CENTRAL TEXAS COLLEGE 2431-IMMUNOHEMATOLOGY Antibody Identification I. INTRODUCTION A. Antibody identification is necessary to: 1. 2. 3. 4.

Assess need to select Ag NEG blood for transfusion Predict and monitor potential cases of HDN Discern allo from auto antibodies Determine clinical significance of positives obtained during antibody screening a. Clinically significant Ab is an Ab capable of decreasing survival of transfused RBCs or has been documented to cause HDN o b. Based on premise that an Ab is clinically significant when it is active at 37 C or detectable by IAT o c. Note: there are Abs active in vitro at 37 C or detectable by IAT that are not clinically significant (ex. Sid)

II.

ANTIBODY IDENTIFICATION PANEL A. Blood samples 1. 2. B. 10 ml clotted blood - usually sufficient for simple Ab ID More may be required if multiple Abs are present

Medical history (Info required for Ab ID) 1. 2. 3. 4. Patient's clinical diagnosis (immune disorders = autoAbs) # of pregnancies (could induce immune Abs) Transfusion history (could induce immune Abs) Recent drug therapy (could cause POS DAT = POS autocontrol)

C.

Reagent RBC Panel 1. Panel cells a. Set of 8 (or more, depending on manufacturer) group O reagent RBCs of known phenotype Should ID clinically significant Abs most frequently encountered Made up of specific RBC samples where some lack & some carry most of clinically significant Ags in very specific sequence to give separate patterns. For example, all of Kell POS cells should not be only ones that are E POS.

b. c.

2.

Antigram a. b. c. d. e. Accompanies each panel and is specific for each lot Lists antigenic makeup of each panel cell Serves as a worksheet to record results Panel cells with rare phenotypes are usually indicated; such as low or high frequency Ags Always match lot# on panel with lot# of antigram

Rh #

Cw

Fya

Fyb

Jka

Jkb

Lea

Leb

r'r R1w

R1

R2

r"r

9 1 0

Ro

+ denotes Ag present; O denotes absence of Ag D. Methodology 1. Patient specimen found to have a positive Ab screen is tested against set of panel cell suspensions at all phases (IS, 37C, & AHG) & reactions are recorded on antigram worksheet. Identity of Abs are determined according to which panel cells have a POS reaction (means the cells possess the Ag the patient Ab is against) Example: If patient has anti-K a. Only panel cells with K Ag will be POS b. All panel cells lacking K Ag will be NEG 3. An autocontrol is tested simultaneously with panel cell testing if not previously tested with Ab screen

2.

E.

Autocontrol 1. Patient's serum is tested against autologous RBCs to detect autoAb a. POS autocontrol may necessitate additional testing, such as elution b. Adsorption techniques may be necessary to establish that autoAbs are not masking presence of alloAbs

Patterns of Serum Reactivity of Allo- & Auto- Antibodies Reagent Red Cells + O + NOTE: Autologous Red Cells O + + Interpretation Alloantibody Autoantibody AutoAb or autoAb & alloAb

Alloantibodies in recently transfused patients may mimic autoantibodies.

III.

INTERPRETING ANTIBODY PANEL RESULTS A. Evaluation of Panel Results 1. Is autocontrol POS or NEG? a. NEG = alloAb b. POS = autoAb (may mask alloAbs) What test phase did the reactions occur? a. Immediate Spin (IS) = IgM antibody b. Albumin / 37C = IgG antibody c. AHG = IgG antibody Is there hemolysis present? a. Indicates a complement binding Ab (ABO,P 1, Lewis, Kidd) What the strengths of the reactions? a. Various strengths may indicate multiple Abs are present, or a Ab showing doseage What Abs can be excluded as the possible Ab? a. Cells that gave NEG reactions at all phases of testing should be ruled out b. c. d. Ags contained in cells are indicated by " " under antigen listing along top of the antigram Cross out Ags corresponding to " " on those cells in which NEG reactions were obtained in all phases of patient testing Abs that would have reacted with these Ags are therefore eliminated as the possible Ab

2.

3.

4.

5.

6.

Does reaction pattern of patient match any of reactions patterns of remaining specificities? a. Is there an "EXACT FIT" or matching reaction pattern to your patient's reaction pattern b. "EXACT FIT" usually indicates the presence of a single alloAb Is patient lacking the Ag corresponding to the suspected Ab? (Confirmation testing)

7.

a.

b.

Individuals do not make alloAbs to Ags they possess (1) Confirmatory test - Test patient's cells for corresponding Ag (2) If NEG for Ag = confirmation of Ab (3) If POS for Ag = misidentification of Ab For recently transfused patients, use pretransfusion sample if available. If not, autologous separation techniques will have to be used

8.

Is there enough statistical evidence to prove suspected Ab? (Confirmation testing) a. b. c. Must ensure the matched pattern of reactivity is not the result of chance Rule of 3: Test patient's serum with at least 3 Ag POS & 3 Ag NEG reagent cells Patient's serum should react as follows: Serum (with suspected X Ab) + 3 X Ag POS cells = POS Serum (with suspected X Ab) + 3 X Ag NEG cells = NEG Indicates a P value = 0.05 or less (< 5% probability that results are due to chance) Indicates there is sufficient evidence to prove suspected Ab is a valid determination

d. e.

B.

Single alloantibodies 1. Single alloantibodies - usually yield clear cut POS & NEG reactions with panel RBCs ("EXACT FIT" PATTERN) a. Example: Compare to Antigram on Page 2
37o O O O O O O O O O O

Sample # 1 2 3 4 5 6 7 8 9 10

IS O O O O O O O O O O

AHG O O O 3+ 3+ O O O O O

Reactions observed on RBC panel (1) (2) (3) b. Cells 4 & 5 were agglutinated by patient's serum Anti-E is likely to be present because both E POS cells were agglutinated by serum & all of E NEG cells were not Requires an additional E pos cell to be tested to satisfy Rule of Three

Reaction should be in a test phase that is characteristic of that particular Ab Ex: Anti-E reacts at 37 C or IAT phase as did patient's Ab
o

2.

Single alloantibodies ID may not be simple. If this happens some important considerations are:

a.

Ab reacting in an unexpected test phase (1) (2) Ex: Most S Abs react only at IAT phase, however they will sometimes react in saline phase While reaction phase is suggestive of Ab specificity, it is important to remember that exceptions will occur

b.

Variation in Ag expression (1) (2) (2) Different RBC samples carrying corresponding Ag may react differently May be due to variation of expression of Ag in population (ex. P1) May be due dosage effect (a) Some Abs prefer to react with RBCs that carry a double dose of Ag (homozygous) (b) Reagent RBCs from heterozygotes carry a single dose of Ag & may be nonreactive Some Ags deteriorate more rapidly than others during storage Enhancement techniques often resolve problems associated with weak Ag expression

(3) (4)

c.

When no discernible specificity on reagent cell panel consider: (1) (2) Incorrect phenotyping of reagent RBCs by manufacturer Serum may react with Ag not listed on Ag profile (a) If reactions are not clear cut, additional phenotyping information may be required from manufacturer (b) If antisera is available, patient's & reagent RBCs may be phenotyped for additional Ags (c) If discrepancy remains unresolved, specimen should be sent to a reference lab

d.

Exclusion of additional Abs Even if the serum displays a reaction pattern of a single Ab, it is important to remember that there may be other Abs present (1) (2) Tests with additional reagent RBCs should be undertaken known as a Select Cell Panel Knowing the phenotype of the patient's RBCs may be helpful in determining the possibilities of another Ab being present

e.

Special considerations with Rh Abs: If anti-E is ID'd in serum of a transfusion candidate, presence of anti-c should be considered: (1) (2) (3) (4) Determine Rh phenotype of patient If patient's RBCs lack both c & E Ags, anti-E will most likely be accompanied by anti-c Anti-c may be less reactive & more sensitive techniques may be necessary to demonstrate its presence Even if anti-c is not detected it is best to select blood that is NEG for both c & E Ags because anti-c is a common cause of delayed HTRs

C.

Multiple Abs 1. Panel interpretation may be difficult when 2 or more Abs are present

2.

Suspect multiple Abs when: a. b. c. d. Reactivities do not match a single Ab Variations in reaction strengths of particular RBCs cannot be explained by dosage Different RBCs react at different test phases Unexpected reactions are obtained when attempting to confirm specificity of a suspected single Ab

3.

To determine IDs of multiple Abs, perform the following evaluations: a. b. c. d. Evaluate reactions of autocontrol, same as previously discussed Eliminate from consideration Ags present on nonreactive cells Perform specific phenotyping tests on the patient's RBCs & eliminate from consideration the Abs to any positive Ag test results Perform enzymes treated panel. (1) Examine panel cells that are reactive when untreated and nonreactive when treated with enzymes

(2)
e.

Interpret changes to panel results

Perform select cell panel to separate Ags occurring on same cells of original panel Examine reactions observed at each test phase, keeping in mind what Abs react best in each phase (1) Reaction phase may help determine specificity (2) Strength of reactions at different phases can be characteristic of some Abs showing dosage Test enough RBC samples of the appropriate phenotype to be 95% confident of the Ab specificity (Rule of Three) Confirm that patient's RBCs lack the Ags for the suspected Abs

f.

g. h. D.

Abs to high incidence Ags 1. 2. 3. 4. High incidence Ag - present on RBCs of 99.9% or > of the general population AlloAbs to high incidence Ags should be suspected when all reagent RBCs are reactive and autocontrol is NEG Best source of donor blood = fellow siblings Helpful in ID of Ab if ethnic origin of patient is known Ex. Most U negative individuals are of African decent

E.

Abs to low incidence Ags 1. 2. 3. 4. 5. 6. Suspected when a single donor unit or reagent RBC sample is reactive with patient's serum and Ab screen was negative Reagent RBC sample or donor sample that reacted with the patient's serum may be Ag tested if rare antisera is available Transfusion of the patient should not be delayed Abs to low incidence Ags often accompany multiple antibodies If available do a Select Cell Panel of low incidence Ags to ID The experience of an immunohematology reference laboratory is needed to confirm the specificity of these Abs (will carry more cells with rare Ag and the antisera)

F.

Procedures for detection of alloantibodies in presence of Cold reactive autoAbs 1. Prewarmed techniques - reagent RBCs & test serum are incubated at 37 C before they are mixed
o

2. 3. 4.

Anti-IgG AHG - eliminates detection of complement components bound to RBCs by cold reacting autoAbs which permits detection of alloAbs. Cold autoadsorption - uses autologous RBCs at cold temp.s to remove autoAb, but not alloAb (only if not recently transfused) Heterologous adsorption (RESt) - uses rabbit RBCs to remove cold autoAb (I, H, IH), but not alloAb

G.

Other POS serological reactions 1. Abs to variety of drugs or additives a. b. Abs to preservatives used in the manufacturing of reagent RBCs can cause agglutination of these cells Reactions rarely occur if reagent cells are washed with saline prior to testing

IV. SPECIAL SEROLOGICAL TECHNIQUES A. Enhancement techniques Ab enhancement techniques are used when weak reactions fail to indicate Ab specificity, or when Ab is suspected but cannot be demonstrated. Use of following techniques may be useful in ID of Abs 1. 2. 3. Alteration of serum pH - Some Abs react more readily by decreasing the pH of reaction medium to a pH of 6.5 Changing potentiator: Albumin, LISS, or PEG procedures - used to increase the Ab uptake of the RBCs Temperature reduction - specificity of some alloAbs that react at RT may only be o demonstrated at temp.s below 22 C (RT panel: set up w/o potentiator and incubate at lower temp) Increased incubation time - used to increase Ab Ag association when weak reactions are observed by normal methods (must stay within limits of potentiator) Enzyme techniques - used to enhance the reactivity of Rh and complement binding Abs but destroy reactivity of MN & Duffy

4. 5.

B.

Enzyme Techniques 1. 2. Principle: Proteolytic enzymes modify RBC Ags in ways that enhance reactivity of some Ag-Ab systems & abolish antigenic configurations of others Enzyme modification also alters physical properties of the cell suspension & can cause spontaneous aggregation of RBCs, must be interpreted carefully

CAUTION:

Enzyme method should never be only technique used for Ab detection because antigenic a b determinants (ex. M, N, S, Fy , & Fy ) are usually destroyed, & Abs recognizing these Ags would not be detected. 3. Enzymes used in blood bank a. Bromelin--pineapple stems b. Papain--papaya plant c. Ficin--figs d. Trypsin--intestinal secretions Techniques of use: a. Two-stage - original (trypsin & ficin) b. One-stage - Low's modification (bromelin & papain) POS, NEG, & AUTO controls must be used regardless of technique

4.

5.

6.

Normal serum should be used as NEG control to check on over-treatment, which may lead to non-specific agglut. b. POS control of dilute anti-D should be used to demonstrate adequate treatment. Advantages of enzymes a. Enhances some reactions b. Useful in detecting low concentrations of Ab

a.

C.

Thiol reagents (such as dithiothreitol (DTT)) 1. Principle: cleave intersubunit disulfide bonds of IgM Abs 2. Applications of thiol reagents: a. Determining Ig class of an Ab b. Dissociating RBC agglutinates caused by IgM Abs c. ID specificities in a mixture of IgM & IgG Abs when an IgM Ab is masking presence of an IgG Ab

D.

Neutralization / Inhibition tests 1. Soluble blood group Ags a. Present in such body fluids as saliva, urine & plasma b. May be used to inhibit reaction of an Ab to an RBC sample 2. Soluble Ag Sources a. Lewis substance - saliva b. P 1 substance - hydatid cyst fluid a c. Sd (Sid) substance - many fluids - most abundant source is urine d. Chido and Rodgers Ags (determinants of C4)- pooled serum / plasma 3. Substances may be used in Ab ID studies: a. Confirm specificity of an Ab by inhibition b. When > 1 Ab is suspected, can neutralize 1 Ab in order to detect other Ab Inactivation of Kell system Ags 1. Most Kell Abs do not react with RBCs treated with 2-aminoethylisothiouronium bromide (AET) Adsorption and elution 1. Adsorption - process of removing an Ab of a particular specificity from a serum by use of Ag POS cells a. WARM - used to remove warm auto Abs b. Can be used to separate 2 Abs, either to prove an Ab (ex. Anti-G) or remove Ab to determine others Elution - process of removing a bound Ab from surface of a RBC. Used to ID specificity of an Ab that is coating patient's RBCs in vivo & detected by DAT

E.

F.

2.

G.

Antibody Titration (Quantification) 1. Principle: Perform serial 2-fold dilutions against RBCs POS for the corresponding Ag a. Ab titer = reciprocal of highest serum dilution showing macroscopic (1+) agglutination Purpose a. Prenatal studies: To monitor quantity of Ab in pregnancies which are at risk for HDN and are used to assess need to perform an amniocentesis b. Determining HTLA Abs: High titer low avidity Abs are weakly reactive undiluted, but unlike other weak Abs they react at high dilutions

2.

ANNEX A ANTIGEN- ANTIBODY CHARACTERISTIC CHART ANTIGENS Antigen System Antigen Frequency W B 85% 70% 30% 80% 92% 34% 21% 97% Demonstrates Dosage no yes yes yes Modified by Enzymes yes yes yes yes

D C E Rh c

e 98% 99% yes yes ---------------------------------------------------------------------------------------------------K 9% very rare occ no Kell k 99.8% 100% no no ---------------------------------------------------------------------------------------------------66% 10% yes decreased Fya Duffy Fyb 83% 23% yes decreased ---------------------------------------------------------------------------------------------------Jka 77% 91% yes yes Kidd Jkb 72% 43% yes yes ---------------------------------------------------------------------------------------------------Lea 22% 23% no yes Lewis Leb 72% 55% no yes ---------------------------------------------------------------------------------------------------P1 79% 94% varies yes P P 100% 100% no yes ---------------------------------------------------------------------------------------------------M 78% 70% yes decreased N MNS S s 55% 31% 89% 97% yes yes no decreased 72% 74% yes decreased

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ANTIGEN-ANTIBODY CHARACTERISTIC CHART ANTIBODY

Serology saline/AHG D C Rh E c occ / yes no/ yes occ/ yes no / yes

Complement binding no no no no

Globulin class IgM IgG occ occ occ occ yes yes yes yes

Optimum Significance Temp HTR HDN warm warm warm warm yes yes yes yes yes yes yes yes

e no/yes no occ yes warm yes yes ----------------------------------------------------------------------------------------------------------------------------K occ /yes some occ yes warm yes yes Kell k no/yes no occ yes warm yes yes ---------------------------------------------------------------------------------------------------------------------------occ/ yes some occ yes warm yes yes Fya Duffy Fyb no /yes some occ yes warm yes yes ---------------------------------------------------------------------------------------------------------------------------Jka occ / yes yes no yes warm yes yes Kidd Jkb occ / yes yes no yes warm yes yes --------------------------------------------------------------------------------------------------------------------------Lea yes / some yes yes v. occ cold yes no Lewis Leb yes/ some yes yes no cold no no -------------------------------------------------------------------------------------------------------------------------P1 yes/ no some yes rare cold yes no P p yes/ ? some yes yes cold no rare -----------------------------------------------------------------------------------------------------------------------M yes/ some no yes occ cold yes yes N MNS S s yes/ no Yes/ some few/ yes no some occ yes occ occ occ yes yes cold cold warm yes yes yes rare yes yes

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