• Total or partial gastrectomy.
• Intestinal stagnant loop syndrome, e.g. jejunal
diverticulosis, blind loops, strictures
• Crohn’s disease and ileal resection
• Congenital selective malabsorption with proteinuria
¾ Therapy with metformin or phenformin.
Special Tests for Diagnosing Folate Deficiency
2. Radioisotope assay: As for vitamin B12 except that Cow’s
milk is used here as the binding protein. Value < 4 μg/L
implies megaloblastic, macrocytic anemia (Normal
red cell folate 160–640 μg/L).
Folate is essential for conversion of histidine to glutamic
acid. Formiminoglutamic acid (FIGLU) is an intermediate
product. In folate deficiency, FIGLU is increased which
appears in urine. This test, however, is not very specific.
Radioactive Folic Acid Test-like Schilling Test
Deoxyuridine (dU) suppression test (for both vitamin B12
1. Short-term in vitro bone marrow cultures are used.
2. In normoblastic cultures, added deoxyuridine enters
DNA thymine pathway and supresses the subsequent
incorporation of subsequently added tritiated thymidine in DNA.
3. In vitamin B12 and folic acid deficiency, the added
deoxyuridine causes less suppression, but the defect
can be corrected by supplying the missing vitamin.
Causes of Folic Acid Deficiency
• Myeloproliferative disorders
6. Anticonvulsant drug therapy and oral contraceptives
Laboratory Findings in Megaloblastic Anemias
¾ Severe anemia (Hb may fall up to 3 g%)
¾ Macrocytosis (MCV 100–140 fl), with anisocytosis
¾ Ovalocytes, and macro-ovalocytes numerous
¾ WBCs, platelets often low in number
¾ Hypersegmented neutrophils > 3%
¾ Reticulocytes disproportionately low vis-a-vis degree of
¾ Marked erythroid hyperplasia
¾ Megaloblastic nuclear appearance in all 3 cell lines
¾ Storage iron normal or increased.
¾ Bilirubin increased (indirect)
¾ Increased LDH, with LDH-1 > LDH-2.
¾ Schilling test abnormal (pernicious anemia). Antibodies
to gastric cells, intrinsic factor (pernicious anemia)
¾ Serum RBC levels of vitamin B reduced (vitamin B12
¾ Urine methylmalonate increased (vitamin B12 deficiency)
¾ Urine FIGLU—folic acid deficiency.
Causes of Bone Marrow Megaloblastosis
¾ Inhibitors of purine or pyrimidine synthesis
¾ Widespread neoplastic disease
¾ Metastatic deposits in bone marrow.
Laboratory Diagnosis of Hemolytic Anemias
Causes and Classification of Hemolytic Anemias
Nonspherocytic congenital hemolytic anemia due to
phosphokinase deficiency or other EM pathway enzyme
defects. Due to G6PD deficiency or other pentose phosphate pathway enzyme defects.
Drug-induced hemolytic anemia—Favism
Paroxysmal nocturnal hemoglobinuria.
¾ Autoimmune hemolytic anemia. Warm antibody type
¾ Hemolytic disease of the newborn
¾ Incompatible blood transfusion
¾ Drug-induced hemolytic anemia
¾ Microangiopathic hemolytic anemia
¾ Hemolytic anemia due to direct action of drugs/
¾ Hemolytic anemia due to infection (Clostridium
¾ Hemolytic anemia due to burns
Increased Breakdown of Hemoglobin
¾ Jaundice and hyperbilirubinemia
¾ Reduced plasma haptoglobin and hemopexin
¾ Increased urinary urobilinogen
¾ Increased fecal urobilinogen
¾ Methemalbuminemia intravascular
Compensatory Erythroid Hyperplasia
¾ Reticulocytosis, erythroblastemia
¾ Erythroid hyperplasia of bone marrow (reversal of M:E
¾ Skeletal X-ray (Widening of marrow space)
¾ Radiological changes in skull and tubular bones (in
congenital hemolytic anemias only).
Demonstration of Shortened Lifespan of Red Cells
Normal plasma haptoglobin level = 1–1.5 g/L. Levels are
assessed by rapid latex agglutination test. Levels under
1 g/L imply two to three times hemolysis or the half-life
of red cells is 17 days or less. Normal plasma hemopexin
level is = 0.5–1 g/L. Its concentration is measured by
radial immunodiffusion technique. In most intravascular
hemolysis, its levels are diminished.
Laboratory Diagnosis of Hereditary Spherocytosis
¾ Anemia with spherocytosis (Hb 9–12 g%)
¾ Osmotic fragility, reticulocyte count (5–7%) and serum
bilirubin (indirect) are raised
¾ Autohemolysis after 48 hours at 37°C—10–50% (normal
¾ Glucose or ATP addition abolishes autohemolysis.
¾ Platelets diminished in number if splenomegaly present
¾ MCHC often increased (34–40%)
¾ Reticulocyte count raised (5–20%).
¾ There can be pancytopenia in aplastic crisis.
¾ 51Cr autologous red cell life reduced with excessive
counting over spleen (the main pitting organ).
¾ Bilirubin slightly (indirect) increased
¾ Urine urobilinogen increased
Laboratory Diagnosis of Hereditary Elliptocytosis
¾ In peripheral smear, ovalocytes are > 50% usually. Both
¾ Osmotic fragility may be raised in anemic patients.
Laboratory Diagnosis of Enzyme
¾ Heinz bodies, 1–2, after commencement of therapy.
Besides methemoglobin reduction test already
described, other tests which can be done are:
1. Brilliant cresyl blue (BCB) reduction test.
3. Fluorescent spot test: NADPH autofluorescences in
Laboratory Diagnosis of Autoimmune
Laboratory Diagnosis of Warm Antibody AIHA
¾ Positive direct antiglobin (Coombs’) test
¾ Monocytosis in peripheral smear ± erythrophagocytosis
¾ Variable total leucocyte count TLC.
¾ Blood withdrawn often shows autoagglutination because
RBCs, are heavily coated with immunoglobulins.
¾ Red cell and serum folate levels are diminished.
¾ Autoantibodies demonstrated in vitro in most cases
¾ Antibody is found on red cell surface and in serum
(surface antibodies are revealed by direct Coombs’ test)
¾ IgA immunoglobulin deficiency.
Laboratory Diagnosis of Cold Antibody AIHA
¾ Cold hemagglutinin disease (CHAD)
¾ Paroxysmal cold hemoglobinuria (PCH).
¾ Red cell agglutination of peripheral (smear) blood
(avoidable by raising temperature to 37°C)
¾ Positive (direct) Coombs’ test (because of C3d on red
cell surface as shown by specific antiglobulin sera).
¾ Donath Landsteiner antibody test, chill the blood, take
back to 37°C, hemolysis occurs.
¾ Positive Coombs’ (direct) test only during the attack.
Laboratory Diagnosis of Paroxysmal
¾ Anemia, macrocytosis, polychromasia
¾ Reticulocytosis, moderate leukopenia, mild thrombocytopenia
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