SOP-H-003 — Peripheral Blood Smear Preparation, Wright-Giemsa Staining, Manual Differential, and Reticulocyte Count SOP

1.0 Purpose

This SOP sets the procedure for peripheral blood smear preparation, Romanowsky staining, manual leukocyte differential counting, microscopic erythrocyte/platelet morphology evaluation, and supravital manual reticulocyte enumeration at Tarangnan Community Hospital. Automated hematology analyzer flags and morphological abnormalities are verified microscopically under this procedure.

2.0 Scope

This procedure applies to all Medical Technologists and Laboratory Aides working in the Hematology Section. It covers manual peripheral blood film preparation by the wedge technique, rapid staining, microscopic scanning across the monolayer in a battlement pattern, manual 100-cell differential reporting, qualitative morphology grading, Pathologist review referral, and supravital New Methylene Blue staining for reticulocyte counting.

3.0 Definitions

• Peripheral Blood Smear (PBS): A thin, monolayer film of anticoagulated whole blood spread onto a glass microscope slide and stained to allow differential leukocyte enumeration and cellular morphology assessment.
• Monolayer: The specific microscopic zone of a blood film adjacent to the feathered edge where erythrocytes lie side-by-side without overlapping, providing optimal optical conditions for cell identification.
• Battlement Pattern: Structured zig-zag microscopic scanning movement across the monolayer from edge to edge to avoid sampling bias caused by unequal distribution of leukocyte classes.
• Reticulocyte: An immature, non-nucleated erythrocyte containing precipitated residual ribosomal ribonucleic acid (RNA) visible microscopically when stained supravitally.
• Supravital Staining: Staining of living, unfixed cellular elements in vitro using vital dyes such as New Methylene Blue or Brilliant Cresyl Blue.
• Reticulocyte Production Index (RPI): A calculated parameter correcting the reticulocyte percentage for severe anemia and premature marrow release (shift reticulocytes), providing an objective measure of effective erythropoiesis.

4.0 Responsibility

4.1 Hospital Director

• Approves this SOP and ensures the laboratory has high-grade optical microscopes, certified staining reagents, and safety materials.

4.2 Pathologist

• Reviews and approves morphological criteria, reporting terminology, and slide-making trigger thresholds.
• Examines referred abnormal smears, hematological malignancies, severe dysplasias, and hemoparasites, issuing formal diagnostic narrative reports.
• Participates in annual Medical Technologist morphology competency assessments.

4.3 Chief Medical Technologist / Laboratory Quality Manager

• Manages smear review protocols, stain quality control logs, and reticulocyte calculation verification.
• Organizes annual inter-observer morphology competency evaluations per Competency Assessment Policy.
• Oversees slide archiving and retention compliance.

4.4 Medical Technologists

• Screen automated analyzer results against slide-making criteria, prepare wedge blood films, perform staining, and verify stain quality.
• Execute 100-cell manual differentials, grade red cell poikilocytosis and platelet morphology, perform manual reticulocyte counts, and calculate ARC, CRC, and RPI.
• Identify Pathologist referral triggers and refer designated slides with complete clinical data.

4.5 Laboratory Aides

• Clean and decontaminate staining troughs, maintain slide drying racks, and restock clean glass slides.
• File dried, labeled slides chronologically into archive boxes under technologist supervision.
• Shall never evaluate cell morphology, perform differential or reticulocyte counts, or enter microscopic findings into reports.

5.0 Policy / Procedure

5.1 Indications and Slide-Making Screening Criteria

• A peripheral blood film shall be prepared, stained, and reviewed microscopically when any of the following clinical or analytical triggers is met:

• Blood films shall be prepared within 4 hours of collection in K2EDTA to prevent storage-induced artifacts.

5.2 Smear Preparation (Manual Wedge Technique)

• Materials: Clean, pre-cleaned, dust-free frosted-end glass slides (25 × 75 mm), smooth-edged spreader slide, graphite pencil, and personal protective equipment.
• Stepwise Procedure: - Invert the K2EDTA whole-blood tube 10 times to achieve complete cell suspension. - Using a plain capillary tube or transfer pipette, place a small drop of blood (approximately 5 to 10 µL, 2–3 mm diameter) onto the midline of a clean slide, approximately 1 cm from the frosted end. - Rest the slide on a flat, non-slip workbench. - Hold the spreader slide at a 30° to 45° angle in front of the blood drop. - Draw the spreader slide backward until it touches the blood drop; allow the blood to spread along the width of the spreader edge by capillary action (75% of slide width). - Push the spreader forward in a single, smooth, continuous, moderate-speed motion toward the opposite end, maintaining uniform contact. - Immediately wave the slide gently in ambient air to promote rapid drying. Rapid drying prevents erythrocyte crenation and water artifact. - Label the frosted end in graphite pencil with patient full name, hospital number, and date. Ink markers and adhesive labels are prohibited.
• Characteristics of an Acceptable Smear: - Covers two-thirds of the slide length with smooth lateral margins narrower than the slide. - Exhibits a gradual transition from thick head to thin, rainbow-sheened feathered edge. - Monolayer zone spans at least 2 cm and is free of waves, streaks, ridges, holes, or chatter marks.

5.3 Staining Technique (Medic Hema-Quick / Wright-Giemsa)

• TCH uses the certified rapid Romanowsky staining method (Medic Hema-Quick) or standard Wright-Giemsa stain.
• Rapid Three-Step Staining Procedure: - Step 1 (Fixation): Immerse the air-dried slide in Fixative Solution (pure absolute methanol with stabilizer) for 5 dips of 1 second each. Drain excess fluid. - Step 2 (Eosinophilic Staining): Immerse the fixed slide into Solution I (buffered Eosin Y) for 5 dips of 1 second each. Drain excess dye. - Step 3 (Basophilic Staining): Immerse the slide into Solution II (buffered Methylene Blue / Azure) for 5 dips of 1 second each. - Rinse: Gently rinse the slide under a slow stream of deionized or phosphate-buffered water (pH 6.8–7.2) for 5 to 10 seconds. - Wipe the back of the slide with a clean tissue to remove excess dye and stand the slide vertically in a drying rack to air-dry completely.
• Quality Control of Staining: - Inspect the smear with each staining batch. Macroscopically, an acceptable smear appears pinkish-purple. - Under 100× oil immersion, verify: - Erythrocytes stain uniform salmon-pink to orange-red without blue tinting or refractile borders. - Neutrophil nuclei stain deep purple with crisp chromatin margins; cytoplasm contains fine lilac-pink granules. - Eosinophil granules stain brilliant orange-red. - Basophil granules stain deep blue-black. - Platelets appear crisp purple with fine azurophilic granulation. - Stain solutions shall be discarded and replaced when precipitate accumulates or staining times drift.

5.4 Microscopic Examination and Morphology Assessment

• Low-Power Examination (10× Objective): - Scan the slide to assess overall cellularity, leukocyte distribution, and staining quality. - Inspect the feathered edge and lateral borders for leukocyte aggregation, platelet clumps, microclots, and large atypical cells.
• High-Power Oil Immersion Examination (100× Objective): - Navigate to the monolayer zone where red blood cells lie side-by-side with minimal touching and no overlap. - Apply one drop of high-grade immersion oil. - Evaluate red blood cell size (anisocytosis, microcytosis, macrocytosis), color (hypochromia, polychromasia), and shape variations (poikilocytosis). - Grade poikilocytes using standard semi-quantitative scoring:

• Critical Morphological Flags: - Presence of schistocytes (fragmented RBCs) ≥ 1% mandates immediate reporting to rule out microangiopathic hemolytic anemia (TTP/HUS/DIC). - Sickle cells, marked spherocytes, intracellular parasites (malaria ring forms), or prominent toxic granulation with cytoplasmic vacuolization require immediate confirmation.

5.5 Manual Leukocyte Differential Count (100-Cell Count)

• Scanning Protocol: - Within the monolayer, count 100 consecutive intact leukocytes using a differential cell counter. - Follow the battlement pattern: move 3 fields horizontally, 3 fields vertically toward the margin, 3 fields horizontally along the edge, and 3 fields back toward the center.
• Enumerate standard differential classes: Segmented Neutrophils, Band Neutrophils, Lymphocytes, Monocytes, Eosinophils, and Basophils.
• Unclassifiable or degenerated leukocytes (smudge cells / basket cells) shall be tallied separately and not included in the 100-cell total. If smudge cells exceed 10%, note on report: "Smudge cells present; suspect chronic lymphocytic leukemia or leukocyte fragility."
• Nucleated Red Blood Cells (NRBC): - Count and record NRBCs encountered during the 100-leukocyte count (expressed as NRBC / 100 WBC). - If NRBC ≥ 5 / 100 WBC, the automated total leukocyte count must be corrected using the formula: Corrected WBC (10³/µL) = [Uncorrected WBC × 100] / (100 + NRBC per 100 WBC) - Report the corrected WBC as the official count, appending: "WBC corrected for presence of nucleated red blood cells."

5.6 Supravital Manual Reticulocyte Count Procedure (New Methylene Blue)

• Principle: Fresh whole blood is mixed with a supravital basic dye (New Methylene Blue). The dye penetrates living, unfixed erythrocytes and precipitates ribosomal RNA into visible dark blue granules, strands, or a reticular network. A smear is prepared and reticulocytes are enumerated microscopically against mature erythrocytes.
• Reagent and Equipment: - Reticulocyte Staining Solution: Certified 1.0% New Methylene Blue solution in buffered saline (1.0 g New Methylene Blue + 0.8 g sodium chloride + 0.4 g potassium oxalate dissolved in 100 mL distilled water, filtered through Whatman No. 1 paper). - Calibrated micropipettes (100 µL). - 12 × 75 mm plastic test tubes. - Clean glass slides and spreader slide. - 37 °C incubator or ambient room temperature workstation. - Binocular microscope with 100× oil immersion objective. - Hand tally counter or Miller disc ocular reticle.
• Stepwise Incubation and Smear Preparation: - Invert the K2EDTA whole-blood specimen 10 times to ensure uniform cell distribution. - Pipette 100 µL of well-mixed whole blood into a 12 × 75 mm test tube. - Add 100 µL of filtered 1.0% New Methylene Blue solution (1:1 volumetric ratio). If the patient is severely anemic (Hct < 20%), increase blood volume to 150 µL with 100 µL dye. - Mix the tube contents thoroughly by gentle tapping. - Incubate the mixture at room temperature (18–25 °C) or in a 37 °C water bath for exactly 15 minutes. - Following incubation, invert the tube 5 times to resuspend settled erythrocytes. - Place a 5–10 µL drop of the dyed cell suspension onto clean slides and prepare two thin wedge smears using the standard wedge technique. - Allow smears to air-dry completely. Do not counterstain with Wright stain.
• Microscopic Counting Protocol: - Focus under 10×, then transition to 100× oil immersion in the monolayer zone. - Mature erythrocytes stain pale greenish-blue without inclusions. - Reticulocytes are identified as erythrocytes containing two or more distinct, dark-blue staining granules, threads, or dense mesh-like reticular networks. - Count a total of 1,000 red blood cells across multiple consecutive fields, enumerating every reticulocyte identified within that population. - Using two separate wedge smears (500 RBCs counted per smear by two technologists or across two areas), verify that counts agree within 15%.

5.7 Reticulocyte Calculations and Clinical Indices

• Relative Reticulocyte Percentage (% Reticulocytes): % Reticulocytes = (Total Reticulocytes Counted in 1,000 RBCs / 1,000) × 100
• Absolute Reticulocyte Count (ARC): Reflects true daily erythroid marrow output independent of hematocrit: ARC (10⁹/L) = (% Reticulocytes / 100) × RBC Count (10¹²/L) × 10³ (Alternatively expressed as: ARC (/µL) = [% Reticulocytes × RBC (10⁶/µL)] / 100 × 10⁶)
• Corrected Reticulocyte Count (CRC): Adjusts reticulocyte percentage for anemia dilution against a standard reference hematocrit of 45%: CRC (%) = % Reticulocytes × [Patient Hematocrit (%) / 45%]
• Reticulocyte Production Index (RPI): Corrects for shift reticulocytes released prematurely from bone marrow into peripheral circulation during erythropoietin stimulation: RPI = CRC (%) / Maturation Factor The Maturation Correction Factor is determined by the patient's measured hematocrit:

• Diagnostic Interpretation of RPI: - RPI > 2.0: Demonstrates adequate, responsive bone marrow erythroid hyperplasia (e.g., active hemolysis, acute blood loss recovery, or successful response to iron/B12 therapy). - RPI < 2.0: Demonstrates hypoproliferative, inadequate erythroid response in the presence of anemia (e.g., aplastic anemia, bone marrow infiltration, erythropoietin deficiency, myelodysplasia, untreated nutritional deficiency).

5.8 Reference Intervals

5.9 Pathologist Consultation and Smear Referral

• The technologist shall refer the blood film, raw CBC printout, and clinical summary to the Pathologist upon finding: - Circulating blasts, promyelocytes, or myelocytes. - Prominent dysplasia across erythroid, myeloid, or megakaryocytic cell lines. - Schistocytes ≥ 1%, sickle cells, or suspected hemoparasites (malaria). - Marked unexplained cytopenias or leukocytosis > 30.0 × 10³/µL. - Discrepancy between automated differential and manual microscopic evaluation.
• Pathologist-reviewed smears shall be stamped "REVIEWED BY PATHOLOGIST", accompanied by a narrative morphology report and signed by the Pathologist.

6.0 Records

7.0 References

• DOH Clinical Laboratory QMS Tool, Section 3 — Service Delivery
• DOH Administrative Order No. 2021-0037, Annex B1 — Technical Standards for Clinical Laboratories
• ISO 15189:2022, Clause 7.3 Examination processes
• CLSI H20-A2, Reference Leukocyte Differential Count (Proportional) and Evaluation of Instrument Methods
• CLSI H44-A2, Methods for Reticulocyte Counting (Automated and Manual)
• Medic Hema-Quick Rapid Staining Product Instructions for Use

Companion policies:

• Hematology Section Workflow Quality Control and Result Validation Sop — Hematology Section quality control, interference, and result validation SOP
• Horiba Abx Pentra Xl 80 Cbc and Maintenance Sop — HORIBA ABX Pentra XL 80 CBC and maintenance SOP
• Laboratory Retention Schedule Policy — Retention schedules for stained slides, worksheets, and patient reports