Tarangnan Community HospitalClinical Laboratory	Document No.: WI-H-003-A-V0	CONTROLLED COPY Do not copy or remove
	Parent Document: SOP-H-003, Sections 5.2–5.7  |  Version: V0
Applies to: Medical Technologists rotating through Hematology  |  Effective Date: MM-DD-YYYY  |  Supersedes: None

Peripheral Blood Smear Wedge, Wright-Giemsa Staining & Reticulocyte Card

Hematology Section  |  Manual Wedge Smear, Rapid Romanowsky Staining, 100-WBC Battlement Track & Reticulocyte Calculation

1. 1
   Slide Labeling in Graphite Pencil Only Label frosted end in graphite pencil: Patient Full Name, Hospital MRN, and Date. Ink pens and adhesive labels are strictly prohibited (dissolved by methanol during fixation). SOP-H-003, Section 5.2.2
2. 2
   Manual Wedge Smear Preparation (30°–45° Spreader Angle) Place 5–10 μL K2EDTA blood 1 cm from frosted end. Hold spreader at 30°–45°; draw back until touching drop; allow blood to spread across 75% of width. Push forward smoothly in one continuous stroke. Wave gently to air-dry immediately (prevents crenation). SOP-H-003, Section 5.2.2, 5.2.3
3. 3
   Rapid 3-Step Romanowsky Staining (Medic Hema-Quick / Wright-Giemsa) Fixation: Pure methanol, 5 dips of 1 sec. Eosin Y (Sol I): 5 dips of 1 sec. Methylene Blue (Sol II): 5 dips of 1 sec. Rinse: Slow stream of deionized / buffered water (pH 6.8–7.2) for 5–10 sec. Air-dry vertically. Inspect for pink-purple macroscopic hue. SOP-H-003, Section 5.3.2, 5.3.3
   5–10 s rinse
4. 4
   100-Cell Leukocyte Differential (Battlement Pattern in Monolayer) Under 100× oil immersion in the monolayer zone, count 100 consecutive intact WBCs following the battlement track: 3 fields horizontal, 3 vertical to margin, 3 horizontal along edge, 3 back toward center. Tally Segs, Bands, Lymphs, Monos, Eos, and Basos. SOP-H-003, Section 5.4, 5.5.1
5. 5
   NRBC Enumeration & Leukocyte Correction Formula Count nucleated red blood cells per 100 WBCs. If NRBC ≥ 5 / 100 WBC, calculate: Corrected WBC = [Uncorrected WBC × 100] / [100 + NRBC]. Report corrected count with mandatory comment. SOP-H-003, Section 5.5.4
6. 6
   Supravital Reticulocyte Incubation (1:1 New Methylene Blue Ratio) Combine 100 μL K2EDTA blood + 100 μL filtered 1.0% New Methylene Blue (1:1 volumetric ratio) in a 12 × 75 mm plastic tube. Tap to mix. Incubate for exactly 15 minutes at room temperature (18–25 °C) or 37 °C water bath. (If Hct < 20%, use 150 μL blood : 100 μL dye). SOP-H-003, Section 5.6.3
   15 min inc
7. 7
   Reticulocyte Smear & 1,000-RBC Microscopic Enumeration Invert incubated tube 5 times; prepare two thin wedge smears; air dry (do not counterstain). Under 100× oil immersion in monolayer, count 1,000 total RBCs. Reticulocytes = erythrocytes containing ≥ 2 dark blue RNA granules or threads. Verify two 500-cell counts agree within 15%. SOP-H-003, Section 5.6.3, 5.6.4