Biologic and Clinical Significance of Red Cell Ferritin

8/8/2019 Biologic and Clinical Significance of Red Cell Ferritin

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1078 Blood , Vo l. 62 , N o . 5 (N ovem ber), 1983 : pp . 1 078 -1087

B io log ic and C lin ica l S ign ificance o f R ed C ell F erritinB y M a rio C azzo la , Laura Dezza , G ae tano Be rgam asch i, G iovann i B aro s i, V itto rio Be llo tti, D an ie la C a lde ra ,

Ma ria M a tilde C irie l lo , S i lv a na Q uag lin i, P ao lo A ro s io , a nd Edoa rdo As ca r i

Re d cel l fe r rit in w as m easu red in no rm al s ub jec ts andp atie n ts w ith d iso rd ers o f iro n m etabo lism , in f lam m ation ,live r dy s fu nc tio n , im p a ired hem og lob in syn th es is . a ndin c re as ed re d ce ll tu rn ove r by mean s o f ra d io im munoas -s ays w ith an tibo d ie s to liv er (ba s ic ) an d h ea rt (ac id ic )fe rr itin s . The no rm al m ean va lues fo r bas ic and a c id icferritin we re 8 .9 an d 22 .7 a ltog ram (ag )/c e Il, res pe ctiv e ly .T he red ce ll fe r ritin con ten t re flec te d ch anges o ccurr ing int iss ue s b o th in iro n d efic ie nc y and iron o ve rlo ad . B as icfe rr itin w as m ore c los e ly re la ted to the b od y iron s ta tusthan ac id ic fe r ritin , an d the a c id ic /ba s ic fe r ritin ra tio w asin creas ed in iron d ef ic ie nc y an d dec re as ed in iron ove rlo ad .T he m a jo r fa c to r de te rm in in g th e red c e ll fe rrit in co n ten ta pp ea red to be th e tran s fe rrin sa tu ra t ion , th a t is , the

S INC E 1972 , w hen th e firs t sensitiv e imm unoassayfo r fe rr itin w as desc ribed , the re h as been animpress ive num ber o f stu d ie s on se rum fe rritin con cen -tra tion in n orm a l and d isea se state s , and the clin ica lusefu ln ess o f the se rum ferr itin as say in eva lua tingbody iro n sta tus is n ow w ell e stab lished .2

A ttem pts have a lso been m ad e to m easu re ferritin inred b lood cell ex trac ts .3 7 R ecen tly , Jacobs and co -w orke rst9 and C azzo la e t a l.#{176 }av e sh ow n tha t the redce lls co n ta in g reate r am oun ts o f ac id ic (h ea rt- typ e)fe rr itin than basic (liv er- o r sp leen -ty pe ) fe rr itin , andth at the red ce ll ferritin con ten t re flec ts the abn orm a lbody iron s ta tus bo th in iro n d efic ien cy and ironoverload .In th e p re sen t w o rk w e have ex tended our p re lim i-n ary stu d ie s# {176}by de te rm in ing th e am oun ts o f basic an dac id ic fe rritin in n orm a l red ce lls and those frompatien ts w ith d iso rd ers o f iron m e tabo lism , in f lam m a-tio n , live r d ysfunc tion , im pa ired h em og lob in sy n thesis ,an d inc rea sed red ce ll tu rno ve r. T h is w ork h as a llow edu s to stud y the re latio nsh ip b etw een red ce ll fe rr itin ,body iro n s ta tus , ra te o f h em og lo b in sy n thesis , an d redce ll tu rnov e r in m an .

Subjec tsMATER IALS AND METHODS

B lood sam ple s w e re obtain ed from norm al sub jects an d hosp ita l-ized pa tie n ts: c lin ic al a nd hem a to lo g ic d ata a re show n in T able I.

From the D ipartim ento d i M edicin a In te rna e Te rap ia M ed icaand D ipartim ento di In form atica e S istem istica , U nive rsi ty o fP avia , Pa via , and Ca tted ra di C him ica B iolo gica V . Un iversity o fM ilan , M ilan , Ita ly .

S ubm itted F eb rua ry 28 . 1 983: a ccepted June 21 . 1983 .Addre ss reprin t requ ests to D r. M ar io (azzo la . Pato lo gia M ed -

ica I, P oliclin ico S . M atteo , 27100 Pav ia , Ita ly .( I 9 83 b y Grune & S tratton , In c.0006-4971 /83 /6205 - -0018$02 .OO/O

dis t r ibut ion o f iro n be tw een m ono fe rric and d ife rric tran s -fer r in . Th is is in keep ing w ith recen t da ta ind ica ting acom pe tit ive ad van tag e o f d ife rric tran s fe rr in in d e liv er ingiron to ery th ro id ce lls . In ad d it ion , the red ce ll fe rritincon ten t w as in crea sed in tha las sem ic pa tie n ts w ith n orm a li ron s ta tus , a ppear ing to be inve rs e ly re la te d to the ra te o fhem og lob in s yn thes is . The d ete rm ina tio n o f re d c e ll fe rn -tin , b ased on a com m erc ia lly a va ilab le b as ic fen n itin as say .can h ave c lin ica l ap p lica tio n . It ca n be used fo r e va lua tin gthe adequ acy o f th e iro n su pp ly to the ery th no id m a rrow ,p ar tic u la rly in pa tie n ts w ith in creas ed red c e ll tu rno ve r.M oreover , it m ay b e u se fu l in ev a lu a ting the body ironsta tus in p at ien ts w ith h em och rom atos is and liver d is -ease .

Normalsubjects. T hir ty -n ine w om en and 29 m en, aged b etw eenI 5 and 65 y r. w e re exam in ed a t the t im e tha t rou tine b loo d sam plesw ere b ein g tak en fo r scre en ing purposes. O n e m ale su b je ct, w ho hadhig h v alu es for red cel l fer ritin (66 an d 70 ag/ce ll, ba sic and ac id icferr itin , resp ect ively ), w as excluded from th is gro up as he w assu bsequently fo und to b e an a lco holic .

Ir on d efic ie nc y. Tw en ty-n in e pa tien ts had iron -de fic ien cy an e-m ia , w hile 20 sub jec ts h ad iron-def ic ie n t e ry th rop oie sis (i.e ., iro nd efic ien cy w ith ou t anem ia). In evaluat ing iron d efic ie ncy , ind iv id-ua l m ea surem ents of iro n sta tus w ere co nsidered to b e ab norm a l atthe fo llow ing levels: tra nsferr in satu rat ion below 16% , red c ellp ro top orphy rin abo ve 100 zg/d l packed red cel ls, an d p lasm aferr itin below I 5 zg / lite r. Iro n d efic ie ncy w as cons ide red to b epre sen t w h en th ere w a s an ab no rm a lity in at leas t tw o of the semea su r emen t s .

Ir on o ve rlo ad . T his gro up in clu ded subjects w ith H LA -re latedhem ochrom ato sis an d p atie n ts w ith iron -lo ad ing anem ia . O f th e 6pa tien ts w ith hom ozygous h em och rom ato sis co nf irm ed by live rb io psy , 4 w e re untrea ted , w h ile the o th er 2 had already en tered aph leb o tom y p rog ram . S ix sub jects w e re cons ide red , o n the ba sis offam ily stud ies,2 to be he terozy gotes for H LA -re lated hem ochrom a-to sis , hav ing b iochem ica l ex pre ssio n of the d ise ase . Iron- load in ganem ia included : (A ) 10 patien ts w ith re frac to ry hy pop las tic ane-m ia, a ll o f w hom had been p rev iou sly g iv en blood tra nsfu sio ns bu tw ere in and ro gen -in duced rem iss ion at the tim e o f stu dy , andth ere fore h ad not be en tra nsfused in th e 4 m o preced in g th e red c ellfer ritin assay ; (B ) 6 p atie n ts w ith con gen ita l dysery thro po ieticanem ia; and (C ) S p atie n ts w ith prim ary acquired siderob lasticanem ia w ho h ad iron o ver load p rob ab ly a s a co nsequ ence o finc rea sed iron absorp tio n . A ll these sub jects had trans ferr in satu ra-tion abo ve 5 0% and/o r p lasm a ferr itin co ncen tratio n ab ov e 300zg/Iiter.T he rem a in ing clin ic al c ate gor ies w e re sele cted w itho ut reg ard toir on s ta tu s.

Pregnancy. Fou rte en pregn ant w om en w ere stu d ied at th e tim eth at b lo od sam ples w ere ta ken fo r hem ato lo g ic eva lua tion .

Po lycyth em ia ve ra . T he se 6 su bje cts w e re re ferred to o ur c lin i-c al dep artm en t for de term in atio n of red cel l vo lum e and ev alu atio no f body iron s tatus. Th ree of them had been alread y vene sec ted .

In f lammation . This g rou p in clu ded 73 p atien ts. In flamm ationw as iden tif ied on th e bas is of c lin ical sym ptom s and on e or m ore ofth e fo l low ing fac tors:3 (A ) W estergren ery th rocy te sed im entatio nra te gre ater than 4 0 mm /hr ; (B ) w hite ce ll co unt o ve r 10 x l0 9 /I ite rin th e ab sen ce of b lood dy scra sia ; (C ) a lph a-2 -g lo bu lin le vel gre ate r


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RED CELL FERR IT IN 1079

th an 0 .85 g/d l; (D ) f ib rinog en con cen tra tion gre ater th an 5 00mg/d l . Of th e 73 pa tie n ts , 2 7 sub jects h ad H odgk ins d isease orn on-H odgk ins lym phom a, 2 0 sub jec ts h ad ch ron ic in fe ction , 1 6sub jects had co llagen disease , an d 10 su bje cts had so lid tum o r.

L iver disease. T his gro up included 1 8 pa tien ts in w hom liv erdysfunct ion w as d iag nosed on the b asis o f c lin ical a nd labo rato ryd ata . In par ticu lar , a ll the p atie n ts had a se rum biliru b in co ncen tra-tion gre ater th an I 5 m g/l iter a nd v aria b le ele vat ion s o f serumtransam ina ses an d alkal ine p hosph atase .

Thalassemia . T his g roup in clu ded : (A ) 8 su b je cts w itha -th ala ssem ia tra it an d o ne su bje ct w ith H b H disease; (B ) 3 4sub jects w ith h ete rozygous fl- tha lassem ia; (C ) I 2 sub jects w ithth ala ssem ia in te rm edia .

S ick le cell tra it. Four su ch sub jects, h av ing n orm al b loo dcoun ts , w ere stu die d.

H em olytic d isea se. T hirteen sub jec ts h ad he red itary sp herocy -to sis and 5 sub jec ts h ad au to imm une hem oly tic anem ia . The irre t icu locy te coun ts ran ged from 5% to 24% , or from 200 to 7 20 x10 / l i ter .

S equ en tia l studies. S equ entia l stu d ie s w e re ca rrie d o ut in : (A ) 5pat ien ts w ith iro n def ic ie ncy an d 5 p atie n ts w ith iro n ov erloadundergo ing app rop ria te trea tm ent; (B ) 4 p atie n ts w ith tha las sem iacom p lica ted by iron d efic ien cy ; (C ) 2 pa tie n ts w ith thalassem iam ajo r , u nde r tra nsfu sio n the rap y , at d iffere n t lev els o f H b .Labora to ry M ethods

Re d cel l ferri tin w as assayed as p rev iou sly d esc ribed in detail.#{17 6}Pu re red cell su spensions w ere prepared b y m ean s o f th e m eth od ofB eutle r et a l .4 S trom a w e re rem oved from red ce ll ly sates b ycen trifug ation , an d fer ritin concen tration w as then m easured u sin grad io imm unoassay s w ith an tibo d ie s to hum an live r an d hea rt fern-t in .0

In o rde r to de riv e th e red cel l fe rn i tin co n ten t, the H b con cen tra -tio n of the red ce ll lysa te w a s m ea sured by the cyanm eth em oglob inm ethod , and red cell num bers ca lcu late d from th is and the prev iousob tain ed m ean corpu scu lar hem oglo b in (M CH ). The red ce ll fern it inco n ten t w as exp res sed a s ag /cel l (ag = a ltog ram , 1 ag = lO#{176}) .P lasm a iro n and to tal iron b in d in g cap aci ty (T IB C ) w ere d ete r-m in ed by colo n im etn ic ana lysis.5 # {1 76} P lasm a fe rn it in w as de term in edin d uplica te u sin g a rad io imm unoassay b ased on an tib od ies aga insthuman liver fe rn it in (sensit iv i ty I ag / lite r). R ed cel l p ro top orh yn inw as m easured by direc t spec tro pho tom etry of ox ygenated b lo odu sin g the m od el 400 0 H em ato fluo rom eter (Environm en tal Scienc esA sso cia tes Inc ., B ed ford , M A ).

In ord er to ev alu ate any e ffec t o f c ell age on th e red ce ll fern itincon ten t , stud ies on ag e-d ep end ent red c ell po pu lations from 6sub jects w ere ca rried o ut. B loo d sam ple s en rich ed in ret icu locy tesw ere prepared as fo llow s. W ashed , leu kocyte -dep le ted red c ells[pa cked cell vo lum e (PCV ) . 80% ] w ere p ip ette d in to po lyethy lenetu bes h av ing in te rna l d im en sio ns of 0 .5 x 1 0 cm . Th ese tube s w erec en trifu ged in a hor izo n ta l ro to r a t 10 ,0 00 g fo r 30 m m at 5#{176}C .f terc en tn ifuga tio n , th e up per and low e r I 0% of the red ce ll co lum n w erer emoved u sin g insu lin syrin ge s and co nsidered a s to p and bottomlayer , re spe ctiv ely . T he rem a in ing w as cons ide red a s m id dle lay er.O n each laye r, re t icu locy te cou nt, py ruv ate k inase ac tiv i ty , and redce ll fern itin w ere est im a ted .

Sta tistica l A na lysisS tatis tica l an aly sis w ere p erfo rm ed using the S PSS p ack age 7

im p lem en ted on a H oneyw e ll 6 03 0 com pu ter .F req uency distribu t ion curv es o f values for H b con cen tra tion ,

p lasm a iro n , T IB C , an d trans fern in satu rat ion show ed a n ear -no rm ald istr ibu tion , w h ile tho se of va lue s fo r p lasm a fern itin , red cel lp ro top orp hyn in , a nd red c ell fe rn it in sh ow ed a sk ew distribu t ion . In

th is la tte r ca se , n orm aliza tion w as obtain ed b y loga rithm ic tran sfo r-m a tion of th e d ata .

S tatis tic al a na lyse s included : sum m a ry s tatistic s, reg res sio n andcorre la t ion ana lys is, var ianc e ana lys is, and discn im inan t analysis.Th e s ign ifican ce of th e d iffe ren ces b etw een m ean s w as te sted by th eF te st o r th e S tud en ts t tes t.

RESULTSThe resu lts ob tain ed a re summ arized in T ab le 1 ,

w h ile the P earson correla tion coe ffic ien ts b etw een theex am ined pa ram ete rs a re rep orted in Tab le 2 . Theda ta show n in th ese 2 tab le s in d icate tha t: (A ) b o th th ered ce ll fe rritin types w ere rela ted to th e b ody ironsta tus, bu t such a corre la tion w as c lose r fo r basicfe rr itin than fo r ac id ic fe rr itin ; (B ) the re w as a sign ifi-can t co rre la tion be tw een th e tw o fe rritin types; (C )ove ra ll, the transfe rr in sa tu ratio n w as the m ajo r d ete r-m inan t o f the red ce ll fe rr itin co n ten t, accou n ting fo r5 4% varia tion in b as ic fe rritin and 41% varia tion inac id ic ferritin .

N o rm a l red ce lls co n ta ined greate r am oun ts o fac id ic ferritin than basic fe rr itin , the m ean ac id ic /basic fe rritin ra tio be ing 2 .53 . W hereas the p la sm afe rr itin concen tra tion w as m arked ly h ighe r in m ale sthan in fem ale s, th e sex d iffe ren ces w ere le ss con sisten tw ith respect to red ce ll fe rr itin , b u t still s ign ifican t(T ab le I) .

B o th in iron -d efic ien cy anem ia and iro n -d e fic ien te ry th rop o ie sis , th e m ean red cell fe rritin con ten t w aslow er than n orm a l. S uch d ec rease w as h ighe r fo r basicferritin than fo r ac id ic fe rritin , and th e re fo re , theac id ic /basic fe rr itin ra tio w as in creased . Th ere w as asig n if ican t inve rse co rrela tion be tw een red ce ll ba sicferritin and red cell p ro top orphy rin in th is g ro up (F ig .I ). T here w as a neg lig ib le o ve rlap b etw een v alu es fo rbasic fe rritin foun d in p a tien ts w ith iro n -d e fic iencyanem ia and th ose fou nd in no rm a l sub jec ts . In fac t, 27of 29 p atien ts w ith iro n -de fic iency anem ia h ad va luesfo r red ce ll b asic fe rritin low er th an 3 .0 ag /ce ll, w h ilea ll no rm al sub jec ts b u t o ne had va lues o ve r th is th resh -o ld .

R ed ce ll ferrit in w as co ns id erab ly in cre ased inpa tien ts w ith iron o ve rlo ad . S uch in crease w as com par-a tive ly h ig he r fo r b as ic fe rr itin than fo r ac id ic fe rr itin ,an d the re fo re , the acid ic/b as ic ferritin ratio w asred uced in th is g ro up . A s show n in F ig . 2, th ere w ereno sig n if ican t d iffe rences be tw een th e v ariou s ironove rload sta tes w ith re sp ec t to the red ce ll ferr itinco n ten t, the m a jo r de te rm inan t o f th is la tte r be ing thetransferrin sa tu ra tion .

Th e rela tion sh ip be tw een red ce ll fe rr itin and ironstatu s w as fu rth er confirm ed by sequ en tial s tu d ie scarried ou t in a few p atien ts . Tw o illu stra tiv e exam p lesare reported in Fig. 3.

P reg nan t w om en and p atien ts w ith po lycy them ia


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N o rm e l a I, ja ct sA S ( 68 1 14.4 18 60 30 21 66 8.9 22.7 2.53

(1 2 .1 -17 .9 ) (1 1-26) (4 0-75) (1 6-47) (1 -6 9 ) (17-300) (3 .0-3 7.3 ) (5 .8 -8 3 .4 ) (0.6 5 -13 .20 )

Tha la saem.aa -Tha l a s semsa (9)

1080 CAZZOLA ET AL .

3) .

Tab le 1 . H em og lob in L eve l. Iron S ta tu s D ata . a n d R ed Ce ll F err itin C o n ten ts in N orm al a nd D ise as e S ta tes . M ean and Rang e

Nb P la sm a ion T IBCTransferr inSaturat ion

Rd C& I #{ 149}Protoporphyr in( g/c I Packd

P ( a smaFerntin

R d C ell F err ,tin Con t en t(ag/Cell)

Basic Ferrit in Ac idic Ferri tin Ac idic/Bas icSi ts (no.) (g/c l) (m o Ie /L ite r) (j m o Ie/L s te r) )% ( Red Ce ls ) (g/Lit&) (L .va r Assay) (H em i A ssay) F err ,te , R at io

Males (29)

F em ale s (3 9)

N /F c om pa is on

I ron d efic ie nc y a nc mia (2 9)

I ro n- de f, ci en t e ry tl so po il si s (2 0 )

I ro n o va rl oa dH om ozygous H IA -re lated hem o-

chromatosis (6)H ete rozygous H LA -re la ted hem ochro-

m ato sis (6)

15.5 19 58 33 20 115 11.1 27.8 2.50(1 3 .3 -17 .9 ) (12 -26 ) (46-71) (20-45) (1 -60 ) (19 -300 ) (4 .0-37.3 ) (7 .5 -83 .4 ) (0 .81 -8.59 )

13 .7 16 60 27 21 44 7 .5 19 .5 2.5 7(12 .1 -14 .9 ) (1 1-25) (4 0-75) (1 6-47) (5 -6 9 ) (1 7 -165) (3 .0-3 0.9 ) (5 .8 -75 .4 ) (0.6 5 -13 .20 )I - 7 .99 t - 2.92 t - 0 .98 t - 3.34 t - 0 .30 t - 7.39 t - 2.26 t - 2.29 t - 0 .16p < 0 . 001 p < 0 . 01 p 0 .0 5 p < 0 .01 p .z 0.05 p < 0 .001 p < 0 .05 p < 0 .05 p > 0.05

8.9 6 80 8 227 7 1 .4 9 .0 6.5 3(36 -12 .1 ) 1-11) (51 -1 14) (1-15) (81-510 ) (3 -15 ) (0 .5 -3 .1) (0 .8 -29 .6 ) (1.00 -34 .99 )

12 .7 8 75 11 89 11 3 .0 11.6 3.8 9(1 1 .7 -14 .11 (412 ) (59-1011 (5-18 ) (46 -221 ) (5 -22 ) (0 .9-13.6 ) (3 .2 -58 .0 ) (0 .93-20.90 )

14.0 39 49 79 27 542 245.2 247.9 1.09(11. 5-15.5) (34 -46 ) (4 5-54) (7 0-87) (7 -4 5 ) (1 32 -1 ,375 ) (1 41 .0 -527 .0) (1 65 .0 -356.0 ) (0.6 3 -2 .9 6)

15.0 32 51 62 6 236 44.8 81.5 1.82(1 1 .9 -16 .7 ) (2 7-35) (4 5-55) (5 5-67) (1 -3 3 ) (9 5 -451 ) (2 9 .6-6 5.0 ) (40 .0 -1 41.3 ) (0 .6 6-3 .7 6 )

Iron -loa din g an em ia (2 1 ) 9.9 34 49 68 12 793 101.4(5 .4-1 2.5 ) (18 -48 ) (3 6 -64 ) (4 7-95) (1 -65 ) (1 99-3,1 50 ) (2 7 .0-6 32 .7 )

128.3 1 .26(35 .0 -5 02.7 ) (0.2 7 -3 .2 1 )

Pregnancy (1 4 ) 12.1 13 68 21 38 22 6 .0 26.8 4 .45(9 .6 -14 .4 ) (5-2 4) (4 8-89) (5-45 ) (1 1-102 ) (5 -1 60) (1 .1 -2 4 .0 ) (10 .0 -5 7 .2 ) (1.2 9 -27 .50 )

Po lyc ythem ia vera (6) 17.8 11 64 18 58 44(1 4 .8 -19 .4 ) (6-1 9) (5 7-75) (9-32 ) (1 4-285 ) 13 -153 )

4 .6 18 .2 3 .9 2(1 . 1 -20. 1) (4 .6 -3 3 .5) ) 1 .1 0 -25 .53 )

In flamm a t,on )73 ( 11 .1 9 52 18 52 228 3 .9 13 .2 3 .41(5 .9 -14 .7 ) (3-2 5) (2 6-84) (4-44 ) (3 -295 ) (3 4 -5 ,2 50 ) (0 .5 -3 8 .0 ) (2.4 -9 5 .0) (0.4 0 -25 .51 )

Liwdeease)18) 13.0 20 49 42 26 229 14.0 38.6 2.77(8 .2 -16 .3 ) (8-3 6) (3 6-69) (1 6-80) (3 -206 ) (5 -6 .8 25) (1 .1 -9 1 .0 ) (3.4 -2 98 .0 ) (0.5 2 -42 .27 )

12.6 19 57 34 48 42 14.2 21.1 1.48(6 .8 - 1 3.9 ) (1 4-27) (4 9 -7 1 ) (2 2-45) (2 3 - 134 ) (15 - 125 ) (3 .8- 1 44 .0 ) (10 .0 - 1 19 .9 ) (0 .5 1-4 .6 8 )

I4etemzygous th a Ia sse r n .a 134 )

tj- th ala ss em ia in te nn ed ia (121

S ic kle c ell tra d (4 )

11 .3 18 59 31 46 53 82 .5 86.1 1 .62(9 .0-1 4.5 ) (9-34 ) (4 3-75) (1 4-49) (5 -1 54 ) (1 5 -280) (1 5 .0-5 71 .9 ) (20 .0 -4 86.9 ) (0 .3 2-9 .7 0 )

9.2 31 52 60 38 628 277.3 163.7 0.72(7. 1 - 1 0.2 ) (2 5-391 (36 - 7 1) (4 5-77) (5 - 1 25 ) (1 05 - 1.8 701 (30 .3- 1 .619 .7 ) (18 .5 -4 52. 1 ) (0.1 3 -13 .65 )

13.4 16 72 25 43 14 3.9 14 .4 3.7 5(11 .9 -14.6 1 (9-24) (4 9-107 ) (8-40 ) (1 4-103 ) (6 -2 7 ) (1 .0 -6 .5) (1 1.5 -1 8 .3) (2.0 2 -11 .50 )

H em oly tic d is ea se (1 8) 11 .7 19 61 32 46 136 7 .8 41.1 2.38(6 .6-1 6.4 1 (9-29) (4 9-89) (1 4-50) (3 -3 28 ) (11-1.100) (3 .2-7 3.3 ) (9.8 -4 12 .0 ) (1 .0 4-13.7 4 )

An thm etsc m ean was ca lc ulated fo r H b. p lasm a won . T IB C. a nd tra n s fernn sa tu ra tio n. w tde geom ethc m ean w a s C a lc u la t ed for the o th er pa rame te rs .

v era sh ow ed , on the av erage , reduced va lues fo r red ce llb asic fe rr itin , w h ich p ara lleled th e reduc tion in trans -ferr in sa tu ra tion (T ab le 1 ). O n the con tra ry , va lues fo rred ce ll ac id ic fe rritin w ere norm a l, an d the acid ic/b asic ferr itin ra tio w as in creased in these sub jec ts .

A s ex pec ted , pa tien ts w ith in f lam m ation had lowse rum iro n w ith low transfe rrin sa tu ra tio n and no rm a l-to - low T IBC , in creased p la sm a ferr itin , an d re ticu -loen do th elia l s ide ro sis . In th is g ro up , the m ean red ce llferr itin co n ten t w as low , w ith a n orm al ac id ic /basicfe rritin ra tio (T ab le 1 ). The red ce ll b as ic fe rr itincon ten t w as d irec tly rela ted to bo th the transferr insa tu ra tion an d p la sm a ferr itin con cen tra tion (T ab le

T ransfe rr in sa tu ra tion w as in creased in 7 o f 18pa tien ts w ith live r d isease, an d p la sm a fe rr itin w ase leva ted in 9 of 1 8 such sub jects . H ow eve r, the re w asno corre la tion be tw een th ese tw o pa ram e te rs (r = 0 .22 ,p = 0 .19 ) . B o th the red ce ll fe rr itin typ es w ereinc rea sed o n th e ave rage (T ab le 1 ). T he b asic fe rritincon ten t w as sig n if ican tly co rre la ted w ith th e trans -fe rr in s atu ra tio n (r = O.6 7 ,p < 0 .00 1) , b u t n o t w ith thep la sm a fe rr itin concen tratio n (r = O.32 ,p = 0 .10 ) .

T ransfe rr in sa tu ra tion ap peared to be an im portan tfac to r in de te rm in ing th e red cell b asic ferritin con ten tw ith in th e tha lassem ia g roup too (r = 0 .4 8 , p < 0 .001) .S uch a re la tionsh ip , how eve r , w as sign if ican tly d if-fe ren t from tha t ob se rv ed in n orm a l su b jects and


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Hb

Transferr in In )Red Cel l In)Plasrna In )RBC Basic In)RBC Acid ic In (A c id ic /B ss icHb P l a sma Iron T IBC Satu ra tio n P ro topo rphyrin) F e rn tin ) F erritin) F erri tin) Fe rr itin R a tio )

1 . 0000 0 .1510 -0 .0270 0 .0928 -0 .3554 0 .0100 0 .0070 -00323 -00646p < 0 .001 p - 0 .003 p - 0 .310 p - 0 .044 p < 00 01 p - 0 .427 p - 0449 p - 0277 p - 0. 1 1 8

P la sma i r on 0 .1510 1 .0000 -0 .2372 0 .9440 -0.5 033 0 .4983 07389 06448 -04357p - 0 . 003 p < 0 . 001 p -z 0 . 001 p < 0.001 p < 0 001 p < 0.00 1 p < 0.0 01 p < 0001 p < 0001

ferr i t in)

0 .5904 0 .7692 06675 -04567p


5/10

a bRBC F t,

l iver0

10

0

U

a

Ft, heart assayRBC Ft,live r assa y

50 0

aUu,Ja50

20

15 H b

9TH ER APY ,1oo m g FSda iIy II_I I I I I I I I I I #{1 49}

*

10 0806040Sn

I 6 1 2 1 6 12

Table 3 . P ea rso n Co rre la tio n C oe ffic ie n ts Be tw een V arious Pa ram e ters o f Iro n S ta tus W ith in P at ien ts W it h In fla m ma tio n (n = 73 )Transfe rr in n )R ed Ce ll In )P la sm a In)RBC Bas ic (n )R BC Aadic In)Acid ic/Basic

H b P lasma Iron T IBC Sa tu ra tio n P ro topo rphyr in ) Fe rritin ) Fe rr itin ) Fe rr itin ) F erri tin R a tio )Hb 1.0000

p 0 .0010 .0881

p - 0 .229-0.2649

p - 0 .012-0.0369

p - 0 .378- 0 .2480

p - 0 .017-0.3109

p - 0 .00 4-0.0712

p - 0 .275-0.1392

p - 0 .120-0.0515

p - 0 .333P la sm airon 0 .0881

p - 0 .2291.0000

p < 0 .0010.3381

p - 0 .0020 .8650

p < 0 .001-0 .3008

p - 0 .0 05-0 .0474

p - 0 .3450 .2975

p - 0 .00 50.1767

p - 0 .067-0.1386

p - 0.12 1T IBC 0 .2649

p - 0.0120 .3381

p - 0 .0021 .0000

p < 0 .001-0.0684

p - 0 .2830 .0234

p - 0 .422--0.3648

p 0 .001-0.2891

p - 0 .00 7-0.2476

p - 0 .0170 .0657

p - 0 .29 1T ransfe rr in -0.0369 0.8650 -0 .0684 1 .0000 -0 .2337 0 .1235 0.4784 0 .2836 -0.2185

saturat ion p - 0 .378 p< 0.001 p - 0.283 p < 0 .001 p - 0 .023 p - 0.149 p< 0.001 p - 0 .00 8 p - 0 .032In)redcell -.2 480 - -0.3008 0.0234 -0.2 337 1 .0000 0 .0775 -0 .3005 -0. 1992 0 .1199p ro toporphyr in ) p - 0 .017 p - 0 .005 p - 0.422 p - 0.023 p < 0 .001 p - 0 .257 p - 0 .0 05 p - 0 .046 p - 0 .156In )p la sm a -0.3109 -0 .0474 -0 .3 648 0 .1235 0 .0775 1 .0000 0 .5093 0 .2344 -0 .2 929

fe rr it in ) p - 0.004 p - 0 .345 p < 0.001 p - 0.149 p - 0 .257 p < 0 .001 p < 0 .001 p - 0 .023 p - 0 .00 6In )RBCbas ic -0 .0 712 0 .2975 -0 .2 891 0 .4748 - 0 .3005 0 .5093 1 .0000 0 .4149 -0 .6141

fe rr itin ) p - 0 .275 p - 0 .005 p - 0.007 p < 0 .001 p - 0.00 5 p -z 0.001 p < 0 .001 p < 0 .001 p < 0 .001In )RBCac id .c -0.1392 0.1767 -0 .2476 0 .2836 -0 .1992 0 .2344 0.4149 1 .0000 0 .4632

fe rr itin ) p - 0 .120 p - 0 .067 p - 0.017 p - 0.0 08 p - 0 .046 p - 0.023 p < 0 .001 p < 0 .001 p < 0.001In)acid ic/basic - 0.0515 -0 .1386 0.0657 -0.2185 0 .1 199 -0.2929 -0.6141 0 .4632 1 .0000

ferritinratio) p - 0 .333 p - 0.121 p - 0.291 p - 0.032 p - 0 .156 p - 0.006 p . 0.001 p < 0 .001 p < 0.001

1 0 8 2 CAZZOLA E T AL .

E# {176} [_ ratIo

-- IDESFERRIOXAMINE 2 g s .c . daliI. lIitiiIiIi.

m onths m onth sF ig . 3 . Changes in hem og lo b in co ncen tra tion . tra ns fe r rin (T f ) sa tu ra t ion . p lasm a fe rr itin (F t) , an d red c e ll fe r rit in (RB C F t) in (a ) a

p at ien t w ith iron d ef ic ie nc y du rin g iro n the rapy and (b ) a p a tien t w ith re frac to ry hypop la s tic a nem ia in rem is s io n d u ring ch e la tiont herapy .

d ifferen t from th at o bse rved in no rm al sub jec ts : a t ag iven va lue fo r tran sfe rrin satu ra tion , th e red ce llfe rr itin co n ten t w as g rea te r in the hem oly tic p atien ts .

T h e p ossib le rela tion sh ip be tw een ce ll age an d redce ll fe rr itin w as in vestiga ted m ore d irec tly by stud ieso f f ractiona tion of red ce ll p opu latio ns. T he re su lts are

sh ow n in F ig . 7 . F rac tion s en riched in re ticu locy te shad m ean va lues fo r py ruva te k inase activ ity th at w eres ig n ifican tly g rea te r th an tho se of the o the r ce ll frac -tions , w hereas d iffe ren ces in red ce ll fe rr itin w ere no tsign if ican t .

A c lin ica l app lica tion of the red ce ll ferr itin a ssay is


6/10

,_ za,:a..E =-a U

100L ._:

a=C-;; .----

10 0

10

U IS

S S

..S... #{149 }# {149 }

#{149} :A S

r=- .0 .67

t , #{14 9} , , #{149}S h et er oz yg ou s 6 -t ha I#{149 }b H diseasea u tha lassem ia tra it

.S#{149}#{149} :.C

:. : LA

10 0

,0 Ths lass#{149}mia

[transferrinsatu r a t Ion ]

---- tS- [25 -so].

,.. [so )_ -----

----

,-P0.001I I I I I I I I I I I I I I I i I I IIronde f i c ienc y0 25 50 75 10 0

T he use o f sen sitive im m unoassays has now a llow edred cell ferritin to b e qu an titated . Th ese inv es tiga tionsrequ ire , a t leas t fo r re sea rch purpo ses, the sim ulta -neou s u se o f tw o im m unoassays, one fo r basic fe rritinand th e o the r fo r ac id ic fe rr itin . It h as been show n , infact, tha t ferr itin is p re sen t in tis sues in m ultip le fo rm sth at d iffe r struc tu rally and imm uno log ica lly ,273 #{176 } andth at red ce ll ferritin is com posed m a in ly by ac id iciso fe rritin s .3 T he fin d ing s ob ta ined by Jacobs an dcow o rk ers8 9 and by us in a p relim ina ry stud y#{1 76}a recom pa tib le w ith the h ypo thesis tha t red ce lls con taintw o ty pes o f ferr itin , on e reac ting w ith an tib od ie saga ins t live r o r sp leen ferritin , and th e o the r reac tingw ith an tibo d ie s ag ain st h ea rt fe rr itin . T he ex istence o ftw o d istinc t fe rr itin ty pes in red ce lls had b een alread ysu gg ested b y W orw ood e t a l.3 2 th rou gh stud ie s o freticu lo cy te fe rritin .

T he re is a rea son ab le ag reem en t be tw een va lues fo rred cell b as ic fe rr itin ob ta in ed in d ifferen t cen te rs .9 # {176} 33In pa rticu lar , V an D er W eyden e t al.,33 using a rap idand s im ple m e tho d , h ave o b ta ined norm a l v alu es ve rysim ilar to tho se ob ta ined in C ard iff9 an d P av ia # {1 76}by

RED C EL L F ER RITIN 1083

T ran sfe rrin satu rat ion , %F ig . 4 . R e la t ion sh ip b etw een tran s fe rrin sa tu ra tio n and re d

c e ll fe rritin (liv e r as sa y ) in n o rm a l s ub jec ts an d p atien ts w ith iro nde fic iency and i ron o ve rlo ad (so lid sym bo ls an d con tinu ou s line )a nd in tha la ss em ic pa tie n ts (o pen sym bo ls an d b ro ken lin e ) .A lth ou gh s ta tis t ica l an a ly se s w e re pe rfo rm ed on 1 50 and 55sub jec ts . respe ctiv e ly , o n ly m ean va lue s are shown fo r c la rity . Th eequa tio ns o f the 2 reg res s io ns curv es w ere : ln (RB C fe rn itin ) =0 .2 02 + 0 .06 36 x tran s fe rrin s a tu ra tio n (c on tin uo us line ), an dIn (R B C fe rr itin ) = 2 .402 + 0 .045 6 x transfe rrin sa tu ra tion (b rokenlin e ) . Th e tw o curv es d iffe red s ign ific an tly w ith respect to bo thloca t ion (F - 1 18 .4 7 , p < 0 .0 01 ) an d s lop e (F = 4 .36 . p < 0 .05 ) .Discr im inan t ana ly s is showed th a t 8 5 .3% o f no rm al s ub jec ts an dpatien ts w ith d iso rders o f iron m etabolism and 7 2 .7% o f tha la s -s em ic sub jec ts cou ld b e com ple te ly sepa ra ted .

sh ow n in T ab le 5. In tw o pa tien ts w ith C oo ley sd isease, no rm al va lues fo r red ce ll fe rr itin w ereo b ta ined o n ly w h en red ce ll p rod uc tion w as com ple te lysup p re ssed by tran sfus io n the rapy .

D ISCUSS IONIron -co n ta in in g granu le s , v is ib le on lig h t m ic ro s-

cop y a fte r P e rls stain ing , h av e b een reco gn ized ine ry th rob la sts and e ry th ro cy te s fo r a t lea st 40 yr .8U sing e lec tron m ic ro scopy , B essis and B re ton -G orius9sh ow ed th at cy top la sm ic iron g ranu le s a re com posed ofagg reg ated ferri tin iro n co re s. S ince th en , a ttem p tshave been m ade to cla r ify the b io lo g ic sign ificance o ffe rritin in e ry th ro id ce lls ,2# {176}26bu t tha t is s till con trov er-sial .

14 ia 22 26 30 34M ean corp uscu la r h em og lo b in , p g

F ig . 5 . R e la tio nsh ip be tw een m ean corpuscu la r hem og lob in(MCH ) and red c e ll fe rr itin (liv e r as say ) in pa tien ts w ith t ha lasse -m ia h av ing no rm al v a lu es fo r tra ns fe rr in sa tu ra tion an d serumfernitin. The sh ad ed a re a rep res en ts the n orm al re fe ren ce rang e .

T ab le 4 . Hb Leve l, Iron S ta tus D ata , an d Red Ce ll F err itin Con ten ts in 4 P at ien ts W ith Th a la ss em ia C om p lica te dby Iron Defic iency . B efo re and A fte r A pp rop ria te T rea tm en t

Hb MCH M CV P lasma iron T1BC Transferr inSattsationR ed C ell

Protoporphyr in)g ,/ cJ P ac ke d P la smaFerr i tinR ed O af F err,iin C on te nt (a g/C ell)B as ic A c id ic A c id ic /B as ic

Pa t i e n t )g /d l) )pg ) fl) ) m o le /L ite r) )uno l e /L i t e r ) )% ) Red Ce lls ) )g /L ite r ) Fe rr itin Fe rr itin F er ri ti n R a ti o(1) a -T ha la ss em ia t ra it

A ssoc ia tedw ith eonde fic ie ncy 10 .1 17 .1 55 7 86 8 168 12 3 .0 6 .0 2.00After rontherapy 13 .9 20 .0 65 17 50 34 40 20 28 .0 10.9 0 .39

( 2) H e te ro zy go us f 3- th al as s em iaAaaoc ia tedwi th r onde f i c i e n cy 8 .9 16 .8 51 3 95 3 230 9 3 .0 16.8 5.6 0After iontherapy 11 .4 21 .0 69 22 64 34 40 45 52 .6 76.3 1 .45

(3) H e te ro zy go us $ -t ha la sa em iaAsaoc ia tedwi thwonde f i c i e n cy 9.0 17.6 56 9 88 10 277 6 4.0 14.6 3.55Afterrontherapy 11 .2 19 .9 65 16 57 28 61 24 85 .3 79.7 0 .93

(4) H e te ro z yg o us f l- th a la s se m iaAssociatedwithpolycythemiavera 16.7 19.5 61 10 59 17 74 13 15.8 34.6 2.19Af te r t2P t r ea tmen t 12 .1 21 .2 70 20 54 37 35 39 30 .9 48 .7 1.58


7/10


8/10

RE D CE LL FERR IT IN 1085

T he second de te rm inan t o f red ce ll fern itin app ea red

red ce ll fe rn itin is h igh er than n orm a l (F ig . 6 ) , thu sind ica ting tha t re ticu locy te s con ta in m ore fe rn itin thanm a tu re red ce lls . O n the o the r hand , ou r stud ie s o n th efern itin co n ten t o f fra c tion ated red ce ll p op u la tio nsshow th at va ria tion d ue to the re ticu locy te cou n t is lesssign ifican t than va ria tion du e to th e iron sta tus (F ig .7) .

In the p re sen t s tudy , tw o fac to rs appea r to beim po rtan t in de te rm in ing the red ce ll fern itin co n ten t:(A ) the transfe rn in sa tu ratio n an d (B ) the ra te o fhem og lob in syn thesis . F u rthe rm ore , such fac to rs m o rec lose ly affec t the basic fern itin than the ac id ic fe rn itin .T hese find ings m ay b e ex p la ined as fo llow s.

Iron is d istr ibu ted to bo dy tissues b y the p lasm atran sfe rrin . It ha s been recen tly show n th at the trans-fe rrin iron poo l is no t hom og en eous b u t is com po sed ofm onofe rn ic and d iferr ic tran sfe rrin , the la tte r hav in g afa r g rea ter capacity to d ispense iron to tissu e.37 3 8 Insu b jects w ith iro n ov erload , transfe rn in sa tu ra tion ish igh an d m ost o f the p la sm a iron is bou nd to d ifern ictransfe rr in ; e ry th ro b la sts o f such sub jec ts lik e ly takeup m ore iro n th an tha t req u ired fo r hem e sy n thesis .2 Instu d ie s o f iron up tak e by no rm a l hum an e ry th ro b la stsin v itro , M ay et a l.39 have foun d tha t in c rea singtransfe rn in sa tu ra tio n from 1 8% to 8 1 % doub led ironup take , w ith ab ou t tw o-th ird s o f such excess iron b e in ginco rp o ra ted in com pounds o th er th an h em e . T hus , th ein c rea sed iron up tak e by th e en y th rob lasts w ou ldex p la in the h ig h va lues fo r red ce ll ferr itin con ten tfou nd in p a tien ts w ith iron ov e rload . O n the o the rh and , p atien ts w ith iron de fic iency have low pla sm airon , a lm os t com ple te ly bound to m onofe rn ic trans -ferr in . T he ir e ry th rob la sts tak e u p le ss iro n than tha trequ ired fo r hem e syn thesis ,2 and the refo re need le ssferr itin a s an in trace llu la r iro n carrie r o r dep o t. T h isw ou ld exp la in th e low fe rn itin co n ten ts o f iron -d efic ien tred ce lls . D u e to a b lo ck in th e re lea se o f iro n from thereticu loendo the lial ce ll to transfern in , pa tien ts w ithin flamm ation h av e low pla sm a iron , m a in ly bo und tom ono fe rr ic transfe rn in . A s ex pec ted , th e ir red -ce llfe rritin co n ten t w as n orm a l to low , w hich is sim ila r toth at o f sub jects w ith iron -d efic ien t ery th ro po ie sis .H ow eve r , the d irec t co rre la tio n be tw een red ce ll an dp la sm a fe rn itin fo und in these p atien ts is no t c lea r . Apossib le exp lana tion is th at fe rn itin syn thesis in in f lam -m ation m ay b e a lso a non specific sys tem ic re sp on se o ft issues. # {17 6}In deed , h ig h leve ls o f fe rn itin have b een fou ndbo th in g ranu lo cy te s and m onocy te s in such cond i-tio ns.5 T hus , in in flamm atio n , th e red ce ll fe rn itinco n ten t m ay be a re su lt o f th e b alan ce be tw een then eg ativ e e ffec t o f red uced iron sup p ly to ery th ro idm arrow and th e n onsp ecif ic stim u lus to fe rn itin syn th e-sis .

to be the ra te o f hem og lob in sy n thesis . In tha la ssem ia ,g lob in cha in sy n th e sis is v ariab ly im pa ired and hem o -g lob in syn th esis is conseq uen tly red uced , the end -re su lt be ing p oorly h em oglo b in ized e ry th ro cy te s .4 W efo und h ig h va lues fo r red ce ll fern itin in tha la ssem icpa tien ts , ev en in tho se w ith norm a l iron sta tus andno rm al transfern in satu ra tion . Th is can b e exp lain edby as sum in g tha t th ala ssem ic e ry th rob la sts tak e upno rm al am oun ts o f iron , b u t pa rt o f the m e ta l cann o tbe u tiliz ed fo r hem og lob in syn thesis . S uch ex cess ironw ou ld be sto red in fe rn itin m olecu le s , thus p rodu cin ginc rea sing am oun ts o f red ce ll fe rn itin . T he inv e rsere la tionsh ip be tw een red ce ll fe rn itin an d m ean corpu s-cu la r h em og lob in (F ig . 5) is in k eep ing w ith th ishyp o thesis . H ow eve r, it sh ou ld be no ted tha t the da tarep o rted in T ab le 4 ind ic ate th at th e am oun t o f iro nen te rin g ery th rob las ts is a lso re levan t to the red ce llfe rritin co n ten t in th ala ssem ic pa tien ts .

A na tu ra l qu es tion is w he the r th e tw o fe rn itin ty pesm easu red by the live r (o r sp leen ) and hea rt as say s haved ifferen t m etabo lic func tion s w ith in e ry th ro id cells .P rev iou s stud ie s by G abuzda and G ardne r42 sh ow edthe ex isten ce o f tw o d istinc t poo ls o f fe rn itin in hum anbone m arrow , and m o re recen t s tu d ie s43 have p ro v idedev id en ce tha t, w ith in e ry th ro id ce lls , o ne poo l co u ld b eused in th e in trace llu la r transpo rt o f iro n fo r hem esyn thes is and the o th e r fo r iron sto rage . B asic fern itinis usua lly foun d in iron sto rage o rg an s ( live r an dsp leen ) , an d in red cells , th is fe rr itin typ e seem s to b ec lose ly re la ted to th e iron sta tu s; the re fo re, it m ayfun c tio n as an in trace llu la r d ep o t o f iron tha t is inexcess o f th at requ ired fo r hem e sy n thesis . A c id icfe rn itin s a re usu ally fo und in o rg ans hav in g h igh ironrequ irem en ts fo r m e tabo lic ac tiv itie s (e .g ., h ea r t) an da re capab le o f tak ing up an d don a tin g th e m e tal m orerap id ly th an basic fe rn itin s .44 In red ce lls , a c id ic fern -tin ap pears to be le ss in f luenced by th e iron sta tus th anbasic fe rn itin . M oreo ve r, th e ac id ic /basic fe rn itin ratiohas been found to d ecrease w ith ery th ro b la st m a tu ra -tion .3 4 Thus, it is p ossib le tha t ac id ic fe rn itin has anactiv e ro le w ith in e ry th ro id ce lls , beh av ing as anin te rm ed iate fo r the tran sfe r o f iron from th e p la sm am em brane to the m itoch ondn ia fo r in co rp oratio n in tohem e . A m ode l o f pos sib le pa thw ay s o f iron w ith inhem e-sy n thesiz ing ery th ro id ce lls is show n in F ig . 8 .

F rom a c lin ica l s tandp o in t, the red ce ll fe rn itin a ssayis m o re cum bersom e than th e p lasm a fe rn itin a ssay andobv iou sly cann o t be em ploy ed in sub jec ts w ho havebeen recen tly transfused . N eve rth ele ss , in sp ite o f th isan d the low num ber o f s tud ie s pe rfo rm ed , a fewc lin ical app licatio ns o f the red ce ll fern itin de te rm ina -tion , based on a comm erc ially av ailab le basic fe rn itinassay , can b e s ing led ou t.

F irs t, the red ce ll fe rn itin co n ten t re flec ts th e ba l-


9/10

1086 CA ZZOLA ET A L.

F ig . 8 . Po ss ib le pa thw ays of ir on w ith in h em e-s yn th es iz in gery thro id ce ll. It m ust be unders tood tha t th is is on ly one o f severa lpo ss ib le exp la na tio ns o f the fin d ing s o b ta in ed in the p re sen twork .

an ce be tw een iron sup p ly to the e ry th ro id m arrow andits u tiliza tion fo r the hem oglob in syn thesis . T hus, itc an b e used to eva lu ate th e adequ acy o f iron su pp lyan d to d iag nose re la tive iron d efic ien cy in pa tien ts w ithinc rea sed red ce ll tu rn ove r .4 5

S econd , it is w e ll know n tha t live r d isease m aye leva te the p la sm a fern itin co ncen tra tio n to a degreed isp rop ortio na te to th at o f iron sto re s .3 S in ce th e redce ll fe rn itin con ten t do es no t ap pea r to be in flu en ced byab norm a lities in live r fu nc tion , it c an b e usefu l in theev alu atio n o f iron s tatu s o f such pa tien ts . It h a s beenrecen tly show n tha t the ra tio o f red ce ll to se rumfe rn itin d isc rim in a tes pa tien ts w ith a lcoho lic liv er d is-ea se an d iro n ov erload from tho se w ith id io pa th ichem och rom a tosis .45 In ad d ition , th e red ce ll fe rr itinco n ten t iden tified pa tien ts w ith id io pa th ic hem och ro -

m a tosis a f ter ph leb o tom y , w ho , desp ite n o rm a l se rumfe rn itin concen tratio ns, had pe rsis ten tly ra ised ironsto re s .46 In the p re sen t stud y , the red ce ll fe rn itincon ten t w as th e b es t m arke r o f iro n ove rload in he tero -zygo tes fo r hem och rom a tosis w ith pa rtia l exp re ssio n o fthe d is ea se .

T he re m ay b e o th e r c lin ical app lica tion s o f the redce ll fern itin a ssay (an exam p le is repo rted in Tab le 5) ,and th e n um be r w ill c erta in ly in cre a se in th e n ex tyears .

NO TE ADDED IN PROOFIn the in te rva l -be tw een in itia l accep tance o f th is

m anu sc rip t an d its pub lica tion , w e h ave used a tw o-siteimm uno rad iom e tn ic as say (IRM A ) based o n a m ousem onoc lon al an tibod y ag ain st hum an hea rt fe rn itin fo rm easu ring red ce ll fe rn itin . Th is IRM A d id no t reco g-n ize hum an live r fe rn itin up to a co ncen tra tion o f1 0 ,0 00 ag /lite r . O n the ave rage , the va lues ob ta inedw ith th is a ssay in norm a l su b jec ts and pa tien ts w ithiro n de ficiency an d iron o ve rlo ad w ere low er than th osefo und w ith the rad io im m unoassay based on po ly clo na lan tib od ie s ag ain st hum an h ea rt fe rn itin . T he m ono-c lon al an tibo dy w ill b e ava ilab le on req ue s t in thefu tu re fo r in te rlabo ra to ry standa rd izatio n and fu rth erstu d ie s on red ce ll fe rn itin .

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