boratory by Patsy Jarreau, MHS, CLS(NCA}, MT(ASCP) roadClinical Laboratory Science Review: A Bottom Line Approach Third Edition Patsy C. Jarreau, MHS, CLS(NCA), MT(ASCP) Associate Professor, Department of Clinical Laboratory Sciences LSU Health Sclences Center; New Orleans, Louisiana ‘CHAPTER AUTHORS Larry Broussard, PhD, DABCC Professor, Department of Clinical Laboratory Sciences LSU Heaith Sciences Center at New Orleans | ‘Angela Faley, MS, CLS(NCA), MT(ASCP)SH ql Associate Professor, Department of Clinica! Laboratory Sciences | iSU Health Sciences Center of New Orleans Daniel Haun, BS, MT(ASCP)H Ditector for Cent Services Medical Center of Louisiana oO A Marcia Firmani, PhD, MI(ASCP) y Assistant Professor, Department af Clinical Laboratory Sciences LSU Health Sciences Center at New Orleans touann Lawrence, DrPH, CLS(NCA), MICASCP)SH Professor, Department of Clinical Laboratory Sciences LSU Health Sciences Center at New Orleans 4 Mary Lux, PhD, CLS(NCA), MT(ASCP) Professor, Depariment of Medicol Technology University of Souther Mississippt Mary Muslaw, MHS,MT(ASCP)SC: Corporate Laboratory Director Wilis Knighton Health Systems Elizabeth Williams, MHS, CLS(NCA), MT(ASCP)SBB Associate Professor, Department of Clinical Laboratory Sciences LSU Health Sciences Center at New Orieans : Michele Zitzmann, MHS, CLS(NCA), MI(ASCP) Associate Professor, Department of Ciinical Laboratory Sciences LSU Health Sciences Center at New Orieans the Fourdétion LSU Heactn Sciences CENTER New Orleans, Louisiana pii : i ‘ yematology ..-- 2-26... ee 1 Donor Safoction and Blood Collection 1 ‘posior Blood Processing 0... 4 “Gomponents/Iranstusion Practice 4 “ABO System : ad Sister so ne ot Other Blood Grou Systems... 12 " Ralibody Seteoning and Kdontifeation 0.14 sonal Technologies %0 Detect Hrigerrifoody ROOCTOTS os. 20 frotorstusion fesing .... +. ve QT ‘povorse Etfects of Fronstusion vs ose 28 Hemolvtic Disease of the Newborn... e 25 28 HA vorcccsrecsssectenetrses 26 Qucity Control. sce? irranenamatoegy Sample estos fee BB ‘Answers and RatONMIE 30 Immunology and Serology ... 33 Typos of munity «55.5 foe Clastes of immunoglobulins eee ee ene 88 Immune Response... 23d Principles of Antigen-Artioody Reactions Used in Serologic Testing...» ve 7 specie Dkeaxe States and Associated Laboratory Tests... vee A Irnmeunology Samolo Questions «+. “9 Answers oF RaHONO 0 Hemotology 53 Hematopoiess + sie ee 6S Hemoglobin... oe ‘Ba Baxle Laboratory Proced utes eee e eee BS Spaciol Homatology --sessseeietsierceeel Red Cel Disease States foes él Winite Cel Disecse States oor 68 Lysosome ond tiple Storage Disorcers 187 Hematology Sample Questions . 8 Anowers and Raonale oe. o Coogulation . .. veveeee Tl Plctelets 1. Un Prosma Coaguioton Foctors. 2... 72 Routine Tests of Hemostattc Function 2... +.2 TA Speco of mest Func 1.0... 78 Homosiast DROIDIS «ee ceecesceesecnsedT Thrombote Dseases |. eB Coagulation Sample Questions ese T8 Answers and Rationale... +. ved BLE OF CONTENTS Urinalysis/Body Fluids . Renat Physiology... . al Specimen Collection ond Handing +++ 83 Physio! Examination : 83 Chemical Exeminotion . 285 Microscopie Examination yore ces 9 Renal Function Tests... : 292 Renal Diseases oo ceee eee pees 93 Body Fluids... 93. Utinal/Body Fukd Sormplo Questions 1... 97 Answers and Rationale’... ce 98 Chemistry . - ‘Sompie Collection and Haneting » Carbohydrates ves Upids and Lipoproteins... Amino Actes Projeins Erayrnes o Electrolytes... veces Acid-Base Balance Hemoglobin Derivatives Toxicology and Therapeutic Brug Montene + Nor-Protein Nizogens . Endocrinology Chemistry Sample Questions Answers cnd Ratlenale Microbiology ... Bacteriol Growth Rocaiternents AntbiotioySusceplbily Testing... Media... Specimen Cotection one Hancting Laboratory Diagnostic Methods ... Gram Postive Coco! sve ev ess Gram Negative Coce! : Gram Pestve Rods a Gram Negative Rost ae Grom Negative Oxidase Postive Fermentors... 151 Zoonotic Deeates vee Fasticious Gramm Negative Rock - ANQOTODES voces sees Sprochetes 6.) isso. Clomydia eit Rexettioe licensees Fungurike tacteta eevee Mycobactera .... x61 Specimen Source and Potential Pahegers «162 Vrolooy : 764 Microdoiogy Sample Questions... 167 Answers ond RatiONOIO ses sc rece es es NDMycology .... : ..+..171 | Loboratory Operations and Characteristics of Fung? 17) | Instrumentation 0... .ceeee ees 221 Laboratory Methods : 21 | Qcaity Cente! one Guaty Assueence 1.” gan Specimen Collection and Haring 171 | teboratery Mathomarics x Supeiticiol Mycoses 172 | Chemisty / imenunology instrumentation ‘Dy ‘Systemic Fungi wT Hematotogy Instumentation 232 Subcutaneous Fungt veer V73 1 Coagulation instrumentation oi 4 Yeasts anc Other Opportunists 376 | Usinalyals instrumentation... 235 Mycology Sample Questions .......2.2.2+..180 | Laboratory Operations andl Instrumentation Sample Anowors ana Retienle ceo Questions 236 § Answers and Rtioncie 231 Porasitology . tees = 181 Speomen Colston cnansieg 128) | Laboratary Satety and Regulations 239° Ove, Cyst and Parasite Examination .......... 18h Blood-borne Pathogens: . HOMIIRS - anticoagulant Sj . Citrate, phosphate, dextrose sxpiration 2. CPDA-D- amicoagulant (a. Adenine added Ssh. -xpiration 3. Gdditive solutiond Ha-clos: a, Contain dextrose; adenine, sodium chloride and other substances ‘VIRAL DISEASES: +e confirmatory test for HBsAg. + Repeatedly © Donated only unit to recipient who developed post transfusion hepatitis, HIV, or HTLV. + PresentPast infection of HCY, HITIY, of HI «Evidence of parenteral drug us OTHER DISEASES: + Received dura mater or pituitary growth hormone ‘of human origin; family history of CID or tisk of vCIC + History of Chagas’ disease, babesiosis b. Added to enhanee ved cell survival: 42-day expiration 4, Rejuvenating solutions? ‘a. Contain pyruvate, inosine, phosphate ‘and adenine b. Restores 2, 3-1 and ¢. Added during storage or up to 3 days after expiration date — Can freeze unit or, if uged in 24 brs., can be stored ay’1-6C yrnust wash fall before transfusion | solution) a. a oi = osuNo bacteremia ae Preoperative collection must he labeled “for autologous use only” and used only for this patient 5. Autologous units — segregate from allogeneic units 6. Low yolume collections 8, Use regular blood bags: volume drawn <10.5 mL/kg hody weight for minimum weight (450 +45 mb plas tosting samples) b, 1£ 300-404 mL drawn, label as Red Blood Cells Low Volume (components may not be made from these units) ¢. If < 300 mL, drawn, use proportionately Jess anticoagalant (see below) Volume to draw: (example: 99 Tb, donor) [901b. 5450 mll(standard donation) » 368 ml 110 tb, —_ If donor weight is given in kg, divide donor “weight by 50, then multiply by 450. (ieecaini sem Yh 1 Amount of anti¢ lant to use: a. 368-x 14% = amt of anticoagulant b, 368.x .14 =51.5 = 52 ml of anticoagulant needed Amount of anticoagulant to remove: 63-52 = Ll; remove Lml of ‘anticoagulant from primary bag into attached satellite bag prior to draw 7. Inoperative collections, “salvaged” blood collected during surgery, washed “on- site” and returned to patient daring 4c Provedare | HEWAPHERESIS/APHERESIS COWECTION) . Donor criteria same as for whole blood, 2. Must wait @hoummafter apheresis procedure to donate whole blood 3. FDA limits to 500 mL/ collection (or 600 mL if weight > 175 pounds) 4, Limits number of donor exposures 5. Methods a. By centrifugation — withdrawal of whole blood, removing selected fraction and reinfusion of the remaining components into the donor b. Filtration ~ removal of only plasma through a membrane for normal plasma collection or for therapeutic purposes ¢. Adsorption — removal of only a selected constituent of plasma with reinfusion of plasma after constituent removed Terms Associated with Apheresis ‘SEPARATION & COLLECTION OF: Cyopherss > ails Phamapheess fs plasma Placlertersis fuse platelets Leukaphesesig Leathe fe Meneseewiocte: HEMATOPOIETIC PROGENITOR AND STEM CELL ‘COLLECTION 1. Used to reconstitute bone marrow post chemotherapy/irradiation or to replace abnormal marrow cells with normal marrow eells (congenital immune deficiencies, anemias, malignantdisorders of bone marrow, red cell disorders, etc.) 2, Cells obtained from bone marrow, umbilical cord blood and peripheral biood (apheresis) 3. Allogeneic marrow — HLA-identical match Towers GVHD link or risk; ABO compati- bility not required ‘Donor Blood Processing , TESTS PERFORMED ON DONOR BLOOD Wesk D determination ‘on D negatives Clinically significa aaodies, FP esonot be cepted rom these uni Phelts and eryoprecipitate can (contain minimum plasma volumes Serofogic test such as RPR HBsAg satis ‘a 12 AnticHTLV-11 NAT Testing for + HIV “HCY + West Nile Components/Transfusion Practice LEBLOOD) ~~ ven in cases of severe shock (blood lass > 25% blood volume) needing rhe’s for oxygen and plasma for volume 2, Rarely used due to increased use and avail- ability of components CRED BLOOD CELL SyPacked cots) 1. Red cells with plasma removed by. s 2. Provides same oxygen caning capacity as ‘whole blood with less volume 3. indicates sufficient plasma rem I, i cused 4. L unit saises hemoglobin (Ab) 1g ot hematocrit (het) 3% sea reo ct {7 Plast removed by successive saline ( washes (automated instrument) Fre al gape nok Plein a anaphylactic shock in IgA) deficient patichts with aniiTga (gd is in normal plasma) 3. Expires 24 hours after seal of original wait broken | LEUKocyTE Re cH “1.” 8546 of red cells retained | 2, Final whe count < $x 10* id for other uses (ei. fa Breve transmission) 3, Preparaton by filtration preferred ‘ a, Washing will remove leukocytes also 4. Used primarily for i nonhemol. (ENE) react ally due to presence of cytokines from white celis or alloimmunization to HLA or leukocyte antigens _— wear te PROTEN cet 1. Cells protected from ultra low temnpertates by eryoprtective agent (glycerol) 3. 80% of original red cells must be recovered 4, _Used for storage of autologous units and “rare” units; expires in 10 years HA 1. Prepared by separating cells and plasma by. centrifugation and freezing plasma within 8 hours of collectionyeow Expires 1 year from date of collection when stored at <-18C or colder or 7 years stored at <-65C CRiorecioTAE> <- (eryoprecipitated antihemophilic fc insoluble portion of plasma forms — CRYO” 2. CRYO is separated from thawed FFP and refrozen immediately Oe aire = 4, Also contains Factor VIC and yon Willebrand facior of Factor VIL smiofeeile- and Factor ST 5. 6. D1. Used for: Ane a. Fibrinogen and Factor XU atitangee ~ b. Severe von Willebrand disease (some Factor Vill eonsenttatss contain ¥ Topical fibrin sealant Seldom used for hemophilia because of Factor VIII concentrates whic! have little or no risk of viral infec transmission (use DDAVP for mild hemophilia A) Be FACTOR CONCENTRATES 1, Recombinant products are prepared from plasma Poole plasma popbe processed to purify and concentrate the proteins and inactivate virnses 2, Dives of spesitic factors with winimal volume compared 10. FFP ‘Volume compa 8 Henepiia treat severe with Factor ‘and mild with DDAVP (stimulates endogenous Factor VIII release} 4.C Hemophilia B> treat with Factor IX ~(béiter than Factor TX complex which can lead to thrombosis) 5. labor a Enctor Vill treat with porcine Fa (ow cross-reactivity) camplex (bypass Factor Vill in cascade) . reat with Factor VIII concentrates that have vWF Tid eases treat with DDAVP Eg Gee Coaguiation section Ifyou need rffeshing!) PLaTELets >> oO-BS°C Prepared from whole blood (stored at 20- 24€ prior to processing) ‘a, First a light spin (io remove red cells) followed by heavy centrifugation (to spin down platelets and white cells) b. Supernaient plasma is expressed into another bag and may he frozen (FEP) ¢, Remaining plasina, platelets and white cells = platelet concentratetant 2. Conditions a. For severe thrombocytopenia and platelet dysfunction b. Prophylactic use of platelets when platelet count is low is controversial (threshold depends on patient's risk of bleeding) ©. Contraindieated in TTP and heparin- indueed thrombocytopenia 3, Platelets from donors who are within 36 hours of taking dr (ie, cass iri bthat iinpaie platelet Tantion shoul wot be ised a3 a “single source” (apheresis product or single unit for a newborn) 4. Platelet refractoriness (lack of expocted response) a. Antibodies to HLA class I antigens b. Platelet antibodies or neutrophil’ lymphocyte antibodies 5. Transfusion a. Lunit of platelets should raise platelet ‘SSS ho 10 im average sed adalt He00-O22), De NOT iransfise through a microaggregate filter Only one ABO type/poal; expires 4 hours after pooling 6 QC pit 26:53 end of storage; stored in ‘Vali of plasma necessary to spaintain pli, usual b. fanit ox 3x 10" plateletsplateletpheresis‘in 90% units test c. Store continuously rotating at 20 — 24C (room temp) d. Outdate depends on type of bag used ¢. Individual leukoredueed platelets - 1. Usually obtained by apheresis 2. Decline in use due to new antibiotics, recombinant growth factors and adverse effects from granulocyte transfusion (acute lung injury) 3. Used for neutropenic patients with documented gram negative sepsis who have not responded to antibiotics 4, Can transmit CMV, induce HLA Sumenization, and cause GVHD, if not radia — 5. Stored for 4 uoure, but should be iraiisfused AP 6. ABO-compatible with recipient IRRADIATED BLOOD. AND COMPONENTS 1. Prevents graft (donor lymphocytes) vs. ipient lymphocytes) disease inactivating donor Iymphocytes 2, Recommended for fetus receiving intrauterine transfusion; immuno- suppresssed or immunocompromised patients, recipients of units ete. 3, Minimum of 25 Gy (2500 cGy) 4. RBCs expired on original ontdate o: days aftr ievadiations whichever is first‘MISCELLANEOUS 1, 63 ml of anticoagulanvbag 2. Expiration based on expectation of 75% of transfased cells will be in eiculation 24 hours after transfusion 3, Changes in plasma during storage (1-6C) NH4 t pH b. Expiration of pooled components K+ Na+ + Platelely- 4 hours (open system) cLiyoprecipitate- 4 hours (open syste) — 4, ‘Transporting blood and components a. Red eells kept at 1 -10C Unit of blood cannot be returned and b. Platelets and granulocytes kept at 20- reissued if >10°C (room temperature 15- 24C 30 minutes) or if seal disturbed c. Frozen components kept frozen 5, Expiration of blood/components when zeal is broken (packing cells or pooling components): a. Prodnets stored at 1-6C = 24 hrs b. Produets stored at 20-24C = 4-hours 6. Pooling components: a. [fred cells visible in pooled produet, component plasma antibodies should be compatible with those red cells 2 Component Quality Control iveweneed) COMPONENT STANDARDS STORAGE Ream | aan ——SSC«R Re CHO PORN 4s ‘Seer 24 Has ee “mnowoxpunceas an bwnidGpaan | IGECSEacae ae open | drone fr EDC ew 2s Oe ab Eat ghee | Dpsweassicuihnsthasdymim — | inure <.wreryeoy tranedlGre eee — rtygon, $C, ast | Aye Cpr S01Nnb< hiro coRBOORN natn | eta give SAE EGOS aes ee awl rin i ts oe ETI Gaterin {SE Day aependson Cescton hgh Sear Ds Hed an Sage Tne TR Core Agson Pages Poled open Seat [ie 20.20 wi agacn ales lelogee taueat | C35 1010 pylabin 9 of Unk ae <8 x Same Feels tao 2 Une Te 10 eked PEED [ZR 108 pein of Urs Ts Saxg]20-20%C wih Comsat Agtaon ee sr Naan rae Tne “Fis (Ope Spier EIKO Genco 75% Uns Teed 2 Haus 20.28 Canviagge 7REMEMBER! bisod s strigerator. The plasma goes: straight neraas to the fteezer and is stored t-180 oF colder The'ted cells go across into the bottonh of the refiigeratsr anid ace stored at 1-66. Like plates in your horme-are stored on a sie at room temperature, plat ‘ona rotating shelf at 220. ° Component Therapy WHAT WOULD YoU DOF. 4. AniO negative patient néeds 6 units of platelets. Only O positive platelets are available. What needs to be-considered in a during storage and ‘ranisportition.Théxe-should be at least 1 segment still attached to the donor bag. 8 -Apatient with a ‘vonWillabracie’s disease sulféred minor cuts and: bruises ip a car accident. What should:the ‘physician consider as the treat- ‘metitof choice in this situation? id case of - Patients with mild vonWillebrand’s disease can usually be treated with DDAVP. This drug causes release of vWF. from endothelial cells causing the plasma level of vWF fo increase. More serious cases usualy require Factoc VIL concenttates {some are available with WF) or eryoprecipitate. 4. What is the component of choive for a patient in DIC "with a low fibrinogen level? ‘Ans. - Cryoptecipitate is used because of the high concentiation of fibrivogen.. FFP cait be'used to restore the depleied ‘coagulation factors, but the:volumne. inceded 10 restore fibrinogen is usually to large. noes 5; -A.95 pound female with a: hemoglobin level of 12.5 g/dL-wiants to donate an autologous for an upcoming surgery. Based on this Information; can she donate? If so, how miich can be drawn and how mich anticoagulant should be removed? ‘Atis.” Hee heitioglobin level is ble for autologous donation. ler weightis under 110.Ibs., so you will need to calculate ‘how much blood {o draw and whether any,“ anticoagulant needs to be removed from. ‘the primary bag prior to,colléction. <-S 95 x 450 = 389 mito.draw: HQ. eee As litte as 300 mf can be drawn without reducitig the anticoagulant. Units fretween 300 and 404 must be labeled ‘Red Blood Cells Low Volume Unit.” Components iiiay’ NOT: be nade from this e unit. Mes 2 6" 433 kg potential donor has completed the ® Hoashden sureenina. How much blood ‘Cah be drawn from this individual and how | much anticoagulant must be:removed? SbAns, Volume (0 déaws. 33x 450 5297 ml Ans. 50. Amount of anticoagulant 297 5 14% = 42 al - Amouiit Of anticoagulant to remove: @-42e2m ‘The unit must be labeled “Red Blood Cells Low Volume.”ABO System ANTIGENS OF ABO SYSTEM 1. Chains of sugar moleculés in which specificity is determined by immunodominant sugar 2. Genetic pathway 3. Subgroups of A ay i rrincipal subgroups of A 4 Serological difference based on reactivity wi b. Other subgroups (43, Ax, ete) contain less A antigen and more H antigen RELATIONSHIP OF ABO, H, SE, AND LE 1. Lack of His genetically bhYBombay phemoypey a. hh has no fucose which is needed for attachment of A or B sugars (Bombay therefor 0) b. Anti-l aggluti agglutinate 0 €@lls 2. Se (seeretor) gene allows expression of A, B, H, and Le in saliva 3. Le antigens are plasma antigens which adsorb onto red cells as individual matures a, Individual with Le gene (and NOT Se gene) will have Le on red cells and in saliva (Se not needed for presence of Let in saliva) b. Individual with Hand Le genes will have H and Let on red cells and Le only in saliva (Se not needed for gyevenee of Le® in saliva, bu itis for c. Individual who has the H, Se and Le genes will have H and Le? on red cells (I and Le have added appropriate fucose molecules to precursor ‘substance) and very little Le* is detectable (however in saliva, H, Le and Le® will all be detectable) CELL GROUPING (FORWARD GROUPING) 1. Reagent anti-A and -B are designed so testing is performed at room temperature REAGENT | REAGENT | INTERPRETATION ANTIA ANTEB. + 0 Tp ACB o + Grow Cals + + ‘raup ABE 0 e ‘rap O Gis 2. Unknown cells + antisera = NO agglutination (cells lack antigen to which antisera [reagent antibody] corresponds) 3. Unknown eells + antisera = agglutination (cells possess antigen to which antisera corresponds) “SERUM GROUPING (REVERSE GROUPING) 1. ‘Testing is performed at room temperature with saline suspended known group Ayand B red vells; optimum reactivity of serum anti-A and -B is 4C [oar] 8 Jantisooy | ntenpacrarion CELL | CELL | IN SERUM opt Tae 7 + 0 Aetiod, o 0 None Ae + Problems with Red Cells: + Rov, ale Wash Call 2. Unknown serum + reagent red cells = NO agglutination (serum lacks antibody to antigen on red cell) b, Unknown serum + reagent red cell agglutination (serum has antibody antigen on red ceils) Discrepancies in ABO‘ Grouping Resolution Techniques Regen ith Saine Washed als 1 inet Cel Types Bama or B Tans nO Gols Chace Taino Histon 1 Subgaps ample A howto any) Tesh ay fo gone + ual Gesgjes BamalsFonBay Tawi Asi Bow Randy de Age as wl nt Agta wh A Baby Se it Asginat BC al ap 0 Seeing C8) + Dsene Roce — Barple Leki Bacon aguind Phen (Check Part gpa 1D Bnd Seo Pl Ren Problems with Serum: Rouse Doe hoe Sern Fos Bae Wades’ or ail yor) + Room Tenet Col Readog Anode LM NPT or de rar ina 2 or A asl eg mh tal Coren rigs on Reve Cals Sane Replace "Wi Cason 6 hl Pw olan Tengen +A — Hy Heo Peon fins Gooner ubamn(bey Pscin Has No Rethed Opti evel Mig oer "CR Pa ge in” Cod Peay Thane Sm Ani e n8 slept wl Age ith Cll Ging 1+ Corps mane Sse — ape: perarmegeinans (hac Palen Digan ink Cali Panel Ree aos) PATIENT SERUM TESTED WITH INTERPRETATION wecers | ocets | o cord Col gravanabey Avec + + a + Anti Unexpected antibody racing t tor + + ord 0 caer tempezatines fat 4 - NP and levis abodes + D 0 D Aner |‘SALINE REPLACEMENT. Principle: Saline replacement can differentiaté rouloaux from agatntination. Ronleaux is-typically deseribed'as having ‘a “stack of coins” appearance when observed in the test mixture is replaced. with ANTIGENS L D.C Eoe 2. Presence of D is only antigen routinely tested D GROUPING 1. Dis most immunogenic of all blood group “antigens 2. D grouping (Rh type) is based on hen tested with anti-D 3. Weak D a, D reactive at antiglobulin phase only b. Weak D is considered D positive ¢. Testing for weak D required on donors and OB patients 4, Monoclonal / Polyclonal Anti-D a, Separate D control not necessary b. Control is a negative reaction with anti-A or -B in ABO cell grouping {patient A and B cells not spontaneously agglutinating) swith a microscope, When the n Rh System €. Ifpatient is AB positive, must mse a 6-8% albumin control, autocontrel or par 5. D control (used with high protein anti-D lust be negative for D negative grouping to he valid; if positive, repeat with another type of anti-D (monoclonal, chemically modified or saline) b, Must contain same media as anti-D reagent without the anti-D (use same manufacturer’ control) ce. Ensures agglutination with anti-D reagent is due to presence of D antigen and NOT due to proteins in reagent or agglutination of in vivo antibody coated eells (positive DAT) d. Most common cause of a positive D control is a positive DAT. UNUSUAL PHENOTYPES: 1. Ith null-no D, C, B, ¢ or e antigens; cells have associated hemolytic: anemia since Rh structure is imegral part of rhe membrane 2. Deleted eells (-D-) - missing one or more of normal Rh alleles RH ANTIBODIES 1. IgG clinically significant 2, May agglutinate at 37C as well as ANG 3. AnticC, -c, -E, -e react stronger with cenzyme-treated cells,__ TeM Antibodies ) REMEMBER! Lewis conte, H ontlM,-N ont) antec, Leb Other Blood Group Systems IgG Antibodies: ‘ont’ tHe anteG, +6. any, Pyts » onthe, 6 tlhe, JRE. 2, Antibodies 1. Plasma antigens that adsorb onte RBCs; a. AntiP, not alleles * & Anii-P; (NOT ani:-P) can be 2. Noton cord celle neutralized to reveal other ‘bedi clinically significant 3 Antibodies Levis ant dlloantibodies (P; substance in . Do NOT cause HDN (Lewis antigens ore goto otal cel ard Lens be Ame etd evs fini) antibodies uswally IgM) ‘ei b. eM antibody eguenty the specificity of the Can be hemolytic Usually only seen in Le(a-b-) persons Often seen in pregnant women. who may temporarily hecome Le(a-b-) me 1. absent or weak. on cord cells 2. ieonverts to 1 as infant matures due to branching of carhohydrate chains; not alleles a. Infants -i positive, | negative b. Adults - I positive, i negative 3. Antibodies a, Anti-l cold antibody Reacts with all adult cells (exeopt rare iadul) lay mask clinically significant alloantibody Remove anti- to detect underlying antibodies by: © an antoadsorption (if not recently transfused) or allogeneic adsorption t= RESt adsorption ®F prewarming serum and using IgG AHG instead of polyspecific + o 1. P, antigen strength deteriorates upon storage KELL Paroxysmal & Reet with ll Bar y poste Mand N are codominant alleles { Antibodies a. Apti-M and -N Usually cold IgM; no HDN & Ofien show dosage (property whereby antibody reacts strongest with cells having a homozygous ‘expression of antigen as opposed to heterozygous cells) Will NOT react with enzyme- treated cells (Mand N antigens are destroyed by enzymes) Anti-M © Many examples are IgG and can cause HDN o May ification of serum to identif Anti. and anti-s - IgG. Anti-U - I, % Formed by black individuals who lack S, sand U * Be K and k (cellano) are codominant alleles a. (next to D) b¢. Antigens inactivated with 2-ME, DTS, or AET 2. Antibodies - Fe DD 1. gk# and Jk? are codominant alleles 2. Antibodies a. 1G B, React STRONGER with enzyme treated cells ¢. Titers rise and fall rapidly GL. Associated with delayed transfusion reactions B DUFFY 1, Fy® and Fy? are codominant alleles 2. 68% African Americans are Fy(0-b-) b. Antigens destroyed by enzymes Antigen typing ~ Fy(atb-) pe % whites ~ homozygous for Fya (PyaFya) & blacks ~ probably heterozygous for Fya (Fyal'y-) ~ dosage problem atta o- alana 2, Antibodies a. laG b. Weak examples may show dosage Negative ‘reaction with enzyme treat ed Parentage Testing ‘Agout PATERNITY TESTING: 8s Jou may have probleine that siclude ABO andlor D grouping. ln pabertiny testing, there ie 4 : ‘chain of zaniple suatody that. must be adhered tom legal eases %, Ethie Y ‘Vielecular techriqiée are. replacing serological methods: DIRECT AND INDIRECT TESTING 1. RBC blood groups with codozsinant alleles can be used for parentage testing along with TILA system and DNA analysis 2. Maternity is assumed 3. Direct exelusion — marker present in child, absent from father and mother Direct Example: ANTEK. Maer + Baby Alleged Father + a | 0 | 4, Indivect exclusion ~ child lacks a marker that the alleged father must transinit Indirect Example: eg Fae we 0 + wy o + Question? Is the alleged father, tested below. exchided? Teetings Father: KK - Mother: kk “Baby: Kk Answer: © fhe alleged faher-ie NOT excluded’4 1. Commercially prepared group O red cells with a specifie distribution of blood group 2. Antibody Screening and Identification ‘SCREENING CELLS AND PANELS antigens (screening cells contain 2-3 different cells: panels vary from ‘Approximately 10 - 20 different cells) ided to the ells Patient serum a, Serum-cell mixture is tested at IAT. a Antibody attaches to corresponding antigen on red cells at 37C (may see hemolysis) 1b. Excess serupvantibody removed by saline washes (failure to adequately wash cells may cause a false negative ~human globulins, i.e., antibodies, proteins, ote., not wash away will neutralize the AHG) ©. Antiglobulin is added and will bind to antibody on the cells 4. Positive reaction js indicated by agglutination or ¥ in size of button due to hemolysis at 37C e. Check cells (Ig sensitized cells) are added; these shonid be positive indicating ANG was actually added in the final step and was not neutralized Notes a. Enzyme treated (ficin, papain, trypsin and bromelin} cells are available to compare with panel reeults of untreated cells D. Panel results + Compare enzyme-treated and untreated cells - enhanced: Kidd, Lewis andi ual destroys M, NS. Fa, a Jo antibody activity: € _} Segregation; if ag-ab reaction occurs during ager Agglutination does notreverse A antibody: intake; removes water which 4 antibody ‘Concerwation which promotes antibody uptake: various temperatures, with different enbancement media, fu with a antiglobulin reagent (indirect sntebutn test (14T]) Patient serum may also be tested against his own cells (auzocontrol) to determine the presence of an autoantibody Uptake if. ifeubalion tine. sitive surface charie ahh 1 feactivity of Rh, Kidd and warm and cold autoantibodies; jh antigens ¢ incbbation ime: causes reversible cell ion, this 4 and seme RA NOTD): lestroyed: Duffy, M,N, and S$ Dosage - Rh (other than D), M, N, Kidd (5k* and Jk) and Duffy (Py* and Fy) & Strength of reaction may separate multiple antibodies (Panel with 1+ and 3+ reaetions may mean two different antibodies) % Phase of reactivity Antibody Characteristics) % Awocontrol - if positive, may indicate a delayed transfusion reaction; if positive along with all panel cells, autoantibody indicated Prewarmed technique - After proving | no clinically significant antibodies tho present, eliminate reactions dae to cold antibodies % Warm serum and cells separately 10 37°C before mixing together % Wash with warm saline prior to further testing (crossmatch, panel, ete.) 5. Determine patient antigen status"REMEMBER! P.B. Ficin The Ss (variable when sing "in use treated els) fy fea example ofthe antibody may show disap) sane is ait Antik ‘Antik ABN Asie Dvni-Lea__AniLe we TRSATbomin ie Po Ca AntbiesReacting ata Higher Thera Rane ra Ani Asc Are Ante Arlbe Akal “ntiDuity anvekild Anti some) ight Complement Binding {using Polyspecicl | Antibodies; Most Common: Anti Aes Ate ——e16 REMEMBER! Antibody Temperatures. of Reactivity - Lefs.go on a joumey to hélp you associate blood rOUp antibodies and their temperatures of reactivity: oe First, we are going to a place thet is very-cold, in your ming, "$68 Yourself putting on a fur Parka, gloves and some very warm _ boots. Lot's get in-our kayak and start Paddling: We paduiie and the biggest brown bag of MaNs you have. vei con. Ths tells Us the P,, M & N antibodies also react in the From our Journey ta the cold, we kriow that Lewis, Hl, Py, Mi _ ARG.N antibodies ‘best react at 4°C, : Jo loath about antbodies that react at 37°C, we must travel to a different part of the world. Let's get in our kayak andpaddie'to |) tur parka’ oloves and boots. “It Finally, we teach land and -982¢ at the jungle that lays before | we See Jungle Jim reading a book about the CVS hé sees in.the jungle. This tells us Rh antibodies are Seen at 87 é : oo “ dungle Jim tous: us there are other things in this en that cagnot be seer unless you look very closely. He takes us to ‘We hack our way through vines and” a clearing. In the middle of the clearing, ‘sty tiny hut. ‘The big huts have the. - them. “Ong belongs io the Kelis, dnd the other 16 Dutlys, and the litle one must be for their Kidtays, So. from our visit with Jungls vim, we know the Rh antibodies Gan -be seen directly at 37°C, but to: see the Kel, Duffy, and Kidd Feactions, we must look further’ and we do this through antigiobulin - testing. DES Quick Review — now state the optimum temperature at which the rad Le8, Congratulations! The story worka! ‘Take 8 minntes and repeat the story out lod! Okay, following anitbodies react: P,, Kell, Jk, M