Week 1: 6/05/18 Q1.The following test is recommended for mass screening of beta thalassemia due to its low cost and simplicity. Not True about the following test is:-
- NESTROFT
- The test result shown here is positive
- Based on decreased red cell osmotic fragility
- The test result shown here is negative
Q2.What next test will you order in a case with above findings?
- Complete osmotic fragility test
- Hemoglobin electrophoresis
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Correct Answers Q1>[4] & Q2>[2]
Principle of NESTROFT
- Normally, red cells put in saline solution begin to lyse at a saline concentration of 0.4-0.5% and lysis is complete at 0.32%.
- However, in beta thalssemia trait, due to alteration in osmotic resistance of the affected RBC’s due to volume/surface area ratio changes, lysis begins at a saline concentration between 0.4-0.35% and it may not be completed even at 0.1% solution.
- NESTROFT is done at a saline concentration of 0.36%.
Procedure of the test
- Two test tubes labelled as buffered saline (0.36%) BS (2ml) and Distill Water (2ml) are taken and a drop of blood is added to each of the tubes and are left undisturbed for half an hour at room temperature.
- Then contents of both tubes are gently shaken and held against a white paper on which a thin black line was drawn.
- The line is clearly visible through DW tube (in distill water red cells are always lysed) and this tube acts as control.
If red cell are lysed in BS the line will be visible and test is considered NEGATIVE.
If the red cells are not lysed (decreased fragility) the line will be blurred/not visible and test is considered POSITIVE
(As in the image in Question)
- SO TEST IS POSITIVE IN QUESTION AND NOT NEGATIVE.
In case the test is positive and red cell indices are microcytic hypochromic HBA2 concentration should be detected by HPLC/Electrophoresis.
NEXT QUESTION COMING UP
Week 2: 13/05/18 Patient recovering from an febrile illness, presents with pallor, mild icterus and increased serum LDH. Have a look at hemogram output of this patient.
Which of the following is likely diagnosis in this case ?
- Microangiopathic Hemolytic anemia
- G-6-P-D deficiency
- Autoimmune Hemolytic Anemia
- Hereditary Spehrocytosis
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Answer = [3]
In the haemogram note that the RBC count is very low as compared to Hb and MCV is very high. (Also note illogical MCH and MCHC values)
Note in Histogram image that many RBCs have size > 250fl (the line is not touching baseline even at 250 fl, it suggests that large particles ie Agglutinated RBC clumps are present in sample s/o Autoimmune Hemolytic anemia.
Autoimmune hemolytic anemia (AIHA) is an uncommon disorder characterized by hemolysis mediated by autoantibodies directed against self-red blood cells (RBC), with the incidence of 1–3 per 100,000/year and mortality rate of approximately 11%.
Serological evaluation of warm AIHA:-
- 95% of warm AIHA cases have a positive DAT. In DAT positive cases, 20-66% have only IgG detected on the RBCs, 24-63% have both IgG and C3 on the surface and 7-14% have only C3 on the surface. (MC type of the IgG autoantibodies are in the IgG1 subclass.)
Serological evaluation of Cold Agglutinin disease:-
- Patients with CAD typically involves IgM autoantibodies and complement, patients almost exclusively have positive DAT with anti-C3 and polyspecific reagents and a negative result with anti-IgG.
Serological evaluation of Paroxysmal Cold Hemoglobinuria:
- PCH is caused by a biphasic IgG autoantibody (Donath-Landsteiner antibody) that fixes complement at low temperature but ultimately dissociates at a higher temperature. As a result, the DAT is positive with anti-C3, but it is generally negative with anti IgG unless performed at colder temperatures.
Note: In mixed-AIHA the serological work-up shows that the DAT is positive for both IgG and C3.
Note: Drug-induced AIHA is serologically indistinguishable from warm AIHA; a presumptive diagnosis can be made only if the patient responds to withdrawal of the drug.
NEXT QUESTION COMING UP
Week 3: 20/05/2018: Q3A. A 13-year-old girl presented with transfusion dependent normocytic anemia with moderate splenomegaly, mild icterus, cholelithiasis and features s/o hemochromatosis. Peripheral blood smear features include anisocytosis, poikilocytosis, basophilic stippling, and some irregularly contracted cells. The bone marrow aspirate image is given below. The karyotype from bone marrow aspirate sample was normal. Which of the following is true about diagnosis ?
- BMA reveals marked erythroid hyperplasia with megaloblastosis
- Lysis of the patient’s red cells can be documented by own serum
- SDS-PAGE of red cell membrane proteins can be of help in diagnosis
- RBCs show very high agglutinability by anti-p sera
Q3B:- Which of the following electron microscopy image would be consistent with your diagnosis ?
Q3C:-Optimal Management of this child would include.
- Splenectomy
- Iron chelation
- Genetic counselling
- All of these
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Answers:-3A=3, 3B=IMAGE 2(RIGHT SIDE), 3C=4
The case describes typical case of Congenital dyserythropoietic anemia-II
- CDA II, also known as hereditary erythroblastic multinuclearity with a positive acidified-serum test (HEMPAS),is the most frequently encountered disorder of the CDA group.
- Anemia is Normocytic (not megaloblastic as in CDA-I) and the leading BMA abnormality is binuclearity or multinuclearity occurring in 10% to 50% of mature erythroblasts (as seen in image), with equal DNA content in both nuclei.
- Electron microscopy (EM) shows a double membrane close to the cell membrane of mature erythroid cells, which is due to residual endoplasmic reticulum. (As in image 2 of Q2, Note that image 1 in Q2 shows swiss chese appearance seen in CDA-I)
- Band 3 appears thinner and shows faster migration SDS-PAGE and hence it can be of help in diagnsis.
- Red cells of patients with CDA II retain throughout life a very high agglutinability by anti-i sera.
To prevent organ damage, lifelong control of iron stores is required, with regular check of ferritin values. Iron depletion (chelation) has to be started at the latest when ferritin approaches a level of 1000 g/mL (1000 ng/mL).
Iron chelation therapy may be indicated, or alternatively, phlebotomy may be considered in patients with mild anemia.
The main utility of splenectomy is abrogation of transfusion requirements in more severe cases and increase of the hemoglobin concentration to render regular phlebotomy possible.
Ref: Review article (click here)
NEXT QUESTION COMING UP
01/07/2018: Author Ekta Jajodia
Q4A. Image 1 represents PBS of a 10 days old African baby presenting with severe anemia and hyberbilirubinemia . Coombs test is negative -ve.
Q4B. Image 2 represents PBS of the same child at 7 months of age; No anemia and no increase in bilirubin. What is diagnosis??
Options Are
- South Asian ovalocytosis
- Hereditary pyropoikilocytosis
- Infantile poikilocytosis
- Rh deficiency syndrome
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Answer: (3) Infantile poikilocytosis
- Aka Hereditary Elliptocytosis With Neonatal Poikilocytosis
- It is a variant of classical hereditary Elliptocytosis (HE)
- Seen in neonates of parents with mild HE
- Inherits 1 mutant alpha spectrin allele
- Neonate: symptomatic hemolytic anemia with marked poikilocytosis
- By 6-12months: hemolysis and poikilocytosis abate; clinically transforms into mild HE
- The severity of the molecular defect, in terms of the percentage of spectrin dimers and the amount of mutant spectrin in the cells, is the same in the neonatal period as it is later in life.
The worsening of hemolysis in the neonatal period has been attributed to the presence of fetal hemoglobin.
CAUSE: neonate has increased HbF >> binds poorly to 2,3 DPG >> increased free 2,3 DPG destabilize the spectrin-4.1-actin interaction.
NEXT QUESTION COMING UP
07/07/2018: Author Ekta Jajodia
Q5. Case Scenario: 47/M complains of chronic thrombocytopenia (80 × 109/L), was treated for ITP without any response. O/E: Splenomegaly and xanthomas on the extensor surface of hands
Lab Investigations: Hb 12gm%, Indirect bilirubin 3 mg/dl, Retic 4% Cholesterol: increased.
Image above shows xanthomas on the extensor surface of hands. The image below shows peripheral smear from same patient. What is your diagnosis??
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- Sitosterolemia is a rare autosomal recessive disorder due to mutation in either ABCG5 or ABCG8 genes.
- These genes encodes for ATP-Binding Cassette G (ABCG) proteins: ABCG5(sterolin-1) and ABCG8 (sterolin-2)
- These proteins prevent plant sterol absorption and promote its excretion.
- Absence of these proteins lead to accumulation of plant sterols in the blood and onto the inner layer of red cell membrane, resulting in stomatocytes.
- The mechanism of macrothrombocytes is not understood.
- Affected patients exhibit xanthomatosis and early-onset premature cardiovascular disease.
- Stomatocytic hemolysis, thrombocytopenia with large platelets, splenomegaly, and abnormal bleeding can be the only clinical sign of sitosterolemia.
