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Hematological tests
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Chapter 2
Tassew Tefera
Hematology
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 Hematology is the study of blood and blood components
 Blood is biological fluid that has the supernatant fluid plasma and
cellular components (RBCs, WBCs and platelets).
 Blood is 6-8% of total body weight and equals approximately 5-6
liters.
 The fluid portion of anticoagulated blood is called plasma.
 The fluid portion of coagulated blood is called serum.
Tassew Tefera
Hematology…
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 Serum vs Plasma
 Both are fluid portions of blood
 Serum:
Is from clotted blood, has no clotting factors (used up in the
clotting process)
Serum = Plasma - clotting factor (fibrinogen)
 Plasma:
 Is from anticoagulated blood, has clotting factors, makes up
about 45-55% of blood’s volume.
 Plasma contains water (95%) and many solutes, including
proteins, mineral ions, organic molecules, hormones,
enzymes, products of digestion, and waste products for
excretion.
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Functions of blood
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 Blood has important transport, distribution, regulatory,
and protective functions in the body.
 Blood assists in regulating the temperature of the body
by absorbing and distributing heat throughout the
body.
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Function of plasma
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 Plasma maintain the pH of the blood between pH 7.35–7.45 and the
pH in body tissues within the physiological limits required for normal
cellular activity.
 Proteins (particularly albumin) and salts (particularly sodium chloride)
regulate plasma osmotic pressure, preventing excessive loss of fluid
from the blood into tissues spaces.
 When a blood vessel is damaged, platelets and blood coagulation
factors interact to control blood loss.
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Functions of RBCs
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 Carry Oxygen from the lungs to the tissues.
 This function is performed by hemoglobin which is present in large
amounts in mature red cells.
 Nutrients absorbed from the digestive tract and are transported to the
cells of the body for use or storage.
 Waste products are transported from the tissues to site of excretion,
e.g. Co2 is carried to the lungs, and the waste products of protein
metabolism (urea, creatinine, uric acid) are transported to the kidneys
 Hormones are carried from endocrine glands to the organs where they
are needed.
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Function of platelets
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 Cause clotting of blood
 When a blood vessel is damaged, platelets and blood
coagulation factors interact to control blood loss.
 Platelets adhere to the damaged tissue and to one another
and activated coagulation factors lead to the formation of
fibrin and a thrombus clot which reinforce the platelet plug.
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Function of WBCs
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 Leukocytes are involved in the body’s defenses
 producing antibodies in response to infection
 protecting the body from damage by viruses, bacteria,
parasites, toxins and tumor cells.
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Blood cell production
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 The process by which blood cells are formed is called hematopoiesis or
haemopoiesis.
 Blood is produced in bone marrow
 Hematopoiesis during fetal life and infancy
 The liver and spleen become the main sites of blood cell
production during the second trimester of pregnancy and fetal bone
marrow in the third trimester.
At birth, hematopoiesis is confined to the bone marrow.
 Hematopoiesis during adult life
 By about 25 years of age, the main sites of hematopoiesis is bone
marrow.
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Blood cell production…
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 Production of RBCs is called erythropoiesis
 Production WBCs is called leucopoiesis
 Production of platelets is called thrombopoiesis
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Erythrocyte(RBCs)
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 It is mainly composed of hemoglobin surrounded by a flexible
protein membrane and outer lipid bilayer.
 Have life-span of about 120 days, degenerated red cells are
removed from the circulation by reticuloendothelial cells, mainly
in the spleen.
 Normal Values:
 Males: 4.5 to 6.0 million/mm3 blood
 Females: 4.0 to 5.5 million/mm3 blood
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Erythrocyte(RBCs)
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 Visual description of RBC morphology useful for diagnostic
information.
1. Normocytic—cells are of normal size.
2. Macrocytic—abnormally large cell.
3. Microcytic—abnormally small cell.
4. Normochromic—cells are of normal color.
5. Polychromatic—variation in color of the cells..
6. Hypochromatic—decrease in staining density of the cells, usually
due to a lack of hemoglobin.
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 The main disorders of red cells are:
 Anemia
 Hemoglobinopathies (thalassemia, abnormal hemoglobin)
 Disorders due to red cell enzyme defects, e.g. G6PD
deficiency
 Disorders due to red cell membrane defects, e.g. hereditary
spherocytosis
 Polycythemia
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 Anemia
 It occurs when the concentration of hemoglobin falls
below what is normal for a person’s age, gender, and
environment,
 Results in reduced oxygen carrying capacity of the blood.
 Sign and Symptoms of anemia
 Fatigue, weakness, breathless on exertion, have
palpitations , headaches and dizziness, pallor of the skin,
mucous membranes and conjunctiva.
 Pregnant women with untreated anemia are at increased
risk of dying during or following childbirth.
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Mechanisms of anemia
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BLOOD LOSS
 Acute bleeding, e.g. accident, surgical, ectopic pregnancy.
 Chronic blood loss, e.g. hookworm infection, schistosomiasis,
gastrointestinal bleeding, menorrhagia
DECREASED RED CELL PRODUCTION
 Lack of essential nutrients,
 Depressed bone marrow activity
 Due to drugs
INCREASED RED CELL DESTRUCTION (HAEMOLYSIS)
 hemolytic anemias:
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Microcytic hypochromic anemia
 Iron deficiency (commonest cause)
 Thalassemia syndromes (see later text)
Hemolytic anemia
Aplastic anemia
Megaloblastic anemia
Normocytic normochromic anemia
Types of anemia
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 RBCs are microcytic (microcytic anemia).
 Ferrous (Fe2+) iron is better absorbed than ferric (Fe3+) iron.
 Total body iron store about 4g.
 Iron deficiency is common at reproductive age since menstrual
losses account for ~20mg Fe/month and in pregnancy an
additional 500–1000mg Fe may be lost (transferred from mother
to fetus).
 Causes of iron deficiency Esophagitis, pregnancy, elderly,
hemorrhoids, hookworm infection.
Iron Deficiency Anemia
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Laboratory diagnosis
 Reduced Hgb and HCT
 Reduced MCV (<76FLz) and MCHC
 Increased Red cell distribution width (RDW) in iron deficiency.
Treatment of iron deficiency
 FeSO4 oral dose of 150–200mg/dl per day for 3-6 months.
 Liquid iron occasionally necessary, e.g. children or adults with
swallowing difficulties.
Iron Deficiency Anemia
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Megaloblastic anemia
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 Macrocytic anemia (MCV usually >110fL).
 RBC become megaloblasts (large in size).
 Hyper segmented neutrophils.
 Leucopenia and thrombocytopenia common.
 Iron stores usually increased.
 Caused by vit B12 and Folate deficiency .
 Treatment-hydroxocobalamin 1mg IM and folic acid PO
should be given immediately.
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Hemolytic anemia
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 Is any situation in which there is a reduction in RBC life-span due
to increased RBC destruction.
 Failure of compensatory marrow response results in anemia.
 Lab diagnosis
 Complete blood count (CBC).
 Peripheral blood film:- polychromasia, spherocytosis,
fragmentation (schistocytes), echinocytes.
 Increased reticulocytes.
 Increased serum bilirubin (unconjugated).
 Treatment
 folic acid and iron supplements.
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 A gross reduction or absence of hemopoietic
precursors in all 3 cell lineages peripheral blood.
 Cause pancytopenia
 It a total reduction in;
 Leukocyte
 Platelets
 Reticulocytes
Aplastic anemia
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 Is an increase in total red cell mass.
 It is suspected by finding a raised hematocrit (Hct)
(packed cell volume, PCV).
 Polycythemia Vera (PV) is a neoplastic clonal disorder of
the BM stem cell causing excessive proliferation of the
erythroid carrying a risk of thrombotic complications.
 Hct >0.48 in adult female and >0.54 in adult male is PV.
Polycythemia Vera (PV)
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WHITE BLOOD CELLS
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 In an adult there are about 4.0-10.0 x 109 WBCs
(leukocytes) per litter of blood.
 The white blood cells consist of the granulocytes
(neutrophils; eosinophils; and basophils) and the
agranulocytes (lymphocytes and monocytes).
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WBCs reference range
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ADULTS
 Neutrophils . . . . . . . . . . . . .1.5– 7.5 x 109 /l (40–75%)
 Lymphocytes.. ... . . . . . . . . .1.2– 4.0 “ “ (21–40%)
 Monocytes . . . . . . . . . . . . . 0.2– 1.0 “ “ (2–10%)
 Eosinophils . . . . . . . . . . . . . 0.02– 0.8 “ “ (2–8%)
 Basophils. . . . . . . . . . . . . . . .0.01– 0.1 “ “ (0–1%)
CHILDREN (2–6 y)
 Neutrophils . . . . . . . . . . . . . 1.5– 6.5 109 /l (20–45%)
 Lymphocytes‡ . . . . . . . . . . ..6.0– 8.5 “ “ (45–70%)
 Monocytes . . . . . . . . . .. . . .0.1– 1.0 “ “ (2–10%)
 Eosinophils . . . . . . . . . . . . . 0.3– 1.0 “ “ (1–8%)
 Basophils. . . . . . . . . . .. . . . . . 0.01– 0.1 “ “ (0.1–1%)
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Neutrophils
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 They are highly mobile phagocytes and important in defending
the body from infection.
 Mature neutrophils have a lobulated nucleus.
 They function as phagocytes and are important in infectious
conditions and in inflammation.
 Increased neutrophil (neutrophilia) are caused by
inflammation, bacterial infection, acute stress, and myeloid
leukemia.
 Decreased neutrophil (neutropenia) are caused by viral
infections, toxin exposure (including foodborne toxins),
autoimmune destruction of neutrophils, and typhoid.
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Eosinophil
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 They inactivate histamine and inhibit edema formation.
 Bi-lobed cell
 Increased eosinophil (eosinophilia) are caused by
parasitic infections, tissue injury, allergic reaction, mast cell
tumors, and pregnancy.
 Decreased eosinophil (eosinopenia) is almost always
caused by the action of glucocorticoids, Stress response;
due to trauma, shock, burns, surgery.
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Basophil
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 They are closely related to mast cells and initiate the
inflammatory response by releasing histamine.
 Increased basophil (basophilia): occur in hypersensitivity
reaction, myeloproliferative disorders (myeloid leukemia).
 Basophils bind IgE on their surface and are involved in
anaphylactic, hypersensitivity, and inflammatory reactions.
 Decreased basophil: occur in miscellaneous disorders;
hyperthyroidism, ovulation, pregnancy, stress.
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Monocytes
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 Monocytes are large cells with a kidney bean-shaped
nucleus.
 Their main function is phagocytosis.
 An increased monocyte count (monocytosis) may occur
in chronic inflammation, lymphoid leukemia, subacute
bacterial endocarditis, tuberculosis, hepatitis, malaria,
systemic lupus erythromatus, rheumatoid arthritis.
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Lymphocytes
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 They are the smallest of the white cells, with a round, evenly
staining nucleus and sparse cytoplasm.
 Their primary function is immunologic, including both antibody
production and cell-mediated immune responses.
 An increased lymphocyte count (lymphocytosis) may occur in
leukemia, viral infection and mycobacterium tuberculosis.
 Decreased lymphocyte counts (lymphopenia) are usually due to
an effect of corticosteroids, stress, and in parvoviruses infections.
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Disorders of white blood cells
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The disorders of white cells could be two types:
 Leukocytosis (increased WBCs)
 Leukopenia (decreased WBCs)
 Absolute increases in leukocyte numbers in response to bacterial,
viral, or parasitic infections, tissue injury, and inflammation.
 Absolute decreases in leukocyte can be caused by some infections
(especially HIV), hypersplenism, immune destruction of cells,
treatment with cytotoxic drugs, bone marrow dysfunction,
megaloblastic anemia, collagen disorders, and malignant conditions.
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Common disorders of white cells include:
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Leukemia
 Acute myeloid leukemia (AML)
 Acute lymphoblastic leukemia (ALL)
 Chronic myeloid leukemia (CML)
 Chronic lymphocytic leukemia (CLL)
 Lymphomas
 Myeloma
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Acute myeloblastic leukemia (AML)
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 Malignant tumor of hemopoietic precursor cells of myeloid
lineage, almost certainly arising in the bone marrow.
 Commonest leukemia in adults.
 Caused by cytotoxic chemotherapy (particularly alkylating agents),
ionizing radiation, chromosomal abnormalities and smoking.
 Diagnosis
 Examination of the peripheral blood film and bone marrow ≥20%
blasts.
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Acute lymphoblastic leukemia (ALL)
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 Malignant tumor of hemopoietic precursor cells of the
lymphoid lineage due to marrow failure in most cases.
 Commonest malignancy in childhood.
 Rare leukemia in adults.
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Chronic myeloid leukemia (CML)
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 Malignant tumor of an early hemopoietic progenitor
cell.
 Rare in children and median age of onset is 50 years.
 Irradiation is the only known epidemiological factor.
Diagnosis and investigations
 Blood film show increased WBC (>25 x 109/L, often
100–300 x 109/L): predominantly neutrophils and
myelocytes; basophilia; eosinophilia.
 Anemia common; platelets typically normal or
increased.
 ESR increased in absence of secondary infection.
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Chronic lymphocytic leukemia (CLL)
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 Progressive accumulation of mature-appearing,
functionally incompetent, long-lived B lymphocytes in
peripheral blood.
 Commonest leukemia in adults.
 Predominantly disease of elderly and median age of 65
years.
 Diagnosis
 CBC shows: lymphocytosis >5.0 x 109/L; usually >20 x
109/L, occasionally >400 x109/L
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Platelets
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 Formed from multinucleate megakaryocytes in the bone marrow.
 They maintain the integrity of the endothelium and act as part of
the clotting process to repair damaged endothelium, where they
ensure mechanical strength of the clot.
 Increased platelet counts (thrombocytosis) occur following
injury, after splenectomy, and in megakaryocytic leukemia.
 Decreased platelet counts (thrombocytopenia) are caused by
autoimmune reactions, thrombotic/thrombocytopenic purpura,
bone marrow suppression and aplasia, bone marrow neoplasia.
 Signs are petechiation and hemorrhage.
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Discussion
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1. Describe the etiologies and types of anemia
2. What are the functions of cells and their reference range?
3. What are the disorder of red blood cells?
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 Blood must be collected with care and adequate safety
precautions to ensure test results are reliable,
contamination of the sample is avoided and infection
from blood transmissible pathogens is prevented.
 Types of blood sample
Capillary blood
Venous blood
Arterial blood
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Capillary blood
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 Collected when the volume of blood required is small, e.g. to
measure hemoglobin, perform a WBC count, and to make
thick and thin blood films.
 Collected when the patient is an infant or young child.
 Thick blood films for malaria parasites are best made from
capillary.
 Capillary blood can be collected from:
 The ‘ring’ finger of a child or adult.
 The heel of an infant up to one year old.
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Technique for collecting capillary blood
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 Make sure the puncture area is warm to allow the blood to flow freely.
On cold days soak the hand or foot of an infant in warm water prior
to collecting a sample.
1. Cleanse the puncture area with 70% ethanol and allow the area to
dry.
2. Using a sterile lancet make a rapid puncture, sufficiently deep to
allow the free flow of blood.
3. Wipe away the first drop of blood with a dry piece of cotton wool
and use the next few drops for the test. Do not squeeze too hard
because this will result in an unreliable test result.
4. When sufficient blood has been collected, press a piece of dry
cotton wool over the puncture area until bleeding stops.
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Venous blood
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 Venous blood is used when more than 100 μl of whole blood is
required or when serum or plasma blood sample is needed.
 Venous blood is preferable to capillary blood particularly when the
patient is an adult and several tests are required.
 Technique for collecting venous blood
1. Select a vein.
2. Apply tourniquet the upper arm of the patient to enable the
veins to be seen. Do not apply the tourniquet too tightly.
3. Using the index finger, feel for a suitable vein, selecting a
sufficiently large straight vein that does not roll.
4. Cleanse the puncture site with 70% ethanol and allow to dry.
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Venous blood…
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5. Make the venepuncture with needle directed upwards in the line of the
vein(with angle of 45 degree) and steadily withdraw required volume
of sample.
6. When sufficient blood has been collected, release the tourniquet and
remove the needle and immediately press on the puncture site with a
piece of dry cotton wool.
7. Remove the needle from the syringe and carefully fill the container(s)
with the required volume of blood. Discard the needle safely.
8. Mix immediately the blood in an EDTA or citrate anti-coagulated
container.
9. Check that bleeding from the venepuncture site has stopped.
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Blood film
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Two types
a) Thin blood film:
 Examination of thin blood films is important in the
investigation and management of anemia, infections, and
other conditions which produce changes in the appearance
of blood cells and differential white cell count.
b) Thick blood film
 For examining blood parasites; e.g. malaria parasites,
trypanosomes, and microfilariae.
 Concentrate parasites.
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Staining of blood films
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Blood films are usually stained by;
 Leishman stain usually used for staining leishmania
parasites.
 Wright’s stain used for morphology blood cells and
rarely for malaria parasites.
 Giemsa stain preferable for staining malaria parasites.
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Hematological tests are mainly used:
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 To investigate anemia,
 To investigate infections and pyrexia (fever) of unknown origin
 To investigate clinically important hemoglobinopathies.
 To monitor patients receiving antiretroviral therapy (ART).
 To investigate a bleeding disorder
 To diagnose and monitor a coagulation disorder.
 To diagnose and treat major blood cell disorders (leukemia or
myelomatosis).
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Hematological tests performed in
district laboratories
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 Measurement of hemoglobin.
 Measurement of packed cell volume (hematocrit).
 Red cell indices (MCV,MCH.MCHC RDW).
 Total White blood cell (WBC) count.
 Platelet count.
 Differential WBC count.
 Erythrocyte sedimentation rate(ESR).
 Reticulocyte count.
 Blood film examination.
 Tests to screen for a bleeding and coagulation disorder
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Measurement of hemoglobin
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Hemoglobin (Hgb)
 Hgb is the pigment part of the erythrocyte.
 It accounts three fourth of the red cell membrane.
 Hgb is made up of iron-containing molecules called heme which
are attached to polypeptide chains called globin.
 It has the ability to combine reversibly with oxygen and carbon
dioxide.
 Oxygen is taken up in the lungs and pumped to the tissues.
 After it has been used, the oxygen is replaced by unwanted carbon
dioxide which is carried back to the lungs by the red cells.
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Measurement of hemoglobin
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Value of test:
 Hemoglobin is measured to detect anemia and its severity and to
monitor an anemic patient’s response to treatment.
 Monitoring the Hgb (or PCV) is also required when patients with
HIV disease are being treated with drugs such as AZT.
 The test is also performed to check the hemoglobin level of a blood
donor prior to donating blood.
 Specimen: Capillary blood or EDTA anticoagulated venous blood
can be used.
Measured by
 Sahli hilage /acid haematin method,
 Haemiglobincyanide (Methemoglobin method),
 Hemoglobino meter method (HemoCue system).
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Normal reference range hemoglobin;
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 Children at birth . . . . . . . . . . . . . . . . . 13.5–19.5 g/dl
 Childrens 2 –5 yrs. . . . . . . . . . . . . . . . . 11.0–14.0 g/dl
 Childrens 6 –12 yrs. . . . . . . . . . . . . . . . 11.5–15.5 g/dl
 Adult men. . . . . . . . . . . . . . . . . . . . . . . 13.0–18.0 g/dl
 Adult women . . . . . . . . . . . . . . . . . . . . 12.0–16.0 g/dl
 (Pregnant women) . . . . . . . . . . . . . . . . 11.0–14.5 g/dl
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Interpretation of hemoglobin results
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 Clinician/health worker at point-of-care should identify
anemia and judge its severity in the following clinical
categories:
 Hgb 12 g/dl Not anemia
 Hgb 10–11 g/dl Mild anemia
 Hgb 8–9 g/dl Moderate anemia
 Hgb 6–7 g/dl Marked anemia
 Hgb 4–5 g/dl Severe anemia
 Hgb 4 g/dl Critical
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Packed Cell Volume (PCV)
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 PCV also referred to as hematocrit
 PCV is that proportion of whole blood occupied by red cells,
expressed as a ratio (%).
 Anticoagulated blood in a glass capillary is centrifuged in a
microhematocrit centrifuge at 1000–5000 rpm for 3–5 minutes
to obtain constant packing of the red cells.
 The PCV value is read from the scale of a microhematocrit
reader or calculated by dividing the height of the red cell
column by the height of the total column of blood.
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Packed Cell Volume (PCV)…
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 Increased PCV(HCT)Polycythemia Vera.
 Abnormally low PCV in anemia may be caused by loss of blood
(hemorrhage), break-down of erythrocytes in circulation
(hemolysis), or lack of production of erythrocytes by the bone
marrow (hypoplasia or aplasia).
 Aplastic anemia is always chronic in onset because anemia
occurs gradually as existing cells reach the end of their lifespan.
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Reference value of HCT
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 PCV values vary according to age, gender, and altitude.
 Normal reference value
 Children at birth . . . . . . . . . . . . . . . . . . . .0.44–0.54
 Children 2–5 y . . . . . . . . . . . . . . . . . . . . . 0.34–0.40
 Children 6–12 y . . . . . . . . . . . . . . . . . . . . 0.35–0.45
 Adult men. . . . . . . . . . . . . . . . . . . . . . . . . 0.40–0.54
 Adult women . . . . . . . . . . . . . . . . . . . . . . 0.36–0.48
 Specimen: EDTA anticoagulated blood or capillary blood
collected into a heparinized capillary .
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RED CELL INDICES
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Red cell indices most frequently used in the investigation of anemia are:
 Mean cell hemoglobin concentration (MCHC)
 Mean cell volume (MCV)
 Mean cell hemoglobin (MCH)
MCHC
 The MCHC gives the concentration of hemoglobin in g/l in 1 littre of packed red
cells.
MCHC= Hgb g/dl x100
PCV (%)
 A normal reference range for MCHC in health is 31.5–36.0 g/dl.
 Low MCHC values are found in iron deficiency anemia.
 An increased MCHC can occur in marked spherocytosis.
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Mean Red Cell Volume (MCV)
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 It provides provides information on red cell size.
 Classify RBCs as microcytic and macrocytic.
 Measured in femtolitres (fl) and is determined from the
PCV and electronically obtained RBC count.
 A normal reference range is 80–100 fl.
 Low MCV values: are found in microcytic anemias particularly
iron deficiency, anemia of chronic disease and thalassemia.
 Raised MCV values: are found in macrocytic anemias, marked
B12 or folate deficiency, acquired sideroblastic anemia,
hypothyroidism, chronic respiratory failure, aplastic anemia
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Mean cell hemoglobin (MCH)
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 The MCH gives the amount of hemoglobin in picograms
(pg) in an average red cell.
 It is calculated from the hemoglobin and electronically
obtained RBC count:
 MCH (pg) = Hgb x10
RBCs
 A normal reference range for MCH in health is 27–32 pg.
 Low MCH values: are found in microcytic hypochromic
anemias.
 Raised MCH values: are found in macrocytic
normochromic anemias.
 MCH is also raised in newborns.
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Factors that affect measurements of
erythrocyte parameters:
59
1. Old samples cause RBC to swell, thus increasing PCV and
MCV and decreasing MCHC.
2. Lipemia causes a falsely high Hgb reading, and hence a
falsely high MCHC.
3. Hemolysis causes PCV to decrease while Hgb remains
unchanged, again leading to a falsely high MCHC.
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WHITE BLOOD CELL COUNT
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 A white blood cell (WBC) count is used to investigate
HIV/AIDS, infections and unexplained fever, and to monitor
treatments which can cause leukopenia or leukocytosis.
Principle of test
 Whole blood is diluted 1 in 20 in an acid reagent which
hemolyzes the red cells (non nucleated red cells), leaving the
white cells to be counted.
 White cells are counted microscopically using an Improved
Neubauer ruled counting chamber (hemocytometer) and the
number of WBCs per littre of blood calculated.
 Blood sample: EDTA anticoagulated blood or capillary blood.
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WHITE BLOOD CELL COUNT
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Methods
A) Thoma pipette(o.5 blood to 11 parts of diluting fluid)
B) Tube method(20 microlittre blood to 380 microlittre
fluid)
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Interpretation of WBC counts
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 Reference ranges vary with gender and age.
WBC reference range
 These are guideline figures which should be checked
locally.
 Children at 1 y . . . . . . . . . . . . . . . . 6.0– 18.0 109/l
 Children 4–7 y . . . . . . . . . . . . . . . . 5.0– 15.0 109/l
 Adults . . . . . . . . . . . . . . . . . . . . . . . 4.0– 10.0 109/l
 Adults women . . . . . . . . . . . . . . . . .4.0– 8.0 109/l
 Pregnant women . . . . . . . . . . . . . . Up to 15 109/l
Tassew Tefera
Leukocytosis
63
 The main causes of a raised WBC count are:
 Acute infections; pneumonia, meningitis, abscess,
acute rheumatic fever, septic abortion.
 Inflammation and tissue necrosis.
 Metabolic disorders; diabetic coma and acidosis.
 Poisoning; chemicals, drugs, snake venoms.
 Acute hemorrhage
 Leukemia
 Stress, menstruation, strenuous exercise.
Tassew Tefera
Leukopenia
64
 The main causes of a reduced WBC count are:
 Viral, bacterial, parasitic infections e.g. HIV/AIDS,
viral hepatitis, measles, rubella, influenza, rickettsial
infections, relapsing fever, typhoid, paratyphoid,
brucellosis etc.
 Drugs (e.g. cytotoxic)
 Aplastic anaemia
 Folate and vitamin B12 deficiencies
 Anaphylactic shock
 Ionizing radiation
Tassew Tefera
PLATELET COUNT
65
 A platelet count may be requested to investigate abnormal
skin and mucosal bleeding which can occur when the platelet
count is very low (usually below 20 x 109/l).
Principle of test
 Blood is diluted 1 in 20 in a filtered solution of ammonium
oxalate reagent which lyzes the red cells.
 Platelets are counted microscopically using an Improved
Neubauer ruled counting chamber.
 Blood sample: Use EDTA anticoagulated venous blood.
Capillary blood should not be used because platelets clumpas
the blood is being collected.
Tassew Tefera
Interpretation of platelets counts
66
 Normal reference value 150–400 x 109 platelets/littre of
blood.
 Thrombocytopenia (reduction in platelet number)is due
to:
 Reduced production of platelets (Aplastic anemia).
 Infections, e.g. typhoid, brucellosis
 Deficiency of folate or vitamin B12
 Drugs (e.g. cytotoxic, quinine, aspirin),
 Leukemia, carcinoma
 Hereditary thrombocytopenia (rare condition).
 Disseminated intravascular coagulation (DIC)
Tassew Tefera
Thrombocytosis
67
 Causes of an increase in platelet numbers include:
 Chronic myeloproliferative diseases, e.g. essential
thrombocythemia, polycythemia Vera, chronic myeloid leukemia,
myelofibrosis.
 Chronic inflammatory disease, e.g. tuberculosis
 Haemorrhage
 Splenectomy.
 Iron deficiency anemia, associated with active bleeding.
Tassew Tefera
Erythrocyte sedimentation (ESR)
68
 ESR is a non-specific test.
 It is raised in a wide range of infectious, inflammatory,
degenerative, and malignant conditions associated with
changes in plasma proteins, particularly increases in
fibrinogen, immunoglobulins, and C-reactive protein.
 The ESR is also affected anemia, pregnancy,
haemoglobinopathies, hemoconcentration and treatment
with anti-inflammatory drugs.
Tassew Tefera
Principle of test of ESR
69
 When citrated blood in a vertically positioned Westergren
pipette is left undisturbed, red cells aggregate, stack together
to form rouleaux, and sediment through the plasma.
 The ESR is the rate at which this sedimentation occurs in 1
hour as indicated by the length of the column of clear
plasma above the red cells, measured in mm/hr.
Tassew Tefera
Interpretation of ESR test results
70
 Reference range
 Men . . . . . . . . . . . . . . . . . . . . . Up to 10 mm/hour*
 Women . . . . . . . . . . . . . . . . . . . Up to 15 mm/hour*
 Elderly . . . . . . . . . . . . . . . . . . . . Up to 20 mm/hour*
 Causes of a significantly raised ESR
 Anemia,
 Acute and chronic inflammatory conditions,
 Infections including: HIV disease, Tuberculosis, Pelvic
inflammatory disease , Ruptured ectopic pregnancy , Systemic
lupus erythematosus.
 Drugs, including oral contraceptives
Tassew Tefera
Reduced ESR
71
 Sedimentation is falsely low in polycythemia, dehydration,
dengue hemorrhagic fever, and other conditions associated
with hemoconcentration.
 Abnormally shaped red cells as in sickle cell disease also
lower sedimentation rate.
Tassew Tefera
72
Thank you
Tassew Tefera
Assignment
73
1. The importance of clinical laboratory in prevention and
control of Disease
2. List and discuss types of laboratory testes
3. What is leukemia and how it is caused
4. Write detail about laboratory and medical laboratory
5. Describe the etiologies and types of anemia
6. Discuss about chemistry tests
7. Discuss about microbiological laboratory examination
8. discuss briefly about sensitivity , specificity , positive
predictive value and Negative predictive value
Tassew Tefera

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Chapter 2. Hematology(1).pptx

  • 2. Hematology 2  Hematology is the study of blood and blood components  Blood is biological fluid that has the supernatant fluid plasma and cellular components (RBCs, WBCs and platelets).  Blood is 6-8% of total body weight and equals approximately 5-6 liters.  The fluid portion of anticoagulated blood is called plasma.  The fluid portion of coagulated blood is called serum. Tassew Tefera
  • 3. Hematology… 3  Serum vs Plasma  Both are fluid portions of blood  Serum: Is from clotted blood, has no clotting factors (used up in the clotting process) Serum = Plasma - clotting factor (fibrinogen)  Plasma:  Is from anticoagulated blood, has clotting factors, makes up about 45-55% of blood’s volume.  Plasma contains water (95%) and many solutes, including proteins, mineral ions, organic molecules, hormones, enzymes, products of digestion, and waste products for excretion. Tassew Tefera
  • 4. Functions of blood 4  Blood has important transport, distribution, regulatory, and protective functions in the body.  Blood assists in regulating the temperature of the body by absorbing and distributing heat throughout the body. Tassew Tefera
  • 5. Function of plasma 5  Plasma maintain the pH of the blood between pH 7.35–7.45 and the pH in body tissues within the physiological limits required for normal cellular activity.  Proteins (particularly albumin) and salts (particularly sodium chloride) regulate plasma osmotic pressure, preventing excessive loss of fluid from the blood into tissues spaces.  When a blood vessel is damaged, platelets and blood coagulation factors interact to control blood loss. Tassew Tefera
  • 6. Functions of RBCs 6  Carry Oxygen from the lungs to the tissues.  This function is performed by hemoglobin which is present in large amounts in mature red cells.  Nutrients absorbed from the digestive tract and are transported to the cells of the body for use or storage.  Waste products are transported from the tissues to site of excretion, e.g. Co2 is carried to the lungs, and the waste products of protein metabolism (urea, creatinine, uric acid) are transported to the kidneys  Hormones are carried from endocrine glands to the organs where they are needed. Tassew Tefera
  • 8. Function of platelets 8  Cause clotting of blood  When a blood vessel is damaged, platelets and blood coagulation factors interact to control blood loss.  Platelets adhere to the damaged tissue and to one another and activated coagulation factors lead to the formation of fibrin and a thrombus clot which reinforce the platelet plug. Tassew Tefera
  • 9. Function of WBCs 9  Leukocytes are involved in the body’s defenses  producing antibodies in response to infection  protecting the body from damage by viruses, bacteria, parasites, toxins and tumor cells. Tassew Tefera
  • 10. Blood cell production 10  The process by which blood cells are formed is called hematopoiesis or haemopoiesis.  Blood is produced in bone marrow  Hematopoiesis during fetal life and infancy  The liver and spleen become the main sites of blood cell production during the second trimester of pregnancy and fetal bone marrow in the third trimester. At birth, hematopoiesis is confined to the bone marrow.  Hematopoiesis during adult life  By about 25 years of age, the main sites of hematopoiesis is bone marrow. Tassew Tefera
  • 11. Blood cell production… 11  Production of RBCs is called erythropoiesis  Production WBCs is called leucopoiesis  Production of platelets is called thrombopoiesis Tassew Tefera
  • 12. Erythrocyte(RBCs) 12  It is mainly composed of hemoglobin surrounded by a flexible protein membrane and outer lipid bilayer.  Have life-span of about 120 days, degenerated red cells are removed from the circulation by reticuloendothelial cells, mainly in the spleen.  Normal Values:  Males: 4.5 to 6.0 million/mm3 blood  Females: 4.0 to 5.5 million/mm3 blood Tassew Tefera
  • 13. Erythrocyte(RBCs) 13  Visual description of RBC morphology useful for diagnostic information. 1. Normocytic—cells are of normal size. 2. Macrocytic—abnormally large cell. 3. Microcytic—abnormally small cell. 4. Normochromic—cells are of normal color. 5. Polychromatic—variation in color of the cells.. 6. Hypochromatic—decrease in staining density of the cells, usually due to a lack of hemoglobin. Tassew Tefera
  • 14. 14  The main disorders of red cells are:  Anemia  Hemoglobinopathies (thalassemia, abnormal hemoglobin)  Disorders due to red cell enzyme defects, e.g. G6PD deficiency  Disorders due to red cell membrane defects, e.g. hereditary spherocytosis  Polycythemia Tassew Tefera
  • 15. 15  Anemia  It occurs when the concentration of hemoglobin falls below what is normal for a person’s age, gender, and environment,  Results in reduced oxygen carrying capacity of the blood.  Sign and Symptoms of anemia  Fatigue, weakness, breathless on exertion, have palpitations , headaches and dizziness, pallor of the skin, mucous membranes and conjunctiva.  Pregnant women with untreated anemia are at increased risk of dying during or following childbirth. Tassew Tefera
  • 16. Mechanisms of anemia 16 BLOOD LOSS  Acute bleeding, e.g. accident, surgical, ectopic pregnancy.  Chronic blood loss, e.g. hookworm infection, schistosomiasis, gastrointestinal bleeding, menorrhagia DECREASED RED CELL PRODUCTION  Lack of essential nutrients,  Depressed bone marrow activity  Due to drugs INCREASED RED CELL DESTRUCTION (HAEMOLYSIS)  hemolytic anemias: Tassew Tefera
  • 17. 17 Microcytic hypochromic anemia  Iron deficiency (commonest cause)  Thalassemia syndromes (see later text) Hemolytic anemia Aplastic anemia Megaloblastic anemia Normocytic normochromic anemia Types of anemia Tassew Tefera
  • 18. 18  RBCs are microcytic (microcytic anemia).  Ferrous (Fe2+) iron is better absorbed than ferric (Fe3+) iron.  Total body iron store about 4g.  Iron deficiency is common at reproductive age since menstrual losses account for ~20mg Fe/month and in pregnancy an additional 500–1000mg Fe may be lost (transferred from mother to fetus).  Causes of iron deficiency Esophagitis, pregnancy, elderly, hemorrhoids, hookworm infection. Iron Deficiency Anemia Tassew Tefera
  • 19. 19 Laboratory diagnosis  Reduced Hgb and HCT  Reduced MCV (<76FLz) and MCHC  Increased Red cell distribution width (RDW) in iron deficiency. Treatment of iron deficiency  FeSO4 oral dose of 150–200mg/dl per day for 3-6 months.  Liquid iron occasionally necessary, e.g. children or adults with swallowing difficulties. Iron Deficiency Anemia Tassew Tefera
  • 20. Megaloblastic anemia 20  Macrocytic anemia (MCV usually >110fL).  RBC become megaloblasts (large in size).  Hyper segmented neutrophils.  Leucopenia and thrombocytopenia common.  Iron stores usually increased.  Caused by vit B12 and Folate deficiency .  Treatment-hydroxocobalamin 1mg IM and folic acid PO should be given immediately. Tassew Tefera
  • 21. Hemolytic anemia 21  Is any situation in which there is a reduction in RBC life-span due to increased RBC destruction.  Failure of compensatory marrow response results in anemia.  Lab diagnosis  Complete blood count (CBC).  Peripheral blood film:- polychromasia, spherocytosis, fragmentation (schistocytes), echinocytes.  Increased reticulocytes.  Increased serum bilirubin (unconjugated).  Treatment  folic acid and iron supplements. Tassew Tefera
  • 22. 22  A gross reduction or absence of hemopoietic precursors in all 3 cell lineages peripheral blood.  Cause pancytopenia  It a total reduction in;  Leukocyte  Platelets  Reticulocytes Aplastic anemia Tassew Tefera
  • 23. 23  Is an increase in total red cell mass.  It is suspected by finding a raised hematocrit (Hct) (packed cell volume, PCV).  Polycythemia Vera (PV) is a neoplastic clonal disorder of the BM stem cell causing excessive proliferation of the erythroid carrying a risk of thrombotic complications.  Hct >0.48 in adult female and >0.54 in adult male is PV. Polycythemia Vera (PV) Tassew Tefera
  • 24. WHITE BLOOD CELLS 24  In an adult there are about 4.0-10.0 x 109 WBCs (leukocytes) per litter of blood.  The white blood cells consist of the granulocytes (neutrophils; eosinophils; and basophils) and the agranulocytes (lymphocytes and monocytes). Tassew Tefera
  • 26. WBCs reference range 26 ADULTS  Neutrophils . . . . . . . . . . . . .1.5– 7.5 x 109 /l (40–75%)  Lymphocytes.. ... . . . . . . . . .1.2– 4.0 “ “ (21–40%)  Monocytes . . . . . . . . . . . . . 0.2– 1.0 “ “ (2–10%)  Eosinophils . . . . . . . . . . . . . 0.02– 0.8 “ “ (2–8%)  Basophils. . . . . . . . . . . . . . . .0.01– 0.1 “ “ (0–1%) CHILDREN (2–6 y)  Neutrophils . . . . . . . . . . . . . 1.5– 6.5 109 /l (20–45%)  Lymphocytes‡ . . . . . . . . . . ..6.0– 8.5 “ “ (45–70%)  Monocytes . . . . . . . . . .. . . .0.1– 1.0 “ “ (2–10%)  Eosinophils . . . . . . . . . . . . . 0.3– 1.0 “ “ (1–8%)  Basophils. . . . . . . . . . .. . . . . . 0.01– 0.1 “ “ (0.1–1%) Tassew Tefera
  • 27. Neutrophils 27  They are highly mobile phagocytes and important in defending the body from infection.  Mature neutrophils have a lobulated nucleus.  They function as phagocytes and are important in infectious conditions and in inflammation.  Increased neutrophil (neutrophilia) are caused by inflammation, bacterial infection, acute stress, and myeloid leukemia.  Decreased neutrophil (neutropenia) are caused by viral infections, toxin exposure (including foodborne toxins), autoimmune destruction of neutrophils, and typhoid. Tassew Tefera
  • 28. Eosinophil 28  They inactivate histamine and inhibit edema formation.  Bi-lobed cell  Increased eosinophil (eosinophilia) are caused by parasitic infections, tissue injury, allergic reaction, mast cell tumors, and pregnancy.  Decreased eosinophil (eosinopenia) is almost always caused by the action of glucocorticoids, Stress response; due to trauma, shock, burns, surgery. Tassew Tefera
  • 29. Basophil 29  They are closely related to mast cells and initiate the inflammatory response by releasing histamine.  Increased basophil (basophilia): occur in hypersensitivity reaction, myeloproliferative disorders (myeloid leukemia).  Basophils bind IgE on their surface and are involved in anaphylactic, hypersensitivity, and inflammatory reactions.  Decreased basophil: occur in miscellaneous disorders; hyperthyroidism, ovulation, pregnancy, stress. Tassew Tefera
  • 30. Monocytes 30  Monocytes are large cells with a kidney bean-shaped nucleus.  Their main function is phagocytosis.  An increased monocyte count (monocytosis) may occur in chronic inflammation, lymphoid leukemia, subacute bacterial endocarditis, tuberculosis, hepatitis, malaria, systemic lupus erythromatus, rheumatoid arthritis. Tassew Tefera
  • 31. Lymphocytes 31  They are the smallest of the white cells, with a round, evenly staining nucleus and sparse cytoplasm.  Their primary function is immunologic, including both antibody production and cell-mediated immune responses.  An increased lymphocyte count (lymphocytosis) may occur in leukemia, viral infection and mycobacterium tuberculosis.  Decreased lymphocyte counts (lymphopenia) are usually due to an effect of corticosteroids, stress, and in parvoviruses infections. Tassew Tefera
  • 32. Disorders of white blood cells 32 The disorders of white cells could be two types:  Leukocytosis (increased WBCs)  Leukopenia (decreased WBCs)  Absolute increases in leukocyte numbers in response to bacterial, viral, or parasitic infections, tissue injury, and inflammation.  Absolute decreases in leukocyte can be caused by some infections (especially HIV), hypersplenism, immune destruction of cells, treatment with cytotoxic drugs, bone marrow dysfunction, megaloblastic anemia, collagen disorders, and malignant conditions. Tassew Tefera
  • 33. Common disorders of white cells include: 33 Leukemia  Acute myeloid leukemia (AML)  Acute lymphoblastic leukemia (ALL)  Chronic myeloid leukemia (CML)  Chronic lymphocytic leukemia (CLL)  Lymphomas  Myeloma Tassew Tefera
  • 34. Acute myeloblastic leukemia (AML) 34  Malignant tumor of hemopoietic precursor cells of myeloid lineage, almost certainly arising in the bone marrow.  Commonest leukemia in adults.  Caused by cytotoxic chemotherapy (particularly alkylating agents), ionizing radiation, chromosomal abnormalities and smoking.  Diagnosis  Examination of the peripheral blood film and bone marrow ≥20% blasts. Tassew Tefera
  • 35. Acute lymphoblastic leukemia (ALL) 35  Malignant tumor of hemopoietic precursor cells of the lymphoid lineage due to marrow failure in most cases.  Commonest malignancy in childhood.  Rare leukemia in adults. Tassew Tefera
  • 36. Chronic myeloid leukemia (CML) 36  Malignant tumor of an early hemopoietic progenitor cell.  Rare in children and median age of onset is 50 years.  Irradiation is the only known epidemiological factor. Diagnosis and investigations  Blood film show increased WBC (>25 x 109/L, often 100–300 x 109/L): predominantly neutrophils and myelocytes; basophilia; eosinophilia.  Anemia common; platelets typically normal or increased.  ESR increased in absence of secondary infection. Tassew Tefera
  • 37. Chronic lymphocytic leukemia (CLL) 37  Progressive accumulation of mature-appearing, functionally incompetent, long-lived B lymphocytes in peripheral blood.  Commonest leukemia in adults.  Predominantly disease of elderly and median age of 65 years.  Diagnosis  CBC shows: lymphocytosis >5.0 x 109/L; usually >20 x 109/L, occasionally >400 x109/L Tassew Tefera
  • 38. Platelets 38  Formed from multinucleate megakaryocytes in the bone marrow.  They maintain the integrity of the endothelium and act as part of the clotting process to repair damaged endothelium, where they ensure mechanical strength of the clot.  Increased platelet counts (thrombocytosis) occur following injury, after splenectomy, and in megakaryocytic leukemia.  Decreased platelet counts (thrombocytopenia) are caused by autoimmune reactions, thrombotic/thrombocytopenic purpura, bone marrow suppression and aplasia, bone marrow neoplasia.  Signs are petechiation and hemorrhage. Tassew Tefera
  • 39. Discussion 39 1. Describe the etiologies and types of anemia 2. What are the functions of cells and their reference range? 3. What are the disorder of red blood cells? Tassew Tefera
  • 40. 40  Blood must be collected with care and adequate safety precautions to ensure test results are reliable, contamination of the sample is avoided and infection from blood transmissible pathogens is prevented.  Types of blood sample Capillary blood Venous blood Arterial blood Tassew Tefera
  • 41. Capillary blood 41  Collected when the volume of blood required is small, e.g. to measure hemoglobin, perform a WBC count, and to make thick and thin blood films.  Collected when the patient is an infant or young child.  Thick blood films for malaria parasites are best made from capillary.  Capillary blood can be collected from:  The ‘ring’ finger of a child or adult.  The heel of an infant up to one year old. Tassew Tefera
  • 42. Technique for collecting capillary blood 42  Make sure the puncture area is warm to allow the blood to flow freely. On cold days soak the hand or foot of an infant in warm water prior to collecting a sample. 1. Cleanse the puncture area with 70% ethanol and allow the area to dry. 2. Using a sterile lancet make a rapid puncture, sufficiently deep to allow the free flow of blood. 3. Wipe away the first drop of blood with a dry piece of cotton wool and use the next few drops for the test. Do not squeeze too hard because this will result in an unreliable test result. 4. When sufficient blood has been collected, press a piece of dry cotton wool over the puncture area until bleeding stops. Tassew Tefera
  • 43. Venous blood 43  Venous blood is used when more than 100 μl of whole blood is required or when serum or plasma blood sample is needed.  Venous blood is preferable to capillary blood particularly when the patient is an adult and several tests are required.  Technique for collecting venous blood 1. Select a vein. 2. Apply tourniquet the upper arm of the patient to enable the veins to be seen. Do not apply the tourniquet too tightly. 3. Using the index finger, feel for a suitable vein, selecting a sufficiently large straight vein that does not roll. 4. Cleanse the puncture site with 70% ethanol and allow to dry. Tassew Tefera
  • 44. Venous blood… 44 5. Make the venepuncture with needle directed upwards in the line of the vein(with angle of 45 degree) and steadily withdraw required volume of sample. 6. When sufficient blood has been collected, release the tourniquet and remove the needle and immediately press on the puncture site with a piece of dry cotton wool. 7. Remove the needle from the syringe and carefully fill the container(s) with the required volume of blood. Discard the needle safely. 8. Mix immediately the blood in an EDTA or citrate anti-coagulated container. 9. Check that bleeding from the venepuncture site has stopped. Tassew Tefera
  • 45. Blood film 45 Two types a) Thin blood film:  Examination of thin blood films is important in the investigation and management of anemia, infections, and other conditions which produce changes in the appearance of blood cells and differential white cell count. b) Thick blood film  For examining blood parasites; e.g. malaria parasites, trypanosomes, and microfilariae.  Concentrate parasites. Tassew Tefera
  • 46. Staining of blood films 46 Blood films are usually stained by;  Leishman stain usually used for staining leishmania parasites.  Wright’s stain used for morphology blood cells and rarely for malaria parasites.  Giemsa stain preferable for staining malaria parasites. Tassew Tefera
  • 47. Hematological tests are mainly used: 47  To investigate anemia,  To investigate infections and pyrexia (fever) of unknown origin  To investigate clinically important hemoglobinopathies.  To monitor patients receiving antiretroviral therapy (ART).  To investigate a bleeding disorder  To diagnose and monitor a coagulation disorder.  To diagnose and treat major blood cell disorders (leukemia or myelomatosis). Tassew Tefera
  • 48. Hematological tests performed in district laboratories 48  Measurement of hemoglobin.  Measurement of packed cell volume (hematocrit).  Red cell indices (MCV,MCH.MCHC RDW).  Total White blood cell (WBC) count.  Platelet count.  Differential WBC count.  Erythrocyte sedimentation rate(ESR).  Reticulocyte count.  Blood film examination.  Tests to screen for a bleeding and coagulation disorder Tassew Tefera
  • 49. Measurement of hemoglobin 49 Hemoglobin (Hgb)  Hgb is the pigment part of the erythrocyte.  It accounts three fourth of the red cell membrane.  Hgb is made up of iron-containing molecules called heme which are attached to polypeptide chains called globin.  It has the ability to combine reversibly with oxygen and carbon dioxide.  Oxygen is taken up in the lungs and pumped to the tissues.  After it has been used, the oxygen is replaced by unwanted carbon dioxide which is carried back to the lungs by the red cells. Tassew Tefera
  • 50. Measurement of hemoglobin 50 Value of test:  Hemoglobin is measured to detect anemia and its severity and to monitor an anemic patient’s response to treatment.  Monitoring the Hgb (or PCV) is also required when patients with HIV disease are being treated with drugs such as AZT.  The test is also performed to check the hemoglobin level of a blood donor prior to donating blood.  Specimen: Capillary blood or EDTA anticoagulated venous blood can be used. Measured by  Sahli hilage /acid haematin method,  Haemiglobincyanide (Methemoglobin method),  Hemoglobino meter method (HemoCue system). Tassew Tefera
  • 51. Normal reference range hemoglobin; 51  Children at birth . . . . . . . . . . . . . . . . . 13.5–19.5 g/dl  Childrens 2 –5 yrs. . . . . . . . . . . . . . . . . 11.0–14.0 g/dl  Childrens 6 –12 yrs. . . . . . . . . . . . . . . . 11.5–15.5 g/dl  Adult men. . . . . . . . . . . . . . . . . . . . . . . 13.0–18.0 g/dl  Adult women . . . . . . . . . . . . . . . . . . . . 12.0–16.0 g/dl  (Pregnant women) . . . . . . . . . . . . . . . . 11.0–14.5 g/dl Tassew Tefera
  • 52. Interpretation of hemoglobin results 52  Clinician/health worker at point-of-care should identify anemia and judge its severity in the following clinical categories:  Hgb 12 g/dl Not anemia  Hgb 10–11 g/dl Mild anemia  Hgb 8–9 g/dl Moderate anemia  Hgb 6–7 g/dl Marked anemia  Hgb 4–5 g/dl Severe anemia  Hgb 4 g/dl Critical Tassew Tefera
  • 53. Packed Cell Volume (PCV) 53  PCV also referred to as hematocrit  PCV is that proportion of whole blood occupied by red cells, expressed as a ratio (%).  Anticoagulated blood in a glass capillary is centrifuged in a microhematocrit centrifuge at 1000–5000 rpm for 3–5 minutes to obtain constant packing of the red cells.  The PCV value is read from the scale of a microhematocrit reader or calculated by dividing the height of the red cell column by the height of the total column of blood. Tassew Tefera
  • 54. Packed Cell Volume (PCV)… 54  Increased PCV(HCT)Polycythemia Vera.  Abnormally low PCV in anemia may be caused by loss of blood (hemorrhage), break-down of erythrocytes in circulation (hemolysis), or lack of production of erythrocytes by the bone marrow (hypoplasia or aplasia).  Aplastic anemia is always chronic in onset because anemia occurs gradually as existing cells reach the end of their lifespan. Tassew Tefera
  • 55. Reference value of HCT 55  PCV values vary according to age, gender, and altitude.  Normal reference value  Children at birth . . . . . . . . . . . . . . . . . . . .0.44–0.54  Children 2–5 y . . . . . . . . . . . . . . . . . . . . . 0.34–0.40  Children 6–12 y . . . . . . . . . . . . . . . . . . . . 0.35–0.45  Adult men. . . . . . . . . . . . . . . . . . . . . . . . . 0.40–0.54  Adult women . . . . . . . . . . . . . . . . . . . . . . 0.36–0.48  Specimen: EDTA anticoagulated blood or capillary blood collected into a heparinized capillary . Tassew Tefera
  • 56. RED CELL INDICES 56 Red cell indices most frequently used in the investigation of anemia are:  Mean cell hemoglobin concentration (MCHC)  Mean cell volume (MCV)  Mean cell hemoglobin (MCH) MCHC  The MCHC gives the concentration of hemoglobin in g/l in 1 littre of packed red cells. MCHC= Hgb g/dl x100 PCV (%)  A normal reference range for MCHC in health is 31.5–36.0 g/dl.  Low MCHC values are found in iron deficiency anemia.  An increased MCHC can occur in marked spherocytosis. Tassew Tefera
  • 57. Mean Red Cell Volume (MCV) 57  It provides provides information on red cell size.  Classify RBCs as microcytic and macrocytic.  Measured in femtolitres (fl) and is determined from the PCV and electronically obtained RBC count.  A normal reference range is 80–100 fl.  Low MCV values: are found in microcytic anemias particularly iron deficiency, anemia of chronic disease and thalassemia.  Raised MCV values: are found in macrocytic anemias, marked B12 or folate deficiency, acquired sideroblastic anemia, hypothyroidism, chronic respiratory failure, aplastic anemia Tassew Tefera
  • 58. Mean cell hemoglobin (MCH) 58  The MCH gives the amount of hemoglobin in picograms (pg) in an average red cell.  It is calculated from the hemoglobin and electronically obtained RBC count:  MCH (pg) = Hgb x10 RBCs  A normal reference range for MCH in health is 27–32 pg.  Low MCH values: are found in microcytic hypochromic anemias.  Raised MCH values: are found in macrocytic normochromic anemias.  MCH is also raised in newborns. Tassew Tefera
  • 59. Factors that affect measurements of erythrocyte parameters: 59 1. Old samples cause RBC to swell, thus increasing PCV and MCV and decreasing MCHC. 2. Lipemia causes a falsely high Hgb reading, and hence a falsely high MCHC. 3. Hemolysis causes PCV to decrease while Hgb remains unchanged, again leading to a falsely high MCHC. Tassew Tefera
  • 60. WHITE BLOOD CELL COUNT 60  A white blood cell (WBC) count is used to investigate HIV/AIDS, infections and unexplained fever, and to monitor treatments which can cause leukopenia or leukocytosis. Principle of test  Whole blood is diluted 1 in 20 in an acid reagent which hemolyzes the red cells (non nucleated red cells), leaving the white cells to be counted.  White cells are counted microscopically using an Improved Neubauer ruled counting chamber (hemocytometer) and the number of WBCs per littre of blood calculated.  Blood sample: EDTA anticoagulated blood or capillary blood. Tassew Tefera
  • 61. WHITE BLOOD CELL COUNT 61 Methods A) Thoma pipette(o.5 blood to 11 parts of diluting fluid) B) Tube method(20 microlittre blood to 380 microlittre fluid) Tassew Tefera
  • 62. Interpretation of WBC counts 62  Reference ranges vary with gender and age. WBC reference range  These are guideline figures which should be checked locally.  Children at 1 y . . . . . . . . . . . . . . . . 6.0– 18.0 109/l  Children 4–7 y . . . . . . . . . . . . . . . . 5.0– 15.0 109/l  Adults . . . . . . . . . . . . . . . . . . . . . . . 4.0– 10.0 109/l  Adults women . . . . . . . . . . . . . . . . .4.0– 8.0 109/l  Pregnant women . . . . . . . . . . . . . . Up to 15 109/l Tassew Tefera
  • 63. Leukocytosis 63  The main causes of a raised WBC count are:  Acute infections; pneumonia, meningitis, abscess, acute rheumatic fever, septic abortion.  Inflammation and tissue necrosis.  Metabolic disorders; diabetic coma and acidosis.  Poisoning; chemicals, drugs, snake venoms.  Acute hemorrhage  Leukemia  Stress, menstruation, strenuous exercise. Tassew Tefera
  • 64. Leukopenia 64  The main causes of a reduced WBC count are:  Viral, bacterial, parasitic infections e.g. HIV/AIDS, viral hepatitis, measles, rubella, influenza, rickettsial infections, relapsing fever, typhoid, paratyphoid, brucellosis etc.  Drugs (e.g. cytotoxic)  Aplastic anaemia  Folate and vitamin B12 deficiencies  Anaphylactic shock  Ionizing radiation Tassew Tefera
  • 65. PLATELET COUNT 65  A platelet count may be requested to investigate abnormal skin and mucosal bleeding which can occur when the platelet count is very low (usually below 20 x 109/l). Principle of test  Blood is diluted 1 in 20 in a filtered solution of ammonium oxalate reagent which lyzes the red cells.  Platelets are counted microscopically using an Improved Neubauer ruled counting chamber.  Blood sample: Use EDTA anticoagulated venous blood. Capillary blood should not be used because platelets clumpas the blood is being collected. Tassew Tefera
  • 66. Interpretation of platelets counts 66  Normal reference value 150–400 x 109 platelets/littre of blood.  Thrombocytopenia (reduction in platelet number)is due to:  Reduced production of platelets (Aplastic anemia).  Infections, e.g. typhoid, brucellosis  Deficiency of folate or vitamin B12  Drugs (e.g. cytotoxic, quinine, aspirin),  Leukemia, carcinoma  Hereditary thrombocytopenia (rare condition).  Disseminated intravascular coagulation (DIC) Tassew Tefera
  • 67. Thrombocytosis 67  Causes of an increase in platelet numbers include:  Chronic myeloproliferative diseases, e.g. essential thrombocythemia, polycythemia Vera, chronic myeloid leukemia, myelofibrosis.  Chronic inflammatory disease, e.g. tuberculosis  Haemorrhage  Splenectomy.  Iron deficiency anemia, associated with active bleeding. Tassew Tefera
  • 68. Erythrocyte sedimentation (ESR) 68  ESR is a non-specific test.  It is raised in a wide range of infectious, inflammatory, degenerative, and malignant conditions associated with changes in plasma proteins, particularly increases in fibrinogen, immunoglobulins, and C-reactive protein.  The ESR is also affected anemia, pregnancy, haemoglobinopathies, hemoconcentration and treatment with anti-inflammatory drugs. Tassew Tefera
  • 69. Principle of test of ESR 69  When citrated blood in a vertically positioned Westergren pipette is left undisturbed, red cells aggregate, stack together to form rouleaux, and sediment through the plasma.  The ESR is the rate at which this sedimentation occurs in 1 hour as indicated by the length of the column of clear plasma above the red cells, measured in mm/hr. Tassew Tefera
  • 70. Interpretation of ESR test results 70  Reference range  Men . . . . . . . . . . . . . . . . . . . . . Up to 10 mm/hour*  Women . . . . . . . . . . . . . . . . . . . Up to 15 mm/hour*  Elderly . . . . . . . . . . . . . . . . . . . . Up to 20 mm/hour*  Causes of a significantly raised ESR  Anemia,  Acute and chronic inflammatory conditions,  Infections including: HIV disease, Tuberculosis, Pelvic inflammatory disease , Ruptured ectopic pregnancy , Systemic lupus erythematosus.  Drugs, including oral contraceptives Tassew Tefera
  • 71. Reduced ESR 71  Sedimentation is falsely low in polycythemia, dehydration, dengue hemorrhagic fever, and other conditions associated with hemoconcentration.  Abnormally shaped red cells as in sickle cell disease also lower sedimentation rate. Tassew Tefera
  • 73. Assignment 73 1. The importance of clinical laboratory in prevention and control of Disease 2. List and discuss types of laboratory testes 3. What is leukemia and how it is caused 4. Write detail about laboratory and medical laboratory 5. Describe the etiologies and types of anemia 6. Discuss about chemistry tests 7. Discuss about microbiological laboratory examination 8. discuss briefly about sensitivity , specificity , positive predictive value and Negative predictive value Tassew Tefera