Standard Tests for the Diagnosis of Anemia

1. Complete Blood Count (CBC)

• Microcytic (MCV <80 fL): Iron deficiency, thalassemia, anemia of chronic disease, sideroblastic anemia
• Normocytic (MCV 80-100 fL): Aplastic anemia, renal disease, endocrinopathies, early iron deficiency
• Macrocytic (MCV >100 fL): B12/folate deficiency, liver disease, hypothyroidism, alcohol use, myelodysplasia

2. Peripheral Blood Smear

3. Reticulocyte Count

• Low/normal reticulocytes → Hyporegenerative (production defect): aplastic anemia, iron/B12/folate deficiency, renal failure, marrow infiltration
• Elevated reticulocytes → Hyperregenerative: blood loss or hemolysis

4. Iron Studies

• Ferritin is the most sensitive single test for iron deficiency (it is a storage protein; low ferritin essentially confirms depleted stores)
• Up to 40% of iron-deficient patients will have a normal MCV, so iron studies should be considered in all anemias unless MCV >95 fL

5. Vitamin B12 and Folate Levels

• Low serum B12 (<200 pg/mL) or low RBC folate confirms deficiency
• Methylmalonic acid (MMA) and homocysteine levels are more sensitive confirmatory tests - MMA is elevated in B12 deficiency only, while homocysteine is elevated in both

6. Hemolysis Workup

7. Bone Marrow Examination

1. Aplastic anemia (pancytopenia)
2. Myelodysplasia
3. Marrow infiltration (malignancy, fibrosis)
4. Unexplained severe anemia
5. Pure red cell aplasia

8. Additional/Specialized Tests

Diagnostic Summary Algorithm

CBC + Blood Smear + Reticulocyte Count
           |
     MCV Classification
    /        |         \
Microcytic  Normocytic  Macrocytic
   |            |           |
Iron studies  Reticulocyte  B12/Folate
(ferritin,    count high?   levels
TIBC)         /     \
           Yes       No
        Hemolysis  Bone marrow
        workup     vs. renal/
                   endocrine

Confirmatory Tests for Malaria

Key principle from Harrison's 22E: "Malaria is not a clinical diagnosis." All suspected cases must be confirmed with parasite-based testing before treatment is initiated. (Harrison's Principles of Internal Medicine 22E, p. 1810)

1. Blood Smear Microscopy (GOLD STANDARD)

Thick Blood Smear

• Red cells are lysed during staining, concentrating parasites by 40-100 fold compared to a thin film
• Used primarily to detect the presence of parasites (higher sensitivity)
• Both parasites and WBCs are counted; parasitemia is expressed as parasites per µL
• At least 100-200 fields must be examined before calling a smear negative
• A minimum of 200 WBCs should be counted under oil immersion

Thin Blood Smear

• RBCs remain intact; examined at the tail of the film where cells are not overlapping
• Used for species identification and quantifying parasitemia (% infected RBCs)
• Parasitemia expressed as number of parasitized erythrocytes per 1000 RBCs

Staining

• Giemsa stain at pH 7.2 is preferred (Romanowsky stain)
• Field's, Wright's, or Leishman's stains can also be used
• Acridine orange (fluorescent dye) allows more rapid diagnosis in low-level parasitemia, but cannot be used for speciation

Microscopy images of Plasmodium falciparum (thin smear):

Thin blood films of P. falciparum: A. Young trophozoite. B. Old trophozoite. C. Trophozoites + pigment in PMNs. D. Mature schizont. E. Female gametocyte. F. Male gametocyte.

Microscopy images of Plasmodium vivax (thin smear):

Thin blood films of P. vivax: A. Young trophozoite. B. Old trophozoite. C. Mature schizont. D. Female gametocyte. E. Male gametocyte.

2. Rapid Diagnostic Tests (RDTs)

Antigens Detected

• ParaSight F (HRP2-based): sensitivity >95% for P. falciparum
• OptiMAL (pLDH-based): distinguishes between P. falciparum and P. vivax

• Cannot quantify parasitemia
• Cannot reliably speciate alone - microscopy should still be performed alongside RDT
• HRP2-based RDTs may remain positive for weeks after treatment due to antigen persistence
• Emerging hrp2/hrp3 gene deletions in P. falciparum (especially in parts of Africa and South America) can cause false-negative HRP2 RDT results - a growing concern per recent WHO guidance

3. PCR (Polymerase Chain Reaction)

• Sensitivity: superior to both microscopy and RDT, especially for low-density parasitemia
• Can precisely identify species and even distinguish mixed infections
• Detects drug resistance markers: dhfr mutations (antifolates), Pfcrt mutations (chloroquine), Pfkelch13 mutations (artemisinin), Pfmdr1 amplification (mefloquine)
• Limitation: not suitable for acutely ill patients because results take days; primarily used for research, epidemiological surveillance, and reference lab confirmation

4. Quantitative Buffy Coat (QBC) Technique

• Involves acridine orange staining of parasites concentrated by centrifugation in capillary tubes
• Requires a fluorescence microscope
• More rapid and sensitive than standard smear for detecting parasites
• Cannot reliably speciate infections
• Mainly used in reference or hospital laboratories that only occasionally diagnose malaria

5. Serology (Antibody Detection)

• Detects anti-malarial antibodies (IgG/IgM), not the parasite itself
• Available only at large reference laboratories
• Used primarily for epidemiological surveys, not for acute diagnosis
• Occasionally helpful for speciation and detecting occult/past infections
• Not used routinely for confirming acute malaria

Comparison Summary

WHO/CDC Diagnostic Algorithm

Suspected malaria (fever + travel to endemic area)
              ↓
   Prepare thick + thin blood smear IMMEDIATELY
              ↓
   Reliable microscopy available?
     YES → Examine smear (detect + speciate + quantify)
     NO  → Perform RDT
              ↓
   If smear negative but clinical suspicion high →
   Repeat smear every 12-24h × 3 (negative ×3 = not malaria)
              ↓
   If low-density parasitemia or species unclear →
   Send for PCR (reference lab)