زائر
MANA F BASI HEMATGY TEHNIQES F ESIDENTS
TEHNIQES AIED T AT TINE HEMATGY ABATY
AI NIVESITY
“ne f the essentia qaities f the iniian is inteest in hmanity; f the seet f the ae f the patient is in aing f the patient”
NTENTS
Sbjet:
Page
e ntes
inipe, intepetatin & pitfas………………………4
Stains in mmn se in hematgy…………………………………...
10
tine stains f bd & bne maw…………………………..
10
Staining f ed e insins……………………………………
10
eyte ythemisty………………………………………….
11
Tests f hemyti anemia……………………………………………...
12
Fagiity tests……………………………………………………...
12
B enzyme assay………………………………………………..
16
Hemgbin stabiity test………………………………………….
19
Hemgbin eetphesis………………………………………
22
Tests f PNH……………………………………………………..
24
Tests f immne hemyti anemia……………………………...
28
the tests………………………………………………………………..
33
Test f ygbins……………………………………………..
34
Pasma vissity…………………………………………………..
35
E NTES
INIPE, INTEPETATIN & PITFAS: “
Sevea types f eetni ntes ae enty aviaabe, many ae apabe f maesing hematgi vaiabes the than e nts. Many f these instements ae sppemented by histgams& sattegam gaphis.
Pinipe
Atmated e nting and sizing f bd es is geneay based n ne f 2 main pinipes; namey the apete impedane & the ight satteing tehngies.
Apete Impedane Tehngy: Fist desibed by te, 1952
It depends n the fat that in any saine-based dient ,es ae eativey bad ndts in eatin t the dient. If the es pass in the sensing zne they an be deteted by inease in the eetia esistane. This tansient eevatin in esistane eates an eeti impse. These eetia pses an then be nted. The magnitde f eah impse is pptina t the e size.
Types f instments: te, Sysmex& e-Dyn.
In patie this type f ntes is fmed simpy f a tbe with a sma ifie (e.g. 100 μm in diamete and 70 μm in ength) immesed in a ditin f bd es. es ae aspiated thgh the ifie. Impedane is meased between a negative eetde inside the ifie tbe and a psitive eetde tside in the ditin f bd es. An extena vam initiates mvement f a mey siphn that ases a meased vme f the sampe t fw thgh the apete tbe.
Athgh the sheath fw was iginay desibed in the ight-satte systems, it an as be appied t apete impedane system t enhane thei abiity t mease e size.
ight satteing tehngy:
It is based n the passage f es thgh a naw beam f ight. The sensing zne is estited paty by nawness f the ight beam and paty by deiveing the es in a vey naw steam. As the es pass thgh the sensing zne they satte ight. The satteed ight an then be eted by a sitabe ptia system and meased by a phtmete. The tansient ineases in satteed ight eate impses fm the phtmete, whih an then be nted eetniay. The e ntes an mease the e size, as the ampitde f the impse is pptina t the size f the e.
In this tehngy it is mandaty t se the sheath fid t btain a naw steam f es. es ente the sensing zne ae snded by the sheath fw whih is adjsted t eate hyddynami fsing effet whih fes the e sspensin t emain in a naw steam as it passes thgh the ente f the sensing zne.
Type: Tehnin.
Diffities and stins
Sme diffities ae met with, in atmated bd e nting, f whih speia designs ae intded:-
· The fist diffity is t nt es ne and ny ne i.e. nt t se a e and nt t nt a e me than ne time. The ss f ne e an in what is aed inidene in whih tw es an pass thgh the sensing zne in the same time and s they ae nted as ne e. The pse an atenativey be geneated at the dead time f the iit the es may aggtinate de t the pesene f d aggtinins. These ate wi give a age impse and nsideed as ne age e. Waming f bd sampes and appiatin f sitabe mathematia agithm t edit t any abnmay age impse measing diffeent ditins fm the same sampe an et this phenmenn. nting f a e me than ne an be de t a phenmenn aed eiatin. This an mainy in apete impedane ntes. nting f bbbes extanes paties as es is anthe ase f fase inease in the nts f bd es. These an be eted by the se f sheath fw, appiatin f a sweep fw (fing fid ass the ea f the ifie t wash its bak) by editing t.
· The send diffity is t disiminate the e f inteest fm the es eetni nise and debis. The stin f the pbem is the pess aed theshding. In ed e nt we set ny a we theshd t disiminate between sma ed es and age pateets. N need f an ppe theshd t disiminate ed es fm eytes de thei eative w nt. As egads t pateets, we set an ppe theshd t eiminate sma ed es and a we theshd t exde eetni nise and debis. White es an be as disiminated fm ed es by appying a we theshd bt afte adding a hemysing agent t desty eythytes. This is appied mainy when thee is sheath fw. When sheath fw is nt sed in apete impedane a theetia distibtin ve is made t pateet vme histgam and pateet nt is extapated fm the aea nde the theetia ve t avid the t mh veap between pateet and sma eythytes and eetni nise and debis. In ight satte it is neessay t add mpnds that stp the debis asing ptia intefeene.
·The thid is the diffity t nt bd es pe nit vme. In stead mst nte nt es pe nit time and then nvet them t es pe nit vme by mtipiatin by speifi fat. This fat is aated by aibatin.
se f e ntes in nting diffeent bd es
1. The ed bd es and pateets ae nted by the same hanne and an be diffeentiated fm eah the by thei size.
2. As egads t ed e indies, the main diffity in measing the mean psa vme (MV) is that the impse pded by any e is ny appximatey pptina t the vme f the e. Abeant impses, whih d nt ty epesent the e size, an bt ften these impses have nsa haateistis, whih enabe them t be identified by sitabe eetni iits and edited t s that they d nt affet aveage impse magnitde that is sed as the mease f MV. This is me imptant in apete impedane witht sheath fw.
eiating es give age impses than es passing in the ente f the ifie. The impses an be edited t easy as they ae nge than they shd be de t nge tansit at the edge f the ifie. Tw es passing thgh tgethe give vesize pse and s an be edited t. When mana MH is eded thee is a tendeny t ndeestimate the MV.
In apete impedane, MH that detemines the intena vissity f the eythytes as detemines its shape as they pass thgh the ifie. They take a iga shape. When the intena vissity f this iga diminishes, it bemes thinne and it eates a smae impse than it shd be (shape fat).
In ight satte, bth the shape f the e and intena efative index affet the ed e vme. In these systems, they sphee and fix the ed es pi t thei enty int the sensing zne. This minimizes these effets. In additin they appy ight fm high ange (5-15°), whih is me affeted by the intena efative index and a w ange (2-3°), whih is me affeted by e vme n e-by-e basis.
3. A ntes an make histgams that ae sef f measing anisytsis and haateizing sitatin in whih tw ppatins ae pesent. In da ange ight satte it is pssibe t mease the individa hemgbin nentatin f a ed e eading t the pssibiity nstting histgam f the distibtin f e hemgbin nentatin.
4. Pateet vme distibtin histgams an be sed t detemine pateet nt, mean pateet vme and pateet anisytsis. ed e and pateet anisytsis and mean pateet vme have itte inia vae de t geat diffeenes between diffeent instments bt they ae imptant in the abaty as they detemine a bd fim shd be examined nt.
5. Hemgbinmety is dne in many instments by a mdifiatin f the yanmethemgbin methd sing Dabkin’s type eagent. This methd has tw disadvantages:
a. Measement f absbane is made afte a set time inteva afte mixing f bd and eagent bt befe eatin is mpeted s a detegent mateia is inded t ense apid e ysis and ede the tbidity de t e membane and pasma ipids. We mst aibate the instment by sing sampes with f knwn hemgbin nentatin.
b. Bihazads methd de t dispsa f yanide ntaining wastes. In sme instments this methd is epaed by the se f a nn-txi hemia” sdim aysphate”.
6. In atmated etiyte nting, these es ae stained by vais dyes and ae nted by etiyte ntes, nventina fwytmete as an ptin in eent bd e ntes in a sepaate hanne.
7. White bd es ae nted by anthe hanne afte ysing eythytes. Tta and diffeentia eyti nts ae aied t at the same hanne in mst e ntes. Sme instments se me than ne hanne f nting eytes.
ight satte ane an ead t miseading ests in diffeentia eyte nt de t the fat that the e vme is nt the ny fat that detemines the appaent e vme. Anthe eqay imptant fat is the ganes, whih in tn detemines the intena efative index f the eyte. F this easn netphis appea age than esinphis despite that they have the same size the esinphis ae even age. They have the same vme if they ae meased by apete impedane. Anthe tehngy is sed nw t et this defet in mbinatin with ight satte, whih is the ight absbane. eytes ae stained by pexidase and the tw tehngies ae appied tgethe at the same time n e-by-e basis. 5 asses f eytes an be disiminated i.e. netphis, ymphytes, mnytes, esinphis and age nstained es (s). This s in the same hanne and s it is aed simtanes mtipaamete measement.
The singe pexidase hanne was pved t be inadeqate f basphi measement. Ths, many e ntes wee pvided with additina hanne f basphis. These instments ae nw in wide se and ae aed mtihanne ntes.
the appahes sing a singe hanne ae sed in the instments. In apete impedane systems the se f high feqeny eetmagneti pbe at the same time as measing vme pvides me infmatin abt the intena nstitents f white es and this impves the diffeentia nts in apete impedane ntes.
In ight satte systems, examinatin f satte at diffeent anges at the same time n e-by-e basis enhanes the diffeentia nt in these ntes. It is nw pssibe t mbine bth ight satte and apete impedane tehngies in the same instment in a singe hanne.
aibatin, nt and intena qaity assessment
aibatin:
In diet measing instments i.e. instments that mease nts pe nit vme the peat annt eaibate the instment. He an ny hek that the instment is wking ety and a the sevie f any pbem. As the majity f instments ae indiet i.e. mease nts pe nit time and need t be aibated t et it t nt pe nit vme. The peat an aibate them by testing bd sampes whse e nts pe nit vme has been detemined independenty. The aibatin fat is then detemined sing the fwing fma:
aibatin fat =
ntnit vme n knwn bd
ntnit time n knwn bd
When this is sted in the memy f the e nte it an then aate nts pe nit vme as sh:
nts nit vme n nknwn=nts nit time n nknwn × aibatin fat
Athgh the impses pded by the passage f a e thgh the sensing zne is appximatey pptina t the e vme, the MV and mean pateet vme annt be meased diety bt aated. The aibatin fat is aated as fws:
aibatin fat= vme f nknwn e size f impse pded by nknwn e
S a instments mst be aibated in espet t e vme measements and the measements based n it i.e. PV and MH
PV = MV × ed e nts
MH = hemgbin PV
In hemgbinmety instments have t be aibated f hemgbin measement bease nne f them pde an abste eading i.e. spetphtmeti absbane f hemgbin that is fy nveted t yanmethemgbin. S MH mst be aibated de its ink t hemgbin, whethe nt the eythytes ae nted diety.
Fesh bd and peseved bd an be sed as aibants. It is me pefeed t se fesh bd aibants. Assigning vaes f fesh bd aibats ae dne by efeene methds by sitabe seeted methds as shwn in tabe:
Measement
Methd
ed e nt
Semi atmated apete- impedane nte aspiating a knwn vme thgh ifie sing aibated manmete and vaidated inidene etin.
PV
aefy pefmed mihaematit
Hemgbin
yanmethemgbin deteminatin
ed e indies
aate fm abve measements
White e nt
The same as sed in ed e bt afte thei ysis
Pateet nt
se a nte with a sheath fw t estimate the eative nmbes f ed es and pateets. ed es ae aated as abve and then pateets ae aated.
The methds emmended by ISH f assigning vaes t fesh bd aibants
Fesh aibants ae stabe ny f f hs. It wi be bth tedis and st ineffetive t pepae fesh aibants daiy. Ths, peat mst se peseved bd aibants, whih ae stabe f 30 days me. They ae expensive and instment speifi.
nts:
They ae stabe peseved bd sampes, easy t manfate in hse and heape t phase bease they d nt need t have pe-assigned vaes. They an be sed as fws:
1. aibate the instment eithe with fesh bd with peseved bd aibants.
2. Make 20 measements n the nt and aate the mean and standad deviatin SD.
3. nstt a shewhat nt hat.
4. Test the nt afte evey 20 patients and aate hw many SD the ests diffe fm the mean.Pt the ests n the shewhat nt hat and k f difts.
5. Make aibatin if the ests f qaity nt shw exessive difts as stated abve.
Patient means:
They ae adjnts t sing peseved bd nts. They ae nsideed smetimes as atenatives.
Extena qaity assessments:
They ae peidia gvenmenta pgams f assessment f the eiabiity f ests f diffeent abaties ading t speifi shemes.
Ses f es:
Disdes and nditins that may Advesey Affet the Aay f Bd e nting
mpnent
Disdenditin
Effet n e nt
atinae
ed es
Mitsis shistytes
May ndeestimate B
we theshd f B nting windw is geate than miyte size
Hwe-Jy bdies
May spisy eevate pateet nt (in whe bd pateet ntes ny)
Hwe-Jy bdies simia in size t pateets
pyythemia
May ndeestimate B
Ineased inidene nting
White es
ekytsis
ndeestimate WB
Ineased inidene nting
Ate ekemia and hni ymphyti ekemia
Via infetins
May spisy we WB
Ineased fagijity f ekytes, inding immate fms
hemtheapy f ate ekemia
May atifatay inease “pateet” nt
ekemi e nea ytpasmi fagments identified as “pateets”
Pateets
Pateet aggtinins
May ndeestimate pateet nt, smetimes with spis inease in WB
Pateet mping. Aggegates may be identified as ekyte
Pasma
d aggtinins
May ndeestimate B with spis miytsis
ed e dbets, tipets, et.have ineased vme
ygbins, yfibingens
Vaiatin in pateet nt
Ptein peipitates may be identified as “pateets”
Sme f these exampes ny affet nts when etain instments ae sed. The sffets depend n ditin, stins
empyed. speimen tempeates.
STAINS IN MMN SE IN HEMATGY
tine stains f bd & bne maw:
Giemsa Stain:
Stk stin: 1 gm Giemsa added t 66 m Gye in a nia fask, heat at 56°f 2 hs, then add 66 m methan. Mix we & eave f 7 days at T, fite & se.
Staining stin: dite 110 with DW.
eishman Satin:
Add 0.2 gm eishman pwde t 100 m methan, wam t 50°f 15 min. & fite.
Biiant esy Be :
add 1 gm BB t 100 m 0.9% saine, ntaining 0.4 gm tisdim itate.
F etiyte staining : inbate 2 vmes f EDTA hepain antiagate fesh bd sampe & 1 vme f the dye at 37°f 20 minte & then spead fims. If the patient is anemi inease the pptin f the bd t the stain.
In Stain:
eagents:
* Fixative: metahn (anaa).
* 1 gm Kfeyanide dissved in 100 m 0.1 N H immediatey befe se.
* Aqas safanin 0.1%.
Methd:
* Fix smeas in methan at T f 15 min. Bt dy.
* Immese in K feyanide H mixte f 30 min. at T.
* Wash in nning tap wate f 20 min.
* inse in DW & nte stain in 0.1% safanin f 3 min.
Staining f ed e insins:
Hemgbin H insins:
Pede:
Mix 2 vmes f feshy bd with 1 vme f biiant esy be staining stin (sed f etiyte satining), inbate at 37°f 2 hs. Mix & spaed fims & examine f Hb H insins whih appea as mtipe geenish-be dts ike the pitted patten f gf ba.
Intepetatin & mments:
In apha tah. tait ny vey asina H bdies ae seen. In Hb H disease insins ae fnd in me than 30% f ed es.Absene f demnstabe insins desn’t pede a diagnsis f apha tha. tait.
Heinz bdy pepaatin:
eagent: Methy viet: dissve 0.5 g methy viet in 100 m f 9 g Na & fite.
Staining: add 1 vme f bd n any antiagant t 4 vmes f the staining stin, aw t stand at T f 10 min., then pepae fims. eave t dy in ai.
Intepetatin: Heinz bdies stain an intense ppe.If negative epeat the staing afte 24 hs inbatin f the bd at 37 ˚.
eyte ythemisty:
Aways se diety spead n-antiagated bd BM smeas & aways n a nt with the test sampe.
Pexidase staining:
eagents:
Bezidine stin: dissve 0.3 gm benzidine in 99 m ethy ah , then add 1 m f 30% satated K Na nitpsside.
H22 : add 3 dps H22 aegent t 12.5 m DW in a test tbe immediatey befe se.
Methd:
* In a staining ak add 10 dps benzidine eagent t ve the sfae f the side. eave f 1.5 min.
* Add ½ the vme i.e 5 dps H22 eagent, mix by genty bwing int the mixte taking ae the smea emains geneay veed a the time. eave f 4.5 min. & tehn wash with tap wate.
* Ai dy, then nte satin with Giemsa f 10 mintes.
the eyte ythemia stains:
mmeiay avaiabe kits ae say sed . Exaty fw the insttins f the kit sed.
TESTS F HEMYTI ANEMIA:
Fagiity Tests:-
smti fagiity test
Sampe: -
§ 2-3 m bd ae withdawn & added t 2 sepaate tbes with hepain ; ne f the fesh test & ne kept at 37 in a steie tbe f the inbated test.
§ The fesh test shd be aied t within 2hs f etin f bd; sted at m tempeate within 6hs ; if the bd has been kept at 4 .
§ The steie tbe is kept f 24 hs at 37 f the inbated test.
Diagnsti signifiane:-
The smti fagiity test gives an indiatin f the sfae aea vme ati f eythytes. Its geatest sefness is in the diagnsis f heeditay spheytsis. The test may as be sed in seening f thaassemia. ed es that ae spheyti f whateve ase, take p ess wate in a hyptni stin befe pting than nma ed es .
Pinipe :-
Sma vmes f bd ae added t age exess f seia hyptni bffeed saine stins. The fatin f ed es ysed at eah saine nentatin is detemined imetiay.
eagents :-
§ 1% bffeed Na stin;
§ Pepaed fm 1m f stk stin f bffeed Na 10% n 9 m distied wate.
* Phsphate bffeed Saine 10% stk stin f F:
* 90gm Na +13.65gm Na2HP4 + 2.43gm NaH2P4.2H2 in DW t a fina vme f 1 .N.B. Aways dissve the peipitated ystas in the stk stin befe making the ditin by inbating f sme time at 37 .
Tehniqe :-
1. Make seia ditins f saine as fws:
D.W.
0.4
0.5
0.6
0.7
0.8
0.9
Na 1%
1.6
1.5
1.4
1.3
1.2
1.1
2 m D.W.
N.
13
12
11
10
9
8
+ ve
1
2
3
4
5
6
7
-ve
Na 1%
0.4
0.5
0.6
0.7
0.8
0.9
1 m
2 m saine
D.W.
1.6
1.5
1.4
1.3
1.2
1.1
1 m
+ Ve nt tbe2m D.W.
- Ve nt tbe2m nma saine (0.9%)
2. Pt I dp f the patient bd in eah tbe by Paste pipette 20 μ and mix immediatey by inveting the tbes.
3. Inbate & eave the sspensins f 30 mins at m tempeate , mix again, and then entifge f 5 min. ( at 1200g ) & mpae with - ve & +ve tbes f haemysis.
4. Expessing the ests f .F. :-
a) Hd - ve nt tbe & stat mpaing the spenatent fm tbe nmbe 13 & ed the fist tbe in whih disatin f the sepnantent stats. Then ntine king in seqene at the est f tbes f esida Bs sediment at the bttm & ed the fist tbe shwing n esida Bs as the nentatin f mpete hemysis.
Nma vaes:Haemysis stats at 0.45 % Na n. & is mpete at 0.35% Na n.
b) Estimate the amnt f ysis in eah tbe spetphtmeteiay at a wave ength f 540 nm. se as a bank the spenatant fm – ve nt tbe & assign a vae f 100 % ysis t the eading with the spenatant f + ve nt tbe then pt the ests against the Na nentatin.
) Fm the .F. ve; we an dede the median psa fagiity (n. At whih 50% hemysis s) as we as any deviatin in the shape f the ve.
Intepetatin:
The smti fagiity f fesh ed es efets thei abiity t take p a etain amnt f wate befe ysing. This is detemined by thei vme t sfae aea ati. The abiity f the nma ed e t withstand hyptniity ests fm its binave shape whih aws the e t inease its vme by abt 70 % befe the sfae membane is stethed; ne this imit is eahed ysis s. Spheytes have an ineased vme t sfae aea; thei abiity t take in wate befe stething the sfae membane is this me imited than nma and they ae theefe patiay sseptibe t smti ysis. The inease in smti fagiity is a ppety f the spheida shape f the e and is independent f the ase f the spheytsis.
Deeased .F. indiates the pesene f nsay fattened ed es (eptytes) in whih the vme t sfae aea ati is deeased. Sh a hange s in in defiieny anemia and thaassemia in whih the ed es with a w MH & MV ae nsay esistant t smti ysis.
etiytes and ed es fm spentmized patients as tend t have a geate amnt f membane mpaed with nma es and ae smtiay esistant. In ive disease, taget es may be pded by passive amatin f ipid & these es, t, ae esistant t smti ysis.
Pitfas :-
1. The bd mst be deiveed int the 13 tbes with bth +ve & -ve nt tbes with geat ae. The itia pint is nt the amnt be exaty 20 bt athe that amnt added t eah tbe mst be eqa .Tw methds ae emmended:-
a) sing an atmati pipette. Afte aspiating the bd genty, the tside shd be wiped with tisse pape taking ae nt t sk t any bd fm the tip by apiay atin.
b) The bd is then deiveed int the saine stin and the pipette insed in and t sevea times nti n bd is visibe inside its tip. The tip has t be hanged befe mving n t the next tbe. This pede takes time and may est in an ineased expse f the fist few tbes. It is theefe advisabe t stat the timing n the additin f the sampe t the fist tbe.
) sing a Paste pipette with a pefety fat end 1mm in diamete. Abt I m f bd shd be sked p, aviding any bbbes, and the tside f the pipette wiped. With the pipette hed vetiay abve tbe1, a singe dp ( abt 20 ) is deiveed witht the bd thing the wa f the tbe. Fthe singe dps ae then deiveed int the emaining tbes.
Methd (b) appeas t be pimitive, bt with patie it is pefety satisfaty. It is as me enmi and mh faste than methd (a).
2. Even when a nma ange has been estabished, it is essentia aways t n a nma nt sampe ang with that f the patients t be tested inde t hek, f exampe, the saine stins.
The sigmid shape f the nma smti fagiity ve indiates that nma ed es vay in thei esistane t hyptni stins; indeed, this esistane vaies gaday as a fntin f ed e age, with the yngest es being the mst fagie. The easn f this is that d es have a highe sdim ntent and a deeased apaity t pmp t sdim.
Inbated smti fagiity
smti fagiity afte inbating the bd at 37˚ f 24 hs. expses the Bs t a metabi stess. The ineased smti fagiity f nma ed es whih s afte inbatin is mainy ased by sweing f the es assiated with an amatin f sdim whih exeeds ss f ptassim. Sh atin exhange is detemined by the membane ppeties f the ed e whih nt the passive fx f ins and the metabi mpetene f the e whih detemines the ative pmping f atins against a nentatin gadient ding inbatin f 24h.,the metabism f the ed e bemes stessed and the pmping mehanisms tend t fai; ne fat being a eative ak f gse. The fagiity f ed es whih have an abnma membane, sh as thse f heeditay speytsis & heeditay eiptysis, ineases abnmay afte inbatin .The ests with ed es with gyyti pathway defiieny sh as thse f pyvate kinase defiieny, ae vaiabe. In sevee defiienies; .F. may inease sbstantiay bt in the ases the fagiity may deease de t a geate ss f ptassim than gain f sdim. In thaassaemia, .F. is feqenty makedy eded afte inbatin de t ss f ptassim. A simia, thgh say ess maked hange is seen in in defiieny.
Aidified Gye ysis time test (A G T)
AGT is a ne tbe test designed t mease the time taken f 50% haemysis f a bd sampe in a bffeed hyptni saine–gye mixte t . It is sef as a seening test f heeditay spheytsis.
Sampe: Bd sampe n EDTA hepain.
Pinipe:-
Gye pesent in a hyptni bffeed saine stin sws the ate f entey f wate mees int the ed es that the time taken f ysis may be me nvienty meased. ike the smti fagiity test, diffeentiatin an be made between spheytes and nma ed es.
eagents :-
1. Phsphate bffeed saine ( PBS): Add 9 vmes f 9 g ( 154 mm ) Na t v f 100 mm phsphate bffe & Adjst the PH t 6.85 +- 0.05 at m temp.
2. Gye eagent (300 mm ):Add 23 m f gye (27.65 g A gade ) t 300 m f PBS & bing the fina V. t 1. by DW.
Tehniqe :
1. Add 20 f whe bd t 5.0m f PBS PH 6.85 & mix the sspensin aefy.
2. Tansfe 1m f the pevis sspensin t a standad vette f a spetphtmete.
3. Adjst the wave ength at 623 nm then add 2m f gye eagent apidy t the vette with a 2m syinge. Immediatey stat a stp wath & simatanesy ed the initia absbane.
4. ed the absbane again at sht intevas nti y eah ½ f the initia absbane & immediatey ed the time. The end pint is 30 mintes if ½ the absbane is nt eahed.
5. The ate f haemysis is meased by the ate f fa f tbidity f the eatin mixte. The ests ae expessed as the time eqied f the ptia density t fa t haf the initia vave (AGT50).
Intepetatin
Nma bd takes me than 1800se ( 30 min ) t eah ( AGT 50). In patients with heeditay spheytis the AGT50 is <180 se. F the fesh test. A sht AGT 50 may as be fnd is hni ena faie, hni ekaemia atimmne haemyti anemia and in sme pegnant wmen.
Signifiane
The same pinipes appy as with the smti fagiity test. es with a high vme t sfae aea ati esist sweing f a shte time than nma es; this appies t a spheytes whethe the spheytsis is ased by heeditay spheytis the mehanims.
Inbated AGT50
This have the same signifiane as the inbated F test. The bd shd be kept steie. A diagnsti est f HS is <120 se.
ed e Enzyme Assay:- “D. Shahia Kama Anis”
G-6-P-D Defiieny
Qaitative Seening Test
Sampe :
Bd sampes may be antiagated with hepain, EDTA AD. In any f these antiagants the enzyme is stabe f 6 days at 4 º & f 24 h at 25 º.
Pinipe:
§ G6PD atayses the xidatin f gse 6 phsphate (G6P) int 6-phsphgnate with the simatanes edtin f NADP int NADPH
G6P + NADP PG + NADPH
§
NADPH is an imptant eding sbstate f the nvesin f xidized gtathine (GSSG) int GSH & nde the nditins f stess the edtin f Hbi t Hb.
§ Seening tests f G.6.PD defiieny depend pn the abiity f Bs t nvet xidized sbstate int a eded state.
The methemgbin edtin test:
Sdim nitite nvets Hb int Hbi. When methyene be is added it ats as an atifiia eetn aept, an intat GP shnt wi envet Hbi int the eded state. If G6PD is defiient, Hbi wi emain in the xidized state.
eagents:
Dextse nitite: 5 gm D-gse + 1.25 gm Na nitite in 1 DW.
Methyene be: 0.4 mm. Dissve 150 mg methythinine hide (methyene be hide, Sigma) in 1 DW.
N.B. Gass tbes ae bette sed bease pasti may adsb sme eagents.
Tehniqe:
1. Add 1 m f bd t the tbe ntaining 0.2 m f the mbined eagent {0. m Na nitite & 0.1 m MB}. se the tbe with a stppe & genty mix the ntents by invesin.
* Pepae a negative nt tbe by adding 1 m f bd t a tbe ntaining 0.2 m saine & a psitive nt tbe by adding 1 m f bd t a tbe ntaining 0.1 m Na nitite& 0.1 m saine (mitting the MB).
* Inbate the 3 tbes at 37 f 3 hs.4. Afte the inbatin, emix the tbes & add 0.1 m fm eah tbe t 10 m distied wate in a sepaate test tbe.
* Mix the ntents genty & mpae s.
Intepetatin:
* Nma bd yieds a simia t the nma efeene tbe ;ea ed.
* Bd fm G6PD-defiient sbjets gives a bwn simia t that in the defiient efeene tbe .
* The degee f defiieny an be semiqantitativey expessed ading t the given by the test. NB: A makedy anemi bd may give fase est witht an ata enzyme defiieny. T veme this, y an adjst the hematit f the bd befe testing.
G6PD Enzyme Assay
Sampe :
Whe bd is eted with EDTA, hepain AD. ed e G6PD is stabe in whe bd f a week when efigeated at 2-8°, bt nstabe in ed e haemysate. Feezing f bd is nt emmended.
Piniipe:
The ate f fmatin f NADPH is pptina t the G6PD ativity& in meased spetphtmetiay at 340 nm.
Pede: a vaiety f mmeiay aviaabe kits ae sed. Fw the insttins f the sed kit exaty.
Nma vae :
146 – 376 (1012 B) 4.6-13.5 g Hb.
Intepetatin f est :
* The gene f G6PD is n the X hmsme & theefe maes an be eithe nma defiient hemizygte. By ntast femaes an be eithe nma, hmzygtes hetezygtes with intemediate enzyme ativity.
* ed es ae ikey t hemyze n ant f G6PD defiieny ny if they have <20% f nma enzyme ativity.
* G6PD ativity fas ff makedy as ed es age ,theefe wheneve a bd sampe has a high ppatin f yng ed es; G6PP ativity wi be highe than nma smetimes t the extenet that a genetiay defiient sampe may yied a vae within the nma ange. This wi be asay, bt nt aways assiated with a high etiytsis.
* A vae is the w nma ange in the fae f etiytsis shd aise the sspiin f G6PD defiieny, bease with etiytsis G6PD ativity shd be highe than nma. In sh ases the defiieny an be nfimed by epeating the assay when the etiytsis has sbsided & sfiinet d ppatin is pesent (abt 6 weeks) atenativey by assaying the d Bs afte fatinatin by denisty.
* In femaes, hetezygzity an be bette defined by ythemia tests athe than G6PD assay, whee the vae may ange between 10 t 90%.Assay f Pyvate Kinase Defieny
Sampe :
ne m. bd n EDTA, hepain, AD.
Sepaatin f Bs fm bd sampes :
eytes and pateets have a high PK enzyme ativity . It is theefe neessay t sepaate the Bs apidy by washing sampe 3 times with nma saine.
Pinipe :
PK is an imptant enzyme f the mebabism f gse in Bs:
PEP +ADP pyvate +ATP
PK ativity is meased as the ate f fa in absbane at 340 nm.
eagents & pede:
The test is ften dne by mmeiay avaiabe kits. Pepaatin f hemysate & enzyme assay ae dne exaty ading t the insttins f the kit ( attahed).
Nma Vaes:
Nma ange at 30 ˚ is 8.3-12.3 eg Hb.
Intepetatin:
Pk defiieny asay pesents as hni hemyti anemia. ne the enzyme ativity is defiient, the diagnsis is estabished. Diffity in diagnsis may aise in:
* Abnma vaiants with we enzyme ativity; whih an be piked by se f a w sbstate nentatin in the test (see insttins).
* High etiyti nt as etiytes have a highe enzyme ativity. In this ase PK defiieny an be sspeted by finding a nma enzyme ativity in spite f high etiytsis. A famiy stdy may hep in this ase.
Tests F nstabe Hemgbin:
Sevea methds ae avaiabe f the demnstatin f hemgbin instabiity, heat instabiity test, isppan stabiity test, n-btan stabiity test.
Samping:
Sampes sed shd be as fesh as pssibe and n me than ne week d. hse sampes f nts f the same age as the test sampe; a nma d bd sampe an be sed as psitive nt.
HEAT INSTABIITY TEST
Pinipe:
When hemgbin in stin is heated the hydphbi van de Waas bnds ae weakened and the stabiity f the mee is deeased. nde nted nditins nstabe hemgbins peipitate whie stabe hemgbin emains in stin.
Sampe:
Sampes taken int any antiagant ae satisfaty. EDTA is the mst nvenient. es feed fm tted bd an as be sed if neessay.
eagent:
Tis-H bffe, pH 7.4, 50 mm. Tis 6.05 g, wate t 1 ite. Adjst the pH t 7.4 with nentated H.
Methd:
* Pepaatin f ysate: (yse 1 vme f washed paked es in 4 vmes f ysing eagent pepaed as fws:
* g EDTA, tetasdim sat.
* 0.7 g ptassim yanide (KN).
* Wate t 1 ite.
* Add 0.2 m f ysate, feshy pepaed t 1.8 m f bffe. Inde a psitive (Hb F) and a negative (Hb A) nt f the same age as the test sampe.
* Sh a ysate wi nt keep f me than 1-2 days at 4 ° as it tends t ge. If neessay it an be fzen at –20 ° f p t 1 mnth. Avid epeated feezing and thawing).
* Pae the tbes in a wate-bath at 50 ° f 120 min. examine the tbes at 60, 90, and 120 min f tbidity and fine fatin.
Intepetatin & mments:
The nma nt may give minima diness at 60 min bt a maj nstabe hemgbin wi have ndegne maked peipitatin at 60 min and gss fatin at 120 min.
ISPPAN STABIITY TEST
Pinipe:
When hemgbin is dissved in a svent sh as isppan, whih is me nn-pa than wate, the hydphbi van de Waa’s bnds ae weakened and the stabiity f the mee is deeased. nde nted nditins nstabe hemgbins peipitate whie stabe hemgbins emains in stin.
eagent:
Tis-H bffe, pH 7.4, 100 mm. Tis 12.11 g, wate t 1 ite. Adjst the pH t 7.4 with nentated H.
Isppan, 17%. 17 vmes f isppan ae made p t 100 vmes with bffe.The estant 17% isppan stin may be sted in a tighty stppeed gass btte f 3 mnths at 4°.
Methd:
· Add 0.2 m f ysate, feshy pepated by the 4 methd given abve, t a tbe ntaining 2.0 m f 17% isppan. Inde a psitive (Hb F) and a negative (Hb A) nt f the same age as the test sampe. Stppe eah tbe and mix by invesin.
· Pae the tbes in a wate-bath at 37 ° f 30 min. examine the tbes at 5, 20 and 30 min f tbidity & fatin.
Intepetatin & mments:
The nma nt wi emain ea at 20 min. At 30 min minima diness shd be appaent, bt signifiant peipitatin wi nt nti 40 min. A maj nstabe Hb wi have ndegne maked peipitatin at 5 min and gss fatin at 20 min. A sighty nstabe hemgbin sh as Hb E wi shw diffse peipitatin at 20 min.
n-BTAN STABIITY TEST
Pinipe:
When hemgbin is dissved in a svent sh as n-btan, whih is me nn-pa than wate, the hydphbi van de Waas bnds ae weakened and the stabiity f the mees is deeased. nde nted nditins nstabe hemgbins peipitate whie stabe hemgbin emains in stin.
eagent:
Stk sdim phsphate bffe, 0.1 m. NaP4 15.6 g, EDTA 3.7 g, wate t 1 ite, adjst the pH t 7.4 with nentated H. The bffe may be sted at m tempeate.
* n-Btan
* Wking stin. The wking bffe is tempeate-dependant; pepae as fws:
18-20 ° n-btan 6.5 m, stk bffe t 1 ite.
21-23 ° n-btan 6.0 m, stk bffe t 1 ite.
24-26 ° n-btan 5.5 m, stk bffe t 1 ite.
Methd:
* Add 0.2 m f washed paked es t a pasti tbe ntaining 2.0m f wking stin. Inde a psitive (Hb F) and a negative (Hb A) nt f the same age as the test sampe.
* Stppe eah tbe and mix by invesin; then emve the stppe. The Bs shd yse giving a ea stin.
* Pae the tbes at m tempeate and examine at 30, 60, 90 and 120 min f tbidity and fine fatin.Intepetatin & mments:
* The nma nt wi emain ea at 120 min.
* A maj nstabe hemgbin wi have ndegne maked peipitatin at 90 min and gss fatin at 120 min.
* A sighty nstabe hemgbin sh as Hb E wi shw diffse peipitatin at 120 min. The test shd be ntined nti the psitive nt shws peipitatin.
* Psitive ests may be given by sampes ntaining as itte as 10% Hb F by sampes ntaining ineased methemgbin as a est f pnged stage.
* Fase negative ests shd be avided by ntining the inbatin venight.
HAEMGBIN EETPHESIS (ESE AETATE,
PH 8.5):
Pinipe:
At akaine PH, Hb is negativey haged & in an eeti fied wi migate twads the ande (+). Stta vaiants with sfae hage diffeenes wi sepaate fm Hb A; thse witht a hange wi nt.
Sampe pepaatin:
* 0.5-1 m bd n EDTA hepain.
* Wash 3 X with nma saine & disad the spenatant.
* Pepae hemysate by adding ne dp f paked Bs t 6 dps hemysate eagent (eady made fm HEENA ). If the patient is makedy anemi, the amnt f hemysate eagent is eded t 4 dps the Hb f the hemysate is meased & adjsted t 10 gmd.Eetphesis tehniqe:
* With the pwe sppy disnneted, fi the 2 mpatements f the hambe with TEB bffe (eady made pwde fm Heena . t be dissved in DW).The bffe shd nt be sed f me than 1 mnth if the PH is hanged at any time. Dn’t se expied bffe pakets.
* Sak 2 stips f fite pape int the bffe & pae ne n eah bidge f the ahmbe.
* In a sepaate dish sak the ese aetate pape (phased fm Heena )swy (t avid bbbe fmatin) in the same bffe stin & eave f at east 5 mintes ( t ense even satatin f the membane).
* Bt the ese aetate membane genty between 2 fite papes, bt d nt et t dy t befe sampe appiatin.
* Pae 10 μ f eah hemysate int a sampe we. Inde a nt sampe with eah n.
* Dip the appiat int the sampe wes & appy t a fite pape.
* Dip the appiat again int sampe wes & appy t ese aetate membane pae n the pape sppt with the edge at the athda appiatin ine & aw the appiat tip t emain in ntat with membane f abt 3 se.
* Pae the membane pside dwn ass the bidge f the tank thing the fite pape stips& appy a gass side n the membane t tighten the ntat.
* se the hambe& nnet t the pwe sppy & adjst the vt t 250-350 f 20 mintes.
* Disnnet fm the pwe sppy& emve the membane, paing it in the Panea S stain ( eady made fm Heena t be dissved in DW), with the ese aetate sfae p & eave f 3-5 min.
* Ete the exess stain by paing in 3 hanges f desatining stin ( 5% aeti aid), 2 mintes eah.
* Dehydate in abste methan f 5 mintes.
* Immese in aeing stin f 6-8 min. nti the pape bemes ea.
* Dy at 56°f 5 mintes then sann the eaed pape.
Intepetatin:
* Types f sepaated HB ae identified in mpaisn t the nt.
* nentatin f eah type is btained as a % n sanning.
eagents:
* Eetphesis bffe: tis EDTAbate (TEB):- PH 8.5 Tis-hydxymethy aminmetahne (Tis), 10.2 g, EDTA 0.6 g, bi aid 3.2 g & DW t 1 ite.Bffe shd be sted at 4 ˚ .
* Panea S: 5 g, tihaeti aid 7.5 g& DW t 1ite.
* Destaining stin (5% aeti aid): 50 m gaia aeti aid t 950 m DW.
* Abste metahn.
* eaing stin:Gaia aeti aid 6 m, abste methan 14 m, 0.8 m ea aid ( phased fm Heena ab. ).
* nt sampe:eithe phased mmeiay avaiabe nt hemysate pepaed fm hemysate with kwn HB vaiants (add few dps f 0.3 m KN ; 20 g t stabiize the HB)
TESTS F PAXYSMA NTNA HEMGBINIA(PNH)
Paxysma ntna hemgbinia is an aqied disde in whih the patient’s ed es ae abnmay sensitive t ysis by nma nstitents f pasma. In it’s assia fm it is haateized by hemgbinia ding seep (ntna hemgbinia), jandie and hemsideinia. Nt nmmny, hweve, PNH pesents as an bse anemia witht bvis evidene f intavasa hemysis deveps in a patient sffeing fm apasti anemia me aey fm myesesis ekemia.
PNH ed es ae nsay sseptibe t ysis by mpement. This an be demnstated in vit by a vaiety f test, e.g. the aidified-sem (HAM), sse,thmbin, d-antibdy ysis, inin and ba-venm tests.
Aidified-Sem Test (Ham Test)
Pinipe:
The patient’s ed es ae expsed at 37 ° t the atin f nma the patients wn sem sitaby aidified t the ptimm pH f ysis (pH 6.5-7.0).
Sampe:
The patient’s ed es an be btained fm defibinated, hepainized, xaated, itated EDTA bd, and the test an be satisfatiy aied t even n es whih have been sted at 4° f p t 2-3 weeks in AD Aseve’s stin, if kept steie. The patient’s sem is best btained by defibinatin, f if in PNH it is btained fm bd awed t t in the dinay way at 37° at m tempeate it wi amst etainy be fnd t be makedy ysed. Nma sem shd simiay be btained by defibinatin, bt sem deived fm bd awed t t spntanesy at m tempeate at 37° an be sed. Nma sem knwn t be stngy yti t PNH ed es is t be pefeed t patient’s sem, the yti ptentiaity f whih is nknwn. Hweve, if the test is psitive sing nma sem it is imptant, patiay if the patient appeas nt t be sffeing fm vet intavasa hemysis, t btain a psitive est sing the patient’s sem, in de t exde HEMPAS. The ativity f a singe individa’s sem as vaies fm time t time and it is aways imptant t inde in any test, as a psitive nt, a sampe f knwn PNH es atifiiay eated “PNH-ike” es.
The sea shd be fesh, i.e. sed within a few hs f etin. Thei yti pteny is etained f sevea mnths at -70°, bt at 4°, and even at -20°, they deteiates within a few days.
Tehniqe:
* Deive 0.5 m sampes f fesh nma sem, gp AB- AB- mpatibe with the patient’s bd, int six (thee pais) f gass tbes.
* Pae tw tbes at 56° f 10-30 mintes in de t inativate the mpement.
* Keep the the tw pais f tbes at m tempeate.
* Add t the sem in tw f the tbes ne-tenth vmes (0.05 m) f 0.2 m H.
* Add simia vmes f aid sbseqenty t the inativated sem sampes.
* Pae a tbes in a 37° wate-bath.
* Wash sampes f the patient’s ed es and f nt nma ed es (mpatibe with the nma sem) twie in 9.0 g Na.
* Pepae 50% sspensins in the saine.
* Add ne-tenth vmes f eah f these e sspensins (0.05 m) t singe tbes ntaining naidified fesh sem, aidified fesh sem and aidified inativated sem, espetivey.
* Mix the ntents aefy and eave the tbes at 37° f ne h.
* entifge.
Tbes
Test (m)
nts (m)
1
2
3
4
5
6
Fesh nma sem
0.5
0.5
0
0.5
0.5
0
Heat-inativated nma sem
0
0
0.5
0
0
0.5
0.2 m H
0
0.05
0.05
0
0.05
0.05
50% patient’s ed es
0.05
0.05
0.05
0
0
0
50% nma ed es
0
0
0
0.05
0.05
0.05
ysis (in a psitive test)
Tae
(2%)
+++
(30%)
N Hemysis
In ase f qantitative measement f ysis
* Add 0.05 m f eah e sspensin t 0.55 m f wate s as t pepae a standad.
* etain 0.5 m f sem t se as a bank.
* Deive 0.3 m vmes f the spenatants f the test and nt seies f e-sem sspensins, and f the bank sem and f the ysed e sspensin eqivaent t 0% and 100% ysis, espetivey, int 5 m f 0.4 m ammnia Dabkin’s eagent.
* Mease the ysis in a phteeti imete sing a yew-geen (e.g. Ifd 625) fite in a spetphtmete at a waveength f 540 nm.
Intepetatins:
If the test es ae fm a patient with PNH:
* They wi ndeg definite, athgh, as aeady mentined inmpete ysis in the aidified sem.
* Vey mh ess ysis, even n ysis at a, wi be visibe in the n- aidified sem.
* N ysis wi be bght abt by the aidified inativated sem.
* The nma nt sampe f e shd nt ndeg ysis in any f the thee tbes.In PNH 10-50% ysis is say btained, when ysis is meased as ibeated hemgbin. Exeptinay, thee may be as mh as 80% ysis as itte as 10%.
The ed es f a patient wh has been tansfsed wi ndeg ess ysis than befe tansfsin, bease the nma tansfsed e, despite iatin in the patient, behave nmay. In PNH, it is haateisti that a yng e (etiyte-ih) ppatin, sh as the ppe ed e aye btained by entifgatin, ndeges me ysis than the ed es deived fm mixed whe bd.
Aidified-Sem Test With Additina Magnesim (Mdified Ham Test)
Pinipe:
The sensitivity f the Ham test an be impved by the additin f magnesim t the test t enhane the ativatin f mpement.
Tehniqe:
The tehniqe is identia t that f the standad Ham test (see abve) with the additin f 10m f 250 mM magnesim hide the aidified-sem test is psitive. The additin f magnesim hide ineases the sensitivity f the aidified-sem test, and it emains speifi f PNH. (Wheneve the aidified-sem test is psitive it is emmended that a diet antigbin test shd be aied t. If this is psitive, it d be de t a yti antibdy, whih has given a fase psitive aidified-sem test. This an be nfimed by apppiate segia stdies. In additin, in sh mpex ases a me definitive test f PNH, whih is nw avaiabe, is fw ytmety afte eatin f the ed es with ante-D59).
The ny disde the than PNH that may appea t give a ea-t psitive test is a ae ngenita dyseythpieti anemia, DA Type II HEMPAS. In ntast t PNH, hweve, HEMPAS ed es ndeg ysis in ny a pptin (abt 30%) f nma sea; meve, they d nt ndeg ysis in the patient’s wn aidified sem and the sse ysis test is negative. The expessin f GPI-inked pteins in HEMPAS is nma. In HEMPAS, ysis appeas t be de t the pesene n the ed es f an nsa antigen whih eats with a mpement-fixing IgM antibdy (anti-HEMPAS) pesent in many, bt nt in a, nma sea.
Heating at 56° inativates the yti system and, if thee is ysis in inativated sem, the test annt be nsideed psitive. Makedy spheyti ed es effete nma ed e may yse in aidified sem, pbaby de t the weed pH, and sh es may yse, t, in aidified inativated sem.
It mst be stessed that PNH ed es ae nt ndy sensitive t ysis by a weed pH pe se. The additin f the aid adjsts the pH f the sem-e mixte t the ptimm f the ativity f the yti system. It is pssibe t nstt pH-ysis ves, if diffeent nentatins f aid ae sed. The ptimm pH f ysis is between pH 6.5 and 7.0 (measements made afte the additin f the ed es t the sem).
Sse ysis Test
Pinipe:
The sse ysis test is based n the fat that ed es absb mpement mpnents fm sem at w ini nentatins. PNH es bease f thei geat sensitivity wi ndeg ysis bt nma ed es d nt.
Sampe:
* Patient sampe n EDTA.
* tted sampe (AB mpatibe with the patient) t have mpatibe sem as nt.Tehniqe:
Istni stin f sse = 0.924 g in 10 m distied wate (92.4 g)(an be sted at 4 ° f p t 2-3 weeks).
* Tw test tbes ae pepaed f the test and the nt.
* Pt the fwing in these tbes.
Test
nt
Ste Saine
850 m
850m
mpatibe Sem
50m
50m
Patient’s washed Bs (50% sspensin)
100m
100m
1) Inbate 30 mintes in 37 °.
2) entifge then examine f hemysis.
3) If ysis is visibe in sse ntaining tbe, mease this in a spetphtmete (W 546), sing a tbe ntaining sem dited in saine as a bank and a tbe ntaining 0.1 m f ed e sspensin in 0.9 m f 0.4 m ammnia in pae f the sse-sem mixte as a standad f 100% ysis.
Intepetatin:
1. ed es fm sme ases f ekemia myesesis may ndeg a sma amnt f ysis, amst aways <10%; in sh ases the aidified-sem test is say negative and PNH shd nt be diagnsed.
* In PNH, ysis vaies fm 10% t 80%, bt exeptinay may be as itte as 5%. Sse ysis and aidified-sem ysis f PNH ed es ae faiy sey eated.
* The sse ysis test is typiay negative in HEMPAS.
Tests F Immne Hemyti Anemia:- “D. Samah Abde-Hamid”
Diet mb’s Test
Sampe:
2 m bd n EDTA
Pinipe:
Demnstatin f inmpete at–antibdies ated t the patient’s ed es, whih beme aggtinated n additin f anti–hman gbin (AHG) sem. The type f the ating gbin an then be detemined by se f speifi anti gbin eagent f IgG, IgM and IgA mpement. It is ths nsideed a test f in viv sensitizatin.
eagent (Anti gbin eagents) :-
a) Py speifi eagents:-
§ These shd ntain bth anti IgG and anti mpement, if pasma is sed, ny anti IgG is neessay as EDTA pevent mpement ativatin.
§ The majity f ed e antibdies ae nn- – mpement – binding IgG, and IgG is theefe an essentia mpnent f any py speifi eagent.
§ Anti IgA is nt eqied as, IgG antibdies f the same speifiity aways in the pesene f IgA antibdies.
§ Anti IgM is as nst eqied bease iniay signifiant IgM a – antibdies that dn’t ase aggtinatin in saine ae mh me easiy deteted by the mpement they bind.
b) Mn speifi eagent: -
§ These an be pepaed against the heavy hains f IgG, IgM and IgA, antibdies &against 3, 4 mpement mpnents.
§ The main inia appiatin f thse antibdies is t define the immne hemia haateistis f antibdies.
Tehniqe:
* The EDTA sampe is washed 3 times (add saine then entifge then p the spenatant fid ti the saine is ea).
* 10% sspensin is pepaed as fws: 1 dp washed B5 + 9 Dps saine 100 washed B5 + 900 saine then
* 2 dps f the sspensin ae added t 2 dps f AHG & the mixte is inbated f 20 min. at 37 ° .
* entifge & examine f aggtinatin bth by naked eye & by mispe.
Intepetatin:-
A psitive DAT desn’t neessaiy mean that the patient has AIHA.A psitive test is seen in:
1. At antibdy n B sfae with witht hemyti anaemia.
2. An a – antibdy n B sfae as in hemyti disease f newbn afte inmpatibe tansfsin.
3. Antibdies pvked by dgs adsbed t the ed es.
4. Nma gbins adsbed t the ed e sfaes as the est f damage by etain dgs.
5. Adsptin f immne mpexes t the ed e sfae as in hspita patients.
6. Sensitizatin in vit, this s if bd is awed t stand in efigeat at 4% even at m tempeate and the test is then dne ;de t adsptin f inmpete d antibdies and mpement .
7. + ve DAT may in a sma % f nma pepe .
8. 20% f patients n ng tem teatment f methydpa devep + ve DAT.
Indiet mbs & d Aggtinin Tests
Sampe :
3 m tted vens bd sampe is entifged and sem is sepaated. This is bette aied t at 37 ° athe than at m tempeate t pevent adsptin f a d at antibdy.
Pinipe:
Detetin f fee at-antibdies in sem ,eithe wam antibdies (whih ae abe t mbine with thei espnding ed e antigen at 37 °) d antibdies (whih mbine with ed e antigen at we tempate)
Tehniqe:
1. Set p a seies f 12 tbes , 10 f the test & 2 f nt.
1
2
3
4
5
6
7
8
9
10
+ ve
- ve
Sem()
200
100
300
200
---
Saine()
-
300
200
200
200
200
200
Washed +ve(50%)
200
200
200
200
2. In ase f demnstatin f d aggtinin inbate the tbes at 4 ° (in efigeat) f venight (N.B a mateias sed in pepaatin as saine, sem, tbes ae paed in 4 ° befe wk).
3. Wash a tbes 3 time with saine & pepae a 10% sspensin f washed Bs.
4. Mix ne dp f the e sspensin & ne dp f A HG .
N.B ( AHG is nt added f demnstatin f d aggtinin & the sspensin is examined diety f aggtinatin).
5. Inbate a tbes at 37 f 10 mintes then entifge & examine f aggtinatin in ase IDAT.
Intepetatin:
The ast tite shwing aggtinatin is eded.
§ psitive test is pesent in :
1- At immne hemyti anaemia inding:
a. Wam at antibdies (f IgG type ;IgA, IgM wam antibdies ae mh ess mmn& if pesent; say in additin t IgG , they ae assiated with mpement adsbtin t the ed es.
b. d at–antibdies ae neay aways IgM in type. Hemysis is de t desttin f the ed es by mpement adsbed t the ed e sfae.
. d type at – antibdies have anti I speifiity (i.e they eat stngy with the vast majity f adt ed es and ny weaky with d bd ed es.
2- Dg inded immne hemyti anemia.
3- H inmpatibiity
§ Fase negative anti-gbin test:-
1- Faie t wash the ed es ppey – the antisea may then be netaized by immn-gbins mpement in the sem pasma.
2- The se f imptent – antisea s that weaky sensitized es ae nt deteted.
3- The se f antisea aking the antibdy espnding t the sbass f immne gbin espnsibe f the ed e sensitizatin.
4- The antibdy being eadiy dissiated in the washing pess.
5- DAT –ve AIHA: In abt 2-6% f patients with AIHA, the DAT is –ve de t w nentatin f antibdy.
the Tests:
Test F ygbins
Sampe:
Sem. 3 m bd shd be withdawn with a wam syinge & kept a 37 ° nti tted.
pinipe:
ygbins ae a gp f pteins that had the mmn ppety f fming a peipitate ge in the d. This phenmen is evesibe by aising the tempate. This gp f pteins ae assified ading t pifiatin & immnhemia anaysis int:-
Type f ygbin
Immnhemia
mpsitin
Assiated diseases
* Type I: mnna ygbin
nsist f a singe mnna Ig
* Type II: mixed ygbin
mnna immngbin with 2 antibdy ativity against a pyna immngin (F ativity)
*Type III: mixed pyna ygabin ae mixed pyna ygbin with F ativity .
· Ig M
· Ig G
· Ig A
· Bene Jnes ptein
· Ig M – Ig G
· Ig G – Ig G
· Ig A – Ig G
Ig M – Ig G
Ig M – IgG- IgA
· Myema
· Wadenstm’s magbin emia
hni ymphyi ekaemia
Myema
wadenstn magbinemia
A
Sjgen’s syndme ix mixed essentia eygdinemia
· Hepatitis
·
SE
A
Sjgen’s syndme
IMN
MV
Ate via hehepatitis
hni ative hehepatitis
Hepatitis
pimay biiay ihsis
p pststepta gmenephitis
gi infetive endaditis
epsy
Kaa – aza
Tpia sp enmegay syndme
Tehniqe :
1- Afte tting the sampe at 37
2- Sepaate the sem by entifgatin at 37 ,then ste it at 4
3- when ygbin is pesent , a white peiptate ge appeas in the sem afte a vaiabe peid, say 24-72 hs, bt the sem shd be bseved f I week t be se that ate ypeipitatin des nt g ndeteted.
4- The evesibiity f ypeipitatin shd be tested by ewaming an aiqt f peipitated sem.
5- The ypeipitate an be qantitated by sevea ways:-
a. entifgatin f the whe sem in a hematit tbe at 4 aws deteminatin f the eative amnt f the ygbin (yit).
b. Atenativey the ptein nentatin in the sem befe & afte ypeipitatin may be mpaed.
. The peipitate fmed in an aiqt f sem may be isated, dissved in an aidi bffe & the ygbin eve estimated by absbane at 280 nm.
Afte isatin & washing f the peipitate the mpnents f the ygbin an be identified by immneetphesis , immnfixatin immndiffsin.These anayses ae pefmed at 37 sing:-
* antisem t whe hman sem
* antisea speifi f apha (α), gamma (γ), m (μ), Kappa (κ), ambda (λ) hains f assifiatin
* antisem t fibingen may be sed t detemine the pesene f yfibingen ppt.Intepetatin & signifiane:
· Type I & II:
ygbins ae say pesent in age amnt in sem (ften me than 5 mgm).
In genea they ae pesent in patients with mnna paapteinemias e.g. in ymphma mtipe myema ;smetimes hweve they ae fnd in patients aking an evidene f ymphid maignany jst ae “benign” paapteins.
· Type III:
ygbins indiate the pesene f iating immne mpexes & ae the est f immne espnses t vais antigens. They ae pesent in eativey w nentatins (say ess then 1mgm) in hematid disease & hni infetins. A Types f ygbins may be espnsibe f speifi symptms that as a est f hanges in the ygbin inded by expse t d; symptms inde:
§ aynad’s phenmenn
§ Vasa ppa
§ Beeding tendenies
§ d – inded tiaia
§ Dista ateia thmbsis with gangene
Sine type II & type III ygbin ae iating sbe imme mpexes, they may be assiated with sem sikness – ike syndme haateized by :
§ pyathitis.
§ vasitis .
§ gmenephitis
§ negi symptms.
In patients with mixed essentia IgM – IgG ygbinemia a athe distitive syndme may that is assiated with athagia, ppa, weakness, ymphadenpahy hepatspenmegay .This synhme may be a seqea f hepatitis B infetin. Gmenephitis is mmn. In sme instanes is s in apidy pgessive fm & is an mins pgnsti signifiane.
ygbins may ase a sis e in a vaiety f abaty tests by peipitating at ambient tempeates & theeby emving etain sbstanes fm sem; mpement fixatin& inativatin& entapment f immngbins in the peipitate ae mmn exampes. edissving the ypeipitate say dse nt fy este ativity t the sem espeiay that f mpement.
Pasma Vissity “
Measement f the ate phase espnse is a hepf indiat f the pesene & extent f infammatin & its espnse t ttt.
sef tests inde:
§ P
§ ES
§ Pasma vissity
Pasma vissity is dependent n the n. f pasma pteins .hange in vissity seems t efet the inia seveity f the disease me than ES.
pinipe:
The time taken f a given vme f pasma t pass thgh a ength f naw tbe is mpaed t the time f an eqa vme f DW.
Sampe :
The test eqies 0.3 – 0.5 m f pasma btained fm EDTA. Antiagated bd.
Tehniqe:
1- Pepaatin:
Bd afte being eted n EDTA, is entifged as sn as pssibe at 3000 pm f 5 min. The pasma an be sted at m temp f p t 1 week witht hange t its vissity.
2- Steps:
* Fi the desending imb f visimete with DW. With stpwath in hand aw the fid t n thgh the desending apiay t the we esevi bb.
* Stat stpwath as fid eve passes the we mak () and stp it when it eahes the ppe mak ().
* ed time taken.3- The pede shd be dne in dpiate f:
* DW & Nma pasma
* DW & test pasma § eative vissity is aated as the time taken f pasma divided by time taken f wate.
ests
Nma ange: 1.4-1.8 Vaes abve 50 may be assiated with sevee symptms eqiing pasmaphaesis.
Intepetatin:
ases with ineased vissity ae:
* Ineased immngbins as a nseqene f pnged antigeni Stimatin:
* h. Athitis
* ive diseases
* Nepasti inease f Igs as in Mtipe myema
