2. Introduction
� The complete blood count (CBC) is one of the most commonly
ordered blood tests.
� The complete blood count is the calculation of the cellular (formed
elements) of blood.
� These calculations are generally determined by special machines that
analyze the different components of blood in less than a minute.
� A major portion of the complete blood count is the measure of the
concentration of white blood cells, red blood cells, and platelets in
the blood.
3. When CBC is ordered?
� CBC may be ordered when a person has any number
of signs and/or symptoms that may be related to disorders that
affect blood cells.
1. When an individual has fatigue or weakness
2. Infection.
3. Inflammation.
4. Bruising, or bleeding.
5. To help diagnose the cause and/or determine its severity.
6. Monitor disease and treatment conditions.
4. Parameters of CBC
The complete blood count, or CBC, lists a number of many important values.
Typically, it includes the following:
1. White blood cell count (WBC or leukocyte count)
2. WBC differential count
3. Red blood cell count (RBC or erythrocyte count)
4. Hematocrit (Hct)
5. Hemoglobin (Hb)
6. Mean corpuscular volume (MCV)
7. Mean corpuscular hemoglobin (MCH)
8. Mean corpuscular hemoglobin concentration (MCHC)
9. Red cell distribution width (RDW)
10. Platelet count
11. Mean Platelet Volume (MPV)
5. White Blood Cells Count
�The normal number of WBCs in the blood is: 4 - 11 X 10 3 /µL,
or 4 - 11 X 10 9 / L
Causes of Leukocytosis Causes of Leukopenia
Infection, most commonly bacterial Bone marrow disorders or damage
Inflammation Autoimmune conditions
Leukemia, Myeloprolifrtive disorders Severe infections (sepsis)
Allergies, asthma cancer that spread to the bone marrow
Tissue death (trauma, burns, heart
attack)
Diseases of immune system (e.g.,HIV)
Intense exercise or severe stress
6. White Blood Cell Differential
WBC Normal range Causes of Increasing Causes of Decreasing
Neutrophils 40-80% of WBCs Neutrophilia mainly associated with
bacterial infections.
Neutropenia may occur due to viral
infections and some medications.
Lymphocytes 20-40% of WBCs Lymphocytosis mainly associated
with acute viral infections,
Lymphocytic leukemia, Connective
tissue diseases
Lymphopenia may due to bacterial,
and fungal infections.
Monocytes 2-10% of WBCs Monocytosis: often occur due to
chronic inflammation, during the
recovery period after severe
infections, infectious mononucleosis.
Monocytopenia may develop due to
acute infections, stress, bone marrow
failure.
Eosinophils 1-6% of WBCs Eosinophilia mainly associated with
allergic diseases, and parasitic
infections.
Eosinopenia found during the acute
stages of all acute infectious diseases.
Basophils 0-1% of WBCs Basophilia caused mainly by allergic
reaction
Basopenia has no diagnostic value.
9. Platelet Count (PLT, Thrombocyte Count)
The normal range = 150 - 400 X 10 3 /µL or 150 - 400 X 10 9 / L
Thrombocytosis caused by:
� Cancer (lung, gastrointestinal, breast,
ovarian, lymphoma)
� Rheumatoid arthritis, inflammatory
bowel disease, lupus
� Iron deficiency anemia
� Hemolytic anemia
� Myeloproliferative disorder (e.g.,
essential thrombocythemia)
Thrombocytopenia caused by
� Viral infection
(mononucleosis, measles, hepatitis)
� Rocky mountain spotted fever, Sepsis
� Platelet autoantibody
� Drugs (acetaminophen, quinidine,
sulfa drugs)
� Leukemia, lymphoma,
Myelodysplasia
� Chemo or radiation therapy
10. Erythrocyte count (RBCs)
�Normal RBC range is:
�Males: 4.5-5.5 X 10 6 c/ µL or 4.5-5.5 X 10 12 c/ L
�Females: 4.0-5.0 X 10 6 c/ µL or 4.0-5.0 X 10 12 c/ L
�RBCs, is generally, decreased in all types of anemia,
and increased in all causes of polycythemia.
11. Hemoglobin
� Hemoglobin measures the amount of oxygen-carrying
protein in the blood.
� Normal values:
� In adult males 13-17 g/dL= 130-170 g/L.
� In adult, non-pregnant females 11.5-15 g/dL=115-150 g/L
�Hb, is generally, decreased in all types of anemia, and
increased in all causes of polycythemia.
12. Hematocrit (Hct), Packed Cell Volume, (PCV)
�Hematocrit is the ratio of the volume of red blood cells
to the volume of blood plasma.
�Normal values (Hct):
�0.40-0.52 L/L= 40-52% in adult males
�0.37-0.47 L/L= 37-47% in adult females.
�Hct, is generally, decreased in all types of anemia,
and increased in all causes of polycythemia.
13. Red Blood Cell Indices
� Red blood cell indices are calculations that provide information on the
physical characteristics of the RBCs:
�Mean corpuscular volume (MCV) is a measurement of the
average size of RBCs.
�Mean corpuscular hemoglobin (MCH) is a calculation of the
average weight (content) of hemoglobin in red blood cells.
�Mean corpuscular hemoglobin concentration (MCHC) is a
calculation of the average percentage of hemoglobin in each
individual RBC.
�Red cell distribution width (RDW), is a calculation of the
variation in the size of RBCs.
14. Mean Corpuscular Volume (MCV)
�MCV = Hct (%) ÷ RBCs count (cell/L) x 10
�Expressed in fl (femtoliter).
�1 liter = 10 15 fl , thus 1 fl= 10 -15 Liter
�e.g.: if the Hct 45%, and the RBCs is 5 X 1012 per liter.
� MCV = 45 ÷ 5 X 10 = 90 fl (femtolitre)
� Normal value for the MCV : 80- 95 fl
� If the MCV is less than 80 fL, means (Microcytic)
� If the MCV is greater than 95 fl, means (Macrocytic)
� If the MCV is within the normal range, the RBCs are Normocytic
15. Mean Corpuscular Hemoglobin (MCH)
� MCH = Hb conc (g/L) ÷ RBCs count (c/L)
Expressed in picogram (pg)
�1 gram = 10 12 pg, thus 1 pg= 10 -12 gram
�e.g: if there are 150g of Hb and 5 X 1012 red cells per litre,
�MCH = 150 ÷ 5 = 30pg (picograms)
� Normal value for the MCH: 27-32 pg
� An MCH lower than 27 pg (Hypochromic)
� An elevated MCH (hyperchromic) occurs in some cases of
spherocytosis.
16. Mean Corpuscular Hemoglobin Concentration
(MCHC)
� MCHC =
e.g: if the Hb concentration is 150g/L and the Hct is 0.45
MCHC = 150 ÷ 0.45 = 333g/L =33.3% (g/dl)
� Normal value for the MCHC : 32-35.5 % or 320-355 g/L
Hemoglobin in g/L
Hematocrit L/L
17. Red cell distribution width (RDW)
� RDW is an index of the variation of red cell size (volume)
in a specimen of blood
Normal range: 13 ± 1.5 %
� Low value indicates uniformity in size of RBCs
� High value indicates a mixed population of small and large
RBCs; (anisocytosis).
18. � Advantages of having RDW :
1. Recognize RBC abnormality from CBC
2. Assist in differential diagnosis
3. Following the course of a disease
20. � Current hematology analyzers use a combination of light
scattering, electrical impedance, fluorescence, light
absorption, and electrical conductivity methods to produce
complete red blood cell, platelet, and leukocyte analyses.
Introduction:
22. � Using this technology, cells are sized and counted by detecting and
measuring changes in electrical resistance when a particle passes
through a small aperture. This is called the electrical impedance
principle of counting cells.
� A blood sample is diluted in saline, a good conductor of electrical
current, and the cells are pulled through an aperture by creating a
vacuum. Two electrodes establish an electrical current.
The Coulter Principle
23. � Low-frequency electrical current is applied to the external
electrode and the internal electrode. Electrical resistance or
impedance occurs as the cells pass through the aperture causing
a change in voltage.
� This change in voltage generates a pulse.
� The number of pulses is proportional to the number of cells
counted.
� The size of the voltage pulse is also directly proportional to the
volume or size of the cell.
24. � Whole blood is aspirated, diluted, and then divided into
two samples.
� One sample is used to analyze the red blood cells and
platelets while the second sample is used to analyze the
white blood cells and hemoglobin.
� Electrical impedance is used to count the white blood
cells, red blood cells, and platelets as they pass through
an aperture.
� The amplitude of each pulse is directly proportional to
the cell volume.
Performance
25. � In the RBC chamber, both the RBCs and the platelets are
counted and discriminated by electrical impedance Particles
between 2 and 20 fL are counted as platelets, and those greater
than 36 fL are counted as RBCs.
� Lyse reagent is added to the diluted sample and used to count
the white blood cells. The lysing reagent also causes WBC's
membrane collapse around the nucleus, so the counter actually
measures the nuclear size.
� After the white blood cells have been counted and sized, the
remainder of the lysed dilution is transferred to the Hgb Flow
Cell to measure Hemoglobin concentration.
26. �Specimen Requirements:
�Whole blood collected in an EDTA tube.
�Samples are stable at room temperature for eight hours.
�Analysis Modes:
�1- Whole blood mode
�2- Pre-diluted mode
27. Sources of Errors in cell count
� 1- Cold agglutinins
� Cause low red cell counts and high MCVs, hematocrits and MCHCs
are also incorrect.
� This can be prevented by warming the sample in a 37°C
28. �2-Fragmented or very microcytic red cells
These may cause red cell counts to be decreased.
Sources of Errors in cell count
29. �3-Platelet clumps and platelet satellitosis:
�These cause falsely decreased platelet counts.
Sources of Errors in cell count
30. �4-Giant platelets:
These are platelets that approach or exceed the size of the red
cells, which cause a decrease in plts count.
Sources of Errors in cell count
31. �4-Nucleated red blood cells:
These interfere with the WBC on some instruments by
being counted as white cells/lymphocytes.
Sources of Errors in cell count
32. �Anything that will cause turbidity and interfere with a
Spectrophotometry method.
�Examples: a very high WBC or platelet count, lipemia,
and hemoglobin's that are resistant to lysis, such as
hemoglobin's S and C.
Sources of Errors in Measuring Hemoglobin
33. �Anything affecting the red cell count or volume
measurement will affect the hematocrit.
Sources of Errors in Hematocrit
34. �Correlating Hemoglobin and Hematocrit Values
�The hemoglobin times three roughly equals the hematocrit in most
patients.
� Example:
�14.8 x 3 = 44 (patient's hematocrit result is 45%)
�11.0 x 3 = 33 (patient's hematocrit result is 32 %)
�The exception to this rule is in patients with hypochromic red
cells. These patients will have hematocrits that are more than three
times the hemoglobin.
35. �In most automated systems, the complete blood count
is numerically reported..
�The differential is numerically recorded and then
graphically displayed
How Data are Reported?
36.
37. RBC and Platelet Histograms
The black line represents normal cell distribution.
The red line on the RBC histogram graphically represents
a Microcytic red cell population.
38. �Red Cells Histogram
�normal red cell histogram displays cells form (36- 360 ) fl
� (24- 36 fl ) flag may be due
1- RBCs fragments
2- WBC's fragments
3- Giant plts
4- Microcyte
� Shift to right :
- Leukemia
- Macrocytic anemia
- Megaloblastic anemia
� Shift to left :
- Microcytic anemia
� Bimodal
- Cold agglutinin
- IDA, Megaloblastic anemia with transfusion.
-Sideroblastic anemia.
� Trimodal
- Anemia with transfusion
45. �The histogram is a representation of the sizing
of the leukocytes. The differentiation is as
follows:
Leukocyte Histogram Analysis
46.
47.
48.
49. � WBC
1. Unusual RBC abnormalities
that resist lysis
2. Nucleated RBCs
3. Fragmented WBCs
4. Unlysed particles greater than
35 fL
5. Very large or aggregated plts
6. Specimens containing fibrin,
cell fragments or other debris
(esp pediatric/oncology
specimens
Interferences that may cause Erroneous results
� RBC
1. Very high WBC (greater than
99.9)
2. High concentration of very
large platelets
3. Agglutinated RBCs.
4. RBCs smaller than 36 fL.
5. Specimens containing fibrin,
cell fragments or other debris
(esp
6. pediatric/oncology specimens
50. Interferences that may cause Erroneous results
� Hgb
1. Very high WBC count.
2. Severe lipemia.
3. Heparin.
4. Certain unusual RBC
abnormalities that resist
lysing.
5. Anything that increases the
turbidity of the sample such
as elevated levels of
triglycerides and High
bilirubin.
� MCV
1. Very high WBC
count.
2. High concentration
of very large
platelets.
3. Agglutinated RBCs
4. RBC fragments
that fall below the
36 fL threshold
5. Rigid RBCs.
51. Interferences that may cause Erroneous results
�RDW
1. Very high WBC
2. High concentration of
very large or clumped
platelets
3. RBCs below the 36 fL
threshold
4. Two distinct
populations of RBCs
5. RBC agglutinates
6. Rigid RBCs
� Plt
1. Very small red
cells near the
upper threshold
2. Cell fragments
3. Clumped platelets
4. Cellular debris
near the lower
platelet threshold
52. Interferences that may cause Erroneous results
�MPV
1. Known factors that interfere with the platelet count and shape
of the histogram
2. Known effects of EDTA
�Hct
Known factors that interfere with the parameters used for
computation, RBC and MCV
�MCH
Known factors that interfere with the parameters used for computation,
Hgb and RBC
�MCHC
Known factors that interfere with the parameters used for computation,
Hgb, RBC and MCV
53. �Plts < 40,000
1. Check the integrity of the specimen (look for clots, short draw, etc.)
2. Confirm count with smear review for clumps, RBC fragments, giant platelets,
very small RBCs
�WBC ++++
Dilute 1:2 with Isoton or further until count is within linearity (for final result, multiply diluted
result by dilution factor); subtract final WBC from RBC; perform spun hct, calculate MCV
from correct RBC & Hct (MCV = Hct/RBC x 10), do not report HGB, MCH, MCHC. Plt
counts are not affected by high WBC. Add comment,
“Unable to report Hgb, MCH, MCHC due to high WBC.”
Handling abnormal results
54. �Plt ++++
� Check smear for RBC fragments or microcytes.
� If present, perform manual plt count.
� If not present, dilute specimen 1:2 with Isoton or further until the count is
within linearity, multiply the diluted result by the dilution factor.
� RBC > 7.0
Dilute 1:2 with Isoton or further until the count is within linearity, multiply
dilution result by dilution factor; perform spun hct, review Hgb, recalculate
MCH, MCHC
Handling abnormal results
56. Mature red blood cells have non nucleus and their colour
cytoplasm is pink, central pallor about 1/3 of the cell. They have
not mitochondria
The red blood cell under the microscope
Image shows Mature red blood cells
Normal RBCs are biconcave, smooth, elastic and allow for
simplicity in movement (i.e. from side to side), in small blood
vessels. The life span of the normal RBC is about 120 days .
57. Blood smear demonstrating features of a patient
with iron deficiency anaemia, including
hypochromic, microcytic RBCs
Blood smear demonstrating features of an individual with
thalassemia demonstrates hypochromic, microcytic RBCs,
variously-shaped (poikilocytosis) RBCs.
* Poikilocytosis = variation in shape of RBCs
Clinical conditions for patients with hypochromic and microcytic picture:-
1. Iron Deficiency
2. Anaemia of chronic disease
3. Sideroblastic anaemia
4. Lead Poisoning
5. Thalassaemia
MCV
58. Iron profile
Disease Serum iron TIBC % Transferin
saturation
Ferritin
Iron Deficiency Low High Low Low
Thalassaemia Normal or
increased
Normal Normal or
increased
Increased
Anaemia of chronic
disease
Low Low Low Normal/High
Sideroblastic anaemia Normal/High Normal/Low High High
Lead Poisoning High Normal High Normal
A low iron with a high transferrin or total iron binding capacity (TIBC) is
usually due to iron deficiency.
In chronic diseases, both iron and transferrin or TIBC are typically low.
High levels of serum iron can occur as the result of multiple blood
transfusions, iron injections into muscle, Lead Poisoning, liver disease or kidney
disease.
59. Blood cells morphology
Morphology Clinical conditions
1. Megaloblastic anaemia
2. Liver disease
3. Alcoholism.
-Nucleus: Round or irregular; -Nucleus/ Cytoplasm: 5:1;
-Chromatin: Fine and closely meshed. This
chromatophilic area is caused by premature Hb; -Nucleoli:
multiple (not visible); -Cytoplasm: deep blue colour,
nongranular with perinuclear hallo
1. Vitamin B12 deficiency
2. Folic acid deficiency
3. Congenital
diserythropnaemia
Picture shows basophilic
Promegaloblast
Picture shows Image shows
Macrocytes
MCV
60. Picture shows basophilic Promegaloblast.
(-Nucleus: Round or irregular
-Nucleus/ Cytoplasm: 5:1
-Chromatin: Fine and closely meshed. This chromatophilic area is caused
by premature Hb.
-Nucleoli: multiple (not visible)
-Cytoplasm: deep blue colour, nongranular with perinuclear hallo
Picture shows Polychromatophilic megaloblast
-Nucleus: Round and central
-Nucleus/ Cytoplasm: 2:1
-Chromatin: Minimal clumbing, loosely defined.
-Nucleoli: Not visible.
-Cytoplasm: Blue-grey to pink-grey. More cytoplasm than in
normoblastic cell.
Clinical conditions for both basophilic Promegaloblast and Polychromatophilic megaloblast
1. Vitamin B12 deficiency
2. Folic acid deficiency
3. Congenital diserythropnaemia
61. RBCs have a hard time making DNA, because there is a lack of B12 and/or folate,
but RNA production proceeds normally. Thus, RBCs have normally maturing
cytoplasm, but slowly-maturing nuclei.
This means that the RBCs grow large before the nucleus becomes mature enough
to signal division and therefore the RBCs end up being larger than normal.
Nucleus looks more immature than the cytoplasm (hence the term “nuclear-
cytoplasmic asynchrony)
Hypersegmented Neutrophil:- Increased size and lobulation, six or more lobes. This
condition is often indicative of reduced DNA synthesis
Clinical conditions :
1. Vitamins B12 deficiency or/and folate deficiency
2. Anti-metabolite therapy or alcoholism
Figures show megaloblastic anaemia.
Macrocytes with Neutrophil hypersegmented.
62. Blood cells morphology
Morphology Clinical conditions
Picture shows Elliptocytes
1. Hereditary
Elliptocyosis
2. Iron deficiency
anaemia (In severe
cases)
Figure shows Heinz bodies (HzB)
Description: Heinz body aggregates of oxidized
denatured precipitated Hb within RBC.
* Heinz body can be easily observed with supra-vital
stains, such as new methylene blue.
1. Glucose-6-Phosphate
Dehydrogenase.
2. Drug-induced anaemia
3. Chronic liver disease .
4. Hyposplenism or a splenia
Description: Elliptocyte is
Oval-shaped (may be slightly
egg, rod, or pencil shaped),
Hb is concentrated at two
ends; normal central pallor.
63. Blood cells morphology
Morphology Clinical conditions
1. Sideroblastic anaemia
2. Lead poisoning
3. Arsenic poisoning
4. Beta-thalassaemia and alpha-
thalassaemia
5. Alcoholism
6. Megaloblastic anaemia
*Description: It is a small fragment of non-functional nucleus
1. Severe iron deficiency anaemia
2. A splenia
3. Hyposplenism
4. Pernicious anaemia
5. Underdeveloped spleen
6. Alcoholism
7. Splenectomized
Images shows Howell-Jolly's body
Basophilic Stippling is also called Punctuate Basophilia
Cell type: Mature RBCs.
Description: Coarse, deep blue inclusions, irregularly aggregated or
clumped ribosomes throughout the cell, mitochnodria and siderosomes
may also aggregate.
Images show
Basophilic Stippling
64. Morphology Clinical conditions
Schistocyte
1. A microangiopathic haemolytic
anaemia (MAHA)
2. Traumatic haemolytic anaemia
.
3. Waring Blender syndrome (the
shearing of erythrocytes by
obstructions of the vascular
bed, such as heartworms and
disseminated intravascular
coagulation
1. Hereditary stomatocytosis
2. Alcoholism
3. Cirrhosis
4. Rh-null disease (D -, C- , E-, c-,
e-)
5. Obstructive liver disease
Stomatocyte
Description Stomatocyte is RBC
with an oval or rectangular area
of central pallor, sometimes
referred to as a "mouth". These
cells have lost the indentation on
one side
Description: Schistocyte is
irregular shape or fragment of cell;
results from damaged membrane.
Some of the irregular shapes appear
as "helmet" cells. Such fragmented
RBC's are known as "schistocytes"
65. Blood cells morphology
Morphology Clinical conditions
Images shows
Acanthocyte
1. Inherited lipid disorder
(Individuals with
abetalipoproteinaemia)
2. Neonatal hepatitis
3. Pyruvate kinase
deficiency
4. Alcoholic cirrhosis
5. Individuals with spur
cell haemolytic anaemia
Picture shows Echinocyte
(Burr cell)
1. Stored blood
2. Severe dehydration
3. Pyruvate kinase
deficiency
4. Burns
5. Renal insufficiency
Description: Echinocytes (also
called burr cells) have serrated
edges over the entire surface of
the cell and often appear
crenated in a blood smear.
Description: The cells appear contracted, dense, and
irregular. Its formation depends on alteration of the
lipid composition and fluidity of the RBC membrane.
Acanthocyte is spherical and densely stained cell
with 3 – 12 spicules of uneven length and width
around the surface.
66. Description: The RBC is shaped like a teardrop. These cells have membrane
abnormalities (Poikilocytosis) and both microcyte and macrocytes
(Anisocytosis).
Clinical conditions
1. Myelofibrosis
2. Megaloblastic anaemia
3. Hypersplenism
4. Metastatic carcinoma.
They are seen when there is extramedullary erythropoiesis or with marrow
disorders or marrow infiltration
Image shows Teardrop cell
(dacrocyte)
Figure shows Red cell
autoagglutination
Description: It is the process whereby RBCs clump
together forming aggregates. This is due to the RBCs
being coated on their surface by antibodies.
Clinical conditions
1. Autoimmune haemolytic anaemia
2. Cold agglutinin disease
3. Waldenström’s macroglobulinemia
4. Trypansomiasis
5. Infection with Mycoplasma Pneumonia or Infectious
mononucleosis.
67. Description: RBC abnormalities in SC include
target cells (double arrow), distorted RBCs due to
the presence of SC crystals (single arrow), and
sickle-like forms (double blunt arrow).
Clinical conditions
1. Haemoglobin SC disease
Picture shows Haemoglobin SC
crystals
Description: It is also called Codocyte. They RBCs that have the
appearance of a shooting target with a bullseye.
In optical microscopy these cells appear to have a dark center (a central,
hemoglobinized area) surrounded by a white ring (an area of relative
pallor), followed by dark outer (peripheral) second ring containing a band
of Hb.
Clinical conditions
1. Haemoglobinopathies (thalassaemia, sickle cell anaemia)
2. Obstructive jaundice
3. Iron deficiency anaemia
Target cells (Codocytes,)
68. Picture shows Pinch cells
Description: it is also called Knizocyte. Pinch cells have two concavities
instead of the one seen with normal RBCs. Like normal erythrocytes,
knizocytes show a clear central area, but this is crossed by a thin strip of
Hb
Clinical conditions of Pinch cells :-
1. Haemolytic anaemia
2. Haemoglobinopathies
3. Pancreatitis
Picture shows Blister cells
Description: it is also called Pyknocyte , acell with a clearing on one side
and a concentrated area of Hb on the other side
Clinical conditions of Blister cells:-
1. Infantile pyknocytosis
2. Infantile viremia.
Description: The hypochromic cell contains small blue granular, irregular-
shaped inclusions on the periphery of the RBC. These iron-containing
(ferritin) granules are called Pappenheimer bodies. The presence of non-heme
iron in the granules is confirmed by A Prussian Blue stain.
Clinical conditions
1. Thalassaemia
2. Sideroblastic anaemia
3. Dyserythropoietic anaemias
4. Myelodysplastic syndrome
Pappenheimer body
69. Bite cells
Description: Bite cell is called Degmacytes. It is RBC with peripheral single or
multiple arcuate defects. Usually associated with spherocytes and blister cells.
Semicircular area of cell removed by spleen
Clinical conditions
1. Normal individuals receiving large quantities of aromatic drugs containing
amino, nitro, or hydroxy groups.
2. Enzymopathies, most notably G6PD deficiency.
3. Drug induced anaemias
4. Thalassaemia and unstable haemoglobinopathies.
Rouleaux Formation.
Description: Stacking of RBCs occurs in the presence of increased amounts of
fibrinogen, immunoglobulin or acute phase reactants in the serum.
In contrast, normal red cell membranes are negatively charged and red cells will
normally "repel" one another. In the presence of positively charged proteins, the red
cells may be brought together in these stacks, known as rouleaux.
Clinical conditions
1. Hyperproteinaemia.
2. Multiple myeloma
3. Macroglobulinaemia
4. Increased fibrinogen (infection, pregnancy
70. � The reticulocyte is the final stage of the development of RBC before full
maturation.
� The presence of reticulocyte in normal blood in very low number (perhaps one
reticulocyte per 100 mature RBCs).
� High number of the reticulocytes may be the product of pathological process or
could be the body’s response to pregnancy, iron, B12 or folic acid therapy,
or to blood loss.
� Reticulocytes are not always part of the FBC and therefore, if required, need to
be specially requested.
� The final step of erythropoiesis is the maturation of reticulocyte into the mature
erythrocyte, which then passes into the blood circulation. However, some
reticulocytes pass directly into the circulation for an additional 1 to 2 days,
following which they differentiate into mature RBCs.
71. � The reticulocyte can be differentiated from the mature erythrocyte not only by
its slightly large size, but also because it contains remnants of messenger RNA
for Hb, detectable by supra vital special stain such as brilliant cresyl blue
(see Figure 1).
� However, although reticulocytes generally can not be specifically identified by
conventional Romanowsky staining, a proportion do stain a slightly bluer
colour than mature RBCs. This bluish tinge is referred to as polychromasia
(see Figure 2).
72. Figure 1. Picture shows Reticulocytes, using vital stain.
Description: They normally make up 1% of the total RBC
count, but may exceed levels of 2.5% when compensating
for anaemia (haemolytic anaemia).
Figure 2. Picture shows Reticulocyte (Poly
chromatophilic ), using Giemsa’s stain.
Description: Non nucleus; Cytoplasm is pink with a
tint of blue. It contains remnants of Golgi and
Mitochondria and residual RNA
Clinical conditions
Low reticulocytes are found in:
1. Chemotherapy
2. Aplastic anaemia
3. Pernicious anaemia
4. BM malignancies
5. Problems of erythropoietin production; various vitamin or mineral deficiencies
High reticulocytes are found in (Reticulocytosis):
1. Increased erythrocyte production (High erythropoiesis)
2. Haemolytic anaemias
73. � As this final step in RBCs maturation can take place in peripheral blood
as well as in the BM, small number of reticulocytes (perhaps 0.5 - 1.5%
of the entire red cell population) may be present in healthy blood.
However, large numbers of reticulocytes, perhaps greater than 2.5%,
imply an abnormality, such as in certain types of anaemia.
� Similarly nucleated red blood cells are seen so rarely in healthy adult
blood that their presence is inevitably the result of a pathological
process, although nucleated RBCs may rarely be seen in healthy neonatal
blood (we will discuss it in detail later).
74. Erythrocyte sedimentation rate (ESR)
This rate of "sinking" is known as the ESR and is an indirect measure of
rouleaux formation.
Conditions that increase the ESR include
1. Infection
2. Inflammation
3. Malignancy (since they are associated with increase in acute phase
reactants)
4. Multiple myeloma (because of elevated immunoglobulin in the serum)
5. Anemia (decrease in the viscosity of blood).
Conditions that decrease the ESR include
1. Polycythemia (because of the increased viscosity of the blood
2. The presence of abnormally shaped red blood cells (sickle cells e.g.
sediment more slowly because of their abnormal shape)
76. A. Red cell membrane defects
1. Hereditary spherocytosis
2. Hereditary elliptocytosis
B. Red cell enzyme defects (Enzymopathies)
1. Glucose-6-phosphate dehydrogenase deficiency
2. Pruvate kinase deficiency
C. Haemoglobinpathies
1. Hb variants ( Hb S, C, E and D)
2. thalassaemia syndrome (α and β thalasaemias)
Classification of inherited haemolytic anaemia
77. Laboratory findings in extravascular
hemolysis
1- Features of increase RBCs breakdown
a- Serum bilirubin raised
b- Urine urobilinogen increased.
c- Fecal strecobilinogen increased.
d- Serum haptoglobin absent.
2- Features of increased red cell production
a- Reticulocytosis.
b- Bone marrow erythroid hyperplasia.
3- Features of damaged red blood cells
a- Red cell morphology ( spherocytes, elliptocytes)
79. 1. Family history: It is an inherited disease
2. Full blood count: Hb concentration and PCV are low in patients with HbSS.
3. Blood morphology: sickle cells, polychromasia, anispoikilocytosis, and target cells are
evident (See the below images)
4. Hb electrophoresis and cation-exchange-high performance liquid chromatography (CE-
HPLC). See the below diagrams
4. Special tests: a. Sickling test: The sickling test is found to be positive in patients with Hb
AS, and it is performed by a powerful reducing agent (sodium dithionate-Na2S2O6).
The test must be done in the deoxygenated condition, decreasing the solubility of Hb S
and inducing the formation of sickle cells.
b. Solubility test (See the below image)
Laboratory diagnosis of sickle cell anaemia
80. � In sickle cell disease
Hb s > 80%,
� Hb F ( 1- 20% ),
� Absence Hb A.
� HB A2 2- 4.5 %
81. Image demonstrates sickle solubility test.
The Sickle Solubility Test (SST) is used to screen for the presence of
sickling Hb.
The SST utilizes a procedure based upon phosphate solubility whereby
RBCs are lysed by saponin and the released Hb is reduced by sodium
hydrosulfite in a phosphate buffer. Reduced HbS is characterized by its very
low solubility and the formation of neumatic liquid crystals (tactoids). The
resulting tactoids of HbS or non-sickling Hb(e.g. HbC-Harlem) causes the
Hb A
(clear red solution) Hb S (Turbid red solution)
Solubility test
84. Hypochromic microcytosis blood picture can be caused by iron deficiency anaemia
(IDA), thalassemia, lead poisoning, sideroblastic anaemia, or anaemia of chronic
disease.
How can you differentiate between these diseases and thalassaemia?
1. Family history: The patient's history can exclude some of these etiologies.
2. Full blood count:
- Low Hb and PCV.
- The MCV is usually less than 75 fl with thalassaemia
- The RDW will be elevated in more than 90% of persons with IDA and sideroblastic
anaemia. , but in only 50% of persons with thalassaemia
3. Blood morphology: There is a slight imbalance in α / β chain synthesis, leading to
hypochromic microcytosis poikilocytosis, and target cells.
Laboratory diagnosis of thalassaemia
85. 4. Iron profile (serum Iron, Transferin saturation, TIBC and Ferritin), as discussed
previously.
5. Hb electrophoresis or CE-HPLC, as shown in the last two images respectively.
6. Genetic analysis
7. BM examination for iron: BM aspirate, stained with Prussian blue or alizarin red dye d
etects the iron in the BM, as shown below:
86. Diagnosis of β-Thalassemia Major
� Hypochromic microcytic
anemia, nucleated red blood
cells and anisocytosis.
� Hemoglobin electrophoresis
in comparison with the
normal control
87. The diagnosis of G-6-PD deficiency is suggested by :
1. Family history
2. Blood morphology: contracted and fragmented cells in the blood film.
3. By the demonstration of Heinz bodies. Reticulocytes have higher levels of G-6-PD than
mature cells
3. Enzyme assay.
Note Its results can be misleading during a haemolytic episode; thus it should be performed
later.
Laboratory diagnosis of G-6-PD deficiency
88. Pruvate kinase deficiency is inherited as autosomal recessive.
The anaemia, which may be moderate to severe, cause relatively mild symptoms as there
is a shift to the right in the O2 dissociation curve due to a rise in intracellular 2,3 DPG.
Pyruvate kinase deficiency presents in childhood with mild jaundice.
1. Family history
2. Full blood count (Hb concentration and PCV will be low)
3. Reticulocyte count will be high because of haemolysis.
4. The diagnosis is also made by measuring erythrocyte pyruvate kinase activity
Pruvate kinase deficiency
Laboratory diagnosis of Pruvate kinase deficiency
90. Disorders of leucocytes may be quantitative or qualitative.
Quantitative disorders
are related to the concentration of leucocytes (leucocyte counts) in
peripheral blood.
Qualitative disorders
refer to the structural or functional abnormalities of white blood
cells.
Leucocytosis is defined as an increase in the number of circulating
leucocytes (total
leucocyte count) above the upper level of normal.
Leucopaenia refers to total leucocyte count below the lower limit of normal.
An absolute rise or fall in the count can affect any white blood cell in
peripheral blood, i.e. neutrophil, eosinophil, basophil, monocyte, or
lymphocyte .
91. Leucoerythroblastic reaction refers to the presence of immature
white blood cells as well as nucleated red cells in peripheral blood.
Leukaemoid reaction
refers to the presence of markedly increased leucocyte count (>50,000/cmm) and immature
white blood cells in peripheral blood resembling leukaemia but occurring
in non-leukaemic conditions.
95. LEUKAEMOID REACTION:
‰
‰
‰
‰
blood picture resembles chronic myeloid leukaemia.
Marked neutrophilic leucocytosis with presence of premature
white cells of all stages (from myeloblasts to segmented
neutrophils) may mimick chronic myeloid leukaemia (CML).
96. DISORDERS OF PHAGOCYTIC LEUCOCYTES CHARACTERISED BY
MORPHOLOGIC CHANGES:
Acquired Morphologic Changes in Neutrophils
Toxic granules
Döhle inclusion bodies and cytoplasmic vacuoles that
are seen in bacterial infections .
Hypersegmentation of nuclei (>5 lobes in >5%
neutrophils) is a characteristic feature of megaloblastic
anaemia
97. Inherited Morphologic Changes
Alder-Reilly Anomaly
Characterized by the presence of abnormally large, darkly
staining granules resembling toxic granules in cytoplasm.
The granules are also variably present in monocytes.
Commonly seen in mucopolysaccharidoses such as Hurler’s
and Hunter’s syndrome.
99. Pelger -Hut Anomaly
In this autosomal dominant disorder, nuclear segmentation
does not occur in granulocytes.
Granulocyte nuclei may be rod-like, round, or at the
most with two
segments (spectacle-like or “pince-nez” nuclei) .
Survival and function of these granulocytes is normal
103. Classification of leukaemia
There are two types of acute leukaemias:
a. Acute Lymphoblastic Leukaemia (ALL)
b. Acute Myeloblastic Leukaemia (AML) or Acute Non Lymphoblastic
Leukaemia (ANLL).
Diagnosis of acute Leukaemia
FAB (French American British system) Classifications
This system classifies patients with acute leukaemia according to:
1. Blood and bone marrow (BM) Morphology: appearence of cell under
microscope.
2. Cytochemistry: chemical activity of the cell.(myeloperoxidase , Sudan Black B)
3. Immunophenotyping: antigen présent in the cell membrane, using flowcytoetry
4. Cytogenetics: Concerning with the study of the structure and function of the cell,
especially the chromosomes.
104. 5. Genetic analysis: Identification of genes and inherited abnormalities
FAB classification for Acute lymphoblatic leukaemia (ALL):
ALL is classified into three categories based on the FAB classification
system.
1. ALL-L1
2. ALL-L2
3. ALL-L3
� This classification according to the cellular morphology.
� L1 and L2 can be either B- or T-lineage, whereas L3 is only B lineage.
� ALL-L1:- It is associated with small homogenous lymphoblasts.
Although the nucleocytoplasmic ratio is high, the scanty cytoplasm
demonstrates variable basophilia.
� Approximately 80% of ALL cases are classified as L1
� ALL-L2:- L2 is associated with much larger, heterogeneous. Although
variable in size, the cytoplasm tends to be more abundant, therefore the
nucleocytoplasmic ratio is lower.
105. � approximately 18% of cases of ALL are classied as L2 categories
� ALL-3:- it constitutes about 2% of all cases ALL. It is essential that
all cases of L3 are correctly claasified as this particular subtype is
associated with specific therapeutic treatment
Differential diagnosis of ALL and AML according to the cell morphology
Morphology ALL ANLL (AML)
Cell size small/moderate Moderate/large
Nuclei usual 1 or 2 nucleoli Frequently more than2
nucleoli.
Cytoplasm Scanty/moderate;
homogeneous
Moderate/abundant; granular,
sometimes with Auer rods
106. Table shows differential diagnosis of ALL and AML according to the
cytochemistry stain
Cytochemistry stain ALL ANLL
1. Peroxidase Negative Positive
2. Sudan black Negative Positive
3. Acid phosphatase Positive Usually negative
Differential diagnosis of ALL and AML according to the immunological
markers
Cluster differentiation (CD) ALL ANLL (AML_
CD19; CD22 Positive (B cell) Negative
CD7, CD2 Positive (T cell) Negative
CD13 and CD33 Negative Positive
CD117 Negative Positive
Glycophorin Negative Positive(M6)
Key:- c= Cytoplasmic
107. FAB
classification
Morphological
features
Immunophenotype Membrane markers
L1 Small; homogenous
cell population
Early B cell: null
(n) cell ALL
CD19
L2 Large;
heterogenous cell
population; one or
two nucleoli
1. Pre B cell:
common (c)
ALL
2. T cell ALL
1. D19 and CD10
2.CD2 and CD7
L3 Large; vaculated
cytoplasm;
heterogenous cell
population; nucleoli
prominent
Mature B cell:
ALL
CD19 and sIg
The FAB morphological classification of ALLwith corresponding
immunophenotype
Key:- sIg = Surface immunoglobulin
108. FAB
Classification
Comments FAB Classification
M0 Undifferentiated myeloblast No cytochemical markers can be defined
M1 Acute myeloblastic leukaemia
without differentiation
M2 Acute myelocytic leukaemia
with differentiation
Also on M2 basophil myeloblasts present.
t(8; 21)
M3 Acute promyelocytic
leukaaemia
Hypergranular promyelocytes. t(15; 17)
M4 Acute myelomonocytic
leukaemia
Myeloid elements resemble M2, peripheral
blood monocytosis. inv
M5 Acute monocytic luekaemia
M6 Acute erythroleukaemia
M7 Acute megakaryocytic
leukaemia
Myelofibrosis may also occur
Table shows The FAB morphological classification of
ANLL (AML)
109. Cytogenetics
In humans, there are two chromosomes that
determine sex: the X and the Y chromosome.
If you have an XX - you are female
If you have an XY - you are male.
Diploid vs Haploid
-Body cells have the full set of chromosomes –
they are --DIPLOID (In humans, 46)
HAPLOID (In humans, 23): Sex cells (sperm
and eggs) have half a set (23).
A karyotype shows all the chromosomes of an
individual, in humans we see 22 pairs of
autosomes, plus 1 pair of sex chromosomes.
(total of chromosomes = 46)
110. Chromosomal abnormalities
It is found in over 80% of cases of ANLL (AML), the commonest
abnormality being trisomy 8.
Some translocations are associated with specific subtypes e.g:
1. t(15; 17) is unique to the M3 type
2. t (8; 21) is found in about 25% of M2 cases, usually young males.
3. Inversion of 16 or deletion of 16q occurs in about 25% of M4 cases
and is associated with eosinophilia.
Chromosomal abnormalities may also influence prognosis for instance :
1. t(8; 21) and inv 16 indicate a favourable diagnosis
2. t(15; 17) and del Y are of intermediate prognosis
3. Others carry a poor prognosis.
111. Flow cytometry
Immunophenotyping by flow cytometry is an important tool in the
diagnosis and staging of patients with a haematological neoplasm.
It is used in conjunction with classical morphology.
At each stage, the cells carry a distinctive set of markers classified by
the CD (cluster of differentiation) nomenclature.
Malignancies can arise at different stages in the development of a cell.
A leukaemia or lymphoma will express a specific set of markers
depending on the stage and pathway of differentiation and they are
classified accordingly (see the below graphs ).
1. B cell: CD5, CD10, CD19, CD20, CD45, Kappa, Lambda
2. T cell: CD2, CD3, CD4, CD5, CD7, CD8, CD45, CD56;
3. Myelomonocytic: CD7, CD11b, CD13, CD14, CD15, CD16, CD33,
CD34, CD45, CD56, CD117, HLA-DR;
4. Plasma cell: CD19, CD38, CD45, CD56, CD138.
113. Chronic Lymphocytic Leukaemia
WBCs count: The outstanding feature is a marked increase in leucocytes,
often 100,000/cmm, or higher
Blood picture: Nearly all are mature small lymphocytes See the below picture.
Variants of Chronic Lymphocytic Leukaemia
1. Prolymphocytic leukaemia
2. Hairy cell leukaemia
3. T cell Chronic Lymphocytic Leukaemia
CLL. The peripheral blood smear shows
an absolute lymphocytosis of small
“mature” lymphocytes with clumped,
smudgy chromatin and scant cytoplasm.
Smudge cells (near top right) are a
frequent finding.
114. Diagnostic and course
Minimum diagnostic criteria are:
(1) a persistent circulating lymphocyte count of >5x103/cmm
(2) BM lymphocytosis > 30%.
The clinical course is extremely variable: it may be rapidly progressive
with a fatal outcome in 1 -2 years or it may be static over decades.
Stage 0 Lyphmphocytosis
Stage 1 Lyphmphocytosis and enlarged lymph nodes
Stage II Lynphmphocytosis and enlarged liver or spleen or both, with or without enlarged nodes
Stage III As with 0, I, II but Hb < 11 g/dl
Stage IV As with 0, I, II or III but platelet count < 100, 000 /cmm
Table shows Clinical staging of CLL
115. Chronic granulocytic leukaemia
While commonest in adults of 30 – 40 years, this disease can occur
at any age.
The clinical picture is usually dominated by growth hepatic and
splenic enlargement.
Signs of impaired marrow function such as anaemia or
thrombocytopenia are in conspicuous until late in the disease.
After a variable period, usually several years, ANLL or common
ALL supervene.
It is generally accepted that CGL arises as a result of a mutation or
series of mutation in a single pluripotential haemopoietic stem cell.
Confirmatory evidence for the monoclonal origin of CGL comes
from the distribution of the Philadelphia chromosome (Ph) in
haemopoietic precursors of CGL patients
Acute transformation in CGL may affect any or several of these
116. Philadelphia chromosome. A piece of chromosome 9 and a piece of
chromosome 22 break off and trade places. The bcr-abl gene is formed on
chromosome 22 where the piece of chromosome 9 attaches. The changed
chromosome 22 is called the Philadelphia chromosome.
117. CML Phases
The clinical courses of CML can be divided into three distinct phases:-
a. Chronic phase
b. Accelerated phase
c. Blast crisis
Chronic phase
The majority of patients who present are in chronic phase, which may last
between 2 and 7 Yrs
Chronic phase is usually associated a hypercellular BM with peripheral
leucocytosis and is responsive to therapy (good prognosis).
The majority of patients at presentation have splenomegaly or
hepatosplenomegaly and this can be reduced in chronic phase, using standard
chemotherapeutic agents.
Accelerated phase
As the disease progresses to accelerated phase, it becomes more difficult to
treat (bad prognosis).
118. The blast (immature) count will increase, although remains less than
20% and the basophil count will equal or exceed 20% (basophilia).
The leucocytosis will become difficult to control and will accompanied
with by either a persistent thrombocytopenia (less than 100x103/cmm)
or a thrombocytosis (in some cases more than 1000 x103/000/cmm)
The presence of thrombocytopenia is part of disease process and is not
secondary to cytoreductive therapy. Additional cytogenetic
abnormalities may be found suggesting disease progression.
Blast crisis describes the transformation to acute leukaemia
The blast count is 20% or higher and immuophenotyping is required
to determine the lineage of the acute leukaemia
119. Laboratory features
The leukocyte count above 250000/cmm (often above 100000/μl),
granulocytes at all stages of development
The basophiles count (basophilia) is increased
The platelet count is normal or increased
It is quite important to distinguish between a pronounced reactive
leucocytosis (leukaemoid reaction), as it is closely similar CML.
Leukaemoid reactions are defined as leucoctosis associated with
immaturity of the WBCs, so that myeloblasts and promyelocytes are
seen in the peripheral blood. This WBC immaturity, which may
resemble CGL.
Leukaemoid reaction is due to causes such as viral infection,
inflammatory reaction or neoplasia.
� The WBCs count in patients with Leukaemoid reaction may reach 50x
103/cmm to 100x 103/cmm with level in excess 100x 103/cmm rarely
reported
120. The diagnosis of CGL is usually easy, but a pronounced reactive
leucocytosis (leukaemoid reaction) can closely mimic CGL.
Features which confirm the diagnosis of CGL include:-
1. A high proportion of myelocytes
2. Elevation of serum vitamin B12, probably due to an increase in
plasma vitamin B12-binding protein.
3. Low to absent levels of leucocyte alkaline phosphatase (LAP) in
patients with CGL
4. At least 85% of patients with CGL will have the Philadelphia
Chromosome, while this chromosomal abnormality is absent in
patients with a leukaemoid reaction
5. An absolute elevation in proliferating marrow cells.
121. � Image shows a small,
hypolobated megakaryocyte
(center of field) in a BM
aspirate, typically of CGL.
Picture shows peripheral
blood (MGG stain): marked
leucocytosis with
granulocyte left shift
123. Abnormal bleeding may result from:
1. Vascular disorders (Inherited and acquired disorders)
2. Platelets disorders which include:
a. Qualitative disorders (Dysfunction platelets )
b. Quantitative disorders (thrombocytopenia)
3. Defective coagulation (coagulopathy) [Inherited and acquired disorders]
Clinical differences between diseases of platelets, vessel wall and coagulation
factors
Finding Platelets/ vessel wall
disorders
Coagulation disorders
Mucosal bleeding Common Rare
Petechiae Common Rare
Deep haematomas Minimal Characteristic
Bleeding from skin cuts Persistent Minimal
124. Inherited Vascular disorders:-
1. Hereditary haemorrhagic telangiectasia. (Dilatation in blood vessels)
The exact causes of telangiectasia are unknow, it may be genetic,
environmental, or a combination of both
2. Connective tissue disorders
3. Giant cavernous haemangioma
Acquired Vascular disorders:-
1. Simple easy bruising
2. Senile purpura
3. Purpura associated with infections.
�Note: Purpura is a condition of red or purple discolored spots on the skin.
Thrombocytopenia and defective platelet function
They are associated with abnormal bleeding and characterised spontaneous
skin purpura, mucosal haemorrhage and prolonged bleeding after trauma.
125. Causes of thrombocytopenia
1. Increased consumption of platelets
a. Immune (drug induced, heparin, autoimmune)
b. Disseminated intravascular coagulation (DIC).
C. Thrombotic thrombocytopenic purpura (TTP)
2. Failure of platelet production
a. Selective megakarocyte depression(drugs, chemical, viruses)
b. Part of BM failure ( Cytotoxic drugs, radiopathy, aplastic
anaemia, leukaemia, HIV, lymphoma and carcinoma)
3. Abnormal distribution of platelets:
Splenomegaly
4. Dilutional loss:
It happens in massive transfusion of stored blood to bleeding patients
126. *Failure of platelet production is the most common thrombocytopenia.
*Rarely, failure of platelet production is congenital as a result of mutation
of the c-MPL thrombopoietin receptor, in association with absent radii, or in
May-Hegglin
Increased destruction of platelets : It is autoimmune (idiopathic)
thrombocytopenic purpura (ITP): ITP is classified into, chronic and
acute.
*Chronic ITP: Its highest incidence occurs among women aged 15 – 50
years.
127.
128. Coagulation factors deficiency
Inherited coagulation disorders
a. Haemophilia A (VIII)
b. Haemophilia B (Chrismus disease) (IX)
c. Von Willibrand disease (VWF)
Acquired coagulation disorders include
a. Liver disease
b. Vitamin K deficiency (II, VII, IX and X). They are called
vitamin K dependant factors.
c. Disseminated intravascular coagulation (DIC)
d. Haemorrhagic disease of the newborn (sterile gut ……..
No bacterial growth. No vitamin K dependant factors. (II,
VII, IX and X)
129. Haemophilia A
Deficiency of FV111 is referred to haemophilia A and
it is inherited as X linked
Laboratory diagnosis of haemophilia A
a. Activated Partial Thromboplastin Time (APTT) is
prolonged
b. Factor VIII clotting assay is low
c. Prothrombin time (PT) and bleeding time are
normal
130. Classes of Haemophilia A based on FV111 activity
Haemophilia B (Christmas disease)
Haemophilia B
Deficiency of 1X is referred to haemophilia B and it is also
inherited as X linked, as haemophilia A
Haemophilia B is called Chrismas disease, as it is named after the
first boy described with it, Stephen Chrismas.
Both diseases, haemophilia A and haemophilia B almost occur
exclusively in males.
Classified as Severe Moderate Mild
FVIII (IU/dl)
Quantity of FVIII
< 1 IU/dl 1 - 5 IU/dl >5 IU/dl
Age at presentation Infancy < 2Yrs >2 Yrs
Bleeding symptoms
and frequency
Frequent spontaneous
bleeds into joints, muscles
and internal organs.
Severe bleeding after
trauma
Much fewer spontaneous
bleeds than patients with
severe disease.
Minor trauma can precipitate a
bleed
No spontaneous bleeds.
Bleeding after significant
trauma/surgery
131. Factor 1X is coded by a gene close to the gene which is coded for
factor V111near the tip of the long arm of the X chromosome.
Carrier detection and antenatal diagnosis is detected the same as
haemophilia A.
Factor 1X have a longer biological half life compared to the factor
VIII.
Laboratory diagnosis of haemophilia B:
a. Activated Partial Thromboplastin Time (APTT) is prolonged
b. Factor 1X clotting assay is low
c. Prothrombin time (PT) is normal
Haemophilia C (Rosenthal’s disease)
Haemophilia C is due to a deficiency of FXI and it is an autosomal
disorders. Rare
132. Von willibrand disease (VWD)
VWD is inherited as autosomal dominant and it is due either a decreased
level or abnormal function of Von Willibrand factor (VWF) as a result of
a point mutation or major deletion.
VWF activates platelets adhesion to damaged endothelium and it the
carrier molecule for factor VIII, preventing it from premature
destruction.
There are three types of VWD, type 1, type 2 and type 3
Approximately 70% of patients with VWD have a quantitative
deficiency of normal functioning VWF (type 1 ) and approximately 25%
have a structural /functional defect (type 2 ). The remainder have severe
VWD where VWF is absent ( type 3).
-VWD type 1 is due to partial quantitative deficiency of VWF.
-VWD type 3 represents a complete deficiency in VWF.
- VWD- type 2 occurs as a result of functional abnormality in VWF
133. Laboratory diagnosis of VWD
1. VWF levels are usually low
2. Activated Partial Thromboplastin Time (APTT) may be prolonged
3. Factor VIII clotting assay is often low
4. Bleeding time can be prolonged
5. The platelet count is normal except for type 2 VWD
6. PT, TT and fibrinogen are unaffected
134. Congenital abnormalities of fibrinogen are divided into 2 types:-
Type I, or quantitative abnormalities (afibrinogenemia and
hypofibrinogenemia):
result from mutations that affect plasma fibrinogen concentration inherited
on both chromosomal alleles and are frequently associated with a bleeding
diathesis but occasionally a thrombotic event.
Type II, or qualitative abnormalities (dysfibrinogenemia and
hypodysfibrinogenemia).
marked by functional abnormalities of fibrinogen who carry one
abnormal allele that may result in either bleeding or thrombosis.
135. Acquired coagualtion disorders
Acquired coagualtion disorders are more common than inherited
coagualtion disorder.
The acquired coagualtion disorders are classified into:
1. Disseminated intravascular coagulation (DIC).
2. Deficiency of vitamin K-dependent factors
3. Malabsorption of vitamin K (e.g. indandion, coumarins)
4. Haemorrhagic disease of the newborn (sterile gut…..No bacterial
growth)
5. Biliary obstruction
6. Inhibition of coagulation
7. Miscellaneous
a. Diseases with M protein
b. Massive transfusion syndrome
c. Therapy with heparin, defibrinating agents or thrombolytic
136. Table shows acquired bleeding disorders
Primary disorder Acquired cause of bleeding
Sepsis, shock, obstetric calamities ,
trauma, surgery, some malignancies,
transplant rejection, recreational
drugs, snake bite.
Acute DIC
Some malignancies, chronic
infections, chronic kidney disease
Chronic DIC (less severe than acute DIC)
Vitamin K deficiency Dietary deficiency or mal-absorption
reduces synthesis of vitamin K dependent
factors.
Newborns are vitamin K deficient and can
present with haemorrhagic disease of the
newborn.
Amyloidosis (extracellular protein deposition) Impaired platelet function, amyeloid binds FX causing plasma
FX deficiency and can interfere with fibrin polymerization
137. Primary disorder Acquired cause of bleeding
Liver disease Reduced synthesis of coagulation factors
and thrombocytopenia
Renal disease Ureamia and increases prostacyclin
release impair platelet function
Autoantibodies to coagulation factors or
platelets
Reduction in affected coagulation factor
or platelet numbers
Dilutional coagulopathy Massive transfusion of stored blood
products and/or volume replacement
with blood substitutes (i.e. they do not
contain coagulation factors and live
platelet) can reduce platelet numbers and
concentrations of circulating coagulation
factors.
Drug therapy Anticoagualnt therapy
Various drugs impair platelet function.
138. Laboratory investigation of a suspected bleeding disorder
Diagnosis of a bleeding disorder is dependent on the presence of bleeding
symptoms in the patient, yet compiling history is subjective. When there is clinical
suspicion of a bleeding disorders, characterization of the presence and nature of any
defect (s) starts with the performance by biomedical scientists of screening tests that
will indicate the area (s) of haemostasis affected (See the blow Table)
Area of haemostasis Tests Result determinants
Platelet /vessels Platelet count
Platelet film examination
Platelet function test
Bleeding time
Platelet count
Platelet morphology
Platelet function; VWF
Platelet function;
Coagulation Prothrombin time (PT)
Activated partial thromboplastin time
(APTT)
Thrombin time (TT)
Fibrinogen activity
Levels of factors II, V, VII, X and
fibrinogen + inhibitors
Levels of factors II, V, VIII, IX, X,
XI, XII, PK, HMWK and fibrinogen
Level of fibrinogen
Direct measurement of fibrinogen
Fibrinolysis D-dimers
* D-dimer test helps in diagnosis patients
with thrombosis
Direct measurement of Fibrin
degradation product (FDPs)
139. Prothrombin time (PT)
In this test the patient’s plasma will form a fibrin clot via the extrinsic pathway
and common pathway and the time taken to clot is recorded as the PT.
The patient’s PT, measured in seconds, is compared to a reference range which
will vary depending on the analytical technique and type of thromboplastin
used.
An elevation of PT may indicate one or more of the following:-
1. Deficiencies of factors II, V, VII, X or fibrinogen.
2. Autoantibodies against the above clotting factors.
3. Anticoagulant drugs affecting the production of vitamin K-dependent factors
(i.e. Warfarin)
4. Anticoagulant drugs directly affecting thrombin
5. Liver disease
6. DIC
Activated partial thromboplastin time (APTT)
In this test the patient’s plasma will form a fibrin clot via the intrinsic and
common pathways
140. An elevation of APTT may indicate one or more of the following:
1. Deficiencies of factors II, VIII, IX, XI, XII, PK, HMWK or
fibrinogen
2. Some subtypes of VWD (due to associated FVIII deficiency)
3. Autoantibodies against the above clotting factors
4. Anticoagulant drugs affecting the production of vitamin K
dependent factors, although the APTT is less affected than the
PT because there are more non-vitamin k dependent factors
contributing to the clotting time.
5. Anticoagulant therapy with heparin
6. Anticoagulant drugs directly affecting thrombin
7. Vitamin K deficiency
8. Liver disease
9. DIC
141. Result interpretation of PT and APTT
Result Interpretation
High PT and APTT Deficiency exists in the common pathway
PT only elevated FVII deficiency
APTT only elevated Deficiency exists in the intrinsic pathway
Thrombin time
The thrombin time (TT) is a simple test, whereby thrombin is added to
patient plasma to directly convert fibrinogen to fibrin.
The elevation of TT above the reference may indicate one or more of
the following:
a. Hypofibrinogenemia –acquired causes such as DIC are more
commonly encountered than hereditary types
b. Dysfibrinogenemia can be hereditary or acquired (i.e. liver disease)
c. Afibrinogenemia.
d. Anticoagulant drugs directly affecting thrombin
e. Hypoalbunaemia, amyloidosis, paraproteins and elevated levels of
fibrin/fibrinogen degradation products (FDP) can interfere with fibrin
polymerization.
