7. If it is abnormal—repeat the said procedure with simultaneous oral administration of intrinsic factor. 8. If excretion increases, it implies lack of intrinsic factor. If it does not, then there is some defect in absorption (Repeat test can be done 48 hours later).
Bring all reagents and samples to room temperature before
1. Retrieve the required number of Sicklevue reaction
tubes, as the number of samples to be tested.
2. Label the reaction tubes appropriately and set on a test
3. Add 2 mL of the buffer to each of the reaction tubes,
using the pipette. Alternately, fill up the tubes by
pouring the buffer up to the 2 mL marking.
4. Mix well and allow to stand for 5 minutes at RT.
5. With the help of a micropipette, add 20 μL of whole
6. Mix and allow to stand for 10 minutes.
7. To read the results, place the tubes into the slots of the
8. Read the turbidity in the tubes by holding the tubes
against a dim illumination and viewing the horizontal
2 mL marking, through the solution, in the Sicklevue
To differentiate between sickle cell trait (Hb AS) and sickle
1. If positive results are obtained during the screening
method, take a fresh Sicklevue reaction tube and
repeat the test procedure as in Screening Method with
2. Centrifuge the reaction tube at 1200 g for 5 minutes in
3. Allow the centrifuge to stop without braking and
carefully remove the reaction tubes without disturbing
4. Observe the pattern formed in the reaction tubes.
a. A turbid solution (horizontal black lines on the Result
Reading Card are barely visible or cannot be seen)
indicates a positive test for sickle cell hemoglobinopathies.
b. A clear solution (horizontal black lines on the Result
Reading Card are clearly visible) indicates a negative
a. Hb AS-Hb S forms a red precipitate at the top and
soluble Hb if present forms a red color solution below
b. Hb SS-A red precipitate at the top and solution is
colorless. If substantial amount of Hb F is present, the
c. All other Hb yield clear red solutions.
1. All positive results should be confirmed on electrophoresis.
2. Severe anemia can cause false negative results. If the
hemoglobin concentration is 8 g% or less the sample
volume for testing should be doubled to 40 μL.
3. Blood samples from patients with multiple myeloma,
cryoglobulinemia and other dysglobulinemias may
4. It is recommended that the performance of reagents
should be verified with known positive and negative
5. As with all tests, the result of the test should be
correlated with clinical findings to arrive at the final
Laboratory Diagnosis of Iron Deficiency Anemia
1. Anisocytosis, microcytic red cells.
2. Poikilocytosis, pencil-shaped cells and target cells.
3. Hypochromia, ring or pessary cells.
5. Reticulocyte count is variable.
6. RBC osmotic fragility is slightly decreased.
7. Hematocrit low, plasma appears paler.
8. Radiochromium—51Cr studies show reduced red cell
span; however, TLC, DLC and platelets have normal
1. Micronormoblastic erythroid hyperplasia.
2. Predominantly intermediate normoblasts are seen.
3. Cytoplasm is decreased and shows differential staining.
4. Cytoplasm matures so slowly that nucleus may be
pyknotic, while cytoplasm is still polychromatic.
5. Bone marrow iron is reduced or absent. [Perl’s reaction
done on fixed bone marrow slide shows absent/
reduced free iron (blue particles) and lack of siderotic
1. Serum iron is reduced (15–16 μg%).
2. Total iron binding capacity is raised (up to 550 μg%).
3. Unsaturated iron-binding capacity is also raised.
4. Percentage saturation is reduced to about 10%.
5. Red cell protoporphyrin increased (no iron available
Normal Values for Iron Metabolism
Serum iron (Fe): 60–170 μg%; Total iron-binding capacity
(TIBC): 300-360 μg%; Saturation: 20–45%; Serum
Laboratory Findings in Iron Deficiency
¾ Microcytic, hypochromic red cells if Hb < 12 g% (men),
¾ Degree of Hb reduced, RBC count diminished depends
on severity. Leukopenia may occur
¾ Platelets increased in number with active bleeding
¾ Reticulocytes lower than expected for degree of anemia.
¾ Serum iron is reduced, iron-binding capacity is
increased % saturation is diminished
¾ Free erythrocyte protoporphyrin increased
¾ RBC survival time slightly increased.
Causes of Iron Deficiency Anemia
Uterine (menorrhagia, metrorrhagia) chronic gastrointestinal
¾ Hookworm infestation (anemia with eosinophilia).
Other Causes of Chronic Blood Loss
¾ Prematurity (decreased iron stores)
¾ Growth (iron deficiency anemia is commonest in
¾ Females in reproductive age group:
¾ Achlorhydria (especially in middle-aged females)
achlorhydria → iron deficiency anemia
¾ Gastrectomy (HCl not available)
¾ Gastroenterostomy (Inflamed anastomosis or intestinal
hurry hence no time for absorption).
¾ Improper feeding in infants and young children
¾ Anorexia (nervosa, of pregnancy or malignancies).
Megaloblastic Macrocytic Anemias
Peripheral Blood Findings in Vitamin B12 or Folic
1. Anemia with macro (ovalo) cytosis.
5. Moderate leukopenia due to neutropenia.
6. Hypersegmented neutrophils, i.e. more than 3 neutrophils
having more than 5 nuclear lobes/100 neutrophils.
7. Macropolycytes (large neutrophils).
8. Mild, usually asymptomatic thrombocytopenia.
1. Megaloblasts (larger normoblasts with large open
sieve-like nucleus) a constant feature.
• Has an eccentric, indented lobulated nucleus
• May show Howell-Jolly bodies.
4. Dissociation of cytoplasmic-nuclear maturation
(hemoglobinization occurs faster than the nuclear
5. Mitoses common, may be abnormal, i.e. tri or quadripolar.
6. Maturation arrest, promegaloblasts and early megaloblasts constitute 50% of the erythroblasts.
3. Absolute number of granulocytes increases but
is not evident because of simultaneous erythroid
1. Number is variable, occasionally diminished.
2. Have deep basophilic cytoplasm.
2. Increased urinary excretion of methylmalonic acid
3. Radioactive vitamin B12 absorption test.
Microorganism + all microorganism +
necessary growth factors all necessary growth
except vitamin B12 factors + known
Both the tubes are inoculated and later the turbidity
developed due to growth of the microorganism is measured
and serum vitamin B12 level is deduced.
¾ Using E. gracilis, normal values are 160–925 ng/L (mean
¾ A value < 100 ng/L implies frankly megaloblastic
Test serum + Vitamin B12 labeled with 57Co
1. Vitamin B12 binding protein or intrinsic factor.
2. Separate free and bound form.
— Normal values = 200–300 ng/L.
Radioactive Vitamin B12 Absorption Test
Principle: Ability to absorb 57Co-labeled vitamin B12 orally,
if simultaneous administration of intrinsic factor improves
absorption, it implies lack of intrinsic factor.
Absorption of radioactive vitamin B12 can be measured in
2. Radioactivity in urine (Schilling test).
3. External counting over liver (chief storage organ).
5. Estimation of plasma radioactivity.
1. Give 1 mg unlabeled vitamin B12 parenterally.
2. Give 1 μg labeled vitamin B12 orally.
3. Within 24 hours 1/3rd of absorbed radioactive vitamin
4. Normal excretion is > 10% of oral dose.
5. Pernicious anemia patients excrete < 5%.
6. If the test is normal no further testing necessary.
7. If it is abnormal—repeat the said procedure with
simultaneous oral administration of intrinsic factor.
8. If excretion increases, it implies lack of intrinsic factor.
If it does not, then there is some defect in absorption
(Repeat test can be done 48 hours later).
Free 58Co-labeled vitamin B, and 57Co-labeled intrinsic factor bound vitamin B
¾ If 57Co-labeled vitamin B is excreted more, there is
deficiency of intrinsic factor.
Causes of Vitamin B12 Deficiency
Comments
Post a Comment
اكتب تعليق حول الموضوع