For PH New

Hematology for PH
By SAMUEL S.(BSc, MSc)

05/31/2024 1
Learning Objectives
Upon completion of this chapter the student will be able to:

• Explain the composition of blood
• Describe the morphology and functions of the formed elements of blood
• Discuss the functions of plasma
• Define hemopoiesis and explain the process of blood cell origin and development
• Indicate the sites of hemopoiesis in infancy, childhood and adulthood
• List at least three hemopoietic growth factors
• Name the cells in the development order that will mature into erythrocytes, thrombocytes
and the five leukocytes
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Learning Objectives
• Discuss how hemopoiesis is regulated
• Describe the morphology of the red blood cell, white blood cell, and platelet precursors
• Define extramedullary hemopoiesis
• Differentiate between intramodulary and intramodulary hemopoiesis
• Define erythropoiesis
• Explain how erythropiesis is regulated and list the effects of the hormone erythropoietin on
erythropoiesis
• Define megaloblastic erythropoiesis and ineffective erythropoiesis
• Define myeloid erythroid ratio
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Outline
• Introduction of blood
• Composition of Blood
• Characteristics of Blood
• Describe the hematopoiesis.
• The regulatory mechanisms in hemopoiesis
• The sites of hemopoiesis in infancy, childhood and adulthood
• Formation of RBC, WBC and platelate

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Blood
• Blood is a constantly circulating fluid providing the body with nutrition, oxygen and
waste removal
 Is the only fluid tissue
 Constitutes 6-8% of the total body weight
• It is mostly liquid with numerous cells and protein suspended in it.
• The average person has about 5 liter of blood
• It is conducted through vessels (arteries and veins)
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Composition of Blood
 Blood is a unique fluid compromised of many
cellular elements and
 Suspended in a fluid called plasma.
• about 45% cells and 55% plasma
• The three main formed elements are:
• Red blood cells (erythrocytes)
• White blood cells (leucocytes)
• Platelets (thrombocytes)
• A plasma consisting of proteins, amino acids,
carbohydrates, lipids and elements.
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Composition cont’d
• Plasma
• Is part of the extracellular fluid
• A complex solution of proteins, salts and numerous metabolic substances
• Acts as a transport medium carrying its constituents to specialized organs of the body.
• Consists of: about 91.5% water and about 8.5% solutes of which about 7% are
proteins
• Out of the 7% protein:
• 54 % Albumin
• 38 % Globulins
• 7 % Fibrinogen
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Composition of Plasma
Constituent Percentage of plasma
Water 90-92 %
Protein 6-8%
Inorganic ions <1% (0.9%)
Organic ions <1% (0.5-0.9%)
Plasma Proteins Plasma concentration

Albumin 4.5 %
Globulin 2.5%
Fibrinogen 0.25%
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Formed Elements
• The three main blood cells/formed elements are:
• Red blood cells (erythrocytes)
• White blood cells (leucocytes)
• Platelets (thrombocytes)
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Erythrocytes (Red Blood Cells)
• Are the most numerous cells in the blood
• The normal RBC count is approximately 4.5 to 6 million cells per microliter.
• Their primary function is gas exchange.
• carry oxygen from the lungs to the tissues

• return CO2, a waste product of metabolism, from the tissues to the lungs to be exhaled.
• are anucleated cells containing few organelles.
• a large proportion of their cytoplasm consists of the iron containing oxygen transport
molecule hemoglobin.
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Erythrocytes cont’d
• Shaped like biconcave disks
• The biconcave disk shape gives RBCs the flexibility to squeeze their way through
capillaries and other small blood vessels.
• approximately 7 to 8 µm in diameter with a thickness of 1.7-2.4m
• In stained smears, RBCs look like a circle with central pallor, which is approximately
one-third the diameter of the cell.
• normally survives in the blood stream for approximately 120 days
• after finishing its life span, it is removed by the phagocytic cells of the reticuloendothelial
system,
• Broken down 05/31/2024
and some of its constituents re utilized for the formation of new cells. 11
Erythrocytes
• Note that the size of the erythrocytes is about the same as the nucleus of the
small resting lymphocyte.
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Function of Blood
• Transportation
• O2 to tissues & CO2 from tissues to lung
• Nutrients from GIT to cells
• Heat and waste products from cells for excretion
• Hormones from endocrine glands to other body cells
• Regulation
• pH
• Temperature
• Osmotic pressure (influence water and ion content of cells)
• Protection
• From bleeding (by the clotting mechanism)
• Immunity (phagocytes, lymphocytes, antibodies, complement proteins, etc)
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Developmental phases of Hematopoiesis
• During fetal development, hematopoiesis occurs in different areas of the developing fetus.
• Where as in adults, all of these processes are restricted primarily to the bone marrow.
• The developmental process is divided into three phases of hematopoiesis :
• The mesoblastic phase

• The hepatic phase, and
• The medullary phase
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Figure 2. An outline of the origin and development of erythropoiesis during embryogenesis
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Mesoblastic Phase (Yolk Sac Phase)
• Begin around the 19th day of development of embryonic fertilization.
• Progenitor cells of mesenchymal origin migrate from the AGM region of the developing
aorta-splanchnopleure to the yolk sac and give rise to HSCs.
• Mesodermal cells, which line the cavity of the yolk sac, migrate to the yolk sac and give rise
to primitive erythroblasts.
• The yolk sac hematopoiesis is characterized by the development of primitive erythroblasts
that produce measurable amounts of Hgbs:
• Portland, Gower-1, and Gower-2
• This phase occurs within a developing blood vessel and cannot contribute to definitive
• The cells surrounding the cavity of the yolk sac called angioblasts will develop into the blood
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vessels
Hepatic Phase
• Begins at 4 to 5 gestational weeks and characterized by recognizable clusters of developing
erythroblasts, granulocytes, and monocytes colonizing the fetal

• The developing erythroblasts signal the beginning of definitive hematopoiesis with a decline in
primitive hematopoiesis of the yolk sac.
• Mesodermal cells migrate to AGM region and give rise to HSCs for definitive or permanent adult
hematopoiesis, but not to primitive erythroblasts.

• Production of megakaryocytes and lymphoid cells begin to appear
• occurs extravascularly, in the liver during the second trimester of fetal life.
• Hematopoiesis in the fetal liver reaches its peak by the third month of development.
• Spleen, kidney, thymus, and lymph nodes contribute to the hematopoietic process.
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Detectable levels of HbF , HbA, and HbA2 may be present 17
Medullary (Myeloid) Phase
• During the 5th month of fetal development, hematopoiesis begins in the developing
bone marrow cavity.
• occurs in the medulla or inner part of the bone marrow.
• Mesenchymal cells migrate into the bone and differentiate into skeletal and
hematopoietic blood cells.
• Myeloid: Erythroid ratio reaches adult level of 3:1 by 21 weeks of gestation.
• Detectable levels of EPO, G-CSF, GM-CSF, HbF, Hb A2, HbA are produced.
• Throughout childhood and adult life, erythrocytes, granulocytes, monocytes, and

platelets
• 05/31/2024
continue to form from stem cells located in bone marrow.
18
• In the bone marrow,hemopoiesis occurs in the extra vascular part of the red marrow
• which consists of a fine supporting reticulum framework interspersed with vascular

channels and developing marrow cells
• When the hemopoietic marrow cells are mature and ready to circulate in the peripheral
blood,
• The cells leave the marrow parenchyma by passing through fine "windows "in the
endothelial cells andemerge into the venous sinuses joining the peripheral circulation.
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Erythropoiesis
• Begins with the development of primitive erythrocytes in the embryonic yolk sac
• RBC are derived from pluripotent HSCs
• share a common precursor (or progenitor cell ) with other myeloid lineage cells
• Megakaryocytes and erythroid cells originate from the CMEP/CFU-GEMM
• The process begins with differentiation of the CMP into the MEP intermediate.
• Progression beyond the MEP stage is associated with lineage commitment to either the
erythroid or megakaryocyte lineages.
• The MEP initially differentiates into a highly proliferative burst forming unit-
megakaryocytic or burst forming unit-erythroid (BFU-Mk or BFU-E),
• Further maturation to colony forming units (CFU-Mk or CFU-E, respectively).
• Either megakaryocyte/platelet formation or erythroid cell production.
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Erythropoiesis
• Specific transcription factors play roles in determining whether MEPs proceed down the
differentiation pathway towards erythropoiesis or megakaryocytopoiesis are:
• Fli-1 and EKLF appear to play antagonistic roles,
• With Fli-1supporting the development of BFU-Mk, and EKLF the formation of BFU-E.
• EKLF expression relies on GATA1 and CP1.

• Cells committed to the megakaryocytic lineage express CD41 and CD61 (integrin aIIb3),
CD42 (glycoprotein I) and glycoprotein V,
• von Willebrandfactor, platelet factor 4 and other platelet proteins.
• MEP maturation along the erythroid pathway occurs, they lose CD41 expression, and
• Express the transferrin receptor (CD71) at the BFU-E stage, and subsequently erythroid
membrane proteins, erythroid enzymes, and hemoglobins
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Figure 2. An outline of the origin and development of erythropoiesis during embryogenesis
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Progenitor cells Blast Forming Unit-Erythroid & Colony Forming Unit-Erythroid
• Colony Forming Unit gives rise to the earliest identifiable colony of RBC Called BFU-E
• BFU-E produces a large multi clustered colony(16 or more clusters)
•Unipotent
•Few receptors for erythropoietin (EPO)
• Develop into CFU-E colonies
• Has many receptor sites for EPO, highly sensitive
• Proliferate into pronormoblasts
• Normal red cells are produced in the bone marrow from erythroid precursors or
erythroblasts.
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Erythroid precursors
• The earliest morphologically recognizable red cell precursor is derived from
an erythroid progenitor cell
• It is derived from a multipotent haemopoietic progenitor cell.
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Erythropoiesis, (Formation/genesis of RBC)
• Erythrocyte maturation is terminal cell differentiation involving hemoglobin synthesis and
formation of a small, anucleated, biconcave corpuscle.
• Major changes that take place during erythropoiesis are:
• Cell and nuclear volumes decrease
• Nuclear: cytoplasmic ratio decreases
• The nucleoli diminish in size and disappear
• Chromatin density increases until the nucleus presents a pyknotic appearance and
• is finally extruded from the cell.
• The number of polyribosomes (Basophilia) gradually decrease,
• Mitochondria and other organelles gradually disappear.
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Pro erythroblast/ Pro normoblast/ Rubriblast
• Earliest recognizable large cell
• 1% of bone marrow cells
• Has homogeneous nucleus with nucleoli and loose lacy(fine) chromatin pattern
• Nucleus stains reddish-blue (dark appearing)
• Size: 12 -20 micrometer in diameter.
• Nucleus large round and not condensed with stippled chromatin
• Mitosis present
• Nucleoli are sometimes apparent.
• Cytoplasm: Scanty, only a rim around the nucleus; deep basophilic
• No hemoglobin (globin production begins)
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• Normal proerythroblast in the bone marrow.
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Basophilic erythroblast/ Early normoblast / prorubricyte
• Nuclear material has begun to coarsen
• Nuclear chromatin shows sharp contrast between light & dark areas
• Spherical nucleus,
• Nucleoli not visible
• Basophilic cytoplasm
• No evidence of pink color that indicate
Hgb development
• Active mitosis
• Slight reduction in size 10 -15μm
• 1-4% of BM cells
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•
Polychromatophilic erythroblast/ Intermediate Normoblast/ Rubricyte
• Cell volume is reduced (10-12m in diameter)

• Nuclear chromatin is thickened and irregularly
condensed
– Pyknotic areas
– Nucleoli no longer visible
– Nucleus is smaller and eccentric
• Decreased N:C ratio of 1:1
• Polysomes decrease
• Cytoplasm: Pink coloration mixed with Basophilia
• Due to increased Hgb appearance
• Light gray blue appearance
• Reduced mitoses
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• 10-20% BM cells
Orthochromatic erythroblast/ Late normoblast/ Metarubricyte
• Size:8-10 micrometer in diameter.
• Incapable of further DNA synthesis
• No longer mitosis
• Acidophilic erythroblast
• Nucleus:
• Nucleus more condensed/Compact
• Chromatin structure less/ condesed
• Small and pyknotic
• Central or eccentric
• At later stage nucleus will be extruded
• Cytoplasm is acidophilic (reddish -pink-
orange)
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• Presence of large quantities of Hgb
Polychromatic Erythrocyte/Reticulocyte
• Size:8-10m in diameter
• Young erythrocytes with a network of clumped ribosomes and RER.
• Nucleus has been extruded
• Has no organelles
• Is not concave
• Cytoplasm is diffusely basophilic
• Polychromatophilic
• RNA causes blue color
• Larger than mature erythrocyte
• Contains 2/3 of total hemoglobin
• Have Transferrin receptors
• 30% of total hemoglobin produced
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• Part of maturation phase occurs in BM and the later part takes phase in circulating blood
Polychromatic Erythrocyte -Reticulocyte
• Supra Vital staining required to make visible.
• When stained with New Methylene Blue are called Reticulocytes
• Ribosomes containing residual RNA
• Reticulocytes are released into the peripheral blood
• Lose RNA and matures into RBC in 1-2 days
• Makes up 1-2% of all erythrocytes
• Provide an index of rate of RBC formation
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Mature erythrocyte
• Size:6-8m in diameter
• Reddish, circular, biconcave cells filled with hemoglobin
• One third of the cell area (central pallor) due to biconcave shape
• No visible internal structure.
• What is its functional advantage?
• Does not synthesize protein

• Transports oxygen to tissue
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Factors regulate Erythropoiesis
General factors Maturation factors
• Erythropoietin • Cobalamin (Vit.B12)
• Thyroxine • Intrinsic factor
• Hemopoietic growth factors • Folic acid (VitB9)
• Vitamins. Vitamins
For Hgb Formation • Vit-C
• First class protein & amino acids • Riboflavin
• Iron, Copper • Nicotinic acid
• Cobalt & Nickel • Pyridoxin (VitB6)
Erythropoietin:
• Produced in kidney (85%) and liver (15%)
• Its action is in the bone marrow
• Regulated by the level of tissue oxygen
• The stimulus to the production of the hormone is the oxygen tension in the tissues
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Factors regulate Erythropoiesis
1.Androgens: increase erythropoiesis by stimulating EPO production but inhibited by Estrogen
2.Thyroid hormones: Stimulate the metabolism of all body cells, increasing erythropoiesis.
• Hypothyroidism is associated with anemia and hyperthyroidism results polcytemia
3.Glucocorticoids: Stimulate the general metabolism and stimulate the bone marrow to produce
more RBCs.
• In Addison’s disease (hypofunction of adrenal cortex) -anemia
• Cushing’s disease (hyperfunction of adrenal cortex) –polycythaemia
4. Pituitary gland:
• Affects erythropoiesis both directly and indirectly through the action of several hormones
5.Haematopoietic growth factors:
• Are secreted by lymphocytes, monocytes & macrophages
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• Regulate the proliferation and differentiation of progenitor stem cells to produce blood cell
State of liver & bone marrow:
1.Liver:
• Healthy liver is essential for normal erythropoiesis
• The liver is the main site for storage of vitamin B12 , folic acid, iron & copper.
• In chronic liver disease anemia occurs.
2.Bone marrow:
• When bone marrow is destroyed by ionizing irradiation or drugs, aplastic anemia occurs.
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1. What is hemopoiesis and how is the process regulated?
2. What are the hemopoietic tissues during fetal life, in infancy, in childhood and in
adulthood?
3. What are the effects of the hormone erythropoietin on red cell development and
maturation.
4. Describe the microenvironment briefly.
5. Explain megaloblastic erythropoiesis.
6. Describe general Characteristic feature of cells during maturation (nuclear ,
cytoplasmic, etc )
7. State the composition of blood.
8. State the main functions of blood.
9. List main characteristics of blood.
10. What is extramedullary hemopoiesis and when does it occur?
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Hemoglobin
• Hemoglobin is normally present in red cells only
• Two primary structures
– Globin
– Heme which is composed of
• Protoporphyrin
• Iron
• The heme structure consists of a ring of C, H and N atoms called
Protoporphyrin IX with an atom of Ferrous ( Fe 2+) iron attached
( ferroprotoporphyrin).
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Methods of Hemoglobin Measurement
•Is the measurement of concentration of Hgb in red cells (whole blood)

•Hgb is reported in g/dL
•There are different methods
(1) Spectrophotometric
a) Cyanmethemoglobin
b) Hemo-Cue
c) Oxyhemoglobin
d) Direct Read- Out
(2.)Visual comparative method
a)Sahli - Hellinge method
b)BMS Hemoglobinometr
( 3) Cu SO4 specific gravity
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I. Spectrophotometric
1. Cyanmet hemoglobin method: reference method

• EDTA whole blood or capillary samples
• Photometric semi or fully automated instruments
• Drab kin's reagent causes red cell lysis, release of hemoglobin and conversion to
cyanmethemoglobin
• Pigment is measured photo metrically at 540 nm
• Proportional to Hgb concentration
• All hemoglobin forms measured EDTA
whole
blood
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Expected Values:
• Adult males: 13-17g/dl
• Adult females: 12-16g/dl
• New borns: 14-23g/dl
• Note: reference values vary with age, sex, physiologic condition, altitude, etc.
Advantages:
• Local reference values should established.
• Stable Hemiglobin cyanide standard available to calibrate instrument
• Convenient method
• Readily available and stable standard solution (readings need not be made
immediately after dilution)
• All forms of hemoglobin except sulfhemoglobin (SHb) are readily converted to
HiCN.
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2. Hemoglobin - HemoCue®
• HemoCue® photometer
• Uses dry reagent system (cuvettes)
• Determines concentration of azide met hemoglobin photo
metrically Specimen
• Electronic check and whole blood control samples must be run to cuvette
monitor instrument function and reagent.

principle
 The hemoglobin concentration in a fresh capillary or ant
coagulated blood sample (EDTA preferred) is determined photo
metrically using a dry reagent system.
 The RBC are lysed and Hgb is converted to
azidemethemoglobin by sodium nitrite and sodium azide. Electronic
calibration red
 This method of Hgb measurement is a widely used point-of-care cuvette
test (POC).
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Hemocue Cont’d
• Advantage HemoCue® system
– No dilution necessary
– The instrument reads the result when it is ready (no need to let stand for lysing of
RBCs).
– Result is reported directly eliminating errors in reading from a calibration chart.
– High accuracy
– No expensive instrument needed

• Disadvantage:
– Test cuvettes are expensive
– Finger prick technique must be good

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3.Oxyhemoglobin Method
• The simplest and quickest method for general use with a photoelectric colorimeter but no longer
widely used.
Method:
• A 1:251 dilution of blood is made with 0.007N NH 4OH with thorough shaking to ensure mixing
and oxygenation of Hb.
• The absorbance of the solution is read at 540nm in a photo-/spectrophotometer against a 0.007N
NH4OH solution as a blank.
Disadvantage:
– Lack of a stable oxyhemoglobin standard.
– Does not measure carboxyhemoglobin, hemiglobin or sulphhemoglobin

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II. Visual comparative method
1. Sahli-Hellige
• Is not recommended because of its imprecision and inaccuracy
• Principle
• 20 L of blood is mixed in a tube containing 0.1mol/l HCl which lyses the RBC and
converts the Hgb to Acid Hematin
• After 10 minutes (more),0.1mol/l HCl or water is added drop by drop, with
mixing ,until the color of the solution matches the color of the glass standard positioned
along side the dilution tube
• The concentration of Hgb is read from the graduated scale on the dilution tube
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Sahli-Hellige cont’d
• It is not recommended method because it has the following dis advantages

• Fading of the color glass standard and
• Difficulty in matching it to the acid hematin solution.
• Conversion to acid –hematin is slow
• Acid Hematin is unstable
• Not suitable for measuring Hgb levels in infants up to 3 months.
• Interpersonal difference in reading the endpoint of dilution.
• Difficulty in matching the acid hematin with the glass standard
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2. Alkaline Hematin Method
• A useful ancillary method under special circumstances .
• It gives a true estimate of total Hb even if HbCO, Hi or SHb is present.
Disadvantage:
• Certain forms of Hb are resistant to alkali denaturation, in particular, Hb-F and Hb Bart.
• More cumbersome and less accurate than the HiCN or HbO2 methods and thus is
unsuitable for use as a routine method.
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3. BMS Hemoglobinometer
• Requires no dilution of blood

• Use in clinics with a few Hb tests are performed
Principle
• A drop of blood is mixed with a saponin impregnated stick.
• This lyses the red cells giving a clear solution of Hb
• The absorption of light by the hemolysed patient blood sample ( existing in one half of
the field of view of the meter ) is matched with a scale on the meter( that of the standard
in the other half)
• The Hb value in gm/dl is obtained from a scale on the meter
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III. Copper Sulphate Densitometry
• is a qualitative method based on the capacity of a standard solution of copper

sulphate to cause the suspension or sinking of a drop of blood.
• The measurement of specific gravity of a sample of blood corresponds to its
hemoglobin concentration.
• The method is routinely utilized in some blood banking laboratories while screening
blood donors for the presence of anemia.
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RBC Count, HGB, HCT
• Each health institution should establish its own reference ranges

• Significance
– Decreased RBC, HGB and/or HCT values….Anemia
• Decreased production, increased loss/destruction
– Increased RBC, HGB and/or HCT values….Polycythemia
• Increased production
• Critical values: HGB <7.0 or >18.5 g/dL
• Relationship: Hb x 3 = HCT + 3% ,RBC x 3 = Hb or RBC x 9 = HCT
• Used to estimate values or check data correlation
• ‘Rules’ only apply if red cells are normal in size and hgb content
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packed cell volume (PCV)
• Commonly referred to as hematocrit

• is a measure of the ratio of the volume occupied by the red cells to the volume of whole
blood in a sample of capillary or venous blood
• The ratio is measured after appropriate centrifugation in the manual methods
• Expressed as a decimal fraction (in l/l) or as a percentage (%) to the nearest 0.5%
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Hematocrit Determination cont’d
Significance of the test

• Enables the calculation of the red cell indices that are widely used in the
classification of anemias.
• These are:
– Mean cell volume (MCV),

– Mean cell Hb concentration (MCHC)
• To screen for anemia when it is not possible to measure hemoglobin.
• To diagnose polycythemia vera and to monitor its treatment.
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Hematocrit Determination
Methods
• Macro method
– Wintrobe
• Micro methods
– Adams microhematocrit method

 Electronic method
– Based on the principle that the average red cell volume is determined,
the red cell count made , and the hematocrit found by calculation e.g.
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coulter counter
Microhematocrit Method
iii. principle of test

• anticoagulated blood in a glass capillary of specified length, bore size, and
• wall-thickness is centrifuged in a micro hematocrit centrifuge at 10000-15000 rpm for 5 minutes to

obtain complete packing of the red cells
• the pcv value is read from the scale of a micro hematocrit reader or calculated by dividing the
height of the red cell column by the height of the total column of blood.
 a small amount of plasma remains trapped between the packed red cells.
iv. Specimen:
• Either well mixed EDTA ant coagulated blood or
• Capillary blood collected into a heparin zed capillary tube

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cont’d
v. Equipment
• Microhematocrit centrifuge
– Fixed speed (10000-15000 rpm)
microhematocrit centrifuge with
essential safety features which
include
• a lid interlock
• metallic casing
• Counter balanced lid and fitted with a
digital timer is required
• HCT reader
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cont’d
• Capillary tubes
– plain (blue band) or heparinized (red band) capillaries
– measuring 75 mm in length(3/4th should be filled)
– internal diameter of 1 mm and
– wall thickness of 0.2 – 0.25 mm
• Sealant
– plastic sealant, modeling clay, or plasticine.
Note: heat sealing should be avoided since it distorts the end of the tube resulting
in breakage, or the heat damages the red cells resulting in an incorrect PCV.
 Do not also use soap or any other sealing substances for sealing Hct tubes since
detergents lyse RBCs
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ix. Interpretation
• Reference range varies between age, gender and altitude
Children at birth 44-54%
Children 2-5 years 34-40%
Children 6-12 years 35-45%
Adult men 40-54%
Adult women 36-46
Ethiopia: Adult Males 41.6 – 55.1%

Adult Females 35.3 – 48.8%
• Decreased in anemia
• Increased in polycythemia
Limitation
• Plasma trapping results in higher values (1-3% higher than those obtained from
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an electronic cell analyzer)
Over view of red cell morphology
• The morphology of blood cells in stained films is the basis of laboratory

diagnosis of hematological disorders such as:
• Anemia
• Systemic diseases
• Infections
• A careful examination of a well-spread and well-stained film can be more

informative than a series of investigations
• The film should be covered with a cover glass using a neutral medium as a
mountant this step is not, however, mandatory
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Morphology of Normal Mature Red cells (Discocytes)
• In health, red cells are said to be normocytic and
normochromic
• In well spread and stained films the great majority of the

cells have:
• Round smooth contours
• Have diameters within the comparatively narrow range
of 6.0-8.0m
• A thickness of 2.5m at the periphery and 1.0m in the
center
• Normal red cell size appears to be the same as the nucleus

of a small lymphocyte
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Size Variation (Anisocytosis)
Macrocytes
• Have diameter greater than 8.0m and the MCV is increased
• Because of their increased hemoglobin content they stain

darker than discocytes
• Macrocytosis is seen in
• stress erythropoiesis as seen in hemolytic anemia
• During recovery from acute blood loss
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Size Variation (Anisocytosis)
Microcytes
• Have diameter less than 6.0m but may appear to have

normal size
• caused by flattening of the cells during smear preparation
• The mean cell volume is decreased to less than 80.0fl
• Area of central pallor usually increases because of the

coexistent hypochromia
• Are seen in iron deficiency anemia and a slight degree of

microcytosis is seen in inflammation
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Variation in Shape (Poikilocytosis)
• Acanthocytes ('spiny cells')
• Spheroidal cells with 3-12 spicules of uneven length irregularly
distributed over the cell surface.
• Seen in:
• disorders of lipid metabolism
• alcoholic liver cirrhosis and Hepatitis
Dacrocytes ('Tear drop cells')
• are tear drop or pear shaped red cells or discocytes with a single
drawn out spicule.
• It is due to overstretching of the red cell membrane in results in
loss of deformability
• Seen in:
• Myelofibrosis,
• Myeloid metaplasia
• Tumour metastases to the bone marrow
• Tuberculosis
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• Drug-induced Heinz body formation
Drepanocytes ('sickle cells')
• are crescent shaped red cells because of the
formation of rod-like polymers of Hb S or
• Have an increased surface area
• increased mechanical fragility which leads to
hemolysis and hence severe anemia
• are primarily seen in sickle cell anemia where there is

substitution of valine for glutamic acid at position 6 of
the beta chain in the hemoglobin molecule
05/31/2024 71
Echinocytes ('crenated cells')
• Red cells showing numerous, short, evenly distributed
spicules of equal length
• These are probably the most common artifacts in a blood
film:
• Consistently found in blood samples that have been
stored for some time at room temperature and
• Because of diffusion of alkaline substances from the
slide into the cells resulting in an increase in pH and
thus crenation of the cells
• In vivo they are seen in uremia, pyruvate kinase deficiency
and neonatal liver diseases
05/31/2024 72
• These are probably the most common artifacts in a blood
film:
• Consistently found in blood samples that have been
stored for some time at room temperature and
• Because of diffusion of alkaline substances from the
slide into the cells resulting in an increase in pH and
thus crenation of the cells
• In vivo they are seen in uremia, pyruvate kinase
deficiency and neonatal liver diseases
05/31/2024 73
• In vivo they are seen in uremia, pyruvate kinase
deficiency and neonatal liver diseases
Elliptocytes /ovalocytes
• Elliptical or oval shaped red cells.
• Normally less than 1% of the red cells are elliptical/oval
shaped. Elliptocytes /ovalocytes
• Found in almost all anemias where approximately 10%

of the red cells may assume elliptical/oval shape and
• in hereditary elliptocytosis where almost all the red
cells are elliptical.
05/31/2024 74
Variation in Shape cont’d
• Schistocytes ('fragmented cells')

• Two types can be distinguished:
• Small fragments of cells of varying shape, sometimes with sharp angles or spines
('spur cells'),
• sometimes round in contour, usually staining deeply but occasionally palely as a
result of loss of hemoglobin at the time of fragmentation
• Larger cells mainly with round contour from which fragments have been split off,
e.g., 'helmet cells'
• They are findings in:
• Certain genetically determined disorders, e.g., The thalassemia's ,
Hereditary elliptocytosis
• Acquired disorders of red cell formation, megaloblastic and iron
deficiency anemias
•05/31/2024
Direct thermal injury as in severe burns 75
Leptocytes ('target cells'/'Mexican hat cells')

Leptocytes ('target cells'/'Mexican hat cells')
• An area of central staining
• They are abnormally thin cells
• common findings in obstructive liver diseases
• Variable numbers are seen in iron deficiency anemia
and thalassemia
• There is gross target cell formation after
splenectomy Stomatocytes
Stomatocytes
• These are cells with a narrow slit like area of central
pallor
• They are common findings in liver diseases
associated with chronic alcohol abuse
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76
Spherocytes / Microspherocytes
• Dense staining spherical cells with smaller
diameter and greater thickness than normal
• They are formed as a result of loss of
membrane due to:
• Genetic lack of structural proteins in the red
cell membrane
• Chemicals
• Bacterial toxins (Clostridium welchii)
• Antibody-mediated hemolytic anemias
• Burn injury
• They are commonly seen in hereditary
spherocytosis that is associated with:
• abnormalities in membrane protein
• lipid loss and
• excessive flux of Na+ across the membrane
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77
Rouleaux formation
• Red cells are aligned in formations resembling stacks of coins
• May be seen as artifacts in the thick areas of the blood film
• They are often associated with:
– Hyperproteinemia
– chronic inflammatory disorders
– multiple myeloma
– macroglobulinemia
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78
Abnormalities in Red cell Hemoglobinization
• Hypochromia/Hypochromasia
• Hypochromic red cells: Hypochromia/Hypochromasia
• Contain less than the normal amount of hemoglobin

• The central pale area is increased to more than one-third of the cell diameter
• In severe hypochromia the hemoglobin appears as a thin rim at the periphery
of the cell
• The cells are usually microcytic and assume target shape
• It is a consistent finding in iron deficiency anemia, thalassemia and Polychromasia/Polychromatophilia
sideroblastic anemia.
• Polychromasia/Polychromatophilia
• As reticulocytes contain residual RNA:
• They have the affinity for the basic component of stain, and
• a degree of blue staining proportional to the amount of RNA
• An increase in reticulocytes in the peripheral blood will be seen as a
polychromatic red cell population which is also macrocytic
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79
Abnormalities in Red cell Hemoglobinization cont’d
• Dimorphism/Anisochromasia
• This is the presence of two populations of red cells, namely
hypochromic and normochromic, in the same film in
• It is a finding in:
• Treated iron deficiency anemia where there is the new
normochromic red cell population and the original
hypochromic population, and
• Patients with hypochromic anemia who have been transfused
05/31/2024
80
Red cell inclusions+
Basophilic stippling / Punctate basophilia
• The red cells contain small irregularly shaped granules with
stain blue distributed throughout the cell surface.
• It is a common finding in:
• lead poisoning
• anemias associated with disorders of hemoglobin synthesis
Howell-Jolly bodies
• Small, round inclusions that contain DNA and are usually
eccentrically located in the cell
• They stain deep purple
• Found:
• In megaloblastic anemia
• In some hemolytic anemias, and
• After splenectomy
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81
Red cell inclusions cont’d
Cabot's rings
• These are incomplete or complete rings, even figures of
'8’
• They appear as reddish - violet fine filamentous
configuration sin Wright- stained films
• They are remnants of the microtubules of the mitotic
spindle
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82
Overview of immunohematology:
• Immunohematology :
• Is more commonly known as "blood banking“
• Deals with the concepts & clinical techniques related to modern transfusion therapy
• Is the area of laboratory medicine dealing with the general procedures involved
• Collecting
• Preparing
• Storing &
• Transfusing of the blood
• Refers to immunologic reactions involving blood components
83
• An application of the principles of immunology to the study of
• Red cell antigens & Solve transfusion reaction
• Corresponding antibodies in blood
• Karl Landsteiner
• Discovered the ABO blood groups in 1900
• Introduced the immunological era of blood transfusion
• It became clear that the incompatibility of many transfusion was caused by the presence
of certain factors on red (blood) cells are known as antigens

84
Overview of immunohematology….
• Two main postulates were drawn:
• Each species of animal or human have certain factors on the red cells that are unique
to that species
• Each species have some common & some uncommon factors to each other
• This landmark event initiated the era of science based transfusion therapy
• Was the foundation of immunohematology as a science
• Blood group antibodies are classified into:
• Natural (None red blood cell)- usually IgM types &
• Immune antibodies- usually IgG types
85
• The anti-serum
• To determine a person’s blood type, some sort of substance must be available to show
what antigens are present on the red cell.
• The substance used for this purpose is referred to as anti serum
• Is highly purified solution of antibody
• Named on the basis of the antibody it contains
• For Example:
• Solution of anti-B antibodies is called anti-B antiserum
86
The ABO blood group system
87
 ABO blood grouping
1. It can be performed on a slide or in a test tube

2. Specimen: whole blood or red cell suspension (in saline, serum, or plasma)
3. Reagents: Anti-A, Anti-B, Anti-A-B (optional)
ABO phenotyping has two components:
1. Forward grouping or Direct (cell grouping)
• Testing of the red blood cells for the presence of ABO Ags or forward grouping.
2. Reverse grouping or Indirect (serum grouping)
• Testing of serum or plasma for the expected ABO Abs or reverse grouping.
88
Routine ABO phenotyping…
• The ABO phenotype is determined when the RBCs are directly tested for the
presence or absence of either A or B antigen.
• Serum testing provides a control for red blood cells testing, since ABO
antibodies would reflect Landsteiner’s rule.
Principle
• When an antigen is mixed with its corresponding antibody under the right conditions it
causes agglutination or haemolysis of the red cells.
Clinical significance
• For safe blood transfusion
• Prevention of Hemolytic Disease of the Fetus and Newborn (HDFN
• For prevention of hemolytic transfusion reaction
89
The methods for grouping
1. Tube method
2. Emergency tile or slide grouping
3. Tile or slide grouping of blood donors
Other methods of Detection of Ag-Ab Reaction
A. Gel Technology method
B. Micro plate testing methods
90
1.Tube grouping method

Specimens
-Patient’s serum .
-Patient’s cells.
Equipment's and reagents
– Anti- A serum and anti- B serum
– Antigen A and antigen B ( Red cell suspension)
– test tube
– Centrifuge
– wash bottles
– normal saline
91
Tube grouping method….
Method
1. Take 4 tubes and label them 1-4.
2. Place each of the following in its numbered tube.
Tube 1: 1 volume of anti- A sera.
1 volume of 3-5% suspension of patient’s washed cells.
Tube 2: 1 volume of anti- B serum.
1 volume of 3-5% suspension of patient’s washed cells.
Tube 3: 1 volume of patient’s serum.
1 volume of 3-5% suspension of washed RBC (reagent RBC) containing antigen A
92
Tube grouping method….
Tube 4: 1 volume of patient’s serum.
1 volume of 3-5% suspension of washed RBC (reagent RBC) containing antigen B.
3.Mix the contents of each by gently taping the tube with the finger and leave for five
minutes at room temp
4. Centrifuge the tube for one minute at 1000 RPM for one minute.
5. Replace the tube in the rack in the same position as before centrifugation, and
read the result by gently tapping each tube, looking for agglutination or heamolysis.
6. Check the tube which shows no visible agglutination by examining the contents
microscopically on a slide using a 10x objective.
93
7. Decide what a blood group of a patient is and record the result in the book provided and one the
patient’s form.
Controls
Anti- A and Anti- B should be controlled using known A (preferably – A2) cells and B cells.
ABO phenotyping Reaction
Phenotype RBC reaction Serum or plasma reaction
Anti-A Anti-B A-cells B-cells
A + O O +
B O + + O
AB + + O O
O O O + +
94
+= agglutination, O= no agglutination
Interpretation of tube method
1. Either hemolysis or agglutination in tests with serum constitute positive test

results
2. A smooth cell suspension after re-suspension of the cell button is a negative test
result
3. Any discrepancy between the results of the tests with serum/plasma & cells
should be resolved before an interpretation is recorded for the patient’s or
donor’s ABO group
95
2. Emergency tile or slide grouping
• Emergency tile grouping is carried out using a white glassed porcelain tile,
Perspex tray or slides.
Specimen
• Patient’s serum
• Patient’s cells
Reagents and equipments:

• Anti- A and anti-B serum
• Group O serum (anti-AB)
• Antigen A and antigen B (20% known RBC suspension)
• Group AB serum( Control)
• Tile or slide
96
Method
1.Divide and mark a white tile as follows
a b c d e f
Anti- A Anti- B Anti- AB A - cell B- cell Control
Then add the following

a. 1 volume of anti- A serum
1 volume of 20% suspension of patient cell
b. 1 volume of anti- B serum

C.1 volume of group O serum

97
d. 1 volume of patient serum
1 volume of 20% suspension of A cell
e. 1 volume of patient serum
1 volume of 20% suspension of B cell
f. control
1 volume of group AB serum
1 volume of 20% suspension of patient washed cells (there should not be agglutination
here).
3. Mix the contents of each division, using a small clean applicator stick for each.
4. After 2-3 minutes read the results and decide the blood group.
98
Illustration of the forward and reverse grouping reaction patterns of the ABO groups
99
3. Tile or slide grouping of blood donors
• This is a rapid method used to group donors , using a white tile or two glass slides
• It consist only cell grouping using anti-A serum and anti-B serum .
Specimen
• Whole blood
Reagents and equipments:

• Anti- A and anti-B serum
• Group O serum (anti-AB)
• Tile or slide
• Applicator stick
100
Method
1. Take two slides or a white tile and mark as follows
anti-A anti-B anti-AB
2. Place in to each marked area (anti-A, anti-B, anti-AB) one drop of

donor capillary blood.
3. In to the division marked
- Anti-A, place one drop of anti-A serum,
- Anti-B, place one drop of anti-B serum,
101
6. Gently tilt the slide continuously for up to 2 minutes
7. Do not place the slide over a heated surface during this period
8. Read, interpret & record the results of the reactions on all slides
• Agglutination-indicates presence of antigen
• Interpretation
1. Strong agglutination of red cells in the presence of any ABO typing reagent
constitutes a positive result
2. A smooth suspension of red cells at the end of 2 minutes is a negative result
3. Samples that give weak or doubtful reactions
 Should be retested using test tube method
102
ABO discrepancies
• Is a condition at which the ABO test result of forward is varied from
reverse result
• May be indicated by the following observations
• Weaker reactions
• Missing reactions
• Extra reactions
Can be technical or sample –related problems
Technical Error
• Identification or documentation error.
• Reagent or equipment errors.
• Standard operating procedure errors
103
Sample- Related ABO discrepancies
• ABO discrepancies that affect the ABO red blood cell testing.
• Extra antigens present

• Missing antigens
• Mixed field reaction
• ABO discrepancies that affect the ABO serum testing.

• Presence of additional antibodies other than anti–A and anti-B or
• The absence of expected antibody reactions
104
Rh blood group system
1. D antigen is the most immunogenic antigen in the Rh system
2. More than 80% of D-negative people receiving a D-positive red blood cell
transfusion produce an antibody with anti-D specificity
3. For that reason, a D-negative patient should receive D-negative red blood cells
4. Most RBC can be typed for the D Ag directly with anti-D reagents
5. When the D-Ag is weakly expressed on the RBC
 Detected by the indirect antiglobulin test (IAT)
6. Red blood cells that are positive for D only by the IAT are referred to as weak D (D u)
105
Rh blood group system
• Rh typing
1. It can be performed on a slide or in a test tube
2. Specimen: whole blood or red cell suspension (in saline, serum, or plasma)
3. Reagents: Anti-D, Rh control reagent
106
Slide method
1. Place 1 drop of anti-D onto a clean, labeled slide
2. Place 1 drop of the appropriate control reagent, if needed, onto a second labeled slide
3. To each slide, add 2 drops of a well mixed 40% to 50% suspension of the red cells to be
tested
4. Thoroughly mix the cell suspension & reagent using a clean applicator stick for each
test, spread it over an area approximately 20 × 40 mm.
5. Tilt the slides gently & continuously observe them for agglutination for not more than 2
minutes
107
Interpretation slide method----
1. Agglutination in the anti-D slide, combined with a smooth suspension in the control
tube
• Indicates that the red cells under investigation are D+
2. A smooth suspension of red cells in both slides
• Indicates negative test result
3. Donor blood must be further tested for the presence of weak D antigen
108
Test tube method
1. Place 1 drop of anti-D in a clean, labeled test tube
2. Place 1 drop of control reagent, if needed, in a second labeled tube
3. Add to each tube 1 drop of a 2-5% suspension of the red cells to be tested
4. Mix gently & centrifuge for the time & speed specified by the manufacturer
5. Gently re-suspend the cell button & examine it for agglutination
6. Record test & control results
• Agglutination with anti-D & a smooth suspension on the control tube constitute a
positive test result indicates Cells being tested are D+
• No agglutination with either anti-D or the Rh control indicates Cells are D–
• If there is agglutination on the negative control slide
• Results of the anti- D test must not be interpreted as positive without further
testing 109
Test for Weak D
1. Some red cells express the D antigen so weakly
• Most anti-D reagents do not directly agglutinate the cells
2. Weak D expression can be recognized most reliably by an indirect antiglobulin
procedure after incubation of the test red cells with anti-D
• Specimen:
• Whole blood (in saline, serum, or plasma)

• red cell suspension
• Reagents:
• Anti-D, Anti-human globulin reagent, IgG coated RBCs, Physiological saline
110
The Red Cell Indices
• Red blood cell indices are measurements that describe the size and Hgb content of RBCs.
• They are also called red cell absolute values or erythrocyte indices.
• The indices are used to help in the differential diagnosis and classification of anemia.
• Red cell distribution width (RDW) is important red cell parameter obtained by electronic
methods
• They are absolute values calculated from: hemoglobin, PCV and RBC count
• Red cell indices include

• Mean corpuscular volume (MCV = HCT/RBC)
• Mean corpuscular hemoglobin (MCH = Hgb/RBC)
• Mean corpuscular hemoglobin concentration (MCHC = Hgb/HCT)
• RDW measures the variation in size of the red blood cells (degree of anisocytosis) 111
Anemia
• Anemia is defined as a reduction in the concentration of circulating Hgb below the level
• that is expected for healthy persons of same age and sex in the same environment.
• Results in impaired oxygen delivery to tissues (hypoxia)
• Anemia is not a diagnosis, but a sign of underlying disease.
• Hence, the evaluation of a patient with anemia is directed at elucidating the causes for the
patient’s decreased red blood cell mass.
• Clinical diagnosis of anemia is made by:
– Patient history
– Physical exam
– Signs and symptoms
– Laboratory findings
05/31/2024 112
Clinical presentation of anemias
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113
Physiologic response to Anemia
• Ability to adapt to anemia depends on:
– Age and cardio/pulmonary function
– Rate at which anemia develops (BM can compensate easier if the onset of anemia is
slow),
– Underlying disease
• Symptoms of hypoxia: decreased oxygen delivery to the tissues/organs causes:
– fatigue, faintness, weakness, dizziness, headaches, dyspnea, poor exercise tolerance,

leg cramps.
• Anemia can be classified based on pathophysiology ,etiology and morphology

114
Methods of Anemia Classification
Several schemes of classifying anemias exist
1. Pathophysiologic
• Anemia is divided using three main causes/mechanisms
I. Impaired erythrocyte formation (Aplastic anemia, IDA, sideroblastic anemia,
II. Increased destruction of RBCs (hemolytic anemias)
III. Increased loss
2. Morphologic
• Based on RBC morphology
• Anemia is divided into three groups mainly on the basis of the MCV
(RBC indices)
– Microcytic hypochromic anemia
– macrocytic anemia
05/31/2024 115
– Normocytic normochromic anemia
Morphologic Categories of Anemia
• a morphologic classification is based on how the cells appear on a

stained smear and should correspond with the red cell indices.
– Normocytic Normochromic (NCNC): anemia due to decrease in the
number of erythrocytes
(e.g. aplastic anemia, or acute blood loss)

– Microcytic hypochromic
• Eg .iron defiency anemia, sideroblastic anemia, thalesemia and ACD
– Macrocytic Normochromic, Eg. Megaloblastic and non megaloblastic

116
Iron Deficiency Anemia (IDA)
• Is a condition in which the total body iron content is decreased below a normal level
• This results in a reduced red blood cell and hemoglobin production
• Major Causes:
– Nutritional deficiency
– Malabsorption (insufficient or defective absorption)
– Increased need
– Chronic blood loss (GI bleeding, ulcer, heavy menstruation)
• Sequence of iron depletion
– Storage iron deplation;1st; Serum Iron depletion ;2nd; cell morphology ;3rd
• Storage iron depletion/ low serum ferritin; serum iron & TIBC are normal, no anemia, .
• Serum iron depletion/TIBC increases (increased transferrin); no anemia, normal red
cells.
117
• Anemia with microcytic/hypochromic red cells = IDA
• Daily Iron Cycle
118
Iron absorption, Transport and storage
• Iron absorbed from duodenum and jejunum in the GI Tract, moves via circulation to the bone ,
• where it is incorporated with protoporphyrin in mitochondria of the erythroid precursor to

make Heme
• Three proteins are important for transporting and storage of iron
– Transferrin: transports iron from the plasma to the erythroblasts in the marrow
for erythropoiesis
– Transferrin will bind to Transferrin receptor on the erythroblast membrane
– Excess iron is stored as Ferritin and hemosiderin in the reticuloendothelial

system
119
Lab Investigation of microcytic hypochromic anemia
• Iron tests
– Used to differentiate microcytic hypochromic anemias or detect iron
overload (repeat transfusion or hemochromatosis)
– Iron circulates bound to the transport protein transferrin which carries iron
to bone marrow red cells for Hgb synthesis or to tissue sites (liver, spleen,
bone marrow) for storage as ferritin
120
Lab Investigation cont’d
1. Serum iron level - measures the amount of iron bound to transferrin; normally
about 33% of transferrin binding sites are occupied with iron (% saturation).
2. Total iron binding capacity (TIBC)

– measures the total amount of transferrin can bind when fully saturated
– Is an indirect measure of the amount of transferrin protein.
– Inversely proportional to the serum iron level
3. Serum ferritin level - indirectly reflects storage iron in tissues without doing a
bone marrow or tissue biopsy.
121
Lab Investigation cont’d
• Serum transferrin receptors (sTfR) reflect the number of receptors in
immature red blood cells and thus the level of erythropoiesis.
• It is unaffected by infection or inflammation .
• Erythrocyte protoporphyrin: protoporphyrin combines with iron to form heme.

• The lack of iron determines an increase in erythrocyte protoporphyrin, which cannot
combine with Iron.
• Transferrin levels are regulated by iron availability
– ↑ synthesis of Transferrin in iron deficient states…↓ serum iron, ↑ TIBC
– ↓ synthesis of Transferrin in iron overload states…↑ serum iron, ↓ TIBC
– ↓ synthesis of Transferrin in inflammatory states or malignancy …↓ serum
iron, ↓ TIBC 122
Blood findings in untreated IDA:
1. Mild to severe anemia (11 to 5 g/dl Hgb); microcytic/hypochromic RBCs.
2. Low RBC indices (MCV 50-80 fl; MCH < 27 pg; MCHC <32%).
3. Aniso with high RDW; ovalocytes /pencil cells, may see target cells, normal or
elevated platelet count.
4. No basophilic stippling - if present, rules out IDA and helps differentiate IDA
from thalassemia minor.
5. No pappenheimer bodies (composed of iron) are seen in IDA.
6. Low retic count – red cell production is restricted by lack of iron.
7. Low serum iron, high TIBC, low serum ferritin (stores), low % saturation of
transferrin. *Increased transferrin synthesis occurs in iron deficient states.
123
Iron Deficiency Anemia cont’d
• RBC morphology
– Hypochromia
– Microcytosis
– Anisocytosis
– Poikilocytosis
• Pencil cells (cigar cells)
• Target cells
– no RBC inclusions Blood smear
• Iron parameters
– Low serum iron,
– High TIBC,
– Low serum ferritin
• Bone marrow (not usually performed)
– No stainable iron
– Increased NRBC
– Erythroid hyperplasia with decreased M:E ratio 124
Typical peripheral blood values in patients with IDA
TEST MILD IDA MODERATE IDA SEVERE IDA
Hemoglobin >11 g/dL Or in normal range >10 g.dL < 10 g/dL
Hematocrit >36% Or in normal range < 30 % < 30%
RBC count May be within normal range May be within normal range Decreased
MCV May be within normal range Microcytic; < 80 fL Microcytic; < 80

fL
MCH May be within normal range < 27 pg < 27 pg
MCHC May be within normal range Normochromic to hypochromic Hypochromic
RDW Slightly elevated Frankly elevated Frankly elevated
125
Sideroblastic Anemia (SA)
• This group of anemias are characterized by defective protoporphyrin synthesis
(blocks) resulting in iron loading and
• a microcytic/hypochromic anemia due to deficient hemoglobin synthesis.
126
Sideroblastic Anemia Continued
• Siderocytes are RBCs in the blood containing abnormal iron granules called Pappenheimer bodies
• Sideroblasts are immature nucleated RBCs in the bone marrow containing iron in the cytoplasm.
• Sideroblastic anemia is characterized by the accumulation of iron in the mitochondria of immature

nucleated RBCs in the bone marrow;
• the iron forms a ring around the nucleus  these are called ringed sideroblasts
• The iron accumulation in the mitochondria is the result of blocks in the protoporphyrin pathway. Lab
findings:
• Microcytic/hypochromic red cells, low MCV and MCHC; variable anemia, low retic.
• RBC inclusions: Basophilic stippling and Pappenheimer bodies (siderocytes). (May see target cells).
• High serum iron and high serum ferritin (stores); low TIBC and high % saturation.
• Decreased transferrin synthesis occurs in iron overload states.
• Bone marrow: ringed syderoblasts (Hallmark of Sideroblastic Anemia) 127
Sideroblastic Anemia (SA)
Bone marrow Bone marrow
Ringed Sideroblast
Sideroblast
NRBC with iron NRBC with ring of iron

Prussian blue stain Prussian blue stain
Be aware that the diagram at the top is similar with the stained preparation at 128
the bottom
Types of Sideroblastic Anemia (SA)
• Blocks in the synthesis of protoporphyrin may be
– primary and irreversible (idiopathic = cause unknown) or
– secondary and reversible
129
Types of Sideroblastic anemia cont’d
• Blocks in the protoporphyrin pathway can be primary or secondary.

• Primary ‑ cause of protoporphyrin blocks are unknown and are not
reversible....called Idiopathic or primary Sideroblastic anemia.
1. Elderly, responds to no treatment. Requires transfusion support if severe anemia.
2. Characterized by a dimorphic red cell population - micro/hypo red cells with
3. normocytic and/or macrocytic red cells....MCV is variable and RDW is high.
4. Primary type of sideroblastic anemia is one of myelodysplastic syndromes called
Refractory Anemia with Ringed Sideroblasts; may terminate in leukemia
130
Secondary Types of SA
• cause of blocks in the protoporphyrin pathway can be identified and are reversible
• Removal of the drug/toxin corrects the anemia.
• Cause include
– Alcohol and anti-tuberculosis drugs inhibits vitamin B6/pyridoxine
– Lead poisoning causes multiple blocks

– Lead intoxication/poisoning – inhaled or ingested and causes multiple blocks:
1. Microcytic/hypochromic RBCs with **coarse basophilic stippling; low MCV.
2. Lead blocks several enzymes in the protoporphyrin pathway causing excesses in RBCs and urine
3. Abnormal lead level; lead line; abdominal pain; neurologic problems.
• Children - serum iron and iron stores
• May not be increased due to accompanying iron deficiency.
• Lead chelation therapy (EDTA).
131
Anemia of Chronic Disease (ACD)
• Associated with systemic diseases, including
– chronic inflammatory conditions such as arthritis

– Chronic infections such as TB
– A long-term chronic illness causes impaired release of iron from storage and
– may impair the response of the bone marrow to erythropoietin stimulation
• ACD – is due to inability to use iron and decreased response of the BM to EPO
– Complex etiology, pts have iron but are unable to use it, history is important
• Associated conditions
– Persistent infection (e.g. HIV),

– Chronic inflammatory or collagen disorders (e.g. RA, SLE)
– Malignant disease (e.g. Hodgkin lymphoma, cancer)
132
ACD pathogenesis
• Lactoferrin is an iron biding protein in the granules of neutrophils
– Its avidity for iron is greater than transferrin
– During infection or inflammation, neutrophil-lactoferrin released into plasma
– Scavenges available iron
– Bind to macrophage and liver cells (because they have receptor for lactoferrin)
• Though iron is present in abundance in bone marrow macrophages,
• its release to developing erythrocyte is slowed

• Cytokines: Produced by macrophages during inflammation and contribute to ACD by inhibiting
erythropoiesis
– Inadequate erythropoiesis
133
Lab Diagnosis/Blood findings
• Early stage: normocytic normochromic and late stage: hypochromic microcytic,
• severity parallels disease
• Very common anemia
• Leukocytosis
• Abundant storage of iron in macrophage (Prussian blue)
• Low serum iron, low TIBC, normal or high serum ferritin
– Decreased synthesis of transferrin occurs due to inflammation.

• Treat underlying disorder if possible
– Iron therapy of no value (harmful), EPO may help in some patients 134
Thalassemia's
• Inherited hemoglobinopathy due to
• Decrease in alpha or beta globin chain synthesis needed for Hgb A; quantitative
defect
– All have microcytic/hypochromic RBCs and target cells
• Genetic mutations classified by:
– ↓ beta chains = beta thalassemia…Greek/Italian
– ↓ alpha chains = alpha thalassemia…Asian
– Both in African ancestries
Beta
Alpha
Target cells/Codocytes
135
Thalassemia's cont…
• A normal adult will synthesize four types of globin chains to produce hemoglobin's
• Normal globin chain production is balanced with alpha and beta chains produced in
about a 1:1 ratio (high α, high β).
• Normal adult Hgb contains
– HgbA (95-98%
– HgbA1 (3-6%)
– HgbA2 (2-3%)
– HgbF <1% 136
• α –Thalassemia is more common than β-Thalassemia
• Severity ranges from lethal, to severe transfusion-dependency, to no clinical abnormalities
– Major types - severe, no alpha or no beta chains made
– Minor/trait types – mild, slight ↓ in normal hgb types

• Impaired alpha or beta globin synthesis results in
• an unbalanced number of chains produced that leads to:
– RBC destruction in beta thalassemia major

– Production of compensatory hgb types in beta thals
– Formation of unstable
– or non-functional hgb types in alpha thals
137
Beta Thal Major (Homozygous)
• Both beta genes abnormal
– Marked decrease/absence of beta chains leads to alpha chain excess…no
Hgb A is produced
– Excess alpha chains are unstable and preciptate in the cell
– Rigid RBCs with Heinz bodies destroyed in bone marrow and blood
(ineffective erythropoiesis)
Heinz bodies Excess alpha chains

Supravital stain
138
• Clinical findings
• Lab findings
– Severe anemia with microcytic hypochromic

– Hgb as low as 2-3gm/dL in homozygous state, and
– markedly reduced red cell indices (MCV, MCH, MCHC),
– anisocytosis, poikilocytosis, many target cells, RBC inclusions
(basophilic stippling),
– increased RDW, polychromatophilia, Reticulocytosis, nRBCs
139
– No hemoglobin A; compensatory Hgb F
• Decreased osmotic fragility,
• Moderately increased bilirubin,
• Increased serum iron,
• Hgb electrophoresis reveals Hgb-F & decreased Hgb-A
• When heterozygous, mild hypochromic microcytic anemia (β-Thalassemia minor)
Treatment
Transfusion
Splenectomy
Iron chelation
140
Beta Thal Minor (Heterozygous)
• One abnormal beta gene
– Slight decreased rate of beta chain production Stippled RBC
– Blood picture can look similar to iron

deficiency but normal Iron test
• Lab findings
– Mild anemia, target cells, no NRBCs, stippled

RBCs Target cell
– No Heinz bodies Ovalocytes
– Normal iron tests

– Compensates with Hgb A2 Wright’s stained blood smear
141
Alpha Thalassemia
• α -Thalassemia: characterized by decreased synthesis of α -globin chains
– classified into 4 types on the basis of deletion of one or more of the 4
alpha genes (2 on each chromosome).
– The severity of disease correlated to the number of genes deleted.
– The deletion of 4 alpha genes is fatal (frequent cause of stillbirth in
AlphaSouth East Asia)
Thal Major/Homozygous
• Deletion of all 4 alpha genes results in complete absence of alpha chain production
• No normal hemoglobin types made
• The fetal Hgb in this situation is composed of 4 gamma chains and is designated as “Bart’s
Hgb”.
• Bart’s Hgb has high affinity for O2 and is unable to release O2 to the fetal tissues
• Known as Barts Hydrops Fetalis
• Die of hypoxia 142
Alpha Thal Intermedia = Hgb H Disease
• Three alpha genes deleted
– Moderate decrease in alpha chains leads to beta chain excess
– unstable Hgb H (has high affinity for O2 )
– Moderate anemia
– Lab findings: Hgb, and red cell indices (MCV, MCH, MCHC) decreased,
– Retics increased, Hgb electrophoresis reveals Hgb-H (4-30%)
143
Alpha Thal Minor (Heterozygous)
• One or two alpha genes deleted (group)
– Slight decrease in alpha chain production
– Mild or no anemia, few target cells
– Essentially normal electrophoresis;
– many undiagnosed
• Thalassemia minor (silent carrier)

• only one functional alpha gene missing the remaining 3 direct normal synthesis of alpha chains
for normal Hgb synthesis—Normal haematologic finding
• α-thalasemia trait : 2 alpha genes missing;
• Imbalance between alpha and beta chain syntheis creats excess beta chains, which joins in
tetrads to form Hgb-H.
144
• mild microcytic hypochromic anemia manifested
Macrocytic Anemia
• Macrocytosis due to a deficiency of Vitamin B12 or Folic Acid (folate )that causes impaired
nuclear maturation –megaloblastic anemia
– Vitamin B12 & folate are DNA coenzymes necessary for DNA synthesis and
normal nuclear maturation
– Results in megaloblastic maturation…nucleus lags behind the cytoplasm

(asynchrony)
• Both deficiencies cause enlarged fragile cells
– Many cells die in the marrow (ineffective)
– Show a similar blood picture and clinical findings
• Only vitamin B12 deficiency causes neurological symptoms…required for myelin synthesis 145
Megaloblastic Anemia
Megaloblastic RBC maturation  macrocytic red cells
Normoblastic RBC maturation  normocytic red cells
RBC maturation in microcytic anemias…IDA
Abbott Manual 146

Megaloblastic anemia Lab findings
• Mild to severe anemia; MCV 100-160 fl; increased MCH, normal MCHC.
• Low RBC and HGB values with mildly decreased WBC and PLT counts (fragile cells)
due to ineffective hematopoiesis
• Low reticulocyte - due to high RBC death in bone marrow
• Macrocytic ovalocytes, teardrops; marked anisocytosis and poikilocytosis is typical
• Schistocytes/microcytes - due to RBC breakage upon leaving the bone marrow
147
Megaloblastic Anemia Lab findings cont’d
• Advanced anemia:
• Howell-Jolly bodies, NRBCs, basophilic stippling, pappenheimer bodies and Cabot rings
• markedly increased LD levels and high bilirubin
• Iron levels due to destruction of fragile red cells in the bone marrow and blood
• Hyper segmented neutrophils (>5 lobes)
• May see giant platelets
Hypersegmented neutrophil >5

lobes
148
Megaloblastic Anemia
• Clinical findings:
– Vitamin B12 and folate deficiency ‑ pale skin, weakness, smooth
sore tongue = glossitis, jaundice.
– Seen in B12 deficiency
– ONLY ‑ CNS damage with symptoms of tingling, numbness, and

difficulty walking or personality changes (megaloblastic madness).
– Important to correctly identify a B12 deficiency.
149
Vitamin B12 (Cobalamin) Deficiency
• Dietary deficiency – rare (except when long term deficiency especially in strict vegetarians)
• Vitamin B12 deficiency develops after 3-6 years; high stores.
Pernicious anemia most common cause

• caused by lack of intrinsic factor (IF)
• IF is a glycoprotein secreted by the parietal cells, along with HCL, and is needed to bind B12 for
absorption into the intestine.
– Acid pH (HCL) is needed for the B12/IF complex to form.
• Total gastrectomy: ‑ removes parietal cells which secrete IF and HCL.
150
• Concurrent IDA can develop due to the lack of an acid pH needed to reduce iron to Fe+2
for absorption.
• May see a dimorphic RBC population due to the production of both microcytic and
macrocytic red cells.
• MCV unreliable (falsely normal) and RDW is high.
• Competition for B12
– Diphyllobothrium latum - fish tapeworm competes for B12
– Intestinal blind loops - bacteria use B12.
• Malabsorption syndromes
– Regional enteritis - inflammation of small bowel.
– Non‑tropical sprue with steatorrhea - chronic wasting disorder.
151
• Pernicious anemia
– defined as the absence of IF and/or the presence of antibodies to IF or parietal cells;

autoimmune factors
– Characterized by achlorhydria (40%) and atrophy of the gastric parietal cells; results
in decreased IF secreted
– Autoimmune factors involved - a high percentage of patients produce

autoantibodies to IF, parietal cells, anti-B12-IF complex
– Low B12 level is followed by antibody tests to IF or parietal cells.
– The clinical history is important 152

Folate (Folic acid) Deficiency
• Dietary deficiency – leading cause, low stores
Two RBC populations
• Increased need Dimorphism
– Pregnancy;
– Growing infants
– Disorders associated with rapid
Macrocytic
proliferation. RBCs
– Alcoholic cirrhosis
– Malabsorption syndromes
Microcytic RBCs
153
Non-Megaloblastic Anemia
• Non-megaloblastic anemia is a macrocytic anemia that is not due to Vitamin B12
or folate deficiency
• Cause: example accelerated erythropoiesis
– Regenerating marrow or marked retic response following recent
blood loss
• Liver disease (including alcoholics)
– Complex & multiple problems
– Degree of anemia varies, round Macrocytes
– Target cells/acanthocytes - due to abnormal lipid metabolism.
– Echinocytes
NRBC are also commonly found on the smear in liver disease
Polychromatophilic RBCs
Wright’s stain 154
lab investigation of anemia
155
Normocytic/Normochromic Anemias
• Is a condition in which the size & Hgb content of RBCs is normal but the number of
RBCs is decreased.
• Due to increased RBC loss or decreased RBC production
• Majority are hemolytic caused by increased destruction due to increased RBC loss or
decreased RBC production
– Majority are hemolytic caused by increased destruction
• It includes
• Aplastic anemia due to BM failure
• Blood loss anemia
• Hemolytic anemia
• Red cell loss due to aging (1%) is compensated by daily bone marrow replacement
Hemolytic Anemias
• Classification of hemolytic anemias
– Mode of transmission:- Hereditary versus acquired
– Type of defect
• Intrinsic, the red cell is abnormal
• Extrinsic, an external agent or trauma destroys red cell
– Site of destruction
• Extravascular or Intravascular
• Compensated hemolytic disease
– RBC replacement = RBC destruction:-anemia does not develop
• Uncompensated hemolytic disease
– RBC destruction > RBC replacement by marrow
• anemia with high retic count but too low to keep up with rate of RBC loss
157
Hemolytic anemias
• Hemolytic anemia has the following features:
• Shortened life span of RBC b/c. , RBC breakdown occurs:
• Elevated erythropoietin that results increased erythropoisis.
• Accumulation of the products of Hgb catabolism.
• More commonly within mononuclear phagocyte system (extravascular hemolysis) which
undergoes work related hyperplasia marked by splenomegaly &
• Less commonly, lysis occur within the vascular compartment
• Anamias result from an increase in the rate of red cell destruction.
• It is characterized by premature destruction of RBC with retention of iron & other products
of destruction.
• Result from an increase in the rate of pre mature red cell destruction
• RBCs are produced in the BM but are destroyed after they enter blood stream.
Hemolytic anemias
• Bone marrow is able to increase production 6 –8 times normal in an attempt to compensate
for the losses.
• Hemolysis can occur in the absence of anemia (compensated production)
• Hemolysis is due to:
• Inherited abnormalities of RBCs
• Acquired causes
Classifications of hemolytic anemia
Mode of transmission: Hereditary versus acquired
–Type of defect:
•Intrinsic -The red cell is abnormal
•Extrinsic -An external agent or trauma destroys red cell
–Site of destruction: Extravascular or Intravascular
Causes of hemolytic anemia can be divided into three groups:
• Loss of structural integrity of red cell membrane and cytoskeleton in hereditary
spherocytosis, elliptocytosis, paroxysmal nocturnal hemoglobinuria (PNH), and immune
and drug-associated antibody damage.
• Defects within red cells from dysfunction of enzyme-controlled metabolism , abnormal
hemoglobin, and thalassemia
• Damage by outside factors such as mechanical trauma, microangiopathic conditions,
thrombotic thrombocytopenic purpura, and chemical toxins
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LABORATORY FINDINGS IN HAEMOLYTIC ANAEMIAS
The laboratory findings in hemolytic anemia are conveniently divided in three groups
1. Features of Increased Hemoglobin Breakdown
i. Jaundice and Hyper-bilirubenemia
ii. Reduced plasma Haptoglobin and Hemopixin
iii. Increased serum LDH
iv. Haemo-globinemia
v. Hemoglobinuria
vi. Meth hemoglobinuria
vii. Haemosidrinuria
Note:Decreased serum haptoglobin is characterics of intravascular hemolysis
2. Features of Increased Red cells Production CompensatoryErythroid Hyperplasia)
i. Reticulocytosis
ii. Macrocytosis and Plychromasia
iii. Erythroid hyperplasia of marrow
iv. Radiological changes in bones (seen only in congenital anaemia)
3. Feature of Damage to Red Cell:
i. Spherocytes
ii. Increased red cells fragility
iii. Red blood cells fragmentation
H Spherocytosis
• Intrinsic defects of the RBC membrane
• Such as Hereditary Spherocytosis Polychromasia
• Hereditary Ovalocytosis / Elliptocytosis Spherocyte
• Hereditary acanthocytosis H Ovalocytosis

• Hemolytic Anemias due to Enzyme Defects
• Pyruvate kinase deficiency
– Most common enzyme deficiency in EMB pathway
• G-6-PD deficiency
Normocytic ovalocytes
– Most common enzyme deficiency in HMP pathway
• PK deficiency PK Deficiency
– ↓ATP impairs cation pump

– Severe hemolytic anemia
– Echinocytes Echinocytes
166
Hemoglobinopathies
• Severity depends on inheritance of homozygous or heterozygous state
– Homozygous = disease; both beta chains abnormal
– Heterozygous = trait; one beta chain is abnormal Sickle cells
• Hemoglobin S disorders are most common
– RBCs contain Hgb S  sickle when oxygen is removed or
low pH
– Rigid sickle cells block vessels leading to organs,
increases tissue hypoxia Target cell
– Irreversibly sickled forms on blood smear Sickle Cell Disease
• Hemoglobin C disease/Hgb CC
– Two C genes inherited
• Lab findings
– Mild anemia
– Many target cells
– Intracellular C crystals
– No Hgb A, >90% Hgb C 167
Hemoglobin S Disorders
Target cell
• Hemoglobin S disease/Sickle cell anemia/Hgb SS
– Two sickle cell genes inherited
– Symptomatic by 6 months of age Sickle cell
• Clinical findings
– Vascular occlusive disease; organ damage & pain
• Laboratory findings HGB S Disease (Hgb SS)
– Severe anemia
– Targets, sickle cells C crystals
– NRBCs, inclusions
– No Hgb A, >80% Hgb S, ↑ F
Target cell
HGB C Disease (Hgb CC)
168
Normocytic Anemias due to Marrow Failure
• Impaired cell production and/or pancytopenia
– Normal or decreased Retic count
– Not hemolytic, normal RBC destruction tests and damaged red cells
are not evident on blood smear
• Marrow replacement anemia
– Infiltration or replacement of normal marrow cells
– Immature WBCs (neutrophils) & immature RBCs (nucleated RBCs)
escape from bone marrow
• Leuko-erythroblastic blood picture
• May result in extramedullary production
169
Aplastic Anemia
– Condition of blood pancytopenia caused by bone marrow failure,decreased
production of all cell lines
– Due to damaged stem cells, damaged bone marrow environment or suppression by
immune mechanisms
– No extramedullary hematopoiesis to compensate for bone marrow failure
• because the injury also affects hematopoietic cells in the liver and spleen
• Types of aplastic anemia
– Primary/idiopathic: about 50%, no cause of injury identified
– Secondary/acquired….
• Chemicals (benzene, insecticides), radiation, drugs(chloramphenicol, sulfonamides), infections
(viral)
– Congenital/hereditary.Fanconi’s Anemia
• Aplasia plus dwarfism, skeletal abnormalities, mental retardation, abnormal skin pigmentation.
High association with leukemia development 170
Aplastic Anemia
• Blood findings Normal RBCs
No Platelets
– Pancytopenia
– Moderate to severe anemia with normocytic
or slight macro RBCs
• Hypocellular marrow < 30% cellularity Blood
– M:E ratio ~3:1 - no change since all cell 10X
precursors are decreased in number.
• Complications
– Bleeding & infection
Bone marrow, decreased #

precursor cells
171
Normocytic/Normochromic Anemias due to blood loss
• Acute post‑hemorrhagic anemia - Normocytic anemia with high retics
• Lab findings:
– Plasma and red cells are lost (surgery/accident) so initially no change in Hgb/Hct values.
– In a few hours, PLTs and WBCs increase
– In 12-24 hrs, fluid replacement occurs....normocytic anemia with low Hgb/Hct/RBC &
relatively normal red cell findings on the smear.
– In 3-5 days following hemorrhage, increased polychromasia and possibly nucRBC's; a

very high # of circulating retics may increase the MCV
– increased WBCs (neutrophilia) and increased PLTs due to bone marrow outpouring
of WBCs and PLTs along with RBCs 172
Polycythemia vera (PV)
• Stem cell disorder, a clonal malignancy with excessive production of
mature hematopoietic cells, especially RBCs
• Refers to a pattern of blood cell changes that includes:
• An increase in Hgb above 17.5g/dl in adult males and 15.5g/dl in females
• An accompanying rise in red cell count (above 6.0 x 1012/l in males and 5.5x1012/l in
females)
• Hematocrit (above 55% in males and 47% in females)
• The increase in red cell volume is caused by endogenous myeloproliferation
• The stem cell origin of the defect is shown in many patients by an over-production of
granulocytes and platelets as well as of red cells
• This is a disease of older subjects with equal incidence in males and females
114
Laboratory findings in PV
• The Hgb, Hct and red cell count are increased
• A neutrophil leukocytosis is seen in over half the patients, and some have
increased circulating basophils
• Increased platelet count is present in about half the patients
• The leukocyte alkaline phosphatase (LAP) score is usually increased above normal
• Increased serum vitamin B12 and vitamin B12-binding capacity due to an

increase in transcobalamin I
115
Laboratory findings in PV cont’d
• The bone marrow is hypercellular with increased presence
of megakaryocytes
• Best assessed by a trephine biopsy
• Clonal cytogenetic abnormalities may occur, but there is no
single characteristic change
• Circulating erythroid progenitors increased and grow in vitro
are independently of added
erythropoietin
• Blood viscosity is increased
• Plasma urea is often increased 116

Differential count
• DLC is the enumeration of the relative proportions (percentages) of the various types of
white cells as seen on stained films of peripheral blood.
• The count is usually performed by visual examination of blood films which are prepared
on slides by the wedge technique.
• For a reliable differential count the film must not be too thin and the tail of the film
should be smooth.
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1. WBC counting…
 Interpreting WBC count
1. Leukocytosis 
• Acute infection, metabolic disorders, inflammation & tissue necrosis, poisoning,
leukemia, pregnancy, acute hemorrhage, etc
1. Leukopenia  production failure, bonemarrow infiltration, Viral, bacterial, parasitic
infections; drugs, radiation, hypersplenism, etc
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Differential counting
 In differential counting the following points should be checked:
1. Erythrocytes:
 Size, shape, distribution, & degree of hemoglobinization
 Presence of nucleated red cells, if so, the total leukocyte count must be corrected
2. Hemoparasites: Malaria, Borrelia, etc.
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Differential counting
Healthy Adult
Cell Type Absolute Relative
Segmented Neutrophil 2.0-7.0 x 109/L 40.0-75.0%
Lymphocyte 1.5-4.5 x 109/L 20-45%

Monocyte 0.2-0.8 x109/L 2-10%
Eosinophil 0.04-0.4 x 109/L 0-7%
Basophil 0.02-0.1 x 109/L 0-2%
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Differential counting….
Neutrophil
 Approximately 40-75% in peripheral blood
 2-5 lobes of nucleus joined by thin filament
 Faint pink cytoplasm sprinkled with pink or
 purple neutrophilic granules
Neutrophilia/neutrophilic leucocytosis:
• an increase neutrophils above normal (>2.0-7.0 x 10 9/L)
• Overwhelming infections
• Metabolic disorders: uremia, diabetic acidosis
• Drugs and chemicals: lead, mercury, potassium chlorate
• Physical and emotional stress
• Hematological disorders: myelogenous leukemia
• Tissue destruction or necrosis: burns, surgical operations
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Reactive Neutrophilia
 Is a condition at which increased neutrophil or abnormal neutrophil
 It coexist with toxic changes and multilineage abnormalities in sepsis
 It occurs in bacterial infections,, drugs and inflammation
 Blood findings with clinical features & chemical analysis, cytogenetic analysis
is
required
 If a neoplastic disorder is suspected as the presumptive diagnostic consideration
• Leukemoid reaction needs to be differentiated from Chronic Myeloid Leukemia
• Leukocytosis with marked left shift of immature neutrophils; bands,
myelocyte, metamyelocyte
• Infection, malignancy, burn, hemolysis, hemorrhage
• Differential: chronic myelocytic leukemia (CML),reactive neutrophilia have
• Increase in granulocytes, Toxic granulation, Dohle body,
vaculation in granulocytes, increased LAP score
1
8
2
Neutropenia:
• a neutrophil count below 2.0 x 109/L
• Myeloid hypoplasia
• Drugs (chloramphenicol, phenylbutazone)
• Ionizing radiation
Hyper granular neutrophils (neutrophils with toxic granules):
• indicative of severe infection or other toxic conditions.
Vacuolation
• seen in progressive muscular dystrophy
Hyper segmentation:
• neutrophils with more than six lobes to their nucleus
• indicative of megaloblastic erythropoiesis (vitamin B12 and/or folic acid deficiency),
iron deficiency anemia and uremia.
A granular Neutrophils:
• neutrophils devoid of granules
• having a pale blue cytoplasm( features of leukemia)
05/31/2024 183
5. Lymphocytes (small & large lymphocytes)
 Approximately 40% in peripheral smear
 Small round nucleus, clumpy, chunk chromatin pattern
Lymphocytosis:
• absolute lymphocyte count above 4.0 x 109/L in adults and above 8.0 x 109/L in
children.
Seen during
• Infectious lymphocytosis associate viral infection
• Acute and chronic lymphocytic leukemia
• Toxoplasmosis
. Lymphocytopenia:
• a lymphocyte count below 1.0 x 109/l in adults and below 3.0 x 109/l in children
• Seen in
• Immune deficiency disorders: HIV/AIDS
• Drugs, radiation therapy
Atypical lymphocytes:
• infectious mononucleosis
• infectious disease of the reticuloendothelial tissues
05/31/2024 184
6.Monocyte
 Approximately 5% in peripheral smear
 Large, convoluted, brainy looking nucleus with lacy looking
chromatin
 Pale, gray blue, ground glass cytoplasm (fine azurophilic granules)
Monocytosis:
• a monocyte count above 1.0 x 109/l
• Occurs during
• Recovery from acute infections
• Tuberculosis
• Monocytic leukemia
Monocytopenia:
• a monocyte count below 0.2 x 109/l
• Occurs during
• Treatment with prednisone
• Hairy cell leukemia
05/31/2024 185
7. Eosinophils
 Bi-lobed or banded nucleus
 Large, red-orange & distinct granules
Eosinophilia
an eosinophil count above 0.5 x 109/L
Occurs during:
• Allergic diseases: bronchial asthma, seasonal rhinitis
• Intestinal parasitic infections: e.g. trichinosis, taeniasis
• Skin disorders
• Chronic myelogenous leukemia
Eosinopenia:
an eosinophil count below 0.04 x 109/L
Occurs during:
• Acute stress due to secretion of adrenal glucocorticoid and epinephrine
• Acute inflammatory states
05/31/2024 186
8. Basophils
 Nucleus is bi-lobed & obscured by granules
 Cytoplasm is pale, washed out & contains intense large blue black granules
 Granules are scattered throughout the cell & it is hard to distinguish any distinct
nuclear characteristics
• Basophilia
• a basophil count above 0.2 x 109/L
• Rare condition
• Occurs during:
• Allergic reactions
• Chronic myelogenous leukemia
• Polycythemia vera
05/31/2024 187
Normocytic/Normochromic Anemias due to blood loss
• Acute post‑hemorrhagic anemia - Normocytic anemia with high retics
• Lab findings:
– Plasma and red cells are lost (surgery/accident) so initially no change in Hgb/Hct values.
– In a few hours, PLTs and WBCs increase
– In 12-24 hrs, fluid replacement occurs....normocytic anemia with low Hgb/Hct/RBC &
relatively normal red cell findings on the smear.
– In 3-5 days following hemorrhage, increased polychromasia and possibly nucRBC's; a very
high # of circulating retics may increase the MCV
– increased WBCs (neutrophilia) and increased PLTs due to bone marrow outpouring
of WBCs and PLTs along with RBCs 188
Leukemia
• The leukemia's are a group of disorders characterized by the accumulation of abnormal

white cells in the bone marrow and hematologic system.
• Results from
• uncontrolled proliferation, and evasion of apoptosis
• inhibition of differentiation.
• These abnormal cells may cause:
– Bone marrow failure

– A raised circulating white cell count
– Infiltration of organs
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05/31/2024 190
Leukemia
• Leukemia results from

1.Genes and environmental factors
• Risk factors for leukemia
• Presence of prooncogens and oncogenes
• Infection
• Epstein-Barr virus, associated with Burkett's lymphoma
• Human T-cell lymphotropic virus type I, HIV
• H-Pylori infection
• Exposure to ionizing radiation
• certain chemicals (e.g., benzene, some anti-neoplastic drugs)
• Some genetic defects
• Epigenetic factors ( DNA methylation , DNA hypoxymethylation , Histone
modification)
05/31/2024 191
Leukaemia results from cont..
2. Clonality of Leukemic cell
• Leukemic stem cells
• lymphoproliferative disorders
• Philadelphia (Ph) chromosome in CML
• X -chromosome inactivation patterns (XCIPs) in female
• Clonal evolution
05/31/2024 192
Classification of leukemia
• The main classification is into acute and chronic leukemia

• On the basis of morphology and cytochemistry, acute leukemia is further subdivided into:
– Acute myeloid (myeloblastic/myelogenous) leukemia (AML)

– Acute lymphoblastic (lymphocytic) leukemia (ALL)
• AML is further subdivided into eight variants on a morphological basis according to the
French-American-British (FAB) scheme (M0 – M7)
05/31/2024 193
Classification of Leukemia cont’d
• ALL is subdivided on a morphological basis according to the FAB classification into L 1,
L2, and L3
• The chronic leukemia comprise two main types:
– Chronic myeloid leukemia (CML)

– Chronic lymphocytic (lymphatic) leukemia (CLL)
• Other chronic types include:
– Hairy cell leukemia

– Prolymphocytic leukemia
– Various leukemia/lymphoma syndromes
05/31/2024 194
Diagnosis of hematological malignancy
• Usually starts from a clinical suspicion,
• A blood count and blood film is an essential first step whenever a hematological
neoplasm is suspected.
• The next step in the diagnostic process depends on the clinical features and the
specific condition that is suspected.
• It is of crucial importance that all laboratory investigations are done with an
awareness of the medical history and physical findings.
• It is also essential that a conclusion as to diagnosis is based on integrating the results
of all laboratory investigations and imaging in the context of the clinical features.
05/31/2024 195
Three important factors contribute to accurate diagnosis:
1.The provision of very detailed clinical information to laboratories
2.The correct sample, e.g. blood or bone marrow aspirate or biopsy
3 The integration of information technology across different sites dealing with diagnostic
samples from a single patient.
• It is also necessary to ensure that a diagnosis is achieved in a timely manner,
• particularly when treatment may be urgent, as in acute leukemia and aggressive
lymphomas.
05/31/2024 196
Blood count
• The most important information that must be extracted from BC
• The red cell indices should also be noted since macrocytosis and, less often, microcytosis can
occur in hematological neoplasms.
• The automated instrument will generally provide a differential count and ‘flags’
• indicating the likely presence of blast cells, abnormal lymphocytes or granulocyte precursors.
• An abnormal total or differential count should always be verified on a blood film.
• Even if the blood count is normal, a blood film should be examined for the presence of
abnormal cells
• it provides strong evidence of a specific diagnosis that can be confirmed on further testing.
• It suggests a differential diagnosis and indicates the appropriate direction of further

05/31/2024 197
Bone marrow
• A bone marrow aspirate is indicated in virtually all patients with suspected ALL, AML ,
CML, myelodysplastic syndrome (MDS) or multiple myeloma
• However, a bone marrow aspirate is not necessary for the diagnosis of CLL.
• Bone marrow aspiration may also be indicated in patients with unexplained cytopenia or
a leucoerythroblastic blood film.
• An aspirate can be useful in lymphoma diagnosis, particularly if accompanied by a
trephine biopsy, and can provide suitable material for
– immunophenotyping and for fluorescence in situ hybridization (FISH).
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CLL CML AML FAB M0 subtype
AML
• A heterogeneous disease with respect to clinical features and acquired genetic aberrations
• Malignant proliferation of early myeloid precursor cells develops as the consequence of a series of
genetic changes in a hematopoietic precursor cell.
• These changes alter normal hematopoietic growth and differentiation, resulting in an accumulation
of large numbers of abnormal, immature myeloid cells
• are capable of dividing and proliferating, but cannot differentiate into mature hematopoietic
cells
• Occurs at all age but more common in adult with exponential increase in incidence with over 40
years
• Only 10-15% of childhood leukemia are AML
• It Can occur either as a primary disease ,secondary to MDS/MPD or anti-neoplastic
treatment
05/31/2024 199
Clinical and laboratory features of AML
• Clinically AML typically presents with Short history of illness, Bruising/hemorrhage,
Serious infection involving lung and Metabolic complication.
• The morphology usually corresponds to acute myeloid leukemia with neutrophilic maturation,
• Represent 5%–12% of AML, predominance in young patients
• Blasts are 20% or greater in the marrow (or blood), and maturing neutrophilic precursors
constitute more than 10% of marrow nucleated myeloid cells
Immuno phenotype and cytochemistry
• On Immuophenotyping, all B and T lineage markers were negative
• CD34, Cd13, Cd33 and sometime CD56
• MPO positive
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Acute myeloid leukemia FAB M0 subtype
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05/31/2024 202
Acute Lymphoblastic leukemia(ALL)
• is the most common childhood cancer

• ALL is the most common subtype, accounting for 75 –80% of all cases
• Childhood ALL comprises different biological subtypes defined by :
– Cell morphology,
– Immunophenotype ,
– Gene expression features
– Genetic abnormalities
• Some of which are associated with disease aggressiveness and treatment response
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Child hood Acute Lymphoblastic leukemia (ALL)
• Childhood ALL is clinically heterogeneous
• In some patients, there is a history of vague illness lasting from week to month other case may
show acute presentation
• ALL can be traced to bone marrow failure and/or leukemic cell infiltration
• The most common presenting features include:
– Bone marrow failure are pallor, petechiae and ecchymoses,

– occasionally mucosal bleeding-secondary to thrombocytopenia
– Persistent fever with non-infectious origin
– Fatigue , lethargy, dizziness, dyspnea-secondary to anemia
– Headache and vomiting-indicate CNS infiltration
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Acute Lymphoblastic leukemia(ALL)
Blood film examination and CBC

• Blood examination typically reveals anemia, neutropenia and thrombocytopenia,
• White cell abnormalities like atypical lymphoid cell
• WBC: low, normal or raised
• Lymphoblast are common when WBC raised
• Leukemic lymphoblast's have chateristics
• Azurophilic granules may be present in some cells
• But the presence of auer rods preclude the diagnosis of ALL
Chemistry profiles:
• Elevated serum LDH, uric acid and phosphorus concentration
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Liver dysfunction due to leukaemic infiltration occurs in 10 –20% of patients, 205
Adult T Cell ALL: CLINICAL PRESENTATION:
• Lymphadenopathy: in cervical, supraclavicular, and axillary regions
• Mediastinal mass: 50-75 is anterior, bulky, and associated with pleural effusions
– can produce such complications: superior vena cava syndrome, tracheal obstruction, pericardial
effusions
• Some patients presenting with symptoms progressing slowly and others presenting more acutely
• Less common presentation:
• patients present with extranodal disease (eg, skin, testicular, or bony involvement).
• Abdominal, liver and spleen involvement is very unusual
• When the disease advances to stage III and IV

– patients develop bone marrow infiltration and a subsequent leukemic phase
– CNS involvement, which is frequently seen at this stage

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Laboratory features
Morphology
• Peripheral blood: lymphoblasts vary from small cells to larger cells (occasional)
• A few azurophilic cytoplasmic granules may be present.
• Auer rods are never seen
• Precursor T cell ALL is morphologically indistinguishable from precursor B cell .
Cytochemistry:
• The blasts often show "chunky" positivity on periodic acid Schiff (PAS) staining,
• variable positivity for nonspecific esterase and sudan black B,
• Negativity for myeloperoxidase.
Immuophenotyping:
• lymphoblasts are typically positive for CD7 and either surface or cytoplasmic CD3, and
variably express CD2, CD5, CD1a, CD4 and/or CD8
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Chronic Lymphocytic Leukemias
• A progressive accumulation of monoclonal, matured-looking, functionally incompetent

B cells
– Site :Peripheral blood, BM,Lymph nodes and spleen

• No defined risk factors for CLL
• Studies failure to show the association between CLL with exposure to ionizing radiation
and antineoplastic drugs
• No genes have been definitively identified that predispose to CLL, thought a familial
association has been identified
• E.g. over-representation of ATM gene mutation and 13q21.33-22.2 abnormalities
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CLL: Clinical presentation
• The most common presenting symptoms:
• Painless lymphadenopathy; often in the cervical, supraclavicular or axillary region
• Most patients do not feel unwell and the lymph node swelling tends to wax and wane over
time, causing delay in presentation
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Laboratory features
• CBC:
• Marked absolute lymphocytosis (20-50X109/L)
• Variable degree of anemia
• Peripheral blood film:
• Confirms lymphocytosis
• Small matured-looking lymphocyte
• Smudge or smear cells
• Between 70% and 99% of white cells in the blood film appear as small lymphocytes
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Laboratory features
Bone marrow Aspirate:
• Normo-or hyper-cellular, with prominent lymphocytosis(typically >30%)
• Erythriod hyperplasia in the bone marrow(AIHA)
• Pure red cell aplasia
BM biopsy:
• Nodular, interstitial or diffused infiltration by small lymphocytes
Immuophenotyping:
• 50-100 times the normal lymphoid mass in the blood, bone marrow, spleen, lymph nodes
and liver may be related to immunological non-reactivity and excessive lifespan
• The cells are a monoclonal population of B lymphocytes
• CLL cells: express CD19, Cd20, CD23, CD5, low surface membrane Ig
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Hairy cell leukemia
• Clinical presentation: Nearly similar to CLL

• Most symptoms are attributable to cytopenia Laboratory findings:
• weakness • 60% to 80 % patients with
• fatigue pancytopenia
• • Common feature of HCL patient

bruising or bleeding
• CBC: WBC low, normal or high
• recurrent infection and • Anemia —85 %,
• Thrombocytopenia —80 %
• splenomegaly • Neutropenia —80%
• About one-quarter of patients have • Monocytopenia —80
• Abnormal liver function tests —20%
hepatomegaly • Hypergammaglobulinemia —20%
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Peripheral lymphadenopathies seen in <10% 214
Hairy cell leukemia
PBF examination:
• Hary cells’-large lymphocyte with eccentric nuclei of variable appearance, reticular
chromatin and indistinct nucleoli
• Bone marrow trephine:
• Infiltration: diffused, focal or interstitial
Immunophenotyping:
• Strongly positive for CD19, CD20, CD22, and CD25, and
• usually lack expression of CD5, CD10, CD21, and CD23
• CD11c, CD103, CD123, cyclin D1
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The Chronic Myeloid Leukemias
• Approximately 20% of all the leukemias and mostly seen frequently in middle age
• In over 95% of patients there is a replacement of normal bone marrow by cells with an
abnormal chromosome-the Philadelphia chromosome
• This is an abnormal chromosome 22 due to the translocation of part of a long (q) arm of
chromosome 22 to another chromosome usually 9, with translocation of part of
chromosome 9 to chromosome 22
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CML: clinical and laboratory features
• A great increase in total body granulocyte

mass is responsible for most of the clinical
features
• Leukocytosis is usually >50x109/l and
sometimes >500x109/l
• A complete spectrum of myeloid cells is
seen in the peripheral blood
• The levels of neutrophils and myelocytes
exceed those of blast cells and
promyelocytes
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CML: clinical and laboratory features
• Philadelphia (Ph) chromosome on cytogenetic analysis of blood or bone marrow
• Hyper cellular bone marrow with granulopoietic predominance
• Variety of granulocyte abnormality
• Leukocyte MPO , AP ….. score is invariably low
• Increased circulating basophils
• Increased Cytoplasmic maturation relative nuclear maturation
• Normochromic, normocytic anemia is usual
• Platelet count may be increased (most frequently), normal or decreased
• Serum vitamin B12-binding capacity are increased
• Serum uric acid is usually raised
• Increased granular promylocyte
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Introduction
• Hemostasis is complex physiological process, that keeps circulating blood in fluid state.
• When injury occurred, it produce clot to stop bleeding and confine the clot to the site of
injury, dissolve the clot as the wound heal.
• When hemostasis systems are out of balance hemorrhage or thrombosis can be life
threating.
• It involve the interaction of vasoconstriction, platelet and coagulation system activation to
stop bleeding.
• Its major component are blood vessel, platelet, coagulation proteins and its inhibitors
and fibrinolysis proteins and its inhibitors.
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Hemostasis
• When blood vessels are damaged, hemostasis (clot formation) will
arrest bleeding.
• This process is divided into three phases :
1.Primary hemoestasis
I. Vascular Phase
 Cutting or damaging blood vessels leads to vascular spasm of the
smooth muscle in the vessel wall.
 Produces a vasoconstriction which will slow or stop blood flow.
 Response will last up to 30 minutes.
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Introduction
• Hemostasis is complex physiological process, that keeps circulating blood in fluid state.
• When injury occurred, it produce clot to stop bleeding and confine the clot to the site of
injury, dissolve the clot as the wound heal.
• When hemostasis systems are out of balance hemorrhage or thrombosis can be life
threating.
• It involve the interaction of vasoconstriction, platelet and coagulation system activation to
stop bleeding.
• Its major component are blood vessel, platelet, coagulation proteins and its inhibitors
and fibrinolysis proteins and its inhibitors.
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Hemostasis
• When blood vessels are damaged, hemostasis (clot formation) will
arrest bleeding.
• This process is divided into three phases :
1.Primary hemoestasis
I. Vascular Phase
 Cutting or damaging blood vessels leads to vascular spasm of the
smooth muscle in the vessel wall.
 Produces a vasoconstriction which will slow or stop blood flow.
 Response will last up to 30 minutes.
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II. Platelet phase
 Damaged endothelial cells lining the blood vessel release von Willebrand's Factor.
 Makes the surfaces of the endothelial cells "sticky“ and close small blood vessels.
 In larger blood vessels, platelets begin to stick to the surfaces of endothelial cells and
form Platelet Adhesion
 The platelets that adhere to the vessel walls secrete ADP and causes the aggregation.
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2.Coagulation Phase
• Begins 30 minute after phases one have commenced.
• The overall process involves the formation of the insoluble protein Fibrin from
Fibrinogen by the action of Thrombin.
• Fibrin forms a network of fibers which traps blood cells and platelets form clot.
3.Fibrinolysis
• The breakdown of the clot is due to the production of a powerful proteolytic enzyme
Plasmin.
• These reactions demonstrate that materials which induce clot formation (Thrombin and
Factor XII) will eventually assist in the breakup of the clot.
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DISORDERS OF COAGULATION
Causes of Bleeding HEREDITARY
 Hemophilia A (factor VIII deficiency)
1.Vessel wall disorders  Hemophilia B (factor IX deficiency)
2. Platelet disorders :  von Willebrand disease
– quantitative or functional.  Disorders of fibrinogen:

 Hypo fibrinogenaemia
3. Coagulation factor:  A fibrinogenaemia
– deficiency or inhibitors.  Dysfibrinogenaemia
ACQUIRED
4. Combination of these  Disseminated intravascular coagulation (DIC)
 Liver disease Vit k deficiency
 Massive transfusion of stored blood
 Acquired inhibitors of coagulation
 Heparin oral anticoagulant therapy
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Bleeding disorders caused by vessel wall abnormalities
 Sometimes called non-thrombocytopenic purpuras
 Relatively common , but do not usually cause serious bleeding
 Most often small bleedings in the skin (petechiae and purpura) or mucus membranes
 Hemorrhage into the joints , muscles, menorrhagia (painful menstruation), epistaxis , GI
bleeding and hematuria can occur
 The platelet count , bleeding time and results of PT and PTT are usually normal!
Petechiae
Clinical conditions related to hemorrhage due to vessel wall abnormalities
 Infections- meningococcemia , rickettsioses …vasculitis

(inflammation of vessels)
 Drug reactions-hypersensitivity ( leukocytoclastic vasculitis)
Clinical conditions related …..cont’d
 Scurvy and the Ehelers-Danlos syndrome… impaired synthesis of collagen
 Cushing syndrome- protein wasting effects of corticosteroid cause lose of perivascular

supporting tissue
 Henoch-Schonlein purpura (HSP) is a disease involving inflammation of
small blood vessels.
 It most commonly occurs in children
 The inflammation causes blood vessels in the skin, intestines, kidneys, and
joints to start leaking.
Clinical conditions related …..cont’d
 Hereditary hemorrhagic telangectasia (lesion formed by dilation of small blood vessels)-

autosomal dominant disorder characterized by dilated , tortuous blood vessels with thin
walls that bleed easily e.g. in the nose , mouth, eyes and GI
 Amyloid infiltration of blood vessels
Thrombocytopenia
• Thrombocytopenia is a condition in which you have a low blood platelet count

(<100x103/μl…thrombocytopenia)
• Platelets (thrombocytes) are colorless blood cells that help blood clot.
• Platelets stop bleeding by clumping and forming plugs in blood vessel injuries.
 <20x103 μl- spontaneous bleeding mucosa and skin in small blood

 20-50x103 / μl- aggravate post-traumatic bleeding
vessels in
 Prolonged bleeding time, normal PT and PTT
 Severe thrombocytopenia is associated with intracranial bleeding

Bleeding related to reduced platelet number :
Thrombocytopenia
Four major categories of causes of thrombocytopenia:
1. Decreased production of platelets… Aplastic anemia , leukemia, ineffective
hematopoiesis
2. Decreased platelet survival… immunologic or non-immunologic

 Anti-platelet antibodies e.g. idiopathic (immune) thrombocytopenic purpura,
HIV-associated thrombocytopenia
 Alloimmune thrombocytopenia- in transfusion
 Mechanical destruction – analogous to RBC destruction

3. Sequestration … in hypersplenism
4. Dilution in massive transfusions- blood stored for longer than

24hrs contains virtually no platelets
Clotting factor abnormalities
 Congenital disorders
 von Willebrand Factor deficiency - Von Willebrand disease
 Factor VIII Deficiency - Hemophilia A or Classic Type
 Factor IX Deficiency – Hemophilia B
 Acquired disorders
 Vitamin-K deficiency = Due to deficient carboxylation of factors II, VII, IX ,X and
protein C.
 Liver diseases ↓ synthesis of factors.
 DIC acquired syndrome characterized by the intravascular activation of coagulation
with loss of localization arising from different causes
 Oral anti coagulants.
Coagulopathy
 Coagulopathy (a bleeding disorder) is a condition in which the blood’s ability to coagulate
is impaired
 This condition can cause a tendency toward prolonged or excessive bleeding (bleeding
diathesis or bleeding disorder), which may occur spontaneously or following an injury or
medical and dental procedures.
 Hemorrhage is a severe form of bleeding that requires intervention.
 Hemorrhage may be:
 acquired or congenital
 Localized or generalized
 mucocutaneous or a soft tissue (anatomic)
23
5
Acquired coagulopathies
 Can be caused by acquired bleeding disorders secondary to:
 trauma, drug exposure, or chronic disease eg.
 liver disease, vitamin K deficiency, and renal failure
Liver Disease
 The bleeding may be localized or generalized, mucocutaneous or anatomic.
 Mucocutaneous bleeding  is mainly due to thrombocytopenia
 Soft tissue bleeding  is due to procoagulant dysfunction and deficiency
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6
Procoagulant deficiency in liver disease
 Liver produces nearly all of the coagulation factors & regulatory proteins.
 Synthetic function of hepatocytes can be suppressed by:
 Hepatitis, cirrhosis, obstructive jaundice, cancer, poisoning, & congenital

disorders of bilirubin metabolism
 Resultantly reducing either the concentrations or activities of the plasma coagulation

factors below hemostatic levels (less than 30% of normal).
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7
Acquired coagulopathies due to Liver Disease
 Liver Disease affects predominantly the production of:
 vitamin K–dependent factors and control proteins C, and S.
 Factor VII is the first factor to exhibit decreased activity
 PTis particularly sensitive to factor VII activity, & will
be prolonged in mild liver disease, serving as a sensitive early marker.
 However, Vitamin K deficiency caused by dietary insufficiency produces
a similar effect on the PT.
 So measuring non–vitamin K dependent factors such as factor V activity is a more
specific marker of liver disease than others.
 to distinguish liver disease from vitamin K deficiency.
45
 Perform factor V activity assay VII assay.
Acquired coagulopathies …. (Liver Disease)
 Fibrinogen is an acute phase reactant that is frequently elevated in early or mild liver disease.
 Moderately and severely diseased liver results in dysfibrinogenemia, in which the fibrinogen
functions poorly.
 It causes generalized soft tissue bleeding associated with a prolonged thrombin time &
Reptilase clotting time.
 In end-stage liver disease, the fibrinogen level may fall to less than 100 mg/dL, which
is a mark of liver failure (Fibrinogen:150-400 mg/dL
46
Platelet abnormalities in Liver Disease
 Moderate thrombocytopenia may occur.
 It results from sequestration & shortened platelet survival associated with:
 Portal hypertension and resultant hepatosplenomegaly.
 Platelet aggregation and secretion properties are often suppressed.
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Disseminated Intravascular Coagulation in Liver Disease

• Chronic or compensated DIC is a significant complication of liver disease that is caused
by:
• Decreased liver production of regulatory antithrombin, protein C, or protein S
• The release of activated procoagulants from degenerating liver cells
• Additionally, due to the inability of the failing liver to clear activated coagulation factors
that are regularly produced by liver.
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1
 In acute, uncompensated DIC,
 the PT, PTT and thrombin time are prolonged;
 the fibrinogen level is reduced to less than 100 mg/dL; and
 fibrin degradation products, including D-dimers, are significantly increased.
 In chronic, compensated DIC:
 the only elevated test result may be the D-dimer assay value.
 DIC may be temporarily corrected by administering:

 RBCs, FP, platelet concentrates, activated protein C, or antithrombin
concentrates.
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2
Hemostasis Laboratory Tests in Liver Disease
 PT& PTT, thrombin time  prolonged
 Fibrinogen concentration  reduced
 Platelet count  reduced, and
 D-dimer concentration  increased
 Factor V and VII assays  to differentiate liver disease from vitamin K

deficiency
 The reptilase time  to confirm dysfibrinogenemia.
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3
Acquired coagulopathies ………
Vitamin K Deficiency
 Vitamin K is a fat soluble compound
 Vitamin K serves as an essential carboxylase
 that catalyzes carboxylation residues on vitamin K-dependent proteins.
 The key vitamin K-dependent proteins include:
 Coagulation proteins: factors II (prothrombin), VII, IX and X.
 Anticoagulation proteins: proteins C, S and Z
 Body stores of vitamin K are limited.
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4
Acquired coagulopathies ………
 Vitamin K types and sources:
1) Vit K1 (Phylloquinone)
 natural form
 found in plants, green leafy vegetables
 provides the primary source of vitamin K to humans through

dietary consumption
2) Vitamin K2 compounds (Menaquinones)
 made by bacteria in the human gut
 provide a smaller amount ofthe human vitamin Krequirement
3) Menadione is synthetic product
52
 Vitamin K deficiency can result from:
 a lack of vitamin k in the diet
 disorders that reduce fat absorption
 taking certain drugs, including antibiotics anticonvulsants and some
 Use of coumarin anticoagulants
 Hepatic insufficiency
246
Hemorrhagic Disease of the Newborn Caused byVitamin K Deficiency
 Newborns are prone to vitamin K deficiency because;
1. The neonatal gut is sterile.
2. Breast milk is not a good source of vitamin K.
 Results in a hemorrhagic disease called vitamin K deficiency bleeding
Laboratory Diagnosis for Vitamin K Deficiency
 Prolonged PT with or without a prolonged PTT.
 In PT and PTT mixing studies, yields normal (corrected) PT and PTT
 Factor assays  depressed activity
Treatment
 Vitamin K administration
57
Congenital coagulopathies Deficiency of Factor
VIII-vWF complex
Haemophilia -A and vWF disease
 Most common inherited disorders of bleeding.
 Caused by qualitative or quantitative defect involving the factor VIII-vWF

complex.
Congenital coagulopathies
Von Willebrand Disease
 Estimated frequency-1%.
 One of most common inherited bleeding disorders in humans
 Most -Autosomal dominant.
 Spontaneous bleeding, excessive bleeding from wounds, menorrhagia.

 Prolonged bleeding time but normal platelet count.
 More than 20 variants, can be grouped into two.

von Willebrand Disease
 It was first described by Finnish Professor Erik von Willebrand in 1926.
 It is Inherited hemorrhagic disorder which;
 Genetically and clinically heterogeneous
 Has autosomal dominant inheritance pattern.
 Caused by a deficiency/dysfunction of VWF
 Most common hereditary bleeding disorder causing a

mucocutaneous bleeding. 61
VWF
 the largest molecule in human plasma, concentration ranging from 0.5 to 1.0 mg/dL.
 synthesized in the ER of endothelial cells & stored in cytoplasmic
Weibel-Palade bodies of endothelial cells.
 also synthesized in megakaryocytes & stored in the α-granules of platelets.
 Performs two major roles in hemostasis
 Mediates adhesion of platelets to sites of vascular injury
 Is a carrier protein for F-VIII
 Inherited defects in VWF may
 Cause bleeding by impairing either platelet adhesion or blood
clotting 62
Von Willebrand Disease

 Hemorrhagic tendency is highly variable
 Depends on the type and severity of disease
 Patients with Type 1 and 2 disease
 May have mild bleeding symptoms
 Characterized by hemorrhage from delicate mucocutaneous tissues
 Epistaxis, easy bruising, GI bleeding, menorrhagia
 Hematoma and hemarthroses, characteristic of hemophilia
A, are not prominent 71

 Patients with severe type 3 VWD

 Severe hemorrhagic tendency
 Spontaneous hemorrhage
 Mucous membranes, GI tract
 Can be frequent and may be life threatening
 Low F-VIII level
 Deep hematomas
 Joint hemorrhages – similar to hemophilia
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3
 Treatment for Von Willebrand Disease
 Correct both bleeding time and coagulation abnormalities
• Raise both F-VIII and VWF to normal levels
 Cryoprecipitate, Source of fibrinogen, factor VIII and VWF
Desmopressin acetate (DDAVP) stimulates release of vWF from

endothelial cells – won’t work for type 3
 Intermediate purity factor VIII which contains intact vWF
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4
Congenital coagulopathies Hemophilia-A (Factor VIII Deficiency)
 Most common hereditary disease with serious bleeding.
 ↓ amount or activity of factor VIII.
 Factor VIII is a cofactor for factor IX in the activation of factor X.

 X-linked recessive.
• Affected males (XY):
–sons unaffected (no male to male transmission)
–daughters obligate carriers
• Carrier female (XX):
–½ sons affected; ½ daughters carriers
• Affected females: very rare.
 In 30% -No family history (new mutations).
 15% of severe cases develop factor VIII inhibitors.

Hemophilia /A “Royal Disease”
Queen Victoria (1837 to 1901) passed hemophilia on to German, Russian and Spanish royal families. Her son,
Leopold, had frequent hemorrhages (British Medical Journal,1868) and died of a brain hemorrhage at 31. His
grandson also died of a brain hemorrhage in 1928.
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6
Hemophilia-A (Factor VIII Deficiency)
 Wide range of clinical severity that correlates with the level of factor VIII activity.
 Explained by heterogeneity in causative mutation.
 Symptoms usually develop in severe cases (factor VIII <1% of normal) – easy
bruising, massive hemorrhage after trauma or surgery
 ‘’Spontaneous’’ hemorrhage in areas subject to
trauma….Hemarthroses
 Petechiae are characteristically absent
• Normal bleeding time, platelet count, PT and a prolonged PTT
• Insufficient generation of thrombin by
– F-IXa/VIIIa complex through the intrinsic pathway of coagulation cascade
– Bleeding severity complicated by excessive fibrinolysis
Hemophilia-B (Christmas Disease)
 Factor IX deficiency.
 X-linked recessive.
 Much less common.
 Clinically= indistinguishable from Hemophilia A with Similar lab findings.

 In 14%- factor IX is present but non-functional.
 Diagnosis by assay of factor IX levels
 PTT is prolonged, PT and bleeding time are normal.
 Treat with recombinant IX.

 Hemophilia C
 Factor XI Deficiency
 Autosomal recessive disorder seen primarily in the Jewish population
 Symptoms range from mild to severe
78
X-Linked Recessive Inheritance
New mutation in
germ cell
New mutation in
maternal or paternal
germ cell
Carrier female
Affected male
Normal male
79
1. Primary: bruising and bleeding
 Minor bleeds:
 early joint and muscle bleeds
 bleeding in the mouth and gums

epistaxis (nosebleed),
hematuria (blood in the urine)
261
Symptoms of hemophilia
 Major bleeds
 central nervous system
 severe injury
 neck/throat, eye, gastrointestinal, hip, iliopsoas, late joint and muscle, testicles, and
retroperitoneum bleeds
262
2. Secondary: bruising and bleeding
 Chronic joint deformities from recurrent bleeding
 Antibodies to transfused factor VIII
 AIDS - Over 60 % of persons with hemophilia treated with

plasma concentrates in the early 1980s became HIV+
263
Clinical severity corresponds with level of factor activity.
 Severe hemophilia
 Factor coagulant activity <1% of normal
 Frequent spontaneous bleeding into joints and soft tissues
 Prolonged bleeding with trauma or surgery
 Moderate hemophilia
 Factor coagulant activity 1-5% of normal
 Occasional spontaneous bleeding
 Excessive bleeding with surgery or trauma
 Mild hemophilia
 Factor coagulant activity >5% of normal
 Usually no spontaneous bleeding
83
 Excessive bleeding with surgery or trauma
Clinical presentation of hemophilia
 Readily diagnosed
 In severe disease and patients with prior family history
 Diagnosis based on
 Unusual bleeding symptoms early in life
 Age of first bleeding varies with severity of disease
 Family history
 Physical exam
 Laboratory evaluation
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5
Laboratory Diagnosis of Hemophilia A
 The laboratory workup starts with the PT, PTT, and thrombin time and continues with factor
assays based on the results of the screening tests.
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6
Disseminated Intravascular Coagulation /DIC
 Can be either an explosive and life-threatening bleeding or a relatively mild or
subclinical disorder.
 Most frequently associated with metastatic malignancy, massive trauma, pregnancy
complications, incompatible blood transfusion or septicemia
 Tentative triggering mechanisms of DIC;
 Tumors and traumatized or necrotic tissue release tissue factor into the
circulation.
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7
DIC…
Endotoxin from gram-negative bacteria activates several steps in the coagulation

cascade.
In addition to a direct effect on the activation of Hageman factor (factor XII)
Endotoxin induces the expression of tissue factor on the surface of monocytes and
endothelial cells, activated cell accelerate coagulation
These activated cell surfaces then accelerate coagulation reactions.
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8
Table. Causes of DIC
26
9
DIC Cont’d…
 Early phase- Potent thrombogenic stimuli cause the deposition of small thrombi and
emboli throughout the microvasculature.
 Followed by a phase of procoagulant consumption and secondary fibrinolysis
 Continued fibrin formation and fibrinolysis lead to hemorrhage from the coagulation factor
and platelet depletion and the antihemostatic effects of fibrin degradation products.
Clinical presentation varies with the stage and severity of the syndrome.
Most patients have extensive skin and mucous membrane bleeding and hemorrhage from
surgical incisions or venipuncture or catheter sites.
Some patients, particularly those with chronic DIC and malignancy, have laboratory
abnormalities without any evidence of thrombosis or hemorrhage 27
0
Fig .Pathophysiology of DIC
27
1
Conditions that may precipitate DIC
27
2
Course of DIC
27
3
DIC: How Does It Occur?
 Step 1: Out of control clotting
 Causes widespread fibrin deposits in vessels of tissues and organs
 Subsequent event: Hemorrhage
• Clotting proteins consumed at a high rate
– Causes multiple factor deficiencies, especially fibrinogen group
• Platelets caught in thrombi and removed
 Step 2: Triggers Fibrinolytic system to remove fibrin
 Results in:
• Circulating degradation products (FDPs) that interfere with
platelet function & normal clot formation
• Degradation of Factor V & VIII
27
4
DIC: How Does It Occur?
 Step 3: Uncontrolled plasmin and thrombin enter circulation
– Why?
• Inhibitors such as AT(Antithrombin ) have been depleted
 Step 4: Appearance of Symptoms
 Bleeding from multiple sites
 Petechiae
 Purpura
 Occlusions in organs
 Oozing from needle puncture sites
 Shock 99
DIC cont’d…
Laboratory manifestations include

 Thrombocytopenia and the presence of schistocytes or fragmented red blood cells that arise
from cell trapping and damage within fibrin thrombi;
 Prolonged PT and PTT and thrombin time
 A reduced fibrinogen level from depletion of coagulation proteins
 Elevated fibrin degradation products (FDP) from intense secondary fibrinolysis.
 The D-dimer immunoassay, which measures cross-linked fibrin derivatives (i.e., those that
have been in blood clots), is a more specific FDP assay.
 Low fibrinogen levels in DIC predict more bleeding
276
DIC
 Treatment
 Goal is to treat the underlying condition
• Remove the triggering process – treat with antibiotics, antineoplasms, remove

dead tissue, treat the diseases or conditions
 Heparin – to prevent or limit further coagulation
 Replace factors, platelets = give FFP
277
Antithrombin (AT) Deficiency
 AT inhibits blood coagulation by inactivating thrombin and the other serine proteases, i.e.,
factors XIIa, XIa, Xa, and IXa.
 Disorder is inherited as an autosomal dominant disorder.
 There are two types of AT deficiencies: type I and type II.
 Type I is a quantitative disorder & Type II is a qualitative disorder
 Deficiency of AT is associated with recurrent venous thrombosis
 Thrombotic event may be primary or may be followed by another risk factor such as
pregnancy, surgery, or any other acquired factors.
 Acquired AT deficiency may be associated with DIC, liver disease, nephrotic syndrome,
oral contraceptives and pregnancy.
278
specimen
• Specimen of choice is venous whole blood collected by venipuncture and mixed 9:1 with
a 3.2% sodium citrate anticoagulant.
• whole blood for platelet function and platelet-poor plasma for coagulation test.
• Patients need not fast for hemostasis testing.
• Drugs such as aspirin &warfarin (Coumadin) affect the outcomes of coagulation tests;
therefore the phlebotomist should attempt to record all drugs the patient is currently
taking.
• Coagulation tests are performed on 3.2% citrated plasma,
• Platelets are not included in most coagulation tests
05/31/2024 279
specimen
• Hemolyzed samples cannot be tested (free hemoglobin activates platelets and in
vitro clotting).
• Lipemic samples, samples from patients receiving intravenous fat emulsions or
from patients with very high bilirubin levels and
– those with low fibrinogen concentrations may give erratic results.
• Do not refrigerate any blood sample sent for coagulation testing other than
homocysteine.
• Serum does not reflect the in vivo state of circulating blood (Serum
contains no fibrinogen, or factors V, VIII or XIII.).
05/31/2024 280
Coagulation and bleeding Tests
Screening tests:
Specific tests:
•Bleeding Time-Platelet & Blood vessel function •Factor assays–hemophilia.
•Prothrombin Time–Extrinsic, •Tests of thrombosis–TT,FDP,

•Platelet function studies:
•Activated partial thromboplastin time–Intrinsic
•Adhesion , Aggregation ,Release tests.
•Thrombin Time–common pathway
•Bone Marrow study
05/31/2024 281
1. Platelet Function Test
Initial approach to diagnosing platelet dysfunction:
• When investigating suspected bleeding disorder, before choosing the laboratory tests to be undertaken,
• it is essential to obtain: a detailed clinical condition, family history and Perform aphysical examination.
• A recent drug history is critically important
• The laboratory evaluation of platelet dysfunction should always include some basic clotting tests:
• e.g.the APTT and PT:to exclude any potential coagulation defects
• Once the clinician suspects a platelet defect, it is always important to exclude thrombocytopenia as a
primary cause of bleeding by performing a full CBC of then in conjunction with ablood-film examination.
• The blood film can give valuable information about platelet size, granule content and number
05/31/2024 282
Bleeding Time (BT)
• The bleeding time is a measure of vascular ,platelet plug formation and platelet integrity.
• It is measured by determining the time required for bleeding to stop from small
subcutaneous vessels that have been severed by a standardized incision.
• It involves making a puncture wound in a superficial area of the skin and monitoring the
time needed for bleeding to stop
• It is an in vivo screening examination for the interaction between platelets and the blood
vessel wall.
Occasionally, the bleeding time test will be ordered on a patient scheduled for surgery.
Clinical significance of Bleeding time
Diagnosis (particularly of platelet disorders)
Prediction of clinically important bleeding
Assessment of the adequacy of various forms of therapy.
05/31/2024 283
Cont……
Four procedures for determining the bleeding time:
1.The Duke method.
• This is the oldest and easiest method which is performed by puncturing the earlobe
or finger with a lancet.
2.The Ivy Method.
• a blood pressure cuff is placed on the upper arm and inflated to 40 mmHg to control
capillary tone and to improve the sensitivity and reproducibility
3.The Mielke Method.
• The blade is placed in a special handle containing a gauge in order to standardize the
depth of the incision.
05/31/2024 284
4.The Simplate or Surgicutt Method.
IV. Template/Simplate / Surgicutt Method
• The Preferred Method and modified From the Ivy Method.

• The first bleeding time device introduced was the Simplate.
• The Simplate device has a trigger and spring method for the blade.
• The blade has a depth of 1.0 mm and a width of 5.0 mm.
• Another brand name is the Surgicutt.
• Instrument is a sterile, standardized, easy to use device that makes a uniform incision.
• Instrument is a spring activated surgical steel blade which is housed in a plastic unit.
• Eliminates variability of blade incision.
• The most standardized method of all the bleeding time procedures.
05/31/2024 285
Procedure
05/31/2024 286
05/31/2024 287
• Normal Values
– 2.5 – 9.5 minutes

– Bleeding time is shorter in men than in women by an average of
about 30 seconds
• The critical Value for bleeding time is > 15 min.
• If the puncture site is still bleeding after 15 minutes, discountinue the test apply pressure
to the site.
• Document and report the results to the clinician.
05/31/2024 288
Cont.……
The bleeding time is dependent upon
 The efficiency of tissue fluid in accelerating the coagulation process
 Capillary function
 The number of blood platelets present and their ability to form a platelet plug
Prolonged bleeding times are generally found when

• When there is platelet dysfunction.
• Thrombocytopenia (Decreased plt. count <50.0 x109/L
• Inadequate function of platelets von Willebrand’s disease
• Poor retractability of capillaries (e.g. scurvy-Vitamin C deficiency)
• Factor deficiency
• Disseminated intravascular coagulation(DIC)
• Granz Mann's thrombasthenia
• hypofibrinogenemia
 .
05/31/2024 289
2. Tests of the Coagulation Cascade:
• Coagulation time is the time required for a measured amount of blood to clot under certain
specified conditions
• Clinical tests of coagulation are functional assays that evaluate the rate of clot formation
from the time that the coagulation cascade is activated
• These tests are commonly used to identify defects of the extrinsic, intrinsic, and final
common pathways of the coagulation cascade so that more advanced testing can be done
to identify specific defects
05/31/2024 290
http://www.blooddiseasehopkins.org/coagulation.htm
05/31/2024 291
Laboratory Investigations of Coagulation System
1. SCREENING TESTS
• Coagulation time
• Prothrombin time (PT)
• Activated partial thromboplastin time (APTT)
• Thrombin time (TT)
• International Normalization ratio (INR)
2. CONFIRMATORY TESTS
• Reptilase time
• Mixing tests.
• Fibrinogen assay
05/31/2024 292
Coagulation time
• is the time required for a sample of blood to coagulate in vitro under standard conditions
Pathophysiology
1. For the clot formation prothrombin is converted into thrombin
2. Thrombin converts soluble fibrinogen into insoluble fibrin
3. For this process clotting factors are needed along with calcium
4. Also assisted by the factors produced by platelets and damaged tissue.
5. So clotting time is the time needed for the generation of thrombin from the complex
system of clotting
6. When there is any deficiency in these factors, will lead to prolonged clotting time
7. Now, it is rarely used because of the variation, instead, the clotting factors are
more accurate.
Specimen
• Clotting Time is done on a fresh sample of blood and the patient needed to be in
the lab.
05/31/2024 293
Methods
Clotting time can be estimated by two methods:

1.Capillary method
e.g. Dale and laid method
2.Venous blood method
e.g. Lee and White
05/31/2024 294
Procedure
 Dale and laid method
This materials needed:

• Capillary tube (non-heparinized),Stop-watch, Sterile pricking needle (disposable Lancet) and
Cotton and spirit.
1.Draw blood into capillary tubes (Non-heparinized).
2. Start stop-watch immediately after puncture happened.
3. Two minute after puncture happened break a small piece of capillary tube and separated the
two halves slowly .
4. Repeated this procedure at 30 second intervals for clotting and see for fibrin threads
In05/31/2024
the normal case clotting begins 1- 8 minutes 295
Procedure
 Lee and White
This method needs:

1. Three test tubes and rack.
2. Water bath and stop-watch.
3. Blood sample by (venipuncture blood collection).
4. Cotton and spirit
05/31/2024 296
Procedure…
1.Fill the water bath at 37 0C.

2. Let the rack with three test tubes in water bath.
3. Draw 3ml of blood by plastic syringe from vein.

4. Start the watch after appearing of blood in the syringe.
5. Transfer 1ml of blood to each test tube.
6. After one minute remove one test tube from water bath and see the fibrin formation.
If you don't see fibrin, leave all tubes in water bath for 1/2 min and see the clot formation.
7. Stop the watch immediately if you see the fibrin (clotting)
Normal range of clotting time is 5-11 minutes (usually 6-7 minute at 37OC).
05/31/2024 297
2.1. Screening Tests
A. Prothrombin time (PT)

• This assay is used to evaluate the extrinsic and common pathways of the
coagulation cascade (Factors VII, X, V, II and fibrinogen).
Significance
• Reflects overall activity of the Extrinsic Pathway.
• Most sensitive to changes in Factor V, VII, X.
• FX, FV, FII, FI of the common pathway and Lesser to Factor I & II.
• The most common use of PT is monitoring OAT
• 3 of the vitamin K-dependent factors (F-II,VII,X)
Principle
• Platelet poor plasma + Tissue Thromboplastin + Calcium
• In presence of F VII, Extrinsic pathway is activated & clot formed
• Normal range: 10-12 seconds
05/31/2024 298
Procedure of PT.…
• Citrated plasma, an activating agent, and phospholipid are added together and incubated at
37°C.
• Calcium is added, and the time necessary for the clumping of kaolin is measured
• The normal time is usually reported as less than 30 to 35 seconds depending on the
technique used.
• Interpretation
Causes of prolonged PT
1. Deficiency of Factor VII,X,V,II,I
2. Vit K deficiency
3. Liver disease esp.Obstructive Jaundice
4. Oral anticoagulants therapy (OAT)
5. DIC
05/31/2024 299
International normalized ratio (INR)
• PT test may also be called an INR test.
• INR stands for a way of standardizing the results of prothrombintime tests, no matter the
testing method.
• Results understood in the same way even when they come from different labs and
different test methods.
05/31/2024 300
International normalized ratio (INR)
05/31/2024 301
Activated Partial Thromboplastin Time (APTT)
• Principle: Measures the clotting time of plasma after the
activation of contact factors and PL & Ca but without added
tissue thromboplastin.
05/31/2024 302
Thrombin Time
Asses the final step of coagulation i.e. conversion of fibrinogen to

fibrinin the presence of thrombin.
• Bypasses Extrinsic & Intrinsic pathway.
• Principle
• Thrombin is added to plasma and the clotting time is measured.
• TT is affected by the concentration and reaction of fibrinogen and
by the presence of inhibitory substances including fibrinogen/fibrin
degradation products(FDPs) and heparin
• •Normal range:15–19 sec.
05/31/2024 303
Clinical Significance
• markedly prolonged in the presence of heparin or direct thrombin inhibitors such as dabigatran. .
05/31/2024 304
05/31/2024 305

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Hematology for PH

By SAMUEL S.(BSc, MSc)

Upon completion of this chapter the student will be able to:

• Describe the hematopoiesis.

• The regulatory mechanisms in hemopoiesis

• The sites of hemopoiesis in infancy, childhood and adulthood

• Formation of RBC, WBC and platelate

Inorganic ions <1% (0.9%)

Organic ions <1% (0.5-0.9%)

Plasma Proteins Plasma concentration

• The three main blood cells/formed elements are:

• Red blood cells (erythrocytes)

• White blood cells (leucocytes)

• carry oxygen from the lungs to the tissues

• The mesoblastic phase

erythroblasts, granulocytes, and monocytes colonizing the fetal

hematopoiesis, but not to primitive erythroblasts.

• Throughout childhood and adult life, erythrocytes, granulocytes, monocytes, and

• which consists of a fine supporting reticulum framework interspersed with vascular

• EKLF expression relies on GATA1 and CP1.

• Nucleoli not visible

• Slight reduction in size 10 -15μm

• Cell volume is reduced (10-12m in diameter)

• Does not synthesize protein

•Is the measurement of concentration of Hgb in red cells (whole blood)

1. Cyanmet hemoglobin method: reference method

monitor instrument function and reagent.

• Advantage HemoCue® system

– No expensive instrument needed

– Test cuvettes are expensive

– Finger prick technique must be good

– Does not measure carboxyhemoglobin, hemiglobin or sulphhemoglobin

• It is not recommended method because it has the following dis advantages

• A useful ancillary method under special circumstances .

• It gives a true estimate of total Hb even if HbCO, Hi or SHb is present.

• Requires no dilution of blood

• is a qualitative method based on the capacity of a standard solution of copper

• Each health institution should establish its own reference ranges

• Used to estimate values or check data correlation

• Commonly referred to as hematocrit

Significance of the test

– Mean cell volume (MCV),

– Adams microhematocrit method

iii. principle of test

• wall-thickness is centrifuged in a micro hematocrit centrifuge at 10000-15000 rpm for 5 minutes to

• Capillary blood collected into a heparin zed capillary tube

Ethiopia: Adult Males 41.6 – 55.1%

• The morphology of blood cells in stained films is the basis of laboratory

• A careful examination of a well-spread and well-stained film can be more

• In well spread and stained films the great majority of the

• Normal red cell size appears to be the same as the nucleus

• Have diameter greater than 8.0m and the MCV is increased

• Because of their increased hemoglobin content they stain

• stress erythropoiesis as seen in hemolytic anemia

• During recovery from acute blood loss

• Have diameter less than 6.0m but may appear to have

• caused by flattening of the cells during smear preparation

• The mean cell volume is decreased to less than 80.0fl

• Area of central pallor usually increases because of the