An unpleasant visitor? The challenge of having a red cell antibody visit the lab

An unpleasant visitor? The challenge of having a red cell antibody visit the lab

Dr. Aseem Kumar Tiwari

Talk - Organization1. Expected Antibodies and ABO blood group system

2. Other blood group systems and unexpected antibodies

3. Detection of unexpected antibodies

4. Antibody identification and Interpretation

5. Rule of Three and Phenotyping

6. Magnitude of thse problem

7. Antibody Screen Negative – Change in algorithm

8. Antibody Screen Positive – Approach to Patient

Expected Antibody

Laboratory Work-up – ABO system

Expected Antibody (Regular Antibody/Typical Antibody)

Forward(cell) Reverse(serum) Interpretation

-A -B AC BC

+ - - + A

- + + - B

+ + - - AB

- - + + O

Slide(1900)/Tube(1960)/Micro-titer plate(1980)/Solid phase adhesion/Column Agglutination Technique

Other Blood Group Systems

Other blood group typing is not done routinely – ‘immunogenicity’ is considerably less than ABO and Rh

Unexpected Antibody – Unpleasant Visitor

Other Blood Group Systems and Unexpected Antibody

Unexpected (Irregular Antibody/Atypical)

Irregular Antibody – Develops either because of Transfusion/Pregnancy

Unexpected Antibody – Detection

Antibody Detection/Screening - Screening Cells

Each vial (donor) has been phenotyped for each antigen

18 antigens are required on at least one of the vials: D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Jka, Jkb, Fya, Fyb

Single Vial (Reagent Pooled ‘O” cells)

Two Cells (Selectogen)

Three Cells (Surgiscreen)

Protocol

Reagent Pooled ‘O” cells for donors

Three Cells (Surgiscreen) for patients

Antigram- An example

Antigens

D C E c e K kFya

Fyb

Jka

Jkb

Lea

Leb

S s M NP1

IS

37

AHG

I + + 0 + + + 0 0 + + + 0 + + + + 0 + - - 2+

II + 0 + + 0 0 + + 0 + 0 0 + 0 + + 0 + - - 0

An antigram (2 or 3 cells) will list the antigens present in each vial

A reaction to one or more cells indicates the presence of an unexpected antibody

Principle of testing ‘Unknown’ with ‘Known’

Antigram

If If an antibody is detected, it must be identified!

Antibody Panel - Identification

An antibody panel usually includes at least 10 panel cells:


Group O red blood cells


Each of the panel cells has been antigen typed (shown on antigram)

+ refers to the presence of the antigen

0 refers to the absence of the antigen

Example: Panel Cell #1 has 14 antigens present: C, c, e, f, M, N, S, s, P1, Lea, k, Fya,, Fyb and Jka


The same phase (s) used in an antibody screen is used in a panel

• AHG

AHG Phase - Identification

0

0

00

03

0

00

000

0 0

330

Interpreting Antibody Panels

Few basic steps to follow when interpreting panels

1. “Ruling out” means crossing out antigens that did not react

2. Circle the antigens that are not crossed out

3. Consider antibody’s usual reactivity

4. Look for a matching pattern

1. Ruling Out

2+

00

2+

002+

0

02+

0

000

00

00

0

00

0

0000

00

00

000

0 0 0

Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage.

2. Circle antigens not crossed out

2+

00

2+

002+

0

02+

0

000

00

00

0

00

0

0000

00

00

000

0 0 0

3. Consider antibody’s usual reactivity

2+

00

2+

002+

0

02+

0

000

00

00

0

00

0

0000

00

00

000

0 0 0

Lea is normally a Cold-Reacting antibody (IgM), so it makes sense that we see the reaction in the IS phase of testing; The E antigen will

usually react at warmer temperatures

4. Look for a matching pattern

2+

00

2+

002+

0

02+

0

000

00

00

0

00

0

0000

00

00

000

0 0 0

…Yes, there is a matching pattern!

E doesn’t match and it’s a warmer rx Ab

Interpretation

anti-Leaa

Rule of three!

Rule of three must be met to confirm the presence of the antibody

Patient serum MUST be:

Positive with 3 cells with the antigen

Negative with 3 cells without the antigen

Same example fulfills the “rule of three”

2+

00

2+

002+

0

02+

0

000

00

00

0

00

0

0000

00

00

000

0 0 0

3 Negative cells

3 Positive cells

Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin

Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at immediate spin

Screening and Identification only in AHG Phase (Customization !)

Antibody Screen

Expected Antibody with 3 cell panel are

(c, k, kpb, Jsb, Leb, N and s)

Antibody Identification

In 11 cell panel identified anti-”c” in the patient.

Confirmed patient red blood cell with rare antisera ”c”

Antibody Panels - Interpretation

Few basic steps to follow when interpreting panels:

• “Ruling out” means crossing out antigens that did not react

• Circle the antigens that are not crossed out

• Look for a matching pattern

Another Example

Phenotyping

Antigen typing the patient red cells can also confirm an antibody

If reagent antisera (of the suspected antibody) is added to the patient RBCs, a negative reaction should result…Why?

Individuals DO NOT make allo-antibodies against antigens they have

Patient has NOT been recently transfused (donor cells could react)

Magnitude of Problem

Scale of Problem

40/32560 (0.12%) or approximately 1 in 1000

15 out of 2026 (0.75%)

18/531 (3.4%) or approximately 34 in 1000

(multi-transfused patients)

Thakral B, Saluja K, Sharma RR, Marwaha N. Red cell alloimmunization in a transfused patient population: a study from a tertiary care hospital in north India. Hematology. 2008 Oct;13(5):313-8.

Rajendra Chaudhary, Nitin Agarwal.Safety of type and screen method compared to conventional antiglobulin crossmatch procedures for compatibility testing in Indian setting. 2011:5 (2):157-15

Thakral B, Saluja K, Sharma RR, Marwaha N. Red cell alloimmunization in a transfused patient population: a study from a tertiary care hospital in north India. Hematology. 2008 Oct;13(5):313-8.

Rh System 14 anti-D (35%) anti-D (5.6%),

6 anti-E (15%) anti-E (22.2%)

3 anti-c (7.5%) anti-c (38.8%)

2 anti-e (5%)

1 anti-C (2.5%)

3 anti-D with anti-C (7.5%)

Kidd System 1 anti-Jk(b) (2.5%) anti-Jk(a) (5.6%)

Lewis System 1 anti-Le(a) (2.5%) anti-Le(a) (11.1%)

1 anti-Le(b) (2.5%) anti-Le(b) (5.6%)

MNS System 5 anti-M (12.5%) anti-M (11.1%)

1 anti-s (2.5%)

INCONCLUSIVE 2 (5%)

Total 40/32560 18/531

Antibody Screen

Antibody Screen Negative999/1000

Type & Screen Vs Cross-match

45373 patients for whom a total of 61668 units of packed red blood cells (PRBC) were cross-matched in the AHG phase using DiaMed; ID cards. An antibody screen was carried out in all the patients using the DiaMed; ID-DiaCell I+II+III. The AHG cross-match was also carried out for all recipients, irrespective of the result of the antibody screen. The results were compared to see if there were any cases where the antibody screening was negative but the AHG cross-match showed incompatibility.

Not a single case was found where the antibody screen was negative and AHG cross-match showed incompatibility.

Type and screen policy can be safe, efficient, cost-effective, and beneficial to the transfusion service in India.

Pathak S, Chandrashekhar M, Wankhede GR. Type and screen policy in the blood bank: Is AHG cross-match still required? A study at a multispecialty corporate hospital in India. Asian J Transfus Sci 2011;5:153-6

Type & Screen Vs Cross-match

11000 patients were cross-matched in the AHG phase

Simultaneous Type and Screen.

Not a single case was found where the antibody screen was negative and AHG cross-match showed incompatibility.

Dr. Dolly Daniel – Personal Communication

Not as yet!

Rajendra Chaudhary, Nitin Agarwal. Safety of type and screen method compared to conventional antiglobulin crossmatch procedures for compatibility testing in Indian setting. 2011:5 (2):157-15

Evaluated safety of T & S procedure for PTT in comparison with conventional test tube cross match.Study did not show the T and S to reach the expected safety level of 99%, it has achieved a reasonable level of safety (91.6%). Only one sample gave a negative antibody screen while conventional crossmatch was incompatible. This may be due to a rare antibody in the patient sera against which the corresponding antigen was not present on the screening cells but present on the donor red cell. A transfusion reaction would have occurred in this case since it was reacting at 37C in AHG. Since, the screening panel used in the present study was not from Indian subcontinent, therefore, it would be advisable to prepare screening cell panels that include RBC antigens which are prevalent within the local/regional population e.g, In, Mi a etc.

Can Cross-match be Omitted?Over the 2-year interval of the study 9128 patients were entered. There were 8936 patients (97.9%) with a negative antibody screen were transfused a total of 10,899 red cell concentrates. Antiglobulin crossmatch performed after the transfusion indicated that 168 red cell concentrates (1.5%) would have been incompatible if the antiglobulin crossmatch had been performed pretransfusion. 79.2% (103/130) were false positive laboratory results. There were 27 transfusion episodes where the antiglobulin crossmatch on blood transfused was positive due to an IgG antibody. Even though these transfused red cell concentrates were designated incompatible by the antiglobulin crossmatch, none of the patients receiving this blood had clinical or serological evidence of haemolysis. We concluded that the antiglobulin phase of the crossmatch can be omitted from pretransfusion testing without putting patients at risk.

Heddle NM, O'Hoski P, Singer J, McBride JA, Ali MA, Kelton JG. A prospective study to determine the safety of omitting the antiglobulin crossmatch from pretransfusion testing. Br J Haematol. 1992 Aug;81(4):579-84

Antibody Screen Positive1/1000

Antigen negative

Coomb’s Cross-match

Antigen negative compatible blood

Antigen Negative Blood?

Case 1

Patient Name: Master S , (5 yr/M)

Hospitsl- RML hospital, New Delhi

Problem: Grouping discrepancy

Blood group: forward- “A”

Reverse- “O”

Case study

Plan of Action

Repeat grouping

Screening with 3 cell panel

Identification with 11 cell panel

Finding ‘antigen negative blood using rare antisera, if red cell blood

transfusion needed

Provisional diagnosis

Findings…Result of repeat blood grouping

=Forward- “A”, Reverse- “O”

Result of antibody detection(screening)

=Probability of Anti-M and Anti- Fyb

Result of antibody identification

= Anti- “M”

Confirmation of absence of M antigen on patients red cells

= No agglutination with Antisera-”M”

Conclusion:

Anti- “M” antibody present in the patient.

Out of 21 unit got only one “M” negative unit, preserved for Master S.

Comparative study of frequency of AgAntigen Pos. donor (%) Neg. donor (%) PGI Chandigarh

(Pos %) PGI

Chandigarh(Neg %)

D 85 93.4 6 6.59 93.39 6.61

C 77 84.60 14 15.38 84.76 15.24

c 57 62.63 34 37.36 52.82 47.18

E 23 25.27 68 74.79 17.9 82.1

e 88 96.79 3 3.29 98.3 1.7

K 4 4.39 87 95.6 5.56 94.44

k 91 100 0 0 100 0

Fya 79 86.81 12 13.18 86.75 13.25

Fyb 62 68.13 29 31.86 56.15 43.85

Jka 72 79.12 19 20.87 82.65 17.35

JKb 53 58.24 38 41.75 66.56 33.44

Lea 13 14.28 78 85.71 20.82 79.18

Leb 54 59.34 37 40.65 60.57 43.53

S 59 64.83 32 35.16 56.47 43.53

s 79 86.81 12 13.18 87.38 12.62

M 81 89.01 10 10.98 75.39 24.61

N 53 58.24 38 41.75 61.51 38.49

P1 62 68.13 29 31.86 71.92 28.08

Acknowledgements

Renee Wilkins, PhD, MLS(ASCP)cm CLS 325/435; School of Health Related Professions; University of Mississippi Medical Center

Thank You

Antibody screen

Crossmatch

Cause Resolution

PosPos NegNeg Antibody directed Antibody directed against antigen on against antigen on screening cellscreening cell

ID antibody, select ID antibody, select antigen negative antigen negative bloodblood

NegNeg PosPos Antibody directed Antibody directed against antigen on against antigen on donor cell which may donor cell which may not be on screening cell not be on screening cell OROR donor unit may have donor unit may have IgG previously attachedIgG previously attached

ID antibody, select ID antibody, select antigen negative antigen negative blood blood OROR perform perform DAT on donor unitDAT on donor unit

PosPos PosPos Antibodies directed Antibodies directed against both screening against both screening and donor cellsand donor cells

Antibody ID, select Antibody ID, select antigen negative antigen negative bloodblood

An unpleasant visitor? The challenge of having a red cell antibody visit the lab

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