¾ Anemia ¾ Red cell agglutination of peripheral (smear) blood (avoidable by raising temperature to 37°C) ¾ Reticulocytosis ¾ Hyperbilirubinemia ¾ Positive (direct) Coombs’ test (because of C3d on red cell surface as shown by specific antiglobulin sera).

 

Gastric causes

• Adult pernicious anemia

• Congenital lack of IF

• Total or partial gastrectomy.

Intestinal causes

• Chronic tropical sprue

• Intestinal stagnant loop syndrome, e.g. jejunal

diverticulosis, blind loops, strictures

• Crohn’s disease and ileal resection

• Congenital selective malabsorption with proteinuria

¾ Fish tapeworm infestation

¾ Severe pancreatitis

Clinical Hematology 251

¾ Celiac disease

¾ Therapy with metformin or phenformin.

Special Tests for Diagnosing Folate Deficiency

Serum Folate Assays

1. Microbiological: As for vitamin B12 except that Lactobacillus casei is used here. Normal values = 6–21 μg/L

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).

FIGLU Test

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

and FA deficiency)

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

Nutritional

¾ Especially old age

¾ Poverty

¾ Scurvy

¾ Partial gastrectomy

¾ Goat’s milk anemia.

Malabsorption

¾ Tropical sprue

¾ Celiac disease

¾ Partial gastrectomy

¾ Extensive jejunal resection

¾ Crohn’s disease.

Increased Demand

Physiological

¾ Pregnancy

¾ Lactation

¾ Prematurity.

Pathological

1. Hematological diseases

• Hemolytic anemias

• Myeloproliferative disorders

• Myelosclerosis

• Sideroblastic anemia

• Multiple myeloma.

2. Various carcinomas

3. Inflammatory diseases

• Crohn’s disease

• Tuberculosis

• Rheumatoid arthritis

• Psoriasis

• Exfoliative dermatitis.

4. Hyperthyroidism

5. Excess urinary loss

• Active liver disease

• Congestive heart failure.

6. Anticonvulsant drug therapy and oral contraceptives

7. Mixed

• Liver disease

• Alcoholism.

Laboratory Findings in Megaloblastic Anemias

Blood Counts

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

anemia.

Bone Marrow

¾ Marked erythroid hyperplasia

¾ Megaloblastic nuclear appearance in all 3 cell lines

¾ Storage iron normal or increased.

Blood Chemistry

¾ Bilirubin increased (indirect)

¾ Serum iron increased

¾ Increased LDH, with LDH-1 > LDH-2.

Other Studies

¾ Schilling test abnormal (pernicious anemia). Antibodies

to gastric cells, intrinsic factor (pernicious anemia)

¾ Serum RBC levels of vitamin B reduced (vitamin B12

deficiency)

¾ Urine methylmalonate increased (vitamin B12 deficiency)

¾ Urine FIGLU—folic acid deficiency.

Causes of Bone Marrow Megaloblastosis

¾ Vitamin B12 deficiency

¾ Folic acid deficiency

¾ Folic acid antagonists

¾ Inhibitors of purine or pyrimidine synthesis

¾ Alcoholism

252 Concise Book of Medical Laboratory Technology: Methods and Interpretations ¾ Genetically determined enzyme defects:

a. Lesch-Nyhan syndrome

b. Orotic aciduria

¾ Cytotoxic drugs

¾ Liver disorders

¾ Myxedema

¾ Sideroblastic anemia

¾ Multiple myeloma

¾ Widespread neoplastic disease

¾ Metastatic deposits in bone marrow.

Laboratory Diagnosis of Hemolytic Anemias

Causes and Classification of Hemolytic Anemias

Intracorpuscular Defects

Hereditary or congenital

Membrane defects

Hereditary spherocytosis

Hereditary elliptocytosis

Hereditary stomatocytosis.

Hemoglobin defects

Hemoglobinopathies

Sickle cell disease

Hb CDE, etc.

Unstable hemoglobin disease.

Thalassemias

β Thalassemia major

HbH disease

Enzyme defects

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

Acquired

Paroxysmal nocturnal hemoglobinuria.

Extracorpuscular Defects

Acquired

Immune mechanism

¾ Autoimmune hemolytic anemia. Warm antibody type

Cold antibody type

¾ Hemolytic disease of the newborn

¾ Incompatible blood transfusion

¾ Drug-induced hemolytic anemia

¾ Non-immune mechanism

¾ Mechanical hemolytic anemia

¾ Cardiac hemolytic anemia

¾ Microangiopathic hemolytic anemia

¾ March hemoglobinuria.

Miscellaneous

¾ Hemolytic anemia due to direct action of drugs/

chemicals

¾ Hemolytic anemia due to infection (Clostridium

welchii)

¾ Hemolytic anemia due to burns

¾ Lead poisoning.

Evidences of Hemolysis

Increased Breakdown of Hemoglobin

¾ Jaundice and hyperbilirubinemia

¾ Reduced plasma haptoglobin and hemopexin

¾ Increased plasma LDH

¾ Increased urinary urobilinogen

¾ Increased fecal urobilinogen

¾ Hemoglobinuria and

hemoglobinemia evidences of

¾ Methemalbuminemia intravascular

¾ Hemosiderinuria hemolysis

Compensatory Erythroid Hyperplasia

¾ Reticulocytosis, erythroblastemia

¾ Macrocytosis, polychromasia

¾ Erythroid hyperplasia of bone marrow (reversal of M:E

ratio)

¾ Skeletal X-ray (Widening of marrow space)

¾ Radiological changes in skull and tubular bones (in

congenital hemolytic anemias only).

Damage to Red Cells

¾ Spherocytosis

¾ Fragmentation of red cells

¾ Heinz bodies.

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

Blood Picture

¾ Anemia with spherocytosis (Hb 9–12 g%)

¾ Osmotic fragility, reticulocyte count (5–7%) and serum

bilirubin (indirect) are raised

¾ Negative Coombs’ test

¾ Autohemolysis after 48 hours at 37°C—10–50% (normal

< 4%)

¾ Glucose or ATP addition abolishes autohemolysis.

Clinical Hematology 253

Peripheral Smear

¾ Spherocytes

¾ Polychromatophils.

¾ Platelets diminished in number if splenomegaly present

¾ MCV normal or reduced

¾ MCH normal

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).

Chemistry

¾ Bilirubin slightly (indirect) increased

¾ Urine urobilinogen increased

¾ Haptoglobin reduced.

Laboratory Diagnosis of Hereditary Elliptocytosis

¾ In peripheral smear, ovalocytes are > 50% usually. Both

MCV and MCH are normal

¾ Osmotic fragility may be raised in anemic patients.

Laboratory Diagnosis of Enzyme

Deficiency Related Anemias

G6PD Deficiency

Blood Picture

¾ Polychromasia

¾ Basophilic stippling

¾ Spherocytosis ±

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.

2. Heinz body test.

3. Fluorescent spot test: NADPH autofluorescences in

long wave ultraviolet light.

4. Enzyme assays.

Laboratory Diagnosis of Autoimmune

Hemolytic Anemia (AIHA)

Laboratory Diagnosis of Warm Antibody AIHA

Blood Picture

¾ Hemolytic anemia

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.

Immunology

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

This has two forms:

¾ Cold hemagglutinin disease (CHAD)

¾ Paroxysmal cold hemoglobinuria (PCH).

CHAD

Blood picture

¾ Anemia

¾ Red cell agglutination of peripheral (smear) blood

(avoidable by raising temperature to 37°C)

¾ Reticulocytosis

¾ Hyperbilirubinemia

¾ Positive (direct) Coombs’ test (because of C3d on red

cell surface as shown by specific antiglobulin sera).

PCH

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

Nocturnal Hemoglobinuria

Blood Picture

¾ Anemia, macrocytosis, polychromasia

¾ Reticulocytosis, moderate leukopenia, mild thrombocytopenia

¾ HbF occasionally raised

¾ Hyperbilirubinemia