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

Chauhan(Tutor)

PRESENTED BY:- Keshav Raj Poudel

DEPARTMENT OF HAEMATOLOGY

PAROXYMAL NOCTURNAL HAEMOGLOBINURIA (PNH)

PNH is the chronic disorder in which I/V haemolysis occurs due to acquired defects in Red cells which renders the membrane highly susceptible to lysis by complement. It is a clonal disorder due to somatic mutation in multipotent haemopoietic stem cells. White cells and platelets are also affected by mutation and thrombosis is a dangerous complication. In many cases the emergence of PNH clone is closely linked to underlying defect of marrow function, particularly aplastic anemia, myelosclerosis and leukemia. Symptom is ordinarily dominated by chronic haemolysis and occasionally terminates as acute myelogenous leukemia.

Etiology and Pathogenesis: PNH is due to the somatic mutation of a gene on x- chromosome which enclose a protein- phosphatidyl glycan protein A (PGPA). Protein A which is essential for the formation of glycerol phosphotidyl inositol (GPI) . It acts as an anchor protein by which no of proteins are attached to RBC membrane S. N
1.

Proteins
Compliment regulatory proteins ENZYMES

Examples
*decay accelerating factor *membrane inhibition of reactive lysis *C8 binding proteins (HRF) Erythrocyte acetyl colin esterase Neutrophil alkaline phosphatase

Designation
*DAF,CD55, MIRL, CD59 C8BP, HRF NAP

2.

3. Immune Lymphocytes function antigen 3 function -Neutrophil Fcy iii receptor proteins -Monocyte endotoxin binding protein receptor - Campath binding protein 4. Other Monocyte urokinase receptor proteins JMH antigen binding protein Four granulocyte surface protein Folate receptor

LFA 3 CD16 a CD14 CD52

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)

CD55 accelerates the conversion of C3b to inactivate C3d . CD59 protects the cell from lysis by the membrane attack complex , the final product of complement activation GPI anchored proteins are also missing from white cells and platelets results thrombotic tendency in PNH patient . There are three population of PNH red cells according to the deficiency of CD59. a) Very sensitive PNH (type III) red cell: 10 -15 times more sensitive than normal. PNH type III red cell have complete deficiency 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 as normal cells and lack enzyme Erythrocyte acetylcolinestarase.

In vivo the proportion of type III cells parallel the severity of patient 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 <20% - Haemolysis is paroxysmal (episodic) proportion of PNH III cells is 20-50 % - Perpetual haemoglobinemia and hemoglobinuria if the proportion of PNH III red cell is > 50% Decay accelerating factor CD55 : It is a 70,000 molecular weight glycoprotein that binds to C3 b and C4b fragments deposit in the cell membranes, blocks assembly of the convertase complex ( C4b2b of classical pathway and C3bBb complex of the alternative pathway )and hasten their decay.

DAF protect normal analogous cells from complement attack. It is absent or markedly reduced on the surface of PNH red cells , leukocytes and platelets. In its absence, convertase complex are stabilized , C3b is fixed more tightly to the cell surfaces, and cell lysis is modestly increased. But its absence is not major cause of haemolysis in PNH. C3b(active) CD55(DAF)___ C3d(inactive) Accelerates Membrane Inhibitor of Reactive Lysis(CD59 ,MIRL) Its mol wt. is 20,000. It inhibit the formation of membrane attack complex ( C5b 9). Its absence is the principle cause of haemolysis in PNH.

Complements and its activation Pathways Complement constitutes of the series of protein, mainly enzymes present in the fresh plasma as inactive precursor, which react sequentially with each other to form product that are important in 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

 It can be activated by antigen antibody complex

Classical pathway

,enzymes(tripsin, plasmin , lysosomal enzyme) , Endotoxin, low ionic strength media etc. Only one molecule of IgM or at least two molecules of IgG on the red cell membrane is necessary to activate the complement system because IgM Ab carries 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 exposed

on the Fc fragment , C1q sub units binds to it and activates C1r which in turns cleaves the third molecule C1s, yielding a active enzyme form of C1 complex which is held together by calcium. In the presence of EDTA or other chelating agent the complex falls apart and whole process of complement fixation will not occur.

This complex activates sequentially C4 ad C2 in the presence of Mg++, generates second enzyme, C4b2b called C3 convertase. The cell bounded C3 convertase optimally activates several thousand mol. Since, large amount of C3 is present in the cell. C3b attach red cell will adhere to the Monocyte and macrophages though their C3b receptors and may be phagocytes or it is rapidly degraded by an enzyme (C3b inactivator, factor I ) to C3d remain in the red cell surface. By occupying the binding C3 sites by C3d , can prevent the further binding of C3b. C3d, unlike C3b is not capable of adhering to the receptor on macrophages and monocyte so that the cell coated with C3d may return to the circulation and will be resistant to the further lysis.

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 or Lipopolysaccharides. It is two step process. 1. Binding of C3b to the activator and interaction of bound C3b with neighboring surface structure initially spontaneously generate fluid phase C3b interact with the factor B to form a complex . 2. Factor B is activated through a cleavage by the protease factor 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 in initiating the alternating pathway, it is essential for preventing the dissociation of C3bBb by factor H.

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 to the insufficient deposition of C3b from plasma. This contrasts with the vast no. of C3b molecules generated by the classical pathway. The classical and alternative pathway cannot be separated from each other in vivo, the alternative pathway amplified the classical pathway because, when C3b is generated, factor B and D may be activated and complex with it to generate further C3b.

The Lytic Phase of complement sequence

It start with the activation of C5 by C3b, yielding membrane bounded C5b and fluid phase C5a. This step is followed by the non enzymatic interaction of C5b with C6, C7, C8 and C9. These molecule adhere to each other to form membrane attack complex (MAC) and insert themselves to the lipid bilayer of the red cell membrane. C8 catalyzed by C9 produced lesion in the membrane .These lesions appears as protein linked cylinders in the red cell membrane. and are about 10 nm in diameter. They form pores through which ions 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 be haemolysed by complement

Clinical Features
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Nocturnal I/v Haemolysis with negative DAT( occasionally positive) Nocturnal Haemolysis with low platelets, low WBC counts. Symptoms of anemia and Nocturnal Hemoglobinuria with dark urine. Recurrent abdominal pain with or without blood in stool. Hepatic vein thrombosis.(Budd-chairi syndrome) Pancytopenia and marrow failure, particularly if there is a reactive reticulocytosis. Abdominal pain, headache. Thrombophlebitis and thromboembolism Mild jaundice. Mild spleenomegaly. Mild hepatomegaly, marked hepatomegaly in hepatic vein thrombosis.

Laboratory Diagnosis

Haemogram : HB decreased PCV decreased RBC count decreased WBC count mild decreased Platelets count mild decreased MCV normal MCH normal MCHC normal MCV,MCH,MCHC decrease in chronic case Reticulocyte count increased but lesser than expected for degree of anaemia because retics are more susceptible to compliment lysis than mature PNH red cell. DLC Mild neutropenia PBF N/N , mild macrocytic and mild microcytic hypochromic RBCs, Neuropenia and Thrombocytopenia

Signs of I/V Haemolysis Plasma Hb High Urine Hb Present Haemosidenuria Positive Heptoglobin Decreased or Absent Haemopexin Decreased Methemealbumin Increased LDH Increased Bilirubin Increased Urine urobilinogen Increased Othre tests Methemoglobin Increased Serum iron Decreased Serum ferritin Decreased DAT Negative , occasionally positive OFT Normal

NAP (Neutrophil Alkaline Phosphate): Reduced PNH patient usually possess a sub population of granulocyte that exhibit impaired phagocytosis and chemotaxis when exposed to activated complement. Mutant granulocyte in PNH are as deficient in leukocyte alkaline phosphatase as the granulocyte of CML. Only those PNH granulocytes that are deficient in LAP are unresponsive to chemoattractants . Lack of LAP in CML granulocyte is associated with absence of L AP mRNA, whereas in PNH LAP mRNA is normal. Bone Marrow: Normoblastic Erythroid Hyperplasia, rarely Megaloblastic , Megakaryocytes are diminished, in pancytopenia , BM is hypoplastic. .

Specific test for PNH : Definitive diag. of PNH in vitro is done by variety of tests1. 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 PNH red 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. When the Hems test is supplemented with Mg , to optimize the activation of compliment, the percentage lysis gives the more accurate estimation of proportion of PNH cells. Methods: 1) Hams test 2) Modified Hams test

principle
Patient 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 collection and 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 veni puncture. Blood is poured into the flask of 20 ml capacity containing 5-10 glass beads. The flask is gently rotated until the blood is clotted(10- 15 mins). Place the flask inside incubator for 5mins. Then the fluid portion is taken out in a clean test tube and serum is separated after centrifugation at low speed.

Preparation of Fresh normal serum: ABO group compatible normal serum There 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 taken whose serum sufficiently lyse PNH RBCs. Required for test: a) Normal fresh serum b) Patients serum c) 50% normal cells suspension d) 50% patients cells suspension preparation 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 like cells(sulphydryl compounds, can act on normal red cells in vitro so as to increase there compliment sensitivity)

Requirements: Apparatus: 1) Flasks(20ml) with some piece of glass beads. 2.) Centrifuge 3) Test tube & test tube rack 4) 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 CONT ROL C ELL 1. 1. 2. Fresh normal serum(ml) Patients serum(ml) 0.5 /CON TROL SERU M 2. 0.5 TEST cells /CONT ROL SERUM 5. 0.5 6. 0.5 TEST /TEST SERU CELLS M 7. 0.5 8. 0.5 9. 0.5

3. 0.5 -

4. 0.5 -

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

3 4 5 6 0.2mol/l HCL (ml) 50% patient RBCs (ml) 50% normal RBCs (ml) 0.05 0.05 0.05 0.01 .05 0.05 0.05 0.05 0.01 0.05 0.05 0.01 0.05 0.01 0.05 0.05 0.01 .05 .05 .01

.05 .01 0.01

250mol/l MgCl2 0.01 (ml)

Mix the content and leave for 1hr at 37C. Centrifuge and look for haemolysis on tube no. 5 and 8. Calculation of % of lysis. Blanknormal serum 0.5ml 100% lysis( standard) - 50% washed red cells 0.05 ml in o.55ml water. Test Supernatant of tube no.5 or 8

 Take 0.3ml of each blank, 100% lysis RBCS and supernatant of

test tube to be tested for haemolysis in 5ml of 0.4ml/l amonia or drabkins 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 with patients serum. In HEMPAS lysis is due to the presence of unusual Ag on red cell surface which react with complement fixing IgM Ab (anti HEMPAS) present in many but not in all sera. Markedly spherocytic red cells may lyse in acidify serum probably due to lowered pH and such cells may lyse too in acidified inactive 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.

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 ionic strength. PNH cells is greater sensitive to complement undergoes lysis but not normal cells. More than 10% lysis implies a positive test. 5-10% lysis is boarder line . The red cells of some cases of Leukemia or myelosclerosis gives less than 10% lysis. The sucrose lysis test is typically neg. in HEMPAS(hereditary erythroid multinuclearity associated with a positive acidified serum) Reagent : a) Iso osmotic solution of sucrose(92.4 g/l) Procedure: Take 2 tubes 12*75 mm and proceed as

Reagents

Tube No 1. (test) 0.85 ml -

Tube No2. (negative control) 0.85 ml 0.05 ml 0.1 ml -

Tube No3. (normal control) 0.85 ml 0.05 ml 0.1 ml

sucrose solution saline

Normal compatible serum 0.05 ml 50% patients cells 50% Normal cells 0.1 ml -

Incubation at 37 0C for 30 , centrifuge the tubes and examine for lysis . Calculation of % of lysis. BlankSupernatant of tube no. 2 100% lysis( standard) 50% washed red cells 0.1ml in 0.9 ml of ammonia 0.4 ml/l . Test Supernatant of tube no.1 Measure the lysis in the photoelectric colorimeter using the yellow green filter or in spectrometer at a wavelength of 540 nm as above. Calculation: % lysis = (Test Blank ) 100 (Std Blank)

Flowcytometry
Principle: Various immunoflurescent dyes coated with Ab can be attached to the Ag present in the cells or particles. As the sample enters the flow channels and the cells are passed through a focused laser beam one cell at a time. As the cells or particles intercept the light source they scatter light and flurochrome are excited to higher energy state. This energy is released as a photon of light with specific spectral properties unique to different fluorochromes. These light are captured and converted to electrical signals. One unique feature of fluorocytometry is that it measure the fluoroscent per cell or particle. This contrast with the spectrophotometry in which the percent abs. and transmission of specific wavelength of light for a bulk volume of sample.

HEMATOLOGY TEST FOR FLOWCYTROMETRY

lympho ma

PNH

Chronic lymphoi d
leukemi a

Mast cell disease

FCM

Plasma cell disorder s

MDS
CMPD

Acute leukemi a

FLOCYTOMETRY ANALYSIS OF PNH CELLS

principle: Patient red cells are stained with a fluorescein labelled antibody that is specific for one of several GPI-linked proteins eg CD55,CD59 etc. which are deficence in PNH red cells. The stained cells are then analyzed with a flow cytometer.

Procedure
Steps for RBCs 1. Three control tubes and three test tubes are taken and labeled as T- Negative , T- CD55, TCD59, C-Negative, CCD55, C-CD59 . 2. The 50 ul of suspension(50ul of whole blood + 500 ul of PBS) 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

Procedure for WBC

1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Four control tubes and four test tubes are taken and labeled as T- Negative , T- CD55, TCD59, TCD16, C-Negative, C- CD55, CCD59, C-CD16. The 50ul of whole blood and 5ul of Ab are mixed in each tube. Incubate in dark for 30 mins at RT. Add 1 ml of FACS lysing solution and mix in vortex mixture and keep it for ten mins. Centrifuge for 5 min at 2000 rpm. Discard the supernatant and tip off on the tissue paper. Add 1 ml of PBS and then centrifuge for 5 min at 2000 rpm. Discard the supernatant. Wash the cell again as in step 7 and 8. Resuspend the deposit in 500 ul of PBS. Cap the tube and keep at 4oC .

Above prepared suspension is taken for flowcytometric analysis. 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 rpm otherwise high speed breaks the cells. 3. Storage the kit at 2-8 0C 4. Once started , the test must be performed without interruption. 5. Avoid carry over contamination.

Side Scatter
Laser Beam

Cell granularity

FSC Detector

Collection Lens

SSC Detector

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