Hematology Course

May 21 2025

LE Cell Test (Lupus Erythematosus Cell Test)

saeedurrahmangroup Hematology Course, Pathology Course 0

Intro:

The LE cell test is a hematological test historically used in the diagnosis of Systemic Lupus Erythematosus (SLE).
LE cells are a specific type of neutrophil or other phagocyte that has engulfed the denatured nuclear material (homogenized chromatin) of another cell, forming a homogeneous, round body known as an LE body or hematoxylin body.
While modern tests like ANA (antinuclear antibody) and anti-dsDNA antibody assays have largely replaced the LE cell test, it still holds historical importance and basic relevance in educational and diagnostic contexts.

About L.E. Cell Test:

LE Cell: A neutrophil or monocyte containing an engulfed, homogenous, smooth, round, purple-staining body (LE body) in its cytoplasm, formed due to phagocytosis of denatured nuclear material coated with autoantibodies.

May 18 2025

Reticulocyte Count

saeedurrahmangroup Hematology Course, Pathology Course 0

About Reticulocyte:

Reticulocytes are immature red blood cells (RBCs) that are recently released from the bone marrow into the bloodstream.
They still contain remnants of ribosomal RNA, which appear as a fine, reticular (mesh-like) network when stained with supravital stains like new methylene blue or brilliant cresyl blue.

Why Reticulocyte Count is Important?

Reticulocyte count helps assess bone marrow function.
It indicates the rate of RBC production.
Used to monitor response to anemia treatment or bone marrow recovery.

Normal Reticulocyte Count

Adults: 0.5 – 2.5% of RBCs
Infants: 2 – 6%
Absolute Reticulocyte Count (ARC): 25,000 – 75,000/µL

Clinical Significance or Indication

Condition	Reticulocyte Count
1. Iron deficiency anemia	Decreased or normal
2. Hemolytic anemia	Increased
3. Aplastic anemia	Decreased
4. After blood loss or therapy	Increased (reticulocytosis)

Types of Reticulocyte Count

Relative Reticulocyte Count – % of total RBCs.
Absolute Reticulocyte Count – Actual number per µL.
Corrected Reticulocyte Count (CRC) – Adjusted for low hematocrit.
Reticulocyte Production Index (RPI) – Assesses bone marrow response in anemia.

Methods of Reticulocyte Count

1. Manual Method (Supravital Staining Method)

Principle:

When blood is mixed with a supravital stain (e.g., new methylene blue), reticulocytes precipitate their residual RNA as dark blue granules or network.

Requirements:

Fresh EDTA blood sample
Supravital stain (e.g., new methylene blue) – Brilliant Cresyl Blue Stain
Microscope
Slides and cover slips

Reagent Preparation:

Reagents Needed:

Brilliant Cresyl Blue (BCB) – 1 g
Sodium Citrate (Na₃C₆H₅O₇) – 0.7 g
Sodium Chloride (NaCl) – 0.9 g
Distilled Water – up to 100 mL

Procedure:

Mix equal parts of blood and stain (usually 2–3 drops).
Incubate at room temperature for 15–20 minutes.
Prepare smears from the mixture.
Allow them to air-dry.
Examine under oil immersion (100x).
Count reticulocytes among 1000 RBCs.

Calculation:

[ratemypost]

May 18 2025

PC – Platelet Count: Methods, Merits & Demerits

saeedurrahmangroup Hematology Course, Pathology Course 0

About Platelet:

Platelets, also known as thrombocytes, are small, anucleated blood cells essential for blood clotting and maintaining vascular integrity.
A platelet count is a part of the Complete Blood Count (CBC) and is vital in diagnosing bleeding disorders, thrombocytopenia, thrombocytosis, and monitoring therapies like chemotherapy or anticoagulant use.

Normal Platelet Count

Adults: 1.5 – 4.5 lakh/cu mm (150,000 – 450,000/µL)
Newborns: 1.0 – 4.5 lakh/cu mm
Critical Values:
- Below 20,000/µL: Risk of spontaneous bleeding
- Above 10 lakh/µL: Risk of thrombosis

Functions of Platelets

Initiate clot formation (hemostasis)
Maintain endothelial integrity
Involved in inflammation and wound healing

Methods of Platelet Count Determination

Manual Method (Rees-Ecker Method / Improved Neubauer Chamber Method.
Automated Platelet Count (Hematology Analyzer)

1. Manual Method (Rees-Ecker Method or Improved Neubauer Chamber Method)

Principle:

A diluting fluid is used to lyse RBCs and stain platelets.
The diluted blood is then charged on a Neubauer counting chamber, and platelets are counted under a microscope.

Rees-Ecker Method (Manual Count)

Reagents:

Rees-Ecker diluting fluid:
- Sodium citrate – prevents clotting
- Brilliant cresyl blue – stains platelets
- Formalin – fixes platelets
- Distilled water – diluent

Procedure:

Mix fresh anticoagulated blood (EDTA) with diluting fluid in a 1:200 dilution.
Fill the Improved Neubauer hemocytometer with the diluted sample.
Allow platelets to settle for 10–15 minutes.
Count platelets in the entire central large square (25 small squares).
Multiply the number of platelets by the dilution factor to get platelet count per µL.

Merits:

Inexpensive
Does not require automated analyzers

Demerits:

Time-consuming
Platelets may clump or be confused with debris
Needs good microscopy skill

Calculation:

PC – Platelet Count: Methods, Merits & Demerits

2. Automated Platelet Count (Hematology Analyzer)

Principle:

Uses electrical impedance or optical flow cytometry.
Platelets are counted based on size and internal complexity.

Procedure:

Whole blood is aspirated by the analyzer.

Platelets are distinguished from RBCs and WBCs by size and counted electronically.

Results displayed along with other CBC parameters.

Merits:

Accurate, fast, and reproducible
No observer bias
Can also provide Platelet Distribution Width (PDW) and Mean Platelet Volume (MPV)

Demerits:

Expensive equipment
May misclassify clumped platelets or large platelets
Requires maintenance and electricity

3. Peripheral Blood Smear (Cross-check Method)

Principle:
Estimate platelet count by comparing them with RBCs under oil immersion in a stained smear (Leishman/Wright).

Procedure:
Prepare and stain a thin blood smear.

Under oil immersion (100x), count platelets in 10 fields.

Average number per field × 15,000 = Approximate count per µL.

Merits:

Simple
Helps detect clumping or morphological abnormalities

Demerits:

Rough estimation only.
Subjective and not suitable for precise counting

Causes of Abnormal Platelet Count:

Condition	Count	Examples
Thrombocytopenia	Low (<150,000)	Dengue, leukemia, bone marrow failure, ITP
Thrombocytosis	High (>450,000)	Infections, iron deficiency, myeloproliferative disorders

Sources of Error

Clotted sample
Platelet clumping (EDTA-induced)
Misidentification of debris or cells
Inadequate mixing of blood before sampling

Precautions

Use fresh EDTA blood (<2 hours old)
Mix well before testing
Avoid hemolysis
Clean counting chamber and cover slip

[ratemypost]

May 17 2025

Hb – Hemoglobin Estimation – Methods, Merits, and Demerits

saeedurrahmangroup All, Hematology Course 0

Introduction

Hemoglobin is a conjugated protein present in red blood cells (RBCs).
It consists of a pigment part called heme which contains iron and a protein part called globin and
Hemoglobin plays a vital role in transporting oxygen from the lungs to tissues and returning carbon dioxide from tissues to the lungs.
After the normal life span of RBC (over 120 days), the red cells are destroyed by the reticuloendothelial cells (specially in the spleen) and the components of the hemoglobin undergo metabolic degeneration.

Normal Hemoglobin Values

Men: 13.5 – 17.5 g/dL
Women: 12.0 – 15.5 g/dL
Children: 11.0 – 13.0 g/dL
Newborns: 14.0 – 24.0 g/dL

Clinical Importance of Hemoglobin Estimation

Diagnosis of anemia and polycythemia.
Monitoring response to treatment in anemic patients.
Evaluation of blood loss.
Pre-operative assessment.
Nutritional assessment in populations.

Methods of Hemoglobin Estimation

Several methods are available for the estimation of hemoglobin. The most commonly used are:

1. Sahli’s (Acid Hematin) Method

2. Cyanmethemoglobin Method (Drabkin’s Method)

3. Hemoglobin Color Scale (WHO Scale)

4. Automated Hematology Analyzer

5. Tallqvist Method (Historical/Obsolete)

1. Sahli’s (Acid Hematin) Method

Principle:

Hemoglobin is converted into brown-colored acid hematin by reacting with hydrochloric acid (N/10 HCl).
The color is matched against a standard comparator.

Procedure:

Add 0.1N HCl into the Sahli’s hemoglobinometer tube up to the mark.
Add blood using a pipette (usually 20 µL).
Mix and let it stand for 10 minutes.
Dilute with distilled water till the color matches the standard.
Read the value on the graduated tube.

Merits:

Simple and inexpensive.
Suitable for fieldwork and small labs.

Demerits:

Subjective color matching.
Less accurate.
Interference by abnormal hemoglobins or lipemia.

2. Cyanmethemoglobin Method (Drabkin’s Method)

Principle:

Blood is mixed with Drabkin’s reagent which contains potassium ferricyanide and potassium cyanide. Hemoglobin is converted into stable cyanmethemoglobin which is measured spectrophotometrically at 540 nm.

Procedure:

Add 20 µL blood to 5 mL Drabkin’s reagent.
Mix and wait for 5–10 minutes.
Measure absorbance at 540 nm.
Compare with standard or calibration curve.

Merits:

Highly accurate and reproducible.
International reference method.
Measures all types of hemoglobin except sulfhemoglobin.

Demerits:

Toxic reagents (cyanide).
Requires spectrophotometer.
Not ideal for field settings.

3. Hemoglobin Color Scale (WHO Scale)

Principle:

Capillary blood is compared with a printed color scale to estimate hemoglobin.

Merits:

Inexpensive.
Easy to use in remote areas.

Demerits:

Highly subjective.
Less accurate.
Not suitable for clinical diagnosis.

4. Automated Hematology Analyzer

Principle:

Measures hemoglobin through photometric or electrical impedance method. Usually uses cyanmethemoglobin or sodium lauryl sulfate (SLS) method.

Merits:

Accurate and fast.
Provides complete blood count (CBC) with hemoglobin.
Ideal for large laboratories.

Demerits:

Expensive instruments.
Requires maintenance and electricity.
Not suitable for small clinics or field use.

5. Tallqvist Method (Historical/Obsolete)

Principle:

Blood on filter paper is compared with a color scale.

Merits:

Very simple and portable.

Demerits:

Crude and inaccurate.
Not used in modern clinical practice.

Conclusion

Hemoglobin estimation is a vital diagnostic procedure. The method chosen depends on the available resources, setting (lab vs. field), and purpose (screening vs. diagnosis). For clinical accuracy, the cyanmethemoglobin method is the most reliable, while Sahli’s method remains a practical tool in limited-resource settings.

May 14 2025

What Is ESR Test?

saeedurrahmangroup All, Hematology Course 0

What Is ESR Test?

The erythrocyte sedimentation rate (ESR or sed rate) is the rate of sedimentation or settlement of RBCs at which red blood cells in anticoagulated whole blood settled in a standardized tube or EDTA tube over a period of one hour.

ESR is a common hematology test, and is a non-specific measure of inflammation.

Clinical Significance

The Erythrocyte Sedimentation Rate (ESR) test is a commonly performed in the Hematology laboratory for the prognostic purpose that may indicate and monitor an increase in inflammatory activity within the body caused by one or more conditions such as chronic inflammatory disease, autoimmune disease, anemia, infections, and malignancies.
The ESR test is not specific for any single disease but is used in combination with other tests to determine the presence of increased inflammatory activity.
The ESR has long been used as a “sickness indicator” due to its reproducibility in both hematological and non hematological.
When anticoagulated blood is allowed to stand undisturbed condition for a period of time, the erythrocytes tends to sink to the bottom.
Two layers are formed, the upper plasma layer and lower sedimented erythrocyte layer. The rate at which the red cells fall is knows as Erythrocyte Sedimentation Rate (ESR).

Determination of ESR is done by several methods and their Specific Values:

Determination of ESR is done by several methods. These are as follows:

Westergren method.
Wintrobe method
Landu method.

In the developing countries former two methods are used for ESR determination in the laboratory.

Wintrobe’s tube :

It is 110 mm long tube with a 3.0 mm internal bore.
It is graduated from ‘0’ upto ‘100’ mm (10 cm).
The scale with the descending order is used for ESR determination.
It is also used for Packed Cell determination.

Westergren’s tube :

It is 300 mm long tube with a 2.5 mm internal bore.
It is graduated from ‘0’ upto ‘200’ mm (20 cm).
The scale with descending order is used for ESR determination.
It is more specific then the Wintrobe’s method and others methods.

Normal value :

By Westergren’s method :
Male : 0-15 mm/1st hour
Female : 0-20 mm/ 1st hour
By Wintrobe’s method :
Male : 0-9 mm/1st hour
Female : 0-20 mm/ 1st hour

High ESR Test Result (Red blood cells sink faster than normal):

May be caused by inflammation from conditions such as:

Giant cell arteritis
Arthritis
Systemic vasculitis
Polymyalgia rheumatica
Inflammatory bowel disease
Kidney disease
Infections
Rheumatoid arthritis and other autoimmune diseases
Heart disease
Certain cancers

Low ESR Test Result (Red blood cells sink slower than normal):

May be caused by conditions such as:

Blood disorders:
Polycythemia (increased red blood cells)
Sickle cell disease (SCD)
Leukocytosis (high white blood cell count)
Heart failure
Certain kidney and liver problems

Additional Notes:

Abnormal ESR results don’t always indicate a medical condition needing treatment.

ESR levels vary based on:

Age and sex

Pregnancy

Menstrual cycle

Aging

Obesity

Alcohol consumption

Exercise

Certain medicines and supplements can affect ESR results.

Determination Of ESR By Westergren’s Method :

Principle of the Test :

When anticoagulated blood is allowed to stand undisturbed condition for a period of time, the erythrocytes tends to sink to the bottom. Two layers are formed, the upper plasma layer and lower sedimented erythrocyte layer. The rate at which the red cells fall is knows as Erythrocyte Sedimentation Rate (ESR) and give the result as mm/1st hour.

Specimen :

Patient should be fasting for 12 to 16 hours.
Collect venous blood.
2.0 ml of blood is collected in a vial containing 0.5 ml of 3.8% Sodium citrate anticoagulant.
Fasting EDTA (Ethyl Di-amine Tetra Acetic acid) anticoagulated whole blood is also used for ESR determination.

Requirements :

Westergren’s tube with stand.
Test tube with test tube rack.
3.8 % Sodium Citrate solution.
Stop watch.
Blood drawer.

Procedure :

Blood drawer attached with clean and dry Westergren’s tube.
Drawn 3.8% sodium citrate upto ‘150’ mark from ‘200’ mark in a test tube.
Drawn well mixed anticoagulated blood upto ‘0’ mark from ‘200’ mark of the tube and mixed with 3.8% sodium citrate present in the test tube.
Mixed well, filled the Westergren tube exactly upto ‘0’ mark (avoid air bubbles formation in the tube).
Placed the tube upright position in the ESR stand, it should fit evenly into the groove of the stand and it allowed standing undisturbed condition exactly for 1 hour.
Set the time in Stopwatch.
Exactly after 1 hour, note the level to which the red cell column has fallen.
Report the result in terms of mm/1st hour.

Diagram : Westergren’s Tube and Wintrobe Tube

Determination Of ESR By Wintrobe Method :

Principle of the Test :

When anticoagulated blood is allowed to stand undisturbed condition for a period of time, the erythrocytes tends to sink to the bottom. Two layers are formed, the upper plasma layer and lower sedimented erythrocyte layer. The rate at which the red cells fall is knows as Erythrocyte Sedimentation Rate (ESR) and give the result as mm/1st hour.

Specimen :

Fresh fasting (12 to 16 hours fasting) Ethyl Di-amine Tetra Acetic acid (EDTA) anticoagulated and undiluted whole blood.

Requirement :

Wintrobe’s tube with stand.
Special Pasteur pipette attached with rubber tied.
Stop watch.

Procedure :

Mixed the blood carefully in the vial.
Filled the Wintrobe’s tube to the ‘0’ mark by using a special Pasteur pipette.
Placed the tube in exact vertical position in the stand.
Set the Stop-watch for 1 hour.
At the end of one hour note the level of erythrocyte column and give the result in terms of mm after first one hour (mm/ 1st hour).

Feature	Westergren Method	Wintrobe Method
Tube Length	300 mm	110 mm
Internal Bore	2.5 mm	3.0 mm
Measurement Range	0-200 mm	0-100 mm
Sensitivity	More sensitive and preferred	Less sensitive

Blood and its composition

saeedurrahmangroup Hematology Course 0

1. Introduction :

Blood is a specialized body fluid that circulates through the blood vessels to supply oxygen and nutrients to the tissues and remove the waste products.

Blood cells produced in the bone marrow.

FUNCTIONS OF BLOOD:

1. Oxygen, carbon dioxide, nutrients, hormones, and waste products transport

2. Temperature regulation in the body, pH balancing, and fluid homeostasis.

3. Immunity to infection and stopping of blood loss via clotting mechanisms.

2. Blood Constituents:

Blood can be divided into two principal parts:

2.1. Plasma: The liquid component, around 55% of the blood.

2. Formed Elements: Comprise 45% of blood; these include erythrocytes, leukocytes and platelets.

2.1. Plasma:

It is the pale yellow liquid that makes up about ~55% of the blood.

Diagram : After centrifugation, Whole Blood ↑

Components:

2.1.1. Water (90-92%): Solvent carrying nutrients, hormones, and waste products.

2.1.2. Proteins (7-8%):

Albumin: Helps to maintain osmotic pressure and acts as carrier protein.

Globulins: Antibodies, and transport proteins.

Fibrinogen: Concerned in blood clotting.

2.1.3. Dissolved Substances (1-2%):

Electrolytes: Sodium, potassium, calcium, chloride, and bicarbonates.

Nutrients: Glucose, amino acids, fatty acids, and vitamins.

Hormones: Insulin, cortisol and other regulatory chemicals.

Metabolic Waste Products: Urea, creatinine, and carbon dioxide.

2.2. Formed Elements or cellular constituents :

The solid part of blood is comprised of three distinct types of cells which serve a different purpose,

A. Red Blood Cells (Erythrocytes) :

– Structure:

– Biconcave disc shape.

– No nucleus and no organelles to create space for hemoglobin.

– Function:

– Transport oxygen from the lungs to tissues and carry carbon dioxide back to the lungs.

– Hemoglobin is an iron-containing protein that binds to oxygen.

– Life Span: ~120 days.

– Normal Count:

– Males: 4.7–6.1 million/µL.

– Females: 4.2–5.4 million/µL.

B. White Blood Cells (Leukocytes)

– Role: Protect the body against infections, foreign invaders, and abnormal cells.

– **Types**:

1. **Granulocytes** (contain granules):

– **Neutrophils**: Most common; first-line defenders against bacterial infections.

– **Eosinophils**: Combat parasitic infections and are involved in allergies.

– **Basophils**: Histamine release during allergic reactions.

2. **Agranulocytes** (no granules):

– **Lymphocytes**:

– T cells: Organizers of immune responses.

– B cells: Produce antibodies.

– Natural Killer (NK) cells: Destroy abnormal cells.

– **Monocytes**: Become macrophages, engulfing pathogens and debris.

– **Lifespan**:

– Neutrophils: Hours to days.

– Lymphocytes and monocytes: Weeks to years.

– **Normal Count**: 4,000–11,000/µL.

###### **C. Platelets (Thrombocytes)**

– **Structure**: Small, disc-shaped fragments derived from megakaryocytes.

– **Function”:

– Facilitate hemostasis by the formation of a platelet plug and release of clotting factors.

– **Duration**: 7–10 days.

– **Normal Count**: 150,000–450,000/µL.

—

### Functions of Blood

1. **Transportation**:

– Oxygen from the lungs to tissues.

– Carbon dioxide from tissues to the lungs.

– Nutrients from the digestive tract to cells.

– Hormones from endocrine glands to target organs.

– Waste products to the kidneys for excretion.

2. **Regulation**:

Distributes heat to maintain body temperature.

Helps in maintaining pH by its buffers, such as bicarbonate.

Maintains osmotic balance and the fluid levels in tissues.

3. **Protection**:

WBCs protect against infections and foreign bodies.

Platelets and clotting proteins help prevent excessive bleeding.

– Antibodies and complement proteins neutralize pathogens.

—

### **Blood Volume**

– **Average Blood Volume**:

Adult male: ~5-6 liters.

Adult female: ~4-5 liters.

– Blood volume accounts for ~7-8% of body weight.

—

### **Hematopoiesis: Formation of Blood Cells**

– **Definition**: The process of blood cell production, which occurs in the bone marrow.

– **Stages**:

1. **Erythropoiesis**: Formation of red blood cells, stimulated by erythropoietin (EPO) from the kidneys.

2. **Leukopoiesis**: Formation of white blood cells.

3. **Thrombopoiesis**: Formation of platelets, regulated by thrombopoietin.

—

### **Disorders Related to Blood**

1. **RBC Disorders**:

– **Anemia**: Low RBC count or hemoglobin (e.g., iron-deficiency anemia, sickle cell anemia).

– **Polycythemia**: Excess RBC production, leading to thickened blood.

2. **WBC Disorders**:

– **Leukopenia**: Decreased WBC count, leading to immune suppression.

– **Leukocytosis**: An elevated WBC count is usually because of an infection.

– **Leukemia**: Cancer of WBCs.

3. **Platelet Disorders**:

– **Thrombocytopenia**: Low platelet count. It results in a propensity to bleed excessively.

– **Thrombocytosis**: Elevated platelet count, raising the chance of thrombus formation.

4. **Plasma Disorders**:

– **Hemophilia**: A genetic disorder that involves the clotting factors.

– **Hypoproteinemia**: Low plasma protein causes edema.

—

### **Conclusion

Blood is a fluid that undertakes the vital functions of transport, regulation, and protection. Knowledge about composition and function is essential in diagnosing various diseases and treating patients affected.

Hematology Course

LE Cell Test (Lupus Erythematosus Cell Test)

Intro:

About L.E. Cell Test:

Reticulocyte Count

About Reticulocyte:

Why Reticulocyte Count is Important?

Normal Reticulocyte Count

Clinical Significance or Indication

Types of Reticulocyte Count

Methods of Reticulocyte Count

Calculation:

PC – Platelet Count: Methods, Merits & Demerits

About Platelet:

Normal Platelet Count

Functions of Platelets

Methods of Platelet Count Determination

1. Manual Method (Rees-Ecker Method or Improved Neubauer Chamber Method)

Principle:

Reagents:

Procedure:

Merits:

Demerits:

Calculation:

2. Automated Platelet Count (Hematology Analyzer)

Principle:

Procedure:

Merits:

Demerits:

3. Peripheral Blood Smear (Cross-check Method)

Causes of Abnormal Platelet Count:

Sources of Error

Precautions

Hb – Hemoglobin Estimation – Methods, Merits, and Demerits

Introduction

Normal Hemoglobin Values

Clinical Importance of Hemoglobin Estimation

Methods of Hemoglobin Estimation

1. Sahli’s (Acid Hematin) Method

2. Cyanmethemoglobin Method (Drabkin’s Method)

3. Hemoglobin Color Scale (WHO Scale)

4. Automated Hematology Analyzer

5. Tallqvist Method (Historical/Obsolete)

Conclusion

What Is ESR Test?

What Is ESR Test?

Clinical Significance

Determination of ESR is done by several methods and their Specific Values:

High ESR Test Result (Red blood cells sink faster than normal):

Low ESR Test Result (Red blood cells sink slower than normal):

Determination Of ESR By Westergren’s Method :

Principle of the Test :

Specimen :

Requirements :

Procedure :

Determination Of ESR By Wintrobe Method :

Principle of the Test :

Specimen :

Requirement :

Procedure :

Related Questions – Answers

Q1. What is the ESR Test?

Q2. Why is the ESR Test done?

Q3. Is ESR specific to any disease?

Q4. How is ESR measured?

Q5. What is the difference between Westergren and Wintrobe methods?

Blood and its composition

1. Introduction :

FUNCTIONS OF BLOOD:

2. Blood Constituents: