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Pathology Course

LE Cell Test (Lupus Erythematosus Cell Test)

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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.

Reticulocyte Count

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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

  1. Relative Reticulocyte Count – % of total RBCs.

  2. Absolute Reticulocyte Count – Actual number per µL.

  3. Corrected Reticulocyte Count (CRC) – Adjusted for low hematocrit.

  4. 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:

  1. Mix equal parts of blood and stain (usually 2–3 drops).

  2. Incubate at room temperature for 15–20 minutes.

  3. Prepare smears from the mixture.

  4. Allow them to air-dry.

  5. Examine under oil immersion (100x).

  6. Count reticulocytes among 1000 RBCs.

Calculation:

Reticulocyte Count
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PC – Platelet Count: Methods, Merits & Demerits

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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

  1. Manual Method (Rees-Ecker Method / Improved Neubauer Chamber Method.
  2. 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:

  1. Mix fresh anticoagulated blood (EDTA) with diluting fluid in a 1:200 dilution.

  2. Fill the Improved Neubauer hemocytometer with the diluted sample.

  3. Allow platelets to settle for 10–15 minutes.

  4. Count platelets in the entire central large square (25 small squares).

  5. 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
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