IMSE_Prelim_Lab: Screening test for Phagocytic engulfment

Phagocytosis

The process by which specialized cells engulf and destroy foreign particles such as microorganisms or damaged cells.

Phagocytic engulfment

The uptake of bacteria or other foreign particles by phagocytic cells.

Major phagocytic cells

Macrophages and neutrophils (PMNs).

Main purpose of the screening test

To demonstrate and observe phagocytic engulfment and provide supporting information about neutrophil function.

Bacterial broth culture used in the laboratory

E. coli culture.

Blood specimen required

At least 2 mL of freshly collected heparinized blood.

Alternative blood collection method

15–20 heparinized capillary tubes.

Preferred anticoagulated specimen

Heparinized blood, collected in a green-top evacuated tube.

Specimen freshness

The blood must be freshly collected because an old specimen may produce false-negative results.

Patient preparation

No special patient preparation is required before blood collection.

Blood collection technique

Aseptic technique.

Quality control

A fresh, heparinized blood sample from a healthy volunteer should be tested simultaneously.

Pre-analytical site preparation

The experimental site should be sterilized before performing the procedure.

Required PPE

Laboratory gown, laboratory goggles or face shield, laboratory mask, and gloves.

Analytical phase

Part of the procedure involving preparation, incubation, staining, and microscopic examination of the specimen.

Test and control tubes

The procedure describes a patient test tube and a control test tube.

Buffy coat volume

4–8 drops of buffy coat are added to the appropriately labeled tubes in the described procedure.

Bacterial broth volume

2–3 drops of bacterial broth culture are added to each tube.

*In this experiment, 5-10 drops was done for a 5mL heparinized whole blood

Incubation conditions

Incubate at room temperature or 37°C for 30 minutes.

Purpose of incubation

Allows interaction between phagocytic cells and bacteria so engulfment can occur.

Blood smear

After incubation, one drop of the specimen is placed on a glass slide and a smear is prepared.

Ideal blood smear appearance

Tongue-shaped, with a thick-to-thin consistency and a feathery edge.

Factors affecting smear quality

The size of the blood drop and the proper angle of the spreader.

Challenge in preparing the smear

The bacterial culture can make the blood viscous or thick.

Wright's stain

A stain used to visualize blood cells and bacteria for microscopic examination.

Components of Wright's stain

Methylene blue and eosin-Y.

Initial Wright's stain time

Cover the smear generously with Wright's stain and allow it to remain for at least 5 minutes.

Buffer step in Wright's stain

Slowly add distilled water or buffer until the buffer begins to overflow the stain and a metallic luster appears.

Buffer incubation time

Allow the buffer to remain on the slide for at least 5 minutes.

Final staining step

Gently wash off the stain and buffer with distilled water, then air-dry or carefully blot the slide.

Microscopic examination

Examine the stained smear using the oil immersion objective (100×).

Mineral oil

Added before microscopic examination to provide a clearer view with the oil immersion objective.

Positive result

WBCs demonstrate engulfment of bacteria.

Negative result

No engulfment of bacteria is observed.

Meaning of bacterial engulfment

Engulfment demonstrates that phagocytic activity has occurred, particularly involving neutrophils in this test.

Neutrophils

Important phagocytic WBCs responsible for engulfing bacteria in this test.

Clinical significance of failed engulfment

Failure of phagocytes to engulf bacteria may support a possible neutrophilic dysfunction.

Clinical interpretation limitation

A negative result alone cannot diagnose neutrophil dysfunction; results must be correlated with patient signs and symptoms.

Important microscopic distinction

Distinguish bacterial cocci from normal cellular granules.

Intracellular bacteria

For a positive result, bacteria must be located inside the WBC rather than outside it.

Extracellular bacteria

Bacteria outside the WBC do not demonstrate phagocytic engulfment.

False-negative result

A result in which engulfment is not observed even though phagocytic function may not actually be impaired.

Causes of false-negative results

Use of a specimen that is not fresh and use of a coagulase-positive Staphylococcus specimen.

Procedure limitation

• The test demonstrates engulfment only

• it does not demonstrate that the bacteria have been destroyed.

Phagocyte destruction of bacteria

Not established by this screening procedure because the procedure only demonstrates engulfment.

Post-analytical phase

Involves appropriate disposal of infectious materials, cleaning the work area, and proper removal of PPE.

Disposal of infectious materials

All infectious materials must be disposed of appropriately.

Work area after testing

Clean the working area with disinfectant before leaving.

PPE after testing

PPE should be removed properly and should not be exposed outside the laboratory premises.