Blood Bank: ABS and AB ID

IAT

ABS involves detection of broad range, unexpected allo- or auto- Abs in patient serum using the (——) procedure

• Column Agglutination Technology (CAT) (gel)

• Solid Phase (Protein A Technologies) (microtiter plates)

What are the automated antibody detection tests? (2)

• Manual: Solid button

• Gel: Layer at the top of gel

• Solid: Complete diffusion

What do 4+ reactions look like in the different Ab detection tests? (3)

• Transfusion anticipated

• Prenatal testing of pregnant women

When are ABS used? (2)

Pooled screening cells

For donors, ABS is performed using (——) reagent

Rh

In the 2nd phase of the manual test, the sample is incubated at 37°C, which usually reacts with which Ag?

• Cannot detect low titer (Kidd)

• Cannot detect low frequency

• Will not detect ABO Abs (reagents are O)

What are the limitations of an ABS (3)

• Every positive ABS

• When screen suggests new Ab

  • Redo ID for every xmat

• Confirm previous Ab (per facility SOP)

When are AB IDs done? (3)

• Donor selection for pt w/ unexpected Abs

• Resolve HDFN

• Resolve transfusion rxn

• Resolve AIHA

What are the applications/uses of an AB ID? (4)

• RhIG administered

• Antibiotic induced warm auto-Ab

• Anti-I or anti-i (consider age)

• AIHA, PCH → Donath Landsteiner

• New Ab or delayed HTR

A medical history is helpful in an Ab ID and provides information about potential Abs a pt may have. Consider the following:

• A pregnant woman with (——) may have passive anti-D

• Bacterial infection may cause (——)

• Viral illness may cause (——)

• Medical conditions like (—2—)

• Recent transfusion can cause (—2—)

(Avoid cold Ab)

• Monoclonal IgG AHG

• Skip IS

(After cold Ab ID’ed)

• Prewarm technique

• Use appropriate enhancement media

• Auto-adsorption if necessary

To avoid cold interference (IgMs at RT) when investigating clinically significant Abs, the following methods may be used (5)

1. Prewarm all materials a 37°C for 10 minutes

2. AHG: (do not remove from incubator) add warmed saline to each tube, centrifuge, and wash 3x

3. Add IgG AHG, centrifuge, read

4. Add check cells to all negative rxns

Describe the prewarm technique procedure

• Ficin (figs)

• Bromelin (pineapple)

• Papain (papaya)

Common proteolytic enzymes used in blood bank (3)

• One step: enzyme added directly to patient serum/panel cell mixture

• Two step (more common): Pretreat panel cells with enzyme, incubate, wash, then add pt serum

Describe the two different enzyme procedures in blood bank

Sialic acid

Enzymes remove the (——) from the RBC membrane, thus “destroying” it and allowing other Ags to be “enhanced”

• MN

• Ss

• Duffy

Which Ags are destroyed by enzymes?

• Rh

• Kidd

• P

• I

• Lewis

Which Ags are enhanced by enzymes?

• Anti-M and anti-P1

• ≤ 22°C

• Auto-control (cold reactive auto Ab)

Temperature reduction helps allo Abs like (—2—) react better; specificity may become apparent at (——); because testing occurs at lower temperatures, it is especially important to use (——)

Adult (I+) and cord (i+) cells at 4°C

• anti-I = (+) rxn w/ adult, (-) rxn w/cord

(temperature reduction) Anti-I specificity is confirmed by testing pt serum w/…

• 4 or more drops of serum

• Increase 37°C incubation

• Additional washes

• Cannot use enhancement media

Increasing the SC ratio increases the amount of Ab in the test system, which may help increase the strength of reactivity of Abs present in low concentrations. How does testing procedure change when increasing SC ratio? (4)

30-60 minutes (do not exceed enhancement media incubation recommendation)

To enhance reactivity, incubation time may be extended to…

• pH=6.5

• 0.2N HCl

• anti-M

Decreasing pH may enhance Ab reactivity, where pH is decreased to (——) by the addition of (——) to the patient serum; weak reactions with (——) are greatly enhanced by this procedure

• Spin and read at 37

• AHG

When using PEG media, ensure that you do NOT (——), instead go directly to (——)

• Dithiothreitol

• 2-mercaptoethanol (2-ME)

• 2-aminoethylisothiouronium bromide (AET)

Strong IgM class Abs may have high thermal amplitude and may mask other Abs. To help differentiate between IgG Abs and IgM Abs, three thiol reagents can be used:

• Cleaves inter-subunit disulfide bonds of IgM

• Kell Ags (except Kx)

Thiol reagents work by (——), but it will also denature all (——)

• Determine Ab class

• Dissociate agglutinates caused by IgM (cold reactive auto Abs)

• ID specificity of masked IgGs

• Adsorption procedures (in combination with enzymes)

Application of DTT and 2-ME (thio reagents) (4)

• Proteolytic enzymes and DTT

• Kell, MNS, Duffy

• Kx

• Frees auto-Ab from pt cell to be adsorbed to another RBC

ZZAP reagent contains a combination of (—2—), and denatures (—3—) with (——) being an exception; this reagent works by (——)`

Neutralization

Technique to confirm Ab identification where commercial substances bind t the Abs in the patient serum, causing them to show no reaction when tested with the corresponding Ag in a panel the use of this technique is to remove specific cold IgM Abs that could mask other clinically significant Abs

• Hydatid cyst fluid → P₁ substance

• Plasma or saliva → Le^a and Le^b (soluble substances)

• Breast milk → I substance

• Human/guinea pig urine → Sd^a substance

• Ch or Rg substance (plasma/serum from (+) individuals)

Common substances used for neutralization technique (5)

two-fold serial dilutions of pt serum against RBC Ag samples

Describe the process of titrating Abs

Reciprocal of the highest dilution that had agglutination

How are Ab titrations results expressed?

• Prenatal studies (most common)

• Ab ID

• HTLA (high titer, low avidity) Abs

3 applications for Ab titration

HTLA Abs

Abs that react very weakly in undiluted serum state at the AHG test phase, but unlike most weak reactive Abs, react at a high dilution (i.e., 1 in 2000)

• Ch

• Rg

• Cs

• Yk

• Kna

• JMH

HTLA Abs include Abs against… (6)

Auto adsorption

Auto Abs are removed using pt’s RBCs so allo-Abs can be identified. After the pt’s serum and RBCs are incubated, the adsorbed serum is tested with panel cells to ID any allo Ab

• R1R1

• R2R2

• rr

• K neg

• Jka neg

• Jkb neg

`If a pt was recently transfused, auto adsorption cannot be done. Instead, perform allogenic adsorption where the patient’s serum is incubated with (—6—) in separate tubes. The resulting serum is then tested with panel cells to determine specificity

Elution

This technique frees Abs that are coating RBCs so that the Ab can be identified in a panel

• ID Ab in baby’s RBC (HDFN)

• ID Ab in acute/delayed HTR

• Investigate (+) DAT

• Concentration and purification of Abs

• Detection of weakly expressed Abs (subgroups)

• ID of multiple Abs

• Preparation of Ab-free RBCs for phenotyping or autoadsorption

Use of elution techniques (7)

• Glycine acid

• Lower pH causing Ab to dissociate

• rapid, sensitive, commercially available

(Elution methods) Acid elution characteristics (method, Ab removal, benefits (3))

• Methylene chloride/chloroform or ether

• Organic solvent dissolves lipid layer of RBC

• Sensitive, inexpensive

• Hazardous/carcinogenic

(Elution methods) organic solvent characteristics (method, Ab removal, benefits (2), limitations`)

• Freeze-thaw → lyses cells (physical)

• Heat → conformational change (physical)

• Rapid, effective for ABO, inexpensive

• Not sensitive for Abs other than ABO

(Elution methods) heat and freeze-thaw characteristics (Ab removal, benefits (3), limitations)

Chloroquine diphosphate and glycine acid EDTA

(—2—) reagents can dissociate IgG Ab from the RBC without damaging the RBC Ags; they are very useful if the RBC needs to be Ag typed, but may not remove auto Abs completely from DAT positive cells (partial removal may be enough to type or use for autoadsorption of warm auto Abs)

Known commercial antiserum or patient’s serum

If a patient has an allo Ab, then pt needs RBC units that are negative for that corresponding allo Ab; select random donor ABO/Rh compatible units and screen for absence of Ag of interest using (—2—)

(# units needed) / (freq of negative)

• If multiple Abs, multiply freq of negs together

How to calculate the number of random units needed to find a compatible donor

AHG crossmatch

If the Ab is clinically significant, Ag negative donors must be found and crossmatched for the patient and (——) must be performed

Polyagglutination

Rare phenomenon where nonspecific agglutination of RBCs occurs with no blood group specificity, usually detected in ABO discrepancies as “extra antigens”

• Cryptantigens

• IgM

• T activation

Acquired polyagglutination is commonly seen in infections, the bacteria enzymes strip off certain body parts of RBC surface antigens exposing the underlying (——) normally hidden by blood group antigens; Normal sera (——) Abs react to these exposed antigens causing agglutination with (——) being the most common type

Tn polyagglutination

Hereditary polyagglutination involving a somatic mutation of a gene found on the X chromosome

Incomplete synthesis of normal residues on glycophorins A and B

(Hereditary polyagglutination) The mutation involved in Tn polyagglutination causes…

Hereditary Erythroblastic Multinuclearity with Positive Acidified Serum (HEMPAS/CDAII)

(Hereditary polyagglutination) A defective glycosylation of the erythrocyte membrane glycoproteins