Direct and Indirect Coombs Tests Blood Grouping

---

Direct an d In d irect Coo m b s T ests Bloo d G ro up in g Q ualit at iv e test for determination of human ant i-I gG and anti-C3d on red blood cells. I VD S tore at 2-8ºC P RI NCIP LE O F THE M ET HOD When used by the recommended techniques, the reagent s will react with immunoglobulins and/or complement at tached t o the red cell s urface, res ult ing in agglut ination (clumping) of adjac ent sensitised c ells. Cells not s ensit ised will not be agglutinat ed (S ee Lim itations ). CL INI CAL S IG NI FICANC E I n 1945, Coombs, Mourant and Rac e desc ribed the use of antihuman globulin serum for detecting red c ell-bound nonagglutinating antibodies. In 1957, Dacie et al s howed t hat the antibodies present in antiglobulin sera were direc ted against certain c omponents of complement. A ntihuman globulin reagent s det ec t non-agglutinating antibody molec ules as well as molecules of c omplement att ac hed to red cells following in vivo or in vitro antigen-antibody reactions. RE AG E NTS P robio Polyspecific A nt i-Human G lobulin Elite Clear and AntiHuman G lobulin Elite Green reagents contain ant i-I gG deriv ed f rom rabbit s with non-spec if ic act iv ity removed by absorption and mouse monoclonal I gM anti-C3d, Clone B RIC-8. The antibodies are diluted in a buff ered solution cont aining bovine albumin. Each reagent is s upplied at opt im al dilution, f or us e with all the recommended techniques s tated below wit hout need for f urt her dilut ion or addit ion. For lot referenc e number and expiry date s ee V ial Label. Reagent A nti-Human Globulin G reen

C olour G reen

Dye Us ed P at ient Blue and Tartrazine

P RE CAUTI ONS 1. The reagents are intended f or in v itro diagnos tic use only. 2. If a reagent v ial is cracked or leak ing, dis card the cont ents immediat ely . 3. Do not us e the reagent s past the ex piration date (see V ial Label). 4. Do not us e the reagent s if a precipitate is pres ent. 5. P rot ec tive c lot hing should be worn when handling the reagents, such as disposable gloves and a laboratory c oat. 6. The reagent s have been f ilt ered t hrough a 0. 2 μm capsule to reduce the bio-burden. O nc e a vial has been opened the contents should remain v iable up unt il the expiry dat e as long as there is no marked turbidit y, whic h can indicate reagent deterioration or contamination. 7. The reagents cont ain < 0. 1% s odium azide. S odium azide may be t ox ic if ingest ed and may reac t wit h lead and copper plumbing t o form explos iv e metal azides . O n dispos al flush away with large volum es of water. 8 . No k nown t es ts can guarant ee t hat products derived from human or animal sourc es are f ree f rom inf ectious agents . Care must be taken in the us e and disposal of each vial and its content s. 9. For information on disposal of the reagent and decontaminat ion of a spillage sit e see Material S afety Data Sheets, available on reques t. NO TE S 1. I t is rec ommended a positiv e cont rol (weak Anti-D 0. 1 I U/ml) and a negative c ontrol (an inert serum) be t es t in parallel with each batch of tests . Tes ts mus t be considered invalid if controls do not s how ex pect ed results. 2. The antiglobulin techniques can only be cons idered valid if A ll negat iv e tests reac t positively with I gG sensitised red

cells. 3. I n the techniques, here mentioned, one volum e is A pproximately 40μl when using the vial dropper provided. 4. Us e of the reagents and t he interpret at ion of res ults must be carried out by properly trained and qualified personnel in accordance wit h requirements of t he c ountry where t he reagents are in use. User must det ermine the suitabilit y of the reagents for use in other techniques S TO RAG E Do not freez e. Reagent v ials should be st ored at 2 - 8ºC on receipt . P rolonged s torage at temperatures outside this range m ay res ult in acc elerat ed loss of reagent react iv ity. MA TE RIA L RE Q UIRE D • Glass test tubes (10 x 75 mm or 12 x 75 mm). • Test tube c ent rif uge. • Volumetric pipet tes. • Phosphat e Buff ered S aline (P BS ): NaC l 0.9%, pH 7.0 ± 0.2 at 22ºC ± 1ºC. • IgG sens itised red c ells . • Inert antibody S erum . • W eak ant i-D. • W at er bat h or dry heat incubator equilibrated t o 37ºC ± 2º C. • Coombs cell was her. • Low I onic S trength S olut ion (LIS S ) , i.e..: P ROB I O’s RE F. 1700080. S AMP LE S amples should be drawn aseptically int o EDT A to prev ent in vitro c omplement binding and t es ted within 24 hours. If EDT A is unav ailable, samples drawn int o ACD , CP D or CPDA -1 are pref erable to c lot ted ones. If only clott ed s amples are available, do not ref rigerat e them bef ore t es ting. A ll blood samples should be washed at least twice wit h P BS before being test ed. PR OCE DURE A. Direct Antiglobulin Technique (DA T) B. 1. Wash t es t red cells 4 t imes with P B S , t ak ing c are to decant saline between washes and res us pend each cell but ton aft er each was h. Completely dec ant saline af ter las t was h. 2. A dd 2 volumes of A nti-Hum an G lobulin to each dry c ell button. 3. Mix thoroughly and c entrifuge all tubes for 20 seconds at 1000 rcf or for a suitable alt ernative t ime and f orc e. 4. G ent ly resuspend red cell but ton and read macros copic ally for Agglutinat ion B. I ndir ect An ti glo buli n Techni que (NIS S IAT ) 1 P repare a 2-3% suspens ion of washed test red cells in P B S . 2. P lac e in a labelled test tube: 2 volum es of t es t s erum and 1 V olume of test red c ell sus pension. 3. Mix thoroughly and incubat e at 37ºC f or 15 minutes. 4. Wash t es t red cells 4 t imes with P B S , t ak ing c are to decant S aline between was hes and resuspend each red cell but ton aft er each wash. Complet ely decant saline aft er last wash. 5 . A dd 2 volumes of A nti-Hum an G lobulin to each dry c ell button. 6. Mix thoroughly and c entrifuge all tubes for 20 seconds at

1 0 00 rcf o r fo r a s uit ab le a lte rna tiv e time a n d fo rce . 7 . Ge n tly re su sp e nd re d ce ll b u tt on a n d rea d ma cro sco p ica lly Fo r ag g lu tina t io n C. LIS S I ndir ec t A ntiglo buli n Te c hni que (LIS S I AT) 1 . Pre p are a 1 .5 -2 % s us p en sio n o f wa sh e d te st re d ce lls in LI SS . 2 . Pla ce in a la b elle d te st tu b e : 2 v olu me s o f te st se rum an d 2 v o lu me s of te st re d ce ll su sp e nsio n . 3 . Mix th oro u gh ly an d in cu ba t e at 3 7º C fo r 1 5 min u te s. 4 . Fo llow ste p s 4 to 7 o f NIS S IAT a bo ve .

Coa t ed with C3 . 5 . The pre se nc e o f c on ta min a tin g he te ro sp ec if ic a g g lut inin s or a nt ibo d ies to C4d h a s be e n ex clu de d in t es ts e mp lo yin g re d ce lls o f a ll A BO g ro up s an d ce lls c o at ed wit h C4 d . 6 . Th e re ac tivity of a n y A n ti-Ig M, An ti-I gA o r A n ti-ligh t ch ain co mp on e n ts th at mig h t b e p res en t h as n ot b e en e st ab lish e d. 7 . Th e Q ua lity Co nt rol of th e re ag e n ts wa s p e rf o rme d u sin g red Ce lls t ha t ha d b e en wa s he d t wic e with P B S p rio r t o us e. 8 . The re a g en ts co mp ly wit h th e rec om men d a tio ns co n ta ine d in Th e la te st issu e o f th e Gu id elin e s f o r th e UK B loo d Tra ns fu sio n Se rvice s. BI BLIO G RAP HY

INT E RPRE TAT IO N O F TE S T RE S ULTS P os itiv e : A gg lu tin at ion o f te st red ce lls co n stit ut es a p os itive t es t re su lt a n d with in th e a cce p te d limita tio ns o f th e te st p ro ce d u re , in dic at es th e p res en ce of Ig G a n d/ or c om ple me nt (C3 ) o n th e te st re d ce lls Ne ga tiv e : No a gg lu tin a tion of th e t es t re d ce lls co n st it u te s a n e ga tiv e res ult a nd wit hin th e ac ce pt ed limit at ion s of th e t es t p roc ed u re, in dic at es th e a b se nc e of I gG a n d/ o r co mple me n t (C3 ) o n th e te st re d ce lls. S tab ili ty of the re a c ti ons

1 . Co om bs RRA , Mou ra nt A E , Ra ce R R. A n e w t es t f o r th e De te ctio n o f wea k a nd “in co mp let e” Rh an t ib o d ies . B rit J E xp P at ho l. 19 4 5 ; 2 6 :2 5 5. 2 . W rig h t MS , Iss it P D. An ti-co mp le men t a nd t he in d ire c t A n tig lob u lin te st. Tra ns fu sio n 1 97 9 ; 1 9 :6 8 8 -6 9 4. 3 . Ho ward J E , W in n L C, Go tt lie b CE , Gru me t FC, Ga rra t ty G, P et z L D. Clinic al sig nific an ce o f th e a n ti - c omp le men t co mp on e n ts of a n tiglo b u lin a n tise ra . T ra n sfu sio n 1 9 82 : 2 2: 26 9 .

1 . W as hin g st ep s sh ou ld b e co mp let ed wit h ou t int e rru p tio n an d Tes ts ce nt rif u ge d a n d rea d imme d ia t ely af te r a d dit io n o f th e re a g en t. De la ys m ay res ult in d isso cia tio n of a n tig en -a nt ibo d y c o mple xe s, ca u sing f a ls e n eg a tive o r we a k po sitiv e re s ults . 2 . Cau tio n sh o u ld be e xe rcise d in t he in te rp re t at ion o f re s ults o f Tes ts pe rfo rme d a t t emp e rat u re s ot h er t h an t ho se rec om men d e d.

4 . Ho well P , G iles CM. A d et aile d se ro lo g ica l st ud y of fiv e an tiJka se ra rea ct ing b y th e a nt iglo b ulin t ec hn iq ue . V ox . S a ng . 1 98 3 ; 45 : 12 9 -13 8 .

LIM ITAT IO NS

6 . Th e De p artm en t of He a lth a n d So cia l S ec u rit y. Hea lt h Se rvice s Man a g eme n t A n tig lob u lin Te st. Fa lse n e ga tiv e res ult s, HN (Ha za rd ) (83 ) 6 2 5 No v 1 9 83 .

1 . Red c ells th a t h a ve a p o sitive DAT d ue t o a co a tin g of I gG Can n o t b e t ype d b y th e Ind ire c t Antig lobu lin Te c hniqu es . 2 . A p os itive DA T du e to c om ple me nt se n sitis at io n ma y no t ref lec t in v iv o co mp leme n t f ixa tio n if t es t c ells a re f rom a ref rige ra te d clo tte d sp e cime n . 3 . In a de q u at e wa sh ing o f red c ells in th e in dire ct an t ig lo b ulin te ch n iqu e s ma y n eu tra lise th e a n ti-hu ma n g lob u lin re a g en t . 4 . Fo llowin g co mp le tio n of th e wa sh ph a se e xce ss res id u a l sa line m ay d ilu t e th e a nt i-h u ma n glo b ulin , red u cin g its p ot en c y. 5 . A n eg a tive d ire ct an tig lo bu lin te st re su lt d o es n ot n e ce ssa rily p re clu d e clin ic al dia g n os is o f A B O Ha emo lyt ic Dise a se o f th e Ne wbo rn o r A u to Imm un e Ha e mo lytic A n a emia . It a ls o d oe s No t ne ce ssa rily ru le o ut HDN , e sp e cia lly if A B O inc om pa tib ility Is su sp e cte d . 6 . Fa lse p os itive o r f alse ne g at ive res ult s ma y a ls o oc cu r d ue t o : • Imp ro pe r s to ra g e , c ell c on ce n tra tio n , in cu b at ion t im e or te mp era tu re • Imp ro pe r o r e xce ss iv e ce n trifu ga t io n 7 . The us er is res p on sib le fo r th e p e rf o rm an ce o f th e rea g e nt s b y an y me th o d o th er th an t h ose he re me n tion e d . 8 . A ny d ev iat ion s f rom th e te ch n iq ue s he re rec om men d e d sh o uld b e va lid at ed prio r to u se 9 . Co n ta mina tio n o f te st mat e ria ls PE RFO RMANCE CHARACTE RI ST ICS 1 . The re a g en ts h av e b ee n ch a rac te ris ed b y a ll th e p ro ce d u re s He re d es cribe d . 2 . P rio r t o rele a se , e a ch lo t o f P ROB IO ’s A n ti-Hu ma n Glo b ulin Is te st ed , b y th e t ec hn iq ue s h ere me n tio ne d , ag a ins t re d ce lls c oa te d wit h A nt i-D, A n ti-K a nd A n ti-Fya t o ch e ck su ita b le re a ctiv ity. 3 . The an ti-Ig G a n d an t i-C3 d p o te nc ies h av e be e n te st ed a ga in st th e fo llo win g min imu m p o te nc y re fe ren ce s ta n da rd o bt ain e d fro m Na tio n a l In stit u te o f B io log ica l S ta n da rd s a n d Co n trols (NIB S C): A n ti-AH G re fe re nc e sta n d ard 9 6 /6 6 6 4 . A nt i-C 3d p o te n cy is d em on st ra t ed in t es ts emp lo yin g ce lls

5 . Iss itt PD , S mith T R. E va lu at ion of a nt iglo b ulin re a ge n ts. A S emin a r o n pe rfo rma nc e ev a lu a tio n . W a sh in gt on , DC. A merica n A sso cia tio n o f B lo od Ba n ks. 1 97 6 ; 25 -7 3.

7 . B ru ce M, W a tt A H, Ha re W , B lue A, Mitc he ll R. A se rio us so u rc e of erro r in a nt iglo b ulin t es tin g. Tra ns fu sio n 19 8 6 ; 2 6 : 1 77 -1 81 . 8 . Th e a nt i-c om ple me nt re ac tivity lo w io nic me th o ds a s p ub lish e d by FDA . Re co mmen d e d Me th od s fo r A nt i - Hu ma n G lo b u lin E va lu a tion (re visio n O cto b er 19 8 4 ). 9 . Dyn a n PK . Ev a lu a tio n o f c omm ercia lly a va ila ble lo w io n ic S tre ng th s alt (L IS S) s olu tio n s. Me d La b S ci (1 9 81 ) 3 8 1 : 1 3 2 0. 1 0 . V o ak D, Do wnie DM, Moo re B PL , Fo rd DS, E ng e lfre it CP , C as e J. Re plica te t es ts fo r th e d e te ctio n a n d co rre ct ion o f e rro rs in a n tih u man g lob u lin (A HG ) te st s: op timu m co nd itio n s a nd q u a lit y c on t ro l. Ha ema to lo gia 1 9 8 8; 2 1 (1 ): 3-1 6 . 1 1 . G uid e lin e s f o r th e B lo o d Tra ns fu sio n Se rvice in th e Un it e d K in g d om . H. M.S .O . Curre n t E d ition . 1 2 . B ritish Co mmit te e fo r S ta n da rds in Ha e ma to lo g y, B lo o d Tra ns fu sio n Ta sk Force . Re co mme nd a tio ns f or e va lu at ion , va lida tio n a n d imp lem en ta tio n o f ne w t ec h niq u es fo r b lo od g ro u p ing , an t ib o d y sc ree n ing a n d cro ss ma tch ing . Tra ns fu sio n Me dic in e , 19 9 5 , 5 , 14 5 - 1 5 0.