Proxymal Nocturnal Haemoglobinuria (Pnh)

7/30/2019 Proxymal Nocturnal Haemoglobinuria (Pnh)

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GUIDES:- Sh. S.K. Sharma(Lecturer)

Sh.A.P. Chauhan(Tutor)

PRESENTED BY:- Keshav Raj Poudel

DEPARTMENT OFHAEMATOLOGY


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PAROXYMAL NOCTURNAL HAEMOGLOBINURIA (PNH)PNH is the chronic disorder in which I/V

haemolysis occurs due to acquired defects in Redcells which renders the membrane highlysusceptible to lysis by complement. It is a clonaldisorder due to somatic mutation in multipotent

haemopoietic stem cells. White cells and plateletsare also affected by mutation and thrombosis is adangerous complication. In many cases theemergence of PNH clone is closely linked to

underlying defect of marrow function, particularlyaplastic anemia, myelosclerosis and leukemia.Symptom is ordinarily dominated by chronichaemolysis and occasionally terminates as acute

myelogenous leukemia.


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Etiology and Pathogenesis:

PNH is due to the somatic mutation of a geneon x- chromosome which enclose a protein- phosphatidyl

glycan protein A(PGPA). Protein A which is essential for theformation ofglycerol phosphotidyl inositol (GPI) . It acts as ananchor protein by which no of proteins are attached to RBCmembrane

S.N

Proteins Examples Designation

1. Compliment

regulatoryproteins

*decay accelerating factor

*membrane inhibition of reactivelysis*C8 binding proteins (HRF)

*DAF,CD55,

MIRL, CD59C8BP, HRF

2. ENZYMES Erythrocyte acetyl colin esteraseNeutrophil alkaline phosphatase

NAP


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3. Immunefunctionproteins

Lymphocytes function antigen 3-Neutrophil Fcy iii receptor-Monocyte endotoxin binding

protein receptor- Campath binding protein

LFA 3CD16 aCD14

CD52

4. Otherproteins

Monocyte urokinase receptorJMH antigen binding protein

Four granulocyte surface proteinFolate receptor

Among these different proteins two proteins normally protect

the cell from lysis by activated complement . They are :1. Decay accelerating factor (CD55)2.Membrane inhibitor of reactive lysis(MIRL or CD59)


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CD55 accelerates the conversion of C3b to inactivate C3d .

CD59 protects the cell from lysis by the membrane attackcomplex , the final product of complement activation

GPI anchored proteins are also missing from white cells andplatelets results thrombotic tendency in PNH patient .

There are three population of PNH red cells according to thedeficiency of CD59.

a) Very sensitive PNH (type III) red cell: 10 -15 times moresensitive than normal. PNH type III red cell have completedeficiency of CD59 and lysed by cobra venom factor.

b) Cells of medium sensitivity(type II) : 3-5 times more sensitive

than normal cells. They have only partial deficiency of CD 59,dont lysed by cobra venom factor.

c) Cells of normal sensitive(type I) : They are equally sensitive asnormal cells and lack enzyme Erythrocyte acetylcolinestarase.


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In vivo the proportion of type III cells parallel the severity ofpatient haemolysis.

78% of PNH patients blood contains a simple mixture of

PNHI and PNH III cells.- Haemolysis is mild when the proportion of PNH III cells is

50%

Decay accelerating factor CD55 :

It is a 70,000 molecular weight glycoprotein that binds to C3b and C4b fragments deposit in the cell membranes, blocksassembly of the convertase complex ( C4b2b of classicalpathway and C3bBb complex of the alternative pathway

)and hasten their decay.


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Complements and its activation Pathways

Complement constitutes of the series of protein, mainly enzymes

present in the fresh plasma as inactive precursor, which reactsequentially with each other to form product that are importantin the destruction of the cells, bacteria etc.

There are total 9 complements denoted by C1-C9. They are

activated in two stages.Stages of complement activation

1. Optionization phase

2. Lytic stage

Optionization phase:

This phase is completed in two pathways

1. Classical pathway

2. Alternative pathway


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Classical pathway It can be activated by antigen antibody complex

,enzymes(tripsin, plasmin , lysosomal enzyme) , Endotoxin,low ionic strength media etc. Only one molecule of IgM or atleast two molecules of IgG on the red cell membrane isnecessary to activate the complement system because IgM Abcarries several C1q binding sites whereas one mol. of IgG carry

only one. First component of the complement activation is formation of

complex of three protein molecules C1q, C1r and C1s

After Abs bound to their Ag, C1 binding sites are exposedon the Fc fragment , C1q sub units binds to it andactivates C1r which in turns cleaves the third moleculeC1s, yielding a active enzyme form of C1 complex which isheld together by calcium. In the presence of EDTA orother chelating agent the complex falls apart and whole

process of complement fixation will not occur.


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This complex activates sequentially C4 ad C2 inthe presence of Mg++, generates secondenzyme, C4b2b called C3 convertase.

The cell bounded C3 convertase optimallyactivates several thousand mol. Since, largeamount of C3 is present in the cell.

C3b attach red cell will adhere to the Monocyteand macrophages though their C3b receptors

and may be phagocytes or it is rapidlydegraded by an enzyme (C3b inactivator,factor I ) to C3d remain in the red cell surface.

By occupying the binding C3 sites by C3d , canprevent the further binding of C3b. C3d,unlike C3b is not capable of adhering to thereceptor on macrophages and monocyte sothat the cell coated with C3d may return tothe circulation and will be resistant to thefurther lysis.


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Alternative pathway Does not necessary involves Ag Ab reaction and represents

non specific innate immunity . The alternative pathway can

be activated by IgA , zymosen, bacterial cell orLipopolysaccharides.

It is two step process.

1. Binding of C3b to the activator and interaction of bound

C3b with neighboring surface structure initiallyspontaneously generate fluid phase C3b interact with thefactor B to form a complex .

2. Factor B is activated through a cleavage by the proteasefactor D releasing a fragment Ba into the plasma and

yielding a transient alternative C3 convertase ( C3bBb)3. C3 convertase ( C3bBb) can be stabilized by properdin.

Although, properdin is no longer implicated ininitiating the alternating pathway, it is essential for

preventing the dissociation of C3bBb by factor H.


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Alternative C3 convertase splits serum C3 into C3a and C3b .

The amount of C3b deposited by alternative pathway is low

due to the small amount of the convertase generated and tothe insufficient deposition of C3b from plasma. Thiscontrasts with the vast no. of C3b molecules generated bythe classical pathway.

The classical and alternative pathway cannot beseparated from each other in vivo, the alternative pathwayamplified the classical pathway because, when C3b isgenerated, factor B and D may be activated and complex

with it to generate further C3b.


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The Lytic Phase of complement sequence

It start with the activation of C5 by C3b, yielding membranebounded C5b and fluid phase C5a. This step is followed by

the non enzymatic interaction of C5b with C6, C7, C8 andC9.

These molecule adhere to each other to form membraneattack complex (MAC) and insert themselves to the lipidbilayer of the red cell membrane. C8 catalyzed by C9produced lesion in the membrane .These lesions appears asprotein linked cylinders in the red cell membrane. and areabout 10 nm in diameter. They form pores through whichions and water cam enter. The osmotic pressure by Hb draw

water into the cell until it swells and burst.Optimum temperature and pH for complement lysis:

Optimum pH 6.8

Optimum temperature 32-37oC, below 15 red cell cant behaemolysed by complement


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Clinical Features

1. Nocturnal I/v Haemolysis with negative DAT( occasionallypositive)

2. Nocturnal Haemolysis with low platelets, low WBC counts.

3. Symptoms of anemia and Nocturnal Hemoglobinuria with darkurine.

4. Recurrent abdominal pain with or without blood in stool.5. Hepatic vein thrombosis.(Budd-chairi syndrome)

6. Pancytopenia and marrow failure, particularly if there is a reactivereticulocytosis.

7. Abdominal pain, headache.

8. Thrombophlebitis and thromboembolism

9. Mild jaundice.

10. Mild spleenomegaly.

11. Mild hepatomegaly, marked hepatomegaly in hepatic vein

thrombosis.


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Laboratory DiagnosisHaemogram :

HB decreased

PCV decreasedRBC count decreasedWBC count mild decreased

Platelets count mild decreasedMCV normal

MCH normalMCHC normal

MCV,MCH,MCHC decrease in chronic caseReticulocyte count increased but lesser than expected for

degree of anaemia because retics are more

susceptible to compliment lysis thanmature PNH red cell.DLC Mild neutropeniaPBF N/N , mild macrocytic and mild microcytic

hypochromic RBCs, Neuropenia and Thrombocytopenia


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Signs of I/V Haemolysis

Plasma Hb High

Urine Hb Present

Haemosidenuria PositiveHeptoglobin Decreased or Absent

Haemopexin Decreased

Methemealbumin Increased

LDH IncreasedBilirubin Increased

Urine urobilinogen Increased

Othre tests

Methemoglobin IncreasedSerum iron Decreased

Serum ferritin Decreased

DAT Negative , occasionally positive

OFT Normal


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NAP (Neutrophil Alkaline Phosphate): Reduced

PNH patient usually possess a sub population of granulocyte thatexhibit impaired phagocytosis and chemotaxis when exposed to

activated complement. Mutant granulocyte in PNH are asdeficient in leukocyte alkaline phosphatase as the granulocyteof CML. Only those PNH granulocytes that are deficient in LAPare unresponsive to chemoattractants . Lack of LAP in CMLgranulocyte is associated with absence of L AP mRNA, whereasin PNH LAP mRNA is normal.

Bone Marrow:

Normoblastic Erythroid Hyperplasia, rarely Megaloblastic ,Megakaryocytes are diminished, in pancytopenia , BM ishypoplastic.

.

Specific test for PNH :


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Specific test for PNH :

Definitive diag. of PNH in vitro is done by variety of tests-

1. By the activation of alternative pathway. Eg

a)HAMS test

2. By the activation of classical pathways Eg.

a) Sucrose lysis test

3.Flowcytometric analysis

HAMS test:

The standard Hams test significantly underestimates proportional PNHred cells. The standard Hams test can be negative when there are less

than 5% PNH type III cells or less than 20% PNH type II cells. Whenthe Hems test is supplemented with Mg , to optimize the activation ofcompliment, the percentage lysis gives the more accurate estimation ofproportion of PNH cells.

Methods: 1) Hams test 2) Modified Hams test


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principlePatient red cells undergo haemolysis when incubated with

compatible acidified serum (PH-6.5-7.0) at 37oC.The serum may

be of the patient serum or from another normal subject.The sensitivity of Hams test can be improved by the addition of

Magnesium to the test to enhance the activation of compliment.

Sample collection:

Every effort must be made to prevent haemolysis during collectionand manipulation of sample.

Defibrinated blood collection:

5-6 ml of patients or normal controls blood is collected in a

disposable syringe having wide bore needle with clean venipuncture. Blood is poured into the flask of 20 ml capacitycontaining 5-10 glass beads. The flask is gently rotated until theblood is clotted(10- 15 mins). Place the flask inside incubator for

5mins. Then the fluid portion is taken out in a clean test tubeand serum is separated after centrifugation at low speed.


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Preparation of Fresh normal serum:ABO group compatible normal serumThere is variability between the sera of individual in there capacity to

lyse PNH Red cells . The activity of a single individual s serum also

varies from time to time. Therefore those normal controls are takenwhose serum sufficiently lyse PNH RBCs.Required for test:a) Normal fresh serumb) Patients serum

c) 50% normal cells suspensiond) 50% patients cells suspensionpreparation of heat inactivated serum:Serum is placed in tube and tubes are placed in water bath at 56oC for

30.

5)Positive control:It is always important to include in any test , as a positive control , a

sample of known PNH cells or artificially created PNH likecells(sulphydryl compounds, can act on normal red cells in vitro so asto increase there compliment sensitivity)


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Requirements:

Apparatus:

1) Flasks(20ml) with some piece of glass beads.

2.) Centrifuge 3) Test tube & test tube rack4) Water bath 5) Pipettes

6) Spectrophotometer

Reagents:

1) 0.2 mole/ l HCL 2) Normal saline

3) 250 m mol/l MgCl2 ( 23.7 g/l)Procedure

S.N REASENT CONTROL CELL

1.

/CONTROLSERU

M2. 3.

TESTcells

4.

/CONTROLSERUM

5. 6.

TESTSERUM

7.

/TESTCELLS

8. 9.

1. Fresh normalserum(ml)

0.5 0.5 0.5 0.5 0.5 0.5 - - -

2. Patients

serum(ml)

- - - - - - 0.5 0.5 0.5


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Heat inactivate the serum of Tube no 3,6 and 9.

3 0.2mol/l HCL(ml)

- 0.05 .05 - 0.05 0.05 - 0.05 .05

4 50% patientRBCs (ml)

- - - 0.05 0.05 0.05 0.05 0.05 .05

5 50% normalRBCs (ml)

0.05 0.05 .05 - - - - - -

6 250mol/l MgCl2(ml)

0.01 0.01 .01 0.01 0.01 0.01 0.01 0.01 .01

Mix the content and leave for 1hr at 37C.Centrifuge and look for haemolysis on tube no. 5 and 8.

Calculation of % of lysis.Blank- normal serum 0.5ml100% lysis( standard) - 50% washed red cells

0.05 ml in o.55ml water.Test - Supernatant of tube no.5 or 8


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Take 0.3ml of each blank, 100% lysis RBCS and supernatant oftest tube to be tested for haemolysis in 5ml of 0.4ml/l amonia ordrabkins reagent.

Measure the lysis in the photoelectric colorimeter using the

yellow green filter or in spectrometer at a wavelength of 540 nm. Calculation: % lysis = (Test Blank ) 100

(Std Blank)

False Positive Result:HEMPAS gives positive Hams test withnormal serum but not withpatients serum. In HEMPAS lysis is due to the presence ofunusual Ag on red cell surface which react with complementfixing IgM Ab (anti HEMPAS) present in many but not in all

sera.Markedly spherocytic red cells may lyse in acidify serum probablydue to lowered pH and such cells may lyse too in acidifiedinactive serum.

If acidify serum test is Positive . It is recommended to carry out

direct AGT . If it is positive the lysis could be due to lytic Ab.


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Sucrose lysis test

It is useful screening test for PNH. It is more sensitive than Hams

test but lack the specificity.Principle:

Red cell absorbed the compliment from serum at low ionicstrength. PNH cells is greater sensitive to complement undergoeslysis but not normal cells.

More than 10% lysis implies a positive test. 5-10% lysis is boarderline . The red cells of some cases of Leukemia or myelosclerosisgives less than 10% lysis. The sucrose lysis test is typically neg. inHEMPAS(hereditary erythroid multinuclearity associated with apositive acidified serum)

Reagent :

a) Iso osmotic solution of sucrose(92.4 g/l)

Procedure:

Take 2 tubes 12*75 mm and proceed as


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Incubation at 37 0C for 30 , centrifuge the tubes and examine for lysis .Calculation of % of lysis.Blank- Supernatant of tube no. 2

100% lysis( standard) - 50% washed red cells0.1ml in 0.9 ml of ammonia 0.4 ml/l .Test - Supernatant of tube no.1Measure the lysis in the photoelectric colorimeter using the yellow green filter or inspectrometer at a wavelength of 540 nm as above.Calculation: % lysis = (Test Blank ) 100

(Std Blank)

Reagents Tube No 1.(test)

Tube No2.(negative control)

Tube No3.(normal control)

sucrose solution 0.85 ml - 0.85 ml

saline - 0.85 ml -Normal compatible serum 0.05 ml 0.05 ml 0.05 ml

50% patients cells 0.1 ml 0.1 ml -

50% Normal cells - - 0.1 ml


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Flowcytometry

Principle:

Various immunoflurescent dyes coated with Ab can beattached to the Ag present in the cells or particles. As thesample enters the flow channels and the cells are passedthrough a focused laser beam one cell at a time. As the cellsor particles intercept the light source they scatter light andflurochrome are excited to higher energy state. This energyis released as a photon of light with specific spectralproperties unique to different fluorochromes. These lightare captured and converted to electrical signals. One

unique feature of fluorocytometry is that it measure thefluoroscent per cell or particle. This contrast with thespectrophotometry in which the percent abs. andtransmission of specific wavelength of light for a bulk

volume of sample.


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FCM

lymphoma Chronic

lymphoid

leukemia

Plasma

celldisorder

s

Acuteleukemi

a

PNH

Mast cell

disease

MDS

CMPD

HEMATOLOGY TESTFORFLOWCYTROMETRY


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FLOCYTOMETRY ANALYSIS OF PNH CELLSprinciple:

Patient red cells are stained with a fluorescein labelled antibodythat is specific for one of several GPI-linked proteins egCD55,CD59 etc. which are deficence in PNH red cells. Thestained cells are then analyzed with a flow cytometer.


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Procedure

Steps for RBCs

1. Three control tubes and three test tubes are taken andlabeled as T- Negative , T- CD55, TCD59, C-Negative, C-CD55, C-CD59 .

2. The 50 ul of suspension(50ul of whole blood + 500 ul ofPBS) and 5ul of Ab are mixed in each tube.

3. Incubate in dark for 30 mins

4. Add 1 ml of PBS and then centrifuge for 5 min at 1000

rpm.5. Discard the supernatant.

6. Wash cell again as step 4 and 5.

7. Resuspend the deposit in 500 ul of PBS.

8. Cap the tube and keep at 4oC


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Procedure for WBC

1. Four control tubes and four test tubes are taken and labeled asT- Negative , T- CD55, TCD59, TCD16, C-Negative, C- CD55, C-CD59, C-CD16.

2. The 50ul of whole blood and 5ul of Ab are mixed in each tube.

3. Incubate in dark for 30 mins at RT.4. Add 1 ml of FACS lysing solution and mix in vortex mixture andkeep it for ten mins.

5. Centrifuge for 5 min at 2000 rpm.

6. Discard the supernatant and tip off on the tissue paper.

7. Add 1 ml of PBS and then centrifuge for 5 min at 2000 rpm.8. Discard the supernatant.

9. Wash the cell again as in step 7 and 8.

10. Resuspend the deposit in 500 ul of PBS.

11. Cap the tube and keep at 4oC .


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Above prepared suspension is taken for flowcytometricanalysis.

Normal Range:

Less than 5% of CD59 cells are considered as normal.

Precaution:

1. PBS is freshly filtered and then be used.

2. Speed of centrifuge should be maintained up to 1200 rpmotherwise high speed breaks the cells.

3. Storage the kit at 2-8 0C

4. Once started , the test must be performed withoutinterruption.

5. Avoid carry over contamination.


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Side Scatter

FSCDetector

CollectionLens

SSCDetector

Laser Beam

Cellgranularity


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Treatment

1.Folic acid should be given prophylactically .2. Blood transfusion:

a) washed red cell

b) saline adenine , glucose, mannitol , preserved blood.

3. Treatment of thrombosis:a)Oral Anticoagulants ( maintain the INR between 2-3.5)

b)Full Heparinization

c)Fibrinolytic therapy (Tissue type plasmin activator)

4.Bone marrow transplantation


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Documents

Proxymal Nocturnal Haemoglobinuria (Pnh)