Stool Microscopy — Image Analysis

What You're Seeing

• Plant fiber/cellulose fragments from undigested vegetable or grain matter
• Pollen grains (some have the characteristic irregular, lobulated outline)
• Possibly starch granules partially digested

Key Distinguishing Features (Why These Are NOT Parasites)

Common Non-Parasitic Findings in Stool Microscopy

• Plant cells / vegetable fibers — very common, often mistaken for parasite fragments
• Pollen — seasonal, rounded/spiky appearance
• Muscle fibers (from meat) — cylindrical with striations
• Fat globules — round, refractile
• Starch granules — oval, stain blue-black with iodine
• Yeast cells — small, budding oval forms (Candida spp. — not always pathogenic)
• Epithelial cells — large, flat polygonal cells

Recommendation

• Repeat stool exam × 3 samples on different days
• Specific antigen tests (e.g., Giardia/Cryptosporidium EIA)
• PCR-based stool pathogen panels for higher sensitivity

Preparing a 0.8% Fecal Suspension for Stool Microscopy

Preparing 0.85% Normal Saline Stock Solution

Making the Stool Suspension

1. Place 1–2 drops of 0.85% saline on a clean glass slide
2. Pick a small amount of stool (~2 mg, approximately the size of a match head) using an applicator stick
3. Emulsify thoroughly into the saline drops — mix well until evenly suspended
4. The suspension should be thin enough to read newsprint through — if too thick, add more saline
5. Apply a coverslip (22×22 mm)
6. Examine under 10× then 40× objective

Formalin-Based Suspension (for concentration)

Key Tips

• Always prepare two slides — one with saline (motility/trophozoites), one with Lugol's iodine (cysts/nuclei detail)
• Use fresh stool within 30 minutes for trophozoite detection
• The suspension should look faintly turbid/cloudy, not opaque

Preparing 0.8% Pooled RBC Suspension

What You Need

Step-by-Step Protocol

Step 1 — Collect & Pool Blood

• Collect blood from 2–3 donors into EDTA tubes (anticoagulated)
• Pool equal volumes together in a clean tube

Step 2 — Wash the RBCs (×3)

1. Add 5–10 mL saline to pooled blood
2. Centrifuge at 1000–1500 rpm for 5 minutes
3. Discard the supernatant + buffy coat (white cell layer) carefully
4. Repeat washing 3 times until supernatant is clear (no hemolysis)

Step 3 — Make the 0.8% Suspension

Simply put: 0.8 mL packed RBCs + 99.2 mL saline = 0.8% RBC suspension

Quick Formula

Quality Check

• The suspension should appear uniformly turbid/pink
• No clumping or hemolysis (clear supernatant after settling)
• Use fresh preparation — ideally same day or store at 2–8°C for up to 1 week

Common Uses of 0.8% RBC Suspension

Unit Conversions

Simple Rule

0.8 mL → Microlitres

99.2 mL → Microlitres

Summary Table

For Smaller Volumes (Practical Bench Use)

For bench work, use a micropipette for the RBC volume (80 µL) and a measuring cylinder for the saline.

More Practical Small-Scale Volumes

Most Common Bench Practice

5–10 mL total — enough for a full day's batch of tests

• Use a micropipette for the 80 µL RBCs
• Use a measuring cylinder or graduated tube for saline
• Gives enough suspension for ~100–200 test drops depending on the assay

Tip: Only prepare what you need for the day — fresh suspension gives better, more reliable results.

Can ICT Be Negative in This Scenario?

Short Answer

Understanding the Scenario

Reasons ICT Can Be Negative

1. Anti-D Prophylaxis (RhoGAM) Given Properly

• If the mother received anti-D immunoglobulin (RhoGAM):
  • At 28 weeks in previous pregnancies
  • After each delivery of Rh+ baby
  • After any sensitizing events (miscarriage, amniocentesis, trauma)
• The administered anti-D is passively acquired and clears within 12 weeks
• No active alloimmunization occurs → ICT stays negative
• This is the most common reason for a negative ICT

2. Insufficient Fetomaternal Hemorrhage (FMH)

• In previous pregnancies, the volume of fetal blood entering maternal circulation may have been too small to trigger an immune response
• Sensitization requires a threshold volume of Rh+ RBCs
• Small FMH → no antibody formation → ICT negative

3. Immune Tolerance

• Some Rh-negative women simply fail to mount an antibody response despite exposure
• Approximately 30% of Rh-negative mothers never become sensitized even without prophylaxis
• Genetic factors influence immune responsiveness

4. Early Gestational Age at Testing

• Anti-D antibodies may be below detectable threshold early in pregnancy
• ICT may turn positive later — repeat testing is essential

5. Previous Children Were Actually Rh Negative

• If prior children were Rh negative (depends on father's genotype), no sensitizing exposure occurred
• Father could be heterozygous (Dd) — 50% chance each child is Rh negative

Clinical Implications — What To Do

Key Takeaway

A negative ICT does not mean the mother is safe to ignore — it means she has not yet formed antibodies. She remains at risk with every Rh+ pregnancy. Prophylaxis must continue.

Weak Positive ICT — What It Means & What To Do

First — Distinguish the Cause

This distinction is the most critical first step

If Passive (Post-RhoGAM)

• Weak positive is expected and reassuring
• Titer usually ≤1:4
• Will disappear within 12 weeks of injection
• No fetal risk
• Continue routine antenatal care

If True Alloimmunization (Active Anti-D)

Step 1 — Perform Antibody Titration Immediately

Critical titer = 1:16 (most centers) — threshold above which fetal hydrops risk rises significantly

Step 2 — Fetal Surveillance

Step 3 — Serial Antibody Monitoring Schedule

• Every 4 weeks up to 28 weeks gestation
• Every 2 weeks from 28 weeks until delivery
• Refer to fetal medicine if titer reaches critical level or is rising significantly

Signs of Fetal Hemolytic Disease to Watch For on USS

• Fetal hydrops (skin edema, ascites, pleural/pericardial effusion)
• Placentomegaly
• Polyhydramnios
• Enlarged fetal liver/spleen (hepatosplenomegaly)

Delivery Planning

After Delivery

• Do NOT give RhoGAM if mother is truly alloimmunized — it has no benefit once sensitization has occurred
• Newborn needs:
  • Direct Coombs Test (DCT)
  • Cord blood Hb + bilirubin
  • Monitor for neonatal jaundice / hemolytic disease of the newborn (HDN)
  • Possible phototherapy or exchange transfusion

Key Takeaway

A weak positive ICT in a G3P2 Rh-negative mother with Rh-positive children means early alloimmunization is likely underway. The priority is to titrate immediately, distinguish passive from active antibody, and begin serial fetal surveillance before the titer climbs to dangerous levels.

Gel Card (Column Agglutination) Grading for ICT

Standard Gel Card Grading Scale

Visual Representation

|  ====  |  ← 4+ (solid band at top)
|  ----  |
|        |

|  ----  |  ← 3+ (large clumps upper half)
|  ----  |
|        |

|  ----  |  ← 2+ (agglutinates throughout)
|  ----  |
|  ----  |

|        |  ← 1+ (small clumps lower half)
|  ----  |
|  ==    |  ← small pellet

|        |  ← W+ (faint, just above pellet)
|       .|
|  ===   |  ← near-complete pellet

|        |  ← Negative
|        |
|  ====  |  ← complete pellet at bottom

In Context of Your ICT Scenario

Important Notes for Gel Card Reading

• Always read in good lighting against a white background
• Read within 10 minutes after centrifugation
• W+ must never be ignored in an Rh-negative pregnant mother
• A result of 1+ and above = report as positive and refer for titration
• Hemolysis in the gel = also a positive result (complement activation)

What To Do Based on Gel Grade in Your Patient

Bottom line: Even a W+ on gel card in a G3 Rh-negative mother with Rh+ children should never be dismissed — it warrants full investigation and titration.

Emperipolesis in Peripheral Smear

Definition

Key distinction from phagocytosis (erythrophagocytosis):

What It Looks Like on Peripheral Smear

• Large histiocyte/monocyte with abundant pale cytoplasm
• Intact lymphocytes, neutrophils or RBCs visible inside the cytoplasm
• Engulfed cells appear normal morphologically — not fragmented
• Engulfed cells sit in a clear halo/vacuole within the host cell

Image — Emperipolesis (Rosai-Dorfman Disease)

Causes / Associated Conditions

Rosai-Dorfman Disease — Key Features

Emperipolesis vs HLH on Smear

Reporting on Peripheral Smear

"Large histiocytes noted with intact lymphocytes/neutrophils within their cytoplasm, consistent with emperipolesis. Findings may suggest Rosai-Dorfman disease or reactive histiocytosis. Correlation with clinical findings, lymph node biopsy and immunohistochemistry (S-100, CD68, CD1a) recommended."

Emperipolesis on Peripheral Blood Smear — Specific Context

What You Actually See on PBF

• A monocyte or large mononuclear cell with
• Intact neutrophil(s), lymphocyte(s), or RBCs sitting inside its cytoplasm
• The engulfed cell has a clear halo around it
• The engulfed cell looks morphologically normal — not digested

Most Common Cells Involved on PBF

Grading on PBF

Key Conditions to Consider by PBF Pattern

1. Emperipolesis + Normal CBC

• Likely reactive — viral infection, post-transfusion
• May be early Rosai-Dorfman

2. Emperipolesis + Pancytopenia + High Ferritin

• HLH until proven otherwise
• Check: Ferritin, triglycerides, fibrinogen, NK cell activity, bone marrow biopsy

3. Emperipolesis + Lymphocytosis + Lymphadenopathy

• Rosai-Dorfman Disease
• Biopsy + S100/CD68/CD1a staining needed

4. Emperipolesis + Blast Cells

• Seen in AML M4/M5 (monocytic leukemia)
• Monocytic blasts can engulf other cells

Differentials — What Can Mimic It on Smear

Practical Tip at the Bench

When you see a cell inside another cell on PBF:

1. Focus up and down — overlapping cells will appear at different focal planes
2. True emperipolesis — engulfed cell stays within the cytoplasm at all focal planes
3. Check if engulfed cell is intact (emperipolesis) or broken down (phagocytosis/HLH)
4. Scan multiple fields — count frequency before reporting

Reporting Template for PBF

"Emperipolesis noted — intact [neutrophils/lymphocytes] observed within the cytoplasm of monocytes/histiocytes. Frequency: [occasional/frequent]. Correlation with clinical history, ferritin, LFTs and bone marrow examination recommended to exclude HLH or Rosai-Dorfman disease."

Gel Card Interpretation — AHG Column

Reading the Image

Interpretation

Left Column — AbId = NEGATIVE

• Complete cell button at bottom
• No agglutinates in gel
• No specific antibody identified in the antibody identification panel
• Suggests the antibody is not one of the common alloantibodies on the panel cells

Right Column — ICT = 1+ WEAK POSITIVE

• Agglutinates scattered in lower-mid portion of column
• Small residual pellet at bottom
• Confirms antibody is present in maternal serum
• But too weak to be identified on AbId panel

Clinical Correlation (Linking Back to Your Patient — G3 Rh Negative)

Possible Explanations for This Pattern

1. Passive Anti-D from recent RhoGAM injection
   • Titer too low to react with all panel cells
   • ICT weakly positive, AbId negative = classic RhoGAM pattern
2. True early alloimmunization
   • Anti-D forming but titer very low
   • Not yet strong enough to identify on panel
3. Antibody to low-frequency antigen
   • Not represented on standard panel cells
   • Rare but possible

Next Steps

Bottom Line

ICT 1+ with negative AbId = antibody is present but weak. In a G3 Rh-negative mother, this must be taken seriously — do not dismiss. Differentiate passive RhoGAM from true alloimmunization by clinical history and serial testing.