Group MembersAqsa AbbasiAyesha SajidAzka KhanMidhat MustafaSadia ArshadFatima RiazRida Rehman

Routine Hematology Tests

What is Hematology

• Full Blood Count (FBC) or Full Blood Examination (FBE) •The study of blood, the blood-forming organs, and blood diseases. •The study of etiology, diagnosis, treatment, prognosis, and prevention of blood diseases that affect the production of blood and its components.• These results are used in conjunction with the history, physical exam and other laboratory findings.•Qualitative and Quantitative Tests

•Hematology tests helps in:

treating bleeding disorderstreating hematological malignaciesScreen, diagnose and measure the severity of diseasein the science of blood transfusion and the work of a blood bankin bone marrow and stem cell transplantation

Hemoglobin value

Hematocrit value

Red Blood Cell Count

White Blood Cell Count

Complete Blood cell

count(CBC)

Platelet Count

Reticulocyte count

Erythrocyte sedimentataion Rate (ESR)

Prothrombin Time

Routine Hematolgy Tests

Complete Blood Cell Count

A complete blood count, often referred to as a CBC, is a common blood test.

A CBC provides detailed information about three types of cells in the blood.

Alexander Vastem is the first person to use the complete blood count for

clinical purposes.

A CBC is routinely performed during annual physical examinations

Blood cell count principles

AUTOMATIC METHOD• automated analyzer, flow-

cytometry• It counts the numbers and

types of different cells within the blood.

• It aspirates a very small amount of the sample through the narrow tubing.

• light detectors and electrical impedance.

• One way the instrument can tell what type of blood cell is present is by size.

• Other instruments measure different characteristics of the cells to categorize them.

MANUAL BLOOD COUNT

• This measurement is made with a microscope and a specially ruled chamber(hemocytometer) using diluted blood.

• Counting chambers that hold a specified volume of diluted blood are used to calculate the number of red and white cells per litre of blood

Thrombocytopenia maybe due to •Cancer chemotherapy•Hemolytic anemia•Idiopathic thrombocytopenic purpura Leukemia•Massive blood transfusion•Thombotic thrombocytopenic purpura (TTP)

Thrombocytosis may be due to:

•Chronicmyelogenousleukemia  (CML)•Polycythemia vera•Primary thrombocythemia•Recent spleen removal

•A platelet count is a test to measure how many platelets you have in your blood. •They are smaller than red or white blood cells.•Normal Range: 150,000 - 400,000 platelets per microliter

Platelet Count

CBC (RBCs, WBCs)

Ayesha Sajid

RED BLOOD CELL TESTNumber of RBCs per volume of whole blood

RANGEMale 4.5-6.4 Mil/c mm

Female 4.0-5.4 Mil/ c mm

HEMOGLOBIN Total amount of oxygen carrying protein inside RBCs

RANGEMale 14-18 Gm/100 mlFemale 12-16.4 Gm/100 ml

HEMATOCRIT Percentage of blood volume made up of RBCs

RANGEMale  42%-54%Female 38%-46%

RED BLOOD CELL INDICES

Mean corpuscular volume (MCV)• Calculates the

size of RBCs• Differential

diagnosis of anemias

Mean corpuscular hemoglobin (MCH)• Amount of

hemoglobin per red blood cell

• Differential diagnosis of anemias

Mean corpuscular hemoglobin concentration (MCHC) • Concentration

of hemoglobin per red blood cell

• Used for laboratory quality control

TYPES OF ANEMIAMicrocytic anemia: small RBCsNormocytic anemia: Normal red blood cellMacrocytic anemia: larger than normal red blood cell

TOO MUCH VS TOO LOWHIGH LEVELS LOW LEVELS

Dehydration Anemia

Polycythemia vera Autoimmune diseases

Kidney disease hemorrhage

heart or lung disease Bone marrow failure

Smoking Hemolysis

High altitude Long-term infections and poor diet and nutrition

WHITE BLOOD CELLS

PRINCIPLEWhole blood is diluted 1 in 20 in an acid reagent and white blood cells are counted

Measures total white blood cell component of whole bloodRANGEMales: 5,000 to 10,000 per microliter (µL) of bloodFemales: 4,500 to 11,000 per microliter (µL) of bloodLeukocytos

is

• Acute infections• Inflammation and

tissue necrosis• Metabolic

disorders• Poisoning• Leukemias

Leukopenia

• Viral, bacterial, parasitic infections

• Drugs• Folate and vitamin

B12 deficiencies• Bone marrow

infiltration

DIFFERENTIAL WBCs COUNT

LEUKEMIATypes of leukemia Description

Acute myelogenous leukemia (AML)

Cancer of myeloid cells. Too many WBCs and not enough RBCs and platelets.

Chronic myelogenous leukemia (CML)

increased and unregulated growth of predominantly myeloid cells.Increased granulocytes 

Acute lymphocytic leukemia (ALL)

 Characterized by excess lymphoblasts.

Chronic lymphocytic leukemia (CLL)

 CLL affects B cell lymphocytes. Lymphocytosis

RETICULOCYTE COUNT

Azka Khan

Reticulocyte Count

Reticulocytes • immature erythrocytes• slightly larger than totally mature erythrocytes• Non-nucleated.• about 1% of the red cells in the human body • develop and mature in the red bone marrow and then circulate for

about a day in the blood stream before developing into mature red blood cells.

• Spend 2 to 3 days in bone marrow.• 1 day in peripheral blood before developing into mature erythrocytes.• Do not have completed production of hemoglobin and consequently

contain residual hemoglobin synthetic machinery, mRNA and rRNA, commonly referred to as “reticulin’’.

• They are called reticulocytes because of a reticular (mesh-like) network of ribosomal RNA that becomes visible under a microscope with certain stains such as new methylene blue.

•

Reticulocytes Count A reticulocyte count measures the percentage of reticulocytes in the blood and assess the erythropoietic activity of the bone marrow.

Why there is a need to perform• As a follow up to abnormal results on a CBC, RBC

count, hemoglobin or hematocrit, to help determine the cause

• To determine if the bone marrow is functioning properly and responding adequately to the body's need for red blood cells (RBCs)

• To help detect and distinguish between different types of anemia

• To monitor response to treatment, such as that for iron-deficiency anemia

• To monitor bone marrow function following treatments such as chemotherapy

• To monitor function following a bone marrow transplant.

High valuesA high reticulocyte count may mean more red blood cells are being made by the bone marrow. This can occur after a lot of bleeding, a move to a high altitude, or certain types of anemia. These conditions cause red blood cells to break down (hemolysis).The reticulocyte count rises after the treatment for pernicious anemia, iron deficiency anemia, or folic acid deficienc anemia starts working.

Low valuesA low reticulocyte count may mean fewer red blood cells are being made by the bone marrow. This can be caused by aplastic anemia or other types of anemia, such as iron deficiency anemia.A low reticulocyte count can also be caused by exposure to radiation, a long-term (chronic) infection, or by certain medicines that damage the bone marrow.

• Whole blood anticougulated with EDTA, is stained with supra-vital dyes (stain living cells) such as methylene blue.

• As reticulocytes are immature cells containing remnant cytoplasmic ribonucleic acid (RNA) and organelles such as mitochondria and ribosomes.

• Reticulocytes are visualized by staining with supra-vital dyes that precipitate the RBC and organelles. This stain causes the ribosomal and residual RNA to coprecipitate with the few remaining mitochondria and ferritin masses in living young erythrocytes to form microscopically visible dark-blue clusters and filaments (reticulum).

• less than 12 hours old is required.

Normal range of reticulocyte Adults 0.5%-1.5%

Newborns Newborns:3%-6%

PRINCIPLE

Prothrombin TimeEvaluating Blood Clotting

Efficiency

Physiology- The Story BehindHemostasis – Prothrombin’s Role in Coagulation

Significance – Why the test is performed?

Evaluation of bleeding disorder- Easy bruising, excessive bleeding after dental appointment, blood in urine, heavy menstrual

cycle, etc.

Testing clotting ability before surgery

Monitor effects of warfarin treatment

Principle: How the test is performed?Blood is

removed from the patient.

A chemical with anti-clotting properties is added to prevent the

withdrawn blood from clotting (oxalated).

Calcium and tissue factor is then added to the blood in order to test the clotting

time. This activates the extrinsic pathway of

coagulation.

The presence of calcium ions and tissue factor should activate the

relevant clotting factors to convert prothrombin into

thrombin.

The time taken for the blood to coagulate is recorded. This is the prothrombin time.

Reference Ranges

•Each laboratory should establish its own normal range but in general the prothrombin time for a normal plasma sample, lies between 12-16 seconds

Source of Tissue Factor e.g.

human, rabbit etc

 - The exact technique used e.g. manual or

automated

  Method of end-point

determination e.g. optical or

mechanical

Atypical Results & Possible Reasons

Foods and substances that may alter PT•Alcohol•Oral contraceptives•Hormone replacement therapy (HRT)•Vitamin K excess – nutritional supplements and foods like broccoli, chickpeas, kale, liver (offal) and soybeans.

IRON PROFILERIDA REHMAN

INTRODUCTION & SIGNIFICANCE

five different types of tests Serum iron level test (SI) total iron-binding capacity (TIBC) test Ferritin tests transferrin test Transferrin saturation test

SIGNIFICANCE differential diagnosis and severity of different types of anemia Protein depletion and malnutrition liver disorders. chronic gastrointestinal bleeding certain unusual disorders▪ iron poisoning▪ Thalassemia▪ hemosiderosis▪ hemochromatosis

PHYSIOLOGY

SERUM IRON (SI) Men: 65 to 176 μg/dL Women: 50 to 170 μg/dL Newborns: 100 to 250 μg/dL Children: 50 to 120 μg/dL

TOTAL IRON BINDING CAPACITY (TIBC) Normal: 240–450 μg/dL

TRANSFERRIN SATURATION Male 20–50% Female 15–50%

FERRITIN LEVEL Men: 18-270 (ng/mL) or 18-270

(mcg/L) Women: 18-160 ng/mL or 18-

160 mcg/L Children: (6 months to 15 years)

7-140 ng/mL or 7-140 mcg/L Infants: (1 to 5 months) 50-200

ng/mL or 50-200 mcg/L Neonates: 25-200 ng/mL or 25-

200 mcg/L

TRANSFERRIN Normal: 204–360 mg/Dl

PATHOLOGY * = or normal

PRINCIPLE

transferrin

Acetic acid

iron

Ferrous state

This change in absorbance is directly proportional to the concentration of iron in the sample.

The system monitors the change in absorbance at 560 nm at a fixed-time interval

Complexes immediately

FerroZine Iron Reagent

ferrous ion

thioglycolate

hydroxylamine

HAMEOGLOBINOPATHY

o Oxygen transport metalloprotein

o In mammals 97% of the RBC’s dry content

o“Pathos” which means sufferingo Suffex path is used to indicate disease like psychopath.

Fatima Riaz

STRUCTURE

o 2 Alpha and 2 Beta subunits- α2ß2 o Each associated with a heme groupo Heme contains iron bound in porphyrin ringoBinds to Histidine 87 in α and Histidine 92 in βoCleavage plane for Oxygen association.

GlobinsPolypep

tide chains

Amino Acid

sequence

Genes

oSequence closely related, difference of one AA in β and α chain of humansand chimpanzees, variants in 7% of the world’s population.oSome produce harmless variants, while some produce hemoglobinopathies

Sickle Cell Disease• SCA or Drepanocytosis is an autosomal recessive genetic

disorder which causes the cells to be rigid and crescent shaped.

• Caused by a point mutation in the -globin chain βof hemoglobin on chromosome 11, causing the hydrophilic amino acid glutamic acid to be replaced with the hydrophobic amino acid valine. 

• The association of two wild-type  -globin subunits with two αmutant -globin subunits forms hemoglobin S (HbS)β

• Homozygous individuals are called HbSS and Heterozygous known HbAS

PATHOPHYSIOLOGY

DIAGNOSIS• Early diagnosis as part of screening tests is vital• Initial test by primary care doctor and successive

other by hematologist

Amniocentesis for prenatal diagnosis

Sickledex Tube Test

Hemoglobin Electrophoresis

Thalassemia

Presented by: Midhat Mustafa

Introduction

Thalassemia is a group of inherited disorders of hemoglobin synthesis characterized by defective globulin chains.

The name is derived from Greek words Thalasso= Sea and Hemia= Blood meaning anemia of sea.

Two types on basis of reduction of respective globulin chain α- Thalassemia β- Thalassemia May involve other chains but alpha & beta are of prime

importance. Demography

Chromosome 16

Smaller blood cells that are lighter In color

Mild anemia

Very lightly colored red blood cells And possible severe anemia

Death before birth (embryonic lethality)

Beta Thalassemia

Mutations on chromosome 11 – 2 gene clusters

Beta thalassemia minor, also called thalassemia minor or thalassemia trait – Mild anemia

Beta thalassemia intermedia, also called thalassemia intermedia or mild cooley’s anemia – Bony deformities, fatigue,increased levels of iron in blood

Beta thalassemia major, also called thalassemia major or cooley’s anemia – jaundice, dark urine, protruding abdomen, defromed bones, fatigue.

Inheritance

Diagnosis:

• Complete blood count (CBC) and special hemoglobin studies.• Tests of the amount of iron in the blood• Family genetic studies, prenatal testing.

Treatment:

• Regular blood transfusions, iron chelation therapy, and bone marrow stem cells transplants

• Surgery of spleen or gall bladder.• Fetal hemoglobin therapy.