BASOPHIL VS. MAST CELL
o Major Basic Protein – basic > absorb red stain Basophil is mistaken as Mast Cell because of its granular
o Color Development: Blue > olive green > deep orange appearance.
MAST cells contain:
> red o Proteolytic Enzyme
Most attractive granulocyte o Serotonin
Parasitic infection
Headphone appearance AGRANULOCYTES: Monocytes and Macrophages
Promonocyte - earliest recognizable precursor of
EOSINOPHIL FUNCTION monocyte
Degranulation
o Classical Exocytosis: ALL granules are released
o Compound Exocytosis: Granules COMBINE before
release
o Piecemeal: secretory vesicle release substance
Regulation of Immune Response
Indicator of Parasitic Infections
o ↑ eosinophils = ↑ parasitemia
Hallmark of Allergic Disorders
MONOCYTE
s
Innate Immunity
Adaptive Immunity
Housekeeping Functions
BASOPHILS - act as scavengers
Least in population -release growth factors affecting hematopoiesis
↑ = leukemia
AGRANULOCYTES: Lymphocytes
Metamyelocyte – earliest recognizable stage
Deep large basophilic
LYMPHOCYTE GROUPS
s
T and B Cells
“metachromatic” – accumulation of heparin, give color to
Natural Killer Cells
basophil
Have folded nucleus
Bilobed, large granules that obscure nucleus
Page 10 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
LYMPHOCYTE INVOLVEMENT CATEGORIES
s
PHASES OF DEVELOPMENT
s
T LYMPHOCYTE DEVELOPMENT
B LYMPHOCYTE DEVELOPMENT
CFU-L
Markers:
1. HLA (Human Leukocyte Antigen)
2. TDT (Terminal deoxynucleotidyl transferase)
Pro B
- becomes Pre B
Pre B
-earliest recognizable pre-cursor of B lymphocyte
Markers:
1. CALLA (common acute lymphoblastic leukemia
antigen) – replaced HLA
2. TDT – still present T CELL BLASTOGENESIS
The majority of lymphocytes that are circulating in the body
Immature B are T lymphocytes.
-exits the bone marrow In Differential Count, 85% of lymphocytes counted are T
-goes to the secondary lymphoid organs to perform its Lymphocytes.
function T cell becomes Reactive or Atypical Lymphocyte once it
- also known as Naïve cell - never been exposed to an undergoes blastogenesis.
antigen
-also known as Hematogones
Marker:
1. sIg (Surface Immunoglobulin)
-No CALLA and TDT Lymphocyte
Development
B CELL BLASTOGENESIS
s
NK Cells
Page 11 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
Large granular lymphocyte
NK Cells comes from T Cell.
Development
LYMPHOCYTE FUNCTION
o Volume = 3.2 mm3
T cells are quite unique because of the clusters of differentiation that Total volume of Fuchs-Rosenthal Hemocytometer: 3.2
are found on the surface. mm3 x 2 (Because it has 2 ruled areas)
CD4+ T lymphocytes
Speirs-Levy Hemocytometer
CD8+ T lymphocytes
LYMPHOCYTE FUNCTION
T Lymphocytes
NK Cells Killing of certain tumor cells and Has 4 ruled areas and each is comprised of 10
smaller squares
virally infected cells 1 square = 1x1 mm2
o Depth is 0.2
o Volume 2 mm3
Total volume: 8 mm3 (2 mm3 x 4 areas)
Glycerin 30mL
Depth of the chamber: 0.1 The diluting fluid for WBC count should be able to
Has 4 corner squares divided into 16 squares lyse the red cell, hence we use of 2% acetic acid.
Central square divided into 25 intermediate squares For the red cell, the intention is to preserve the
and each is further divided into 16 smaller squares morphology of the right cell.
o Ideal diluting fluid: NSS
GENERAL PRINCIPLES
WHY DO WE PERFORM MANUAL CELL COUNT?
Distilled Water
RBC Count
DILUTING PIPETTES
Gower’s Anhydrous sodium sulfate (12.5g)
Solution
Glacial Acetic Acid (33.3mL)
Page 13 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
● Cells touching the innermost third line on the lower
and right grid SHOULD NOT BE INCLUDED (boxed
cells)
ROUTINE COMPUTATION
Page 14 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
WBC is 30 x 10⁹ L (NV: 5-10 Lymphocyte is 50 (NV: 45),
x 10⁹ L) and segmenter is segmenter is 40, eosinophils
90% (NV: 65%). are 10, and WBC count is 6
x 10⁹ L.
Production of segmenter is
increased, thereby the total Ratio is diferent from the
WBC count also increased. normal one because of an
increase in one or more
types of WBC but not
necessarily the whole WBC
count.
TECHNIQUES
s
Page 15 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
5 or more nuclear divisions 2% Distilled Water (40 mL)
the number of segments they have. ● WBC pipette: blood to 1 mark, DF to 11 mark
● It is emphasizing that as neutrophils mature, their ● Charge hemocytometer, stand for 15 mins to allow lysis of
segments increase. cells
● If there are 5 or more segments, it is bound to undergo ● LPO; Count cells in all 9 squares
pyknosis (if it hasn’t encountered any bacteria).
EOSINOPHIL COUNT
CORRECTED WBC COUNT
THORN TEST
● Fasting patient at 8 am: Eo count is 180/cumm
● Diurnal Trend
● 20% midmorning decrease below the 8 AM level
● 30% increase at night compared to the 8AM level
BASOPHIL COUNT
COPPER AND CRUICKSHANK METHOD
● It ia performed when high levels of nucleated RBCs are ● Based on hemocytometry
observed to get WBC count in SI unit. ● Fuchs-Rosenthal or Speirs-Levy Hemocytometer
● Increased nRBCs are normally demonstrated by ● NV for adults: 0-200/cumm
newborns and geriatric patients but adult patients
should have no nRBCs.
MEGAKARYOPOIESIS AND THROMBOPOIESIS
● Corrected WBC is performed when:
○ ≥ 5 nRBCs in adults
○ > 10 nRBCs in newborns or geriatric patients
● Platelets
○ comes from CFU-GEMM which will develop into CFU-
MEG-E
● Same formula is also used for eosinophil. ○ “Kapag nag-lyse yung red cell mo sa Rees and Ecker prep
● ANC (x 10⁹ L) = WBC count in SI units x total neutrophil mo, alam mo na na wala ka na ring platelet kasi pareho
in decimal
sila ng leading(?) susceptibility.”
● Total neutrophils would includeboth segmenters and band
○ BFU-MEG to CFU-MEG undergoes mitosis
cells
○ LD-CFU-MEG
▪ beginning of terminal differentiation
EOSINOPHIL COUNT
● It is NOT routinely done. ▪ no longer performs mitosis
● Diluting Fluids: Randolph’s Solution, Pilot’s Solution ▪ undergoes endomitosis
▪ Ex. CFU-MEG undergoes mitosis producing 16 LD-
Pilot’s Solution CFU-MEG (terminal number which will become
megakaryocytes, does not undergo division
Propylene Glycol (50 mL) lyses RBCs (act as stain anymore)
transporter due to its ▪ Undergoes endomitosis
viscosity and does not ⮚ Division of the nucleus of the cell instead of
evaporate quickly)
the entire cell itself
○ Megakaryocyte
10% Aqueous Solution lyses all WBCs, othrr than
▪ Largest most mature cell in the bone marrow
Na2CO3 (1 mL) the base- resistant
eosinophils PLATELET QUANTITATION
Page 16 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
PBS: PLATELET ESTIMATION PERIPHERAL BLOOD SMEAR REPORTING
PERIPHERAL BLOOD SMEAR ● Ex. Smear shows normocytic normochromic (size and
color) erythrocytes. WBC count is the reference range
with a differential count of:
PERIPHERAL BLOOD SMEAR PREPARATION ○ Size and color: based on MCV and MCH
▪ Normocytic: size of red cell is the same with
the size of nucleus of the small lymphocyte
▪ Normochromic: only ⅓ of the cell exhibits
central pallor
○ Note for the presence of anisocytosis, variation in
size and staining
○ Presence of poikilocytes
▪ Large number: single population
⮚ Few: <5%
⮚ Moderate: 6-25%
⮚ Many: >25%
▪ Small number: more than one population [Ex.
SS (smear shows) moderately microcytic
hypochromic red cells with moderate
poikolicytosis.]
⮚ Slight: 6-10%
⮚ Moderate: 11-15%
⮚ Marked: >15%
● Ex. WBC count is above the reference range with a
differential count of:
○ Focus under HPO, obtain count in 10 fields, average
then multiply by two (Ex. WBC ct in 10 fields = 200,
200/10 (fields) = 20 x 2 = 40x109)
○ Reference range: 5-10x109
○ Differential count:
▪ Reported in percentage
● Ex. Platelets are above in number:
● More than ½ of the slide
○ Focus under OIO, obtain count in 10 fields, average
● NOT too thin or too thick
(Ex. PLT in 10 fields = 70, 70/10 (fields) = 7
● Staining should not have any irregularities
○ Decreased: <6
● Should have a feathery edge
○ Adequate: 8-20
○ When focused under OIO, locate the zone of
○ Increased: >25
morphology and write the report for PBS
● Ex. Atypical/immature cells are present/seen or
absent/not seen.
PERIPHERAL BLOOD SMEAR EVALUATION ○ Present: immature cells are included in the
differential count.
● Ex. Smear is positive/negative for malarial parasites.
o Positive: identify the genus, species, and form.
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LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
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LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB