Bull Vet Inst Pulwy 52, 125-130, 08 RETICULOCYTE INDICES IN THE DIAGNOSIS OF IRON DEFICIENCY IN SUCKLING PIGLETS MARTIN SVOBODA, RADEK FICEK, AND JOSEF DRÁBEK Clini of Pig Diseses, University of Veterinry nd Phrmeutil Siene, 612 42 Brno, Czeh Repuli svoodm@vfu.z Reeived for pulition June 02, 07 Astrt The im of the study ws to evlute the usefulness of the retiuloyte indies in the dignosis of nemi in piglets. The piglets in group I (n=16) were treted with iron dextrn (0 mg of Fe, i.m.) t the ge of 16 d. In group II (n=16), piglets t the ge of 3 d reeived 100 mg of Fe nd then t the ge of 22 d - 0 mg of Fe. The piglets in group III (n=16) reeived 0 mg of Fe t the ge of 3 d. From the 7 d of ge, ll the piglets hd ess to reep feed. In group I, there ws derese in red lood ell indies nd the piglets developed nemi. These piglets hd lower growth intensity ompred to piglets of groups II nd III. In omprison with H, PCV, nd RBC, the retiuloyte indies (hemogloin onentrtion in retiuloyte nd index RET-Y) reflexed more rpidly impired erythropoiesis nd in the se of mild iron defiieny (group II) they reted more effetively to iron tretment. Key words: piglets, iron, hemogloin, retiuloytes, nemi. Iron defiieny nemi represents serious prolem in swine prodution. The reserve of iron in neworn piglets is limited (50 mg of Fe) (26). The sow s milk provides the piglet only with 1-2 mg of Fe per dy (3), while the dily requirement of iron for norml development is 7-10 mg (24). Therefore, without dditionl iron supplementtion, the piglets develop nemi within 10-14 d fter irth (10). The most ommon method of nemi prevention is i.m. dministrtion of 0 mg of Fe 3+ in the form of iron dextrn to two to three-dy-old piglets (25). The primry dignosis of iron defiieny nemi involves the exmintion of red lood ell indies, i.e. hemogloin onentrtion, hemtorit, nd erythroyte ount. The dignosis of the ltent iron defiieny is sed on the determintion of iohemil indies in lood plsm. However, these indies n e influened lso y other ftors thn iron defiieny (8, 18). With the introdution of utomted flow ytometry, humn lortories strted to use new retiuloyte indies (hemogloin onentrtion in retiuloytes nd index Ret-Y) s sensitive inditors of iron-defiient erythropoiesis (2). These indies reflet the tul stte of mrrow erythroid tivity, nd re not influened y ftors ffeting trditionl iohemil mrkers (14). To the uthors knowledge, the new retiuloyte indies hve not een yet tested s inditors of iron defiient erythropoiesis in piglets. The im of our study ws to evlute these indies in the dignosis of iron defiieny nemi in sukling piglets. Mteril nd Methods Experimentl design. The piglets were divided into three groups using split litters. All the groups reeived iron in the form of iron dextrn t different doses. The piglets in group I (n=16) serving s the ontrol nemi group were given intrmusulrly (i.m.) 0 mg of Fe 3+ t the ge of 16 d. The piglets in group II (n=16) were injeted i.m. with 100 mg of Fe 3+ t the ge of 3 d nd then, t the ge of 22 d, were given the seond dose of iron (0 mg of Fe 3+ ). The piglets in group III (n=16) reeived 0 mg of Fe 3+ t the ge of 3 d. The linil sttus of the nimls ws monitored dily. Clinil signs of nemi (ple muosl memrnes, ple skin on the whole ody surfe) in piglets of group I were pprent t the ge of 16 d. The piglets reeived sow s milk until they were wened. From dy 7 of the ge until the end of the tril (dy 35), the piglets hd ess to reep feed (SKS wening pellets, Slvkovské krmné směsi. s). This reep feed ontined 2 mg of Fe/kg nd ws offered to the piglets d liitum. The piglets were wened t the ge of 29 d. Smpling nd nlyses. Blood ws olleted from the ven v rnilis on dys 3, 8, 16, 22, 29, nd 36 of the piglets life. In groups I nd II, the lood ws lso tken 48 h fter iron dministrtion. EDTA (ethylenediminetetreti id) ws used s n ntiogulnt for the hemtologil exmintion nd heprin ws used s n ntiogulnt for the determintion of iron onentrtion in lood plsm. The piglets were weighed on the dys of the lood olletion.
126 The hemtologil indies were mesured using utomti nlyser Sysmex EX-2100 nd inluded: hemogloin onentrtion (H), pked ell volume (PCV), red lood ell ount (RBC), men orpusulr volume (MCV), men orpusulr hemogloin (MCH), men orpusulr hemogloin onentrtion (MCHC), retiuloyte ount (RET), perentge of retiuloytes (RET %), hemogloin onentrtion in retiuloytes (RET-He), nd index RET-Y. The iron onentrtion in the lood plsm ws determined photometrilly y mesuring the iron omplex with ferrozin (Iron liquid 917, Rohe Dignosti, Germny). The differenes etween groups were evluted y non-prmetri Kruskl-Wllis ANOVA test t the level of signifine P<0.05. The hnges within groups were evluted y non-prmetri Friedmn ANOVA nd susequently y pired Wilox test t the level of signifine P<0.01. The hemtologil indies re presented in Figs 1-9. The iron onentrtion in the lood plsm nd ody weights re presented in the form of tles. Results Differenes mong groups. At the ge of 16 nd 22 d, the indies of H, PCV, MCV, MCH, RET- He, RET-Y, nd Fe in group II (100 mg of Fe 3+ ) were signifintly lower thn in group III (0 mg of Fe 3+ ). In group I, the indies of H, PCV, RBC, MCV, MCH, RET, RET %, RET-He, RET-Y, nd Fe strted to e signifintly lower s erly s from the 8 d of ge. At the ge of 16 d, the ody weight in group I ws signifintly lower thn in groups II nd III. Chnges in the exmined indies within groups: Group I. The indies of H, PCV, RBC, MCV, MCH, MCHC, RET, nd RET % deresed signifintly t the ge of 16 d. After iron dministrtion t the ge of 16 d, H, PCV, MCV, MCH, RET, RET %, RET-He, RET-Y, nd Fe inresed signifintly within 48 h. Group II. At the ge of 16 d, there ws deline in the indies of MCV, MCH, MCHC, RET, RET %, Ret-He, RET-Y, nd Fe. After iron dministrtion t the ge of 22 d, there ws signifint rise of RET, RET %, RET-He, RET-Y, nd Fe indies within 48 h. Group III. At the ge of 22 d, the indies of MCV, MCH, RET-He, nd RET-Y delined signifintly. The iron onentrtion in lood plsm delined erlier, i. e. t the ge of 16 d. One week fter wening, the indies of RET, RET %, RET-He, RET-Y, nd Fe inresed signifintly. Tle 1 Men iron onentrtion in lood plsm (µmol/l) Dys of ge 3 8 16 22 29 36 Group I 4.9 ± 2.2 3 ± 0.7 3.1 ± 0.6 24.0 ± 7 23.6 ± 7.2 27.1 ± 5.6 Group II 4.8 ± 2.6 24.8 ± 5.1 7.6 ± 3.9 4.6± 1.6 22.2 ± 7.5 26.2 ± 6.4 Group III 4.0 ± 1.6 26.0 ± 5.4 15.8± 5.2 13.0 ± 6.9 13.5 ± 7.4 23.5 ± 7.3 48 h fter tretment (dy 16 in group. I, dy 22 in group. II): Group I 36.6 ± 12.2, Group II.8 ± 6.9 Letters,, nd express signifint differenes etween the groups (P<0.05). ± SD. Tle 2 Men ody weight during the tril (kg) Dys of ge 3 8 16 22 29 36 Group I 2.2 ± 0.5 3.3 ± 0.8 5.2 ± 1.2 6.4 ± 1.4 7.1 ± 1.5 9.1 ± 2.1 Group II 2.5 ± 0.5 3.8 ± 0.8 6.4 ± 1.4 8.1± 1.9 8.9 ± 1.7 11 ± 2.2 Group III 2.4 ± 0.4 3.9 ± 0.7 6.4± 1.5 8.7 ± 1.9 9.5 ± 1.5 11.1 ± 1.5 Letters,, nd express signifint differenes etween the groups (P<0.05). ± SD.
127 130 0.45 1 110 100 0.40 0.35 90 H (g/l) 80 70 PCV (l/l) 0.30 0.25 60 50 0. 40 30 Medin 0.15 Medin dy 3 dy 8 dy 16 * dy 22 * dy 29 dy 36 Dys of ge Fig. 1. Hemogloin onentrtion. 0.10 dy 3 dy 8 dy 16 * dy 22 * dy 29 dy 36 Dys of ge Fig. 2. Pked ell volume. RBC (10 12 /l) 8 7 6 5 4 MCV (fl) 90 85 80 75 70 65 60 Medin 3 Medin 55 50 2 dy 3 dy 8 dy 16 * dy 22 * dy 29 dy 36 Dys of ge Fig. 3. Red lood ell ount. 45 dy 3 dy 8 dy 16 * dy 22 * dy 29 dy 36 Dys of ge Fig. 4. MCV. 24 22 310 300 MCH (pg) 18 16 MCHC (g/l) 290 280 270 260 14 12 Medin 250 240 Medin 10 dy 3 dy 8 dy 16 * dy 22 * dy 29 dy 36 Dys of ge Fig. 5. MCH. 230 dy 3 dy 8 dy 16 * dy 22 * dy 29 dy 36 Dys of ge Fig. 6. MCHC. RET (10 12 /l) 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Medin dy 3 dy 8 dy 16 * dy 22 * dy 29 dy 36 Dys of ge Fig. 7. Retiuloyte ount. RET % (%) 28 26 24 22 18 16 14 12 10 8 6 4 2 0 dy 3 dy 8 dy 16 * dy 22 * dy 29 dy 36 Dys of ge Fig. 8. Perentge of retiuloytes. Medin
128 26 160 24 140 RET-He (pg) 22 18 16 14 RET-Y (h) 1 100 80 12 Medin 60 Medin 10 dy 3 dy 8 dy 16 * dy 22 * dy 29 dy 36 Dys of ge Fig. 9. Hemogloin in retiuloytes. dy 3 dy 8 dy 16 * dy 22 * dy 29 dy 36 Dys of ge Fig. 10. Index Ret-Y. Disussion Iron defiieny is reognised y orrelting the red lood ell indies with iohemil mrkers of negtive iron imlne. Trditionlly, the stndrd iohemil mrkers of iron metolism hve een serum or plsm iron, trnsferring (Tf), trnsferring sturtion (TfS), ferritin, nd more reently, mesurement of serum solule Tf reeptor (stfr). Iron defiieny nemi is hrterised y hypohromi nd miroytosis in onjuntion with low serum iron nd ferritin onentrtions, deresed TfS, or inresed stfr (12, 23). When inflmmtory disorders our, they our onomitntly with iron defiieny, serum iron, trnsferring, nd ferritin vlues my e unrelile (8, 19). Prussin lue stining of the mrrow iopsy is onsidered s the gold stndrd for the ssessment of iron sttus, ut this invsive test is rrely used. An inresed trnsferring reeptor hs een used s sensitive inditor of iron defiieny. As solule trnsferring reeptor is not ffeted y ute or hroni inflmmtion, the test n e used s relile index of mrrow iron stores (21, 23). However, for sreening purposes, methods of mesuring ferritin nd serum trnsferring reeptors re reltively expensive, ompred with RBC indies. As erythroytes hve lifespn of 1 d, the trditionl red lood ell indies nnot provide informtion on the rpid hnge in erythropoieti tivity (16). Therefore, there is need to seek n erlier nd more sensitive mrker for funtionl iron defiieny. Hemogloin onentrtion in retiuloytes reflets the mount of ville iron during RBC prodution, s retiuloytes exist in the irultion system for only 1-2 d. The usefulness of this index s n erly mrker of iron defiieny ws demonstrted y the studies evluting the iron sttus in humn ptients (1, 4). With the introdution of utomted flow ytometry nd fluoresent dyes, it is possile to nlyse retiuloyte indies inluding their volume (1, 5). The prmeter Ret-Y mesured on the Sysmex XE 2100 provides reltive mesure of the equivlent of men orpusulr volume of retiuloytes. Kikler t l. (14) found in humn ptients tht Ret-Y prmeter hs the highest sensitivity nd speifiity mong tests used for the dignosis of iron defiieny nemi. The retiuloyte indies n e used lso for n erly reognition of non-responsiveness to iron dministrtion. Fishne et l. (9) nd Mdougll et l. (15) reported in humn ptients tht t lest 4 or 8 weeks re neessry to oserve signifint response to iron dministrtion y monitoring the erythroyte indies, inluding hemogloin nd hemtorit. The min reson for the lte dignosis is the long life spn of mture RBC. In the se of retiuloyte indies, Fishne et l. (9) nd Mittmn et l. (17) found in humn ptients tht these indies inrese signifintly within 48 h fter iron dministrtion. The nemi limit, i.e. the point when nemi egins to exert detrimentl effet on weight gin nd gives linil symptoms, is set y most uthors t H onentrtion elow 80 g/l (7, 11). In group I (0 mg of Fe 3+, dy 16), there ws derese in RBC indies nd the piglets developed nemi (H elow 80 g/l). These piglets hd lower growth intensity ompred to piglets of groups II nd III. This is in greement with findings of other uthors (7, 22). Forty-eight hours fter iron dministrtion, there ws signifint inrese in H, PCV, MCV, MCH, RET, RET %, RET-He, RET-Y, nd Fe indies. This demonstrtes omprle retion of the exmined indies fter the iron tretment in the se of severe iron defiieny. In group II (100 mg of Fe 3+, dy 3) the indies of MCV, MCH, RET, RET %, RET-He, RET-Y, nd Fe deresed signifintly t the ge of 16 d. The ft tht there ws not sttistilly signifint derese in H, PCV, nd RBC demonstrtes lower sensitivity of the respetive indies for the detetion of iron-defiient erythropoiesis. Forty-eight hours fter the iron dministrtion the indies of RET, RET %, RET-He, RET-Y, nd Fe inresed signifintly; while the indies of RBC, H, PCV, MCV, nd MCH did not. This
129 indites their lower retion to iron tretment in the se of mild iron defiieny. There re onfliting dt onerning the effiy of 100 mg of Fe 3+ dose. Ky et l. (13) found this dose s pproprite for the norml development of sukling piglets. On the ontrry, Dykin et l. (6) do not onsider suh low dose s suffiient nd reommend the dose of 0 mg of Fe 3+. It is ovious from our results tht the dose of 100 mg of Fe 3+ does not provide suffiient hemogloin onentrtions (dys 16 nd 22) nd thus it should not e used in the prtie. Even in group III (0 mg of Fe 3+, dy 3) there ws deline in MCV, MCH, RET-He, RET-Y, nd Fe indies. The lk of derese in H, PCV, nd RBC indites low sensitivity of these indies for the detetion of impired erythropoiesis. The rise of retiuloyte indies one week fter wening ould e explined y the ft tht piglets strted to feed intensively on reep feed. Reltively rpid inrese in hemogloin onentrtion fter iron tretment (in group I fter 48 h in group II fter 1 week) differs from the dt otined in humn ptients. Fishne et l. (9) nd Mdougll et l. (15) reported signifint rise of hemogloin s lte s fter 4-8 weeks fter iron dministrtion. The rpid inrese in hemogloin ontent fter iron tretment ould e explined y n intensive erythropoiesis, whih is relted to high growth intensity of sukling piglets (). In omprison with H, PCV, nd RBC vlues, the retiuloyte indies reflet more rpidly n impired erythropoiesis. The retion of MCV nd MCH to impired erythropoiesis is omprle with retiuloyte indies. When the iron ws given to piglets with severe iron defiieny (group I), there ws signifint inrese fter 48 h, oth in trditionl (H, PCV, MCV, nd MCH) nd retiuloyte indies. In the se of mild iron defiieny (group II), the retiuloyte indies reted to iron tretment more rpidly thn trditionl RBC indies. 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