LABORATORY TESTING OF SPHEROCYTIC ANAEMIA

KGMU LUCKNOW

Dr Brijendra Bahadur Singh

SpherocyteSpherocytes are round, slightly smaller than normal red blood cells, and lack central pallor

•Decreased surface area to volume ratio.•Most surface tension efficient and least flexible.

+ve -ve

Family history +ve family history -ve

Immune mediated hemolytic anemia

OFT

Coombs test

High retic count

Spherocytes in PBS

Retic. count

Heinz bodies+

HS

HS confirmed

EMA, dye binding test, SDS PAGE

Bite cellUnstable

Hb

G6PD assay

G6PD def.

Isopropanol test, Heat

denaturation test

Unstable Hb disease

DD of Spherocytic anemia• Hereditary Spherocytosis• Immune Hemolytic Anemia• Oxidant damage - G6PD deficiency - Unstable Hemoglobin• Thermal injury• Toxin - Clostridium - Bee, Spider, Snake venom

Normal RBC Skeleton

Hereditary Spherocytosis• Caused by defect in proteins that disrupt the vertical

interactions between the transmembrane protein and underlying protein cytoskeleton.

• 75% of families inherited as autosomal dominant trait • 25% of cases inherited as non dominant

Pathophysiology• Gene mutations where the defective proteins disrupt the

vertical linkages include:• ANK1 – ankyrin (40 - 50% of cases)• SPTA1- α spectrin( ≤ 5 % of cases)

• SPTB- β spectrin(15 -30 % of cases)• EPB42- protein 4.2(≤ 5 % of cases)• SLC4A1- band 3(20 -30 % of cases)

Mechanism in HS

Clinical & Laboratory Findings In HS• Clinical manifestations : - Splenomegaly - Anemia - Jaundice • Complete blood count result : -↓ hemoglobin -↑ MCHC -↑RDW -↑ reticulocyte count • PBS finding: Spherocytes, polychromasia• Direct antiglobulin test result – negative• Indicators of hemolysis – ↓ serum haptoglobin, ↑ serum LDH, ↑ serum indirect bilirubin.• Bone marrow shows erythroid hyperplasia

Peripheral Blood Picture

Additional Tests For Atypical Cases Of HSOsmotic fragility test

Eosin-5’-maleimide binding test

SDS-PAGE

Ektacytometry

Acidified glycerol lysis test

Autohemolysis test

Cryohemolysis test

Osmotic Fragility Test

• It gives an indication of the surface area/volume ratio of the erythrocytes. As this ratio falls cells become more sensitive to osmotic lysis.

• Red blood cells that are spherocytic , for whatever cause, take up less water in a hypotonic solution before rupturing than do normal blood cells.

Principle of OFT

• When RBCs are placed in hypotonic solution water is drawn into RBC

• This result in swelling of cell, which acquire a spherical shape

• After a critical volume is reached membrane first leaks small molecules like potassium ions

• Membrane pores increase in diameter, larger molecules like Hb leave the cell

• The amount of Hb in supernatant is measured colorimetrically and compared with sample of completely lysed cells

Reagents used in OFT:

• Stock solution of 10% Nacl (100gm in 1 ltr)

• Defibrinated or heparinzed blood sample( as other anticoagulants likely to induce morphological changes which may affect the result)

• The test should be set up within 2 hours of collection, or within 6 hours if the blood is refrigerated

Technique of OFT• Prepare a series of saline dilutions of 0.90%, 0.70%, 0.60%, 0.55%,

0.50%, 0.45%, 0.40%, 0.35%, 0.20% & 0.10%. (more convenient to make 1% saline solution by taking 1 part of stock solution + 9 parts of distilled water and then make further dilutions)

• Add 5 ml of each saline solution to respective test tubes & add 5 ml of distilled water to last test tube

• Add 50 μliter of defibrinated or heparinized blood to each tube & mix immediately by inverting tubes several times, avoiding foam

TEST TUBE1% buffered NaCl in ml. D.W in ml

Final conc(%)% hemolysis

1 0.00 10 0.002 1.0 9.0 0.103 2.0 8.0 0.204 3.0 7.0 0.305 3.5 6.5 0.356 4.0 6.0 0.407 4.5 5.5 0.458 5.0 5.0 0.50910

5.56.0

4.54.0

0.550.60

11 7.0 3.0 0.7012 8.0 2.0 0.8013 9.0 1.0 0.90

Technique of OFT• Wait for 30 mins at room temperature• Mix again and centrifuge for 5 mins @1200rpm• Remove the supernatant and estimate the amount of lysis in the supernatant

using spectrophotometer at 540 ηm• Use tube 13(or with 0.90% saline) as blank and assign value of 100% lysis

to 1st tube(or tube DW ) – Express the readings of other tube as % of value of 1st tube

• Percent of hemolysis = O.D. of supernatant x 100 O.D. supernatant 1st tube

Eosin-5'-maleimide test

• EMA is fluorescent dye that binds to transmembrane protein band 3, Rh, RhAg and CD47in the RBC membrane

• HS patients show a lower mean fluroscence intensity( MFI) than RBCs from normal control and from patients with spherocytes due to immune-mediated hemolysis

• Sensitivity- 93% - 96%• Specificity- 94% - 99%

Ektacytometry-• Variation in membrane surface area and cell water content can

be determined by osmotic gradient ektacytometry • Ektacytometer is a laser-diffraction viscometer that records the

laser diffraction pattern of a suspension of RBCs exposed to constant shear stress in solutions of varying osmolality from hypotonic to hypertonic

• An RBC osmotic deformability index is calculated and plotted against the osmolality of the suspending solution

Additional tests for HS

Cryohemolysis Test- HS cells are particularly sensitive to cooling at 0⁰C in hypertonic solution. % hemolysis is calculated after patient’s RBCs are incubated in buffered 0.7mol/l sucrose first at 37 ⁰C for 10 minutes and then at 0⁰C for 10 mins. – Normal cells show 3% - 15% hemolysis – RBCs in HS greater than 20% hemolysis

• SDS-PAGE- Identify membrane protein deficiencies by separation of various proteins in solubilized RBC membranes with quantitation of the proteins by densitometry

Additional tests for HS• Acidified Glycerol Lysis test- measures the amount of hemolysis after

patient RBCs are incubated with a buffered glycerol solution at an acid pH

• Autohemolysis Test- Patient RBCs and serum are incubated for 48 hours with and with out glucose. – Normal controls : - <5% hemolysis at the end of 48 hours incubation period - < 1% hemolysis if glucose is added. – In HS hemolysis : 10% - 50%

Immune Hemolytic Anaemia Autoimmune Drug induced Alloimmunehemolytic anemia hemolytic anemia hemolytic anemia

-Warm AIHA - Drug dependent -Hemolytic transfusion -Cold agglutinin reaction disease - Drug independent -Hemolytic disease of -Paroxymal cold new born hemoglobinuria -Mixed AIHA

Warm AIHA Cold agglutinin disease

Paroxymal cold hemoglobinuria

Mixed-Type AIHA

Ig Class IgG IgM IgG IgG, IgM

Optimum reactivity temp

37⁰C 4⁰C 4⁰C 4-37⁰C

Sensitization detected by DAT

IgG or IgG +C3d

C3d C3d IgG and C3d

Complement activation

Variable Yes Yes Yes

Hemolysis Extravascular primarily

Extravascular; rarely intravascular

Intravascular Both

Auto Ab specificity

Pan reactive or Rh complex

I(most), i(some)Pr(rare)

P Panreactive; unclear specificity

Warm AIHA• May be either primary or secondary in etiology

– Primary-idiopathic in nature– Secondary-due to an underlying disease

• Lymphoproliferative disorders-CLL, B lymphocytic lymphoma• Non lymphoid neoplasm- Thymoma, Ca-lung, colon, kidney,ovary• Autoimmune diseases- SLE, RA, PAN• Infection- Viral• Immunodeficiency disorder

Mechanism of Destruction• Primarily due to extravascular hemolysis

• Antibodies bind to surface of RBC membrane

• Fc portion of antibody binds to macrophages– Interaction → spherocytes

• Spherocytes become trapped in spleen and are destroyed

Cold Agglutinin Disease• Due to development of an IgM antibody• Antibody active at cold temperature (4°C) and not usually

physiologically significant• Either primary or secondary in etiology

– Primary-idiopathic in nature– Secondary-due to an underlying disease

• Infection- Mycoplasma pneumoniae, Infectious mononucleosis• Lymphoproliferative disorder- CLL, B lymphocytic lymphoma

Mechanism of destruction• Intravascular hemolysis

– IgM antibodies activate the compliment system →cytolysis

• Extravascular hemolysis (predominantly)– C3b and iC3b rather than the Fc portion of IgM are recognized– Hemolysis occurs in the liver via action of Kupffer cells

Laboratory findings in immune mediated hemolytic anemia• ↓hemoglobin• ↓ serum haptoglobin level• ↑reticulocyte count• ↑ indirect serum bilirubin and LDH• MCV may be ↑• Hemoglobinuria(intravascular hemolysis or severe extravascular

hemolysis)• PBS- Polychromasia, spherocytes, occasionally RBC agglutination,

nucleated RBCs, schistocytes and erythrophagocytosis

Coomb's Test(Antiglobulin Test)Principle of Antiglobulin Test • The incomplete antibodies (IgG) attach to red cell membrane

by the Fab portion of the immunoglobulin molecule (IgG)

• The IgG molecules attached to the red cells are unable to bridge the gap between sensitized red cells which are separated from each other by the negative charge on their surface and the sensitized red cells do not agglutinate

Coombs Test

Glucose-6-phosphate dehydrogenase deficiency• Important intracellular enzyme for protecting hemoglobin &

other cellular protein and lipids from oxidative denaturatation• Gene located on X chromosome• Deficiency is the most common RBC enzyme defect• G6PD-deficient RBCs can’t generate sufficient NADPH to

reduce glutathione and thus can’t effectively detoxify hydrogen peroxide

Clinical and laboratory findings in G6PD deficiency• History: Recent infection, administration of drugs, ingestion of

fava beans• Clinical manifestation: Chills, fever, headache, nausea, backpain

and abdominal pain, jaundice and dark urine• Indicator of hemoylsis: ↓serum haptoglobin, ↑serum LDH, ↑

serum indirect bilirubin, hemoglobinemia and hemoglobinuria

Clinical and laboratory findings in G6PD deficiency• CBC results: ↓ hemoglobin, ↑reticulocyte count• PBS finding: Polychromasia, RBC morphology varies from

normal to marked anisocytosis, poikilocytosis, spherocytosis or schistocytes, bite cells and heinz bodies

• DAT result: Negative

PBS of G6PD deficient patient

Heinz bodies

Tests for G6PD deficiency

Quantitative spectrophotometric assays

Fluorescent spot test.

Dye reduction assays.

Quantitative Spectrophotometric Assays• Gold standard for determining G6PD deficiency• Based on direct measurement of NADPH generated • G-6-P+ NADP G6PD 6-Phosphogluconate + NADPH

• Rate of NADPH formation ≈ G6PD activity and measured as increase

in absorbance at 340nm• Activity is measured as a ratio of G6PD activity per gram of

hemoglobin(g Hb)• Cut off point for G6PD deficiency set as < 20% of normal activity (<

4.0 IU/g Hb)

Fluorescent Spot Test

• Based on the principle that NADPH generated is fluorescent• Blood and glucose-6-phosphate/NADP reagent are incubated

and spotted on filter paper at timed intervals• Normal G6PD activity – moderate to strong fluorescent spots

under UV light• Decreased or no activity – display weak or no fluorescence

Dye Reduction Assay

• NADPH produced by enzymatic reaction reduces a dye, giving a visually observed colour change

• In normal G6PD activity, NADPH generated reduces the dye to formazan product, brown black in colour

• Senstivity of 98% in detecting deficient specimen with G6PD deficiency < 4% IU/g Hb

•Peripheral blood film of microspherocytes seen in Clostridium perfringens sepsis

•regular spherocytes are usually smaller than normocytic red blood cells, microspherocytes are even smaller than that

•usually seen in critically ill, septic patients with severe C. perfringens infection

MCQ

1. In hemolysis mediated by IgG antibodies, which abnormal RBC morphology is typically observed on the peripheral blood film?

a- Spherocytes b- Nucleated RBCs c- RBC agglutination d- Macrocytes

2- In autoimmune hemolytic anemia, a positive DAT is evidence that an:

A- IgM antibody is in the patient’s serum B- IgG antibody is in the patient serum C- IgM antibody is sensitizing the patient’s RBCs D- IgG antibody is sensitizing the patient’s RBCs

3- The most important finding in the diagnostic investigation of a suspected autoimmune hemolytic anemia is:

a- Detection of a low hemoglobin and hematocrit b- Observation of hemoglobinemia in a specimen c- Recognition of a low reticulocyte count d- Demonstration of IgG and /C3d on the RBC surface

4- In HS a characteristic abnormality in the CBC results is: a- increased MCV b- increased MCHC c- decreased MCH d- decreased platelet and WBC count

5- An altered shape of spherocyte in HS is due to : a- An abnormal RBC membrane protein affecting vertical

protein interactions b- Defective RNA synthesis c- An extrinsic factor in Plasma d- Abnormality in the globin composition of the hemoglobin

molecule

6- Which of the following results are consistent with HS? a- Increased osmotic fragility, negative DAT result b- Decreased osmotic fragility, positive DAT result c- Increased osmotic fragility , positive DAT result d- Decreased osmotic fragility, negative DAT result

7- Increased osmotic fragility is seen in all of the following except a- Hereditary spherocytosis b- Autoimmune immune hemolytic anemia c- Hereditary elliptocytosis d- Thalassemia

8- A patient experiences an episode of acute intravascular hemolysis after taking primaquine for the first time. The physician suspects that the patient may have G6PD deficiency and orders an RBC G6PD assay 2 days after the hemolytic episode begins. How will this affect the test result?

a- No effect b- False increase due to reticulocytosis c- False decrease due to hemoglobinemia d- Absence of enzyme activity

9- All of the following are indicators of hemolysis except a- Increased serum haptoglobin b- Decreased hemoglobin c- Increased serum indirect bilirubin d- Increased LDH

10- Immune mediated hemolytic anemia is due to a(n): a- Structural defect in the RBC membrane b- Allo- or autoantibody against an RBC antigen c- T cell immune response against an RBC antigen d- Obstruction of blood flow by intravascular thrombi

ANSWERS 1- a 2- d 3- d 4- b 5- a 6- a 7- d 8- b 9- a 10- b