hemovigilance & blood banking

Hemovigilance & Blood Banking

Dr. M.Edalati

Ph.D. of Hematology and Blood Banking

Hemovigilance

تعریف

جمغ و سيستماتيك صورت به تشریق اس ناشی ػوارض گشارش-1 واحد یك در آوري

اثزات به مزبوط هاي داده تحليل و تجشیه و گزدآوري- 2

اخذ و تصحيح منظور به خطز اػلام و خون انتقال ناخواسته آنها مجدد وقوع اس جلوگيزي بزاي لاسم اصلاحی اقدامات

بزرسی و بيمارستان یك در خون تشریق موارد ساسي مستند - 3 متوالی هاي سال در آن اي مقایسه

ها بيمارستان در خون تشریق ارتقاي و هدایت - 4

:یک واحد خون کامل

RBC (1 FFP (2

(3 CP Plt (4

IG , Alb plasma

Packed red blood cells

(RBC)

از %۲۰ حدود شده شسته قرمز گلبول فرآوردۀ در

به نزدیک و سفید گلبولهای از %۸۵ ، قرمز گلبولهای .یابد می کاهش اولیه پلاسمای از ۹۹%

Fresh Frozen Plasma

(FFP)

Cryo precipitate

VIIIفاکتور

واحد بین المللی 80-120میزان به

فیبرینوژن 150 -300mg

VWF

مقدار اولیه% 40-%70به میزان

XIII فاکتور

اولیه مقدار % 20 -% 30میزان به

فیبرونکتینمقادیر قابل توجهی

Platelet

Whole blood

مثلث مرگ

نمونه گیری

تزریق بانک خون

فرم ها

BLood Banking

ABO DISCREPANCIES & OTHER

PROBLEMS

DISCREPANCIES

A discrepancy occurs when the red cell testing

does NOT match the serum testing results

In other words, the forward does NOT match

the reverse

WHY?

Reaction strengths could be weaker than

expected

Some reactions may be missing in the reverse

or forward typings

Extra reactions may occur

Patient Anti-A Anti-B A1 Cells B Cells

1 4+ 1+ 0 4+

2 0 4+ 1+ 0

3 4+ 4+ 1+ 0

4 0 3+ 0 0

WHAT DO YOU DO?

Identify the problem

Most of the time, the problem is technical

Mislabeled tube

Failure to add reagent

Either repeat test on same sample, request a new sample, or wash cells

Other times, there is a real discrepancy due to problems with the patient’s red cells or serum

TECHNICAL ERRORS

Clerical errors Mislabeled tubes

Patient misidentification

Inaccurate interpretations recorded

Computer entry error

Reagent or equipment problems Using expired reagents

Using an uncalibrated centrifuge

Contaminated or hemolyzed reagents

Incorrect storage temperatures

Procedural errors Reagents not added

Manufacturer’s directions not followed

RBC suspensions incorrect concentration

Cell buttons not resuspended before grading agglutination

CONTAMINATED SAMPLES OR REAGENTS

Sample contamination

Microbial growth in tube

Reagent contamination

Bacterial growth causes cloudy or discolored

appearance…do not use if you see this!

Reagents contaminated with other reagents (don’t

touch side of tube when dispensing)

Saline should be changed regularly

HEMOLYSIS

Detected in serum after centrifugation (red)

Important if not documented

Can result from:

Complement binding

Anti-A, anti-B, anti-H, and anti-Lea

Bacterial contamination

Red

supernatant

ABO DISCREPANCIES

ABO DISCREPANCIES

Problems with RBCs

Weak-reacting/Missing antigens

Extra antigens

Mixed field reactions

Problems with SERUM

Weak-reacting/Missing antibodies

Extra antibodies

Grouping

Forward Reverse

Missing/Weak Extra Mixed Field Missing/Weak Extra

A/B Subgroup

Disease

(cancer)

Acquired B

B(A) Phenotype

O Transfusion

Bone Marrow

Transplant

Young

Elderly Immunocompromised

Cold

Autoantibody

Anti-A1

Rouleaux

Cold

Alloantibody

Rouleaux

May cause all + reactions

Forward Grouping Problems

Red Cell Problems

• Affect the forward grouping results

– Missing or weak antigens

– Extra antigens

– Mixed field reactions

Forward Grouping:

Missing or Weak antigens

Anti-A Anti-B A1 Cells B Cells

0 0 0 4+

• ABO Subgroups

• Disease (leukemia, Hodgkin’s disease)

• Since the forward and reverse don’t match, there must be a discrepancy (in this case, a missing antigen in the forward grouping)

Group O Group A

Subgroups of A (or B)

• Subgroups of A account for a small portion of the A population (B subgroups rare)

• These subgroups have less antigen sites on the surface of the red blood cell

• As a result, they show weakened (or missing) reactions when tested with commercial antisera

• Resolution: test with Anti-A1, Anti-H, and anti-A,B for A subgroups

Forward Grouping:

Extra Antigens

Anti-A Anti-B A1

Cells

B

Cells

4+ 1+ 0 4+

• Acquired B

• B(A) phenotype

• Rouleaux

• Polyagglutination

• Wharton’s Jelly

EXAMPLE

Acquired B Phenotype

• Limited mainly to Group A1 individuals with:

– Lower GI tract disease

– Cancer of colon/rectum

– Intestinal obstruction

– Gram negative septicemia (i.e. E. coli)

Acquired B

• Bacteria (E. coli) have a deacetylating enzyme that effects the A sugar….

Group A individual

N-acetyl galactosamine

Acquired B Phenotype

Bacterial enzyme removes acetyl group

Galactosamine now resembles D-

galactose (found in Group B)

Resolving Acquired B

• Check patient diagnosis: Infection?

• Some manufacturers produce anti-B reagent that does not react with acquired B

• Test patients serum with their own RBCs

– The patients own anti-B will not react with the acquired B antigen on their red cell (autologous testing)

B(A) phenotype

• Similar to acquired B

• Patient is Group B with an apparent extra A antigen

• The B gene transfers small amounts of the A sugar to the H antigen

• Sometimes certain anti-A reagents will detect these trace amount of A antigen

• Resolution: test with another anti-A reagent from another manufacturer

Other reasons for “extra” antigens

• Polyagglutination – agglutination of RBCs with human antisera no matter what blood type – Due to bacterial infections

– Expression of hidden T antigens react with antisera

• Rouleaux – extra serum proteins

• Wharton’s Jelly – gelatinous substance derived from connective tissue that is found in cord blood and may cause false agglutination (Remember: only forward typing is performed on cord blood) – Wash red cells or request new sample from heel, etc

Forward Grouping:

Mixed Field Agglutination


0 2+ mf 4+ 0

• Results from two different cell populations

• Agglutinates are seen with a background of unagglutinated cells

– All groups transfused with Group O cells

– Bone marrow/stem cell recipients

– A3 phenotype (sometimes B3)

Mixed Field Agglutination (Post transfusion) ~ (ABO Testing) Can be seen in A, B and AB individuals who have received O units. The antisera reacts with the patient’s RBCs, but not with the transfused O cells.

~ (Antibody screen) Can also be seen post transfusion if a person makes an antibody to antigen on donor cells; antibody agglutinates with donor cell, but not their on cells.

Reverse Grouping Problems

Reverse Grouping

• Affect the reverse grouping results

– Missing or weak antibodies

– Extra antibodies

Reverse Grouping:

Missing or Weak antibodies

• Newborns – Do not form antibodies until later

• Elderly – Weakened antibody activity

• Hypogammaglobulinemia – Little or no antibody production (i.e.

immunocompromised)

• Often shows NO agglutination on reverse groupings

Resolving Weak or Missing antibodies

• Determine patients age, diagnosis

• Incubate serum testing for 15 minutes (RT) to enhance antibody reactions

• If negative, place serum testing at 4°C for 5 minutes with autologous control (a.k.a. Autocontrol, AC)

• This is called a “mini-cold” panel and should enhance the reactivity of the antibodies

Reverse Grouping:

Extra Antibodies

• Cold antibodies (allo- or auto-)

– Cold antibodies may include anti-I, H, M, N, P, Lewis

• Rouleaux

• Anti-A1 in an A2 or A2B individual

Cold antibodies

• Sometimes a patient will develop cold-reacting allo- or auto-antibodies that appear as “extra” antibodies on reverse typing

• Alloantibodies are made against foreign red cells

• Autoantibodies are made against ones own red cells. Cold reacting antibodies cause agglutination with red cells at room temperature and below. The autocontrol will be positive. – Resolution: warming tube to 37° and washing red cells

can disperse agglutination; breaking the IgM bonds with 2-ME will also disperse cells

Cold agglutinins

RBC-Histogram Results

RBC

HGB

HCT

MCV

MCH

MCHC

RDW

2.23 x1012/L

14.4g/dl

24.9%

111.7fl

64.6pg

57.8g/dl

25.4fl

(x 1000)

Results RBC-Histogram

RBC

HGB

HCT

MCV

MCH

MCHC

RDW

4.35 x1012/L

14.5g/dl

43.5%

100.0fl

33.3pg

33.3g/dl

14.7fl

Incubation 30 min

(x 1000)

Rouleaux

• Can cause both extra antigens and extra antibodies • “stack of coins” appearance • May falsely appear as agglutination due to the

increase of serum proteins (globulins) • Stronger at IS and weak reaction at 37°C and no

agglutination at AHG phase • Associated with:

– Multiple meloma – Waldenstrom’s macroglobulinemia (WM) – Hydroxyethyl starch (HES), dextran, etc

Resolving Rouleaux

• Remove proteins! • If the forward grouping is affected, wash cells to

remove protein and repeat test • If the reverse grouping is affected, perform saline

replacement technique (more common) – Cells (reagent) and serum (patient) centrifuged to allow

antigen and antibody to react (if present) – Serum is removed and replaced by an equal volume of

saline (saline disperses cells)* – Tube is mixed, centrifuged, and reexamined for

agglutination (macro and micro) *some procedures suggest only 2 drops of saline (UMMC)

True Rouleaux

True Rouleaux

most of the red cells, in the proper viewing area, are stacked together like coins.

Four or more cells make up each formation, leaving much of the field empty of cells (increased white space).

Rouleaux is clinically significant when increased globulins are present, as in multiple myeloma.

False rouleaux

True rouleaux

Artifactual

Autoagglutination

• Cells clumping

together rather than

stacked like coins.

• Autoagglutination is

caused by the

presence of antibody

in the plasma.

Anti-A1

• Sometimes A2 (or A2B) individuals will develop an anti-A1 antibody

• A2 (or A2B) individuals have less antigen sites than A1 individuals

• The antibody is a naturally occurring IgM

• Reacts with A1 Cells, but not A2 Cells

Anti-A1 from patient

+ A1 cells

+ A2 cells

AGGLUTINATION

NO AGGLUTINATION

http://www.jdaross.cwc.net/humoral_immunity.htm

Resolving anti-A1 discrepancy

Anti-A Anti-B A1

Cells

B

Cells

4+ 0 2+ 4+

• 2 steps:

– Typing patient RBCs with Anti-A1 lectin

– Repeat reverse grouping with A2 Cells instead of A1 Cells

– Both results should yield NO agglutination

Others…

• The Bombay phenotype (extremely RARE) results when hh is inherited

• These individuals do not have any antigens and naturally produce, anti-A, anti-B, anti-A,B, and anti-H

• Basically, NO forward reaction and POSITIVE reverse

• Resolution: test with anti-H lectin (Bombay’s don’t have H and will not react)

Resolving ABO Discrepancies

• Get the patient’s history:

– age

– Recent transplant

– Recent transfusion

– Patient medications

….

Agglutination

Passive Agglutination/Hemagglutination

• Definition - agglutination test done with a soluble antigen coated onto a particle

+

• Applications

– Measurement of antibodies to soluble antigens

Coombs (Antiglobulin)Tests

• Incomplete Ab • Direct Coombs Test

– Detects antibodies on erythrocytes

+

Patient’s RBCs Coombs Reagent (Antiglobulin)


• Indirect Coombs Test

– Detects anti-erythrocyte antibodies in serum

Patient’s Serum

Target RBCs

+ Step 1

+

Coombs Reagent (Antiglobulin)

Step 2


• Applications

– Detection of anti-Rh Ab

– Autoimmune hemolytic anemia

Agglutination/Hemagglutination Inhibition

• Definition - test based on the inhibition of agglutination due to competition with a soluble Ag

+

Prior to Test

+ +

Test

Patient’s sample

Let’s practice !

Example 1


3+ 0 0 1+

Problem:

Causes:

Resolution:

Example 2


3+ 1+ 0 4+

Problem:

Causes:

Resolution:

Example 3


2+ 0+ 1+ 4+

Problem:

Causes:

Resolution:

Example 4


0 0 0 3+

Problem:

Causes:

Resolution:

Example 5


0 2+mf 3+ 0

Problem:

Causes:

Resolution:

Example 6


4+ 4+ 0 1+

Problem:

Causes:

Resolution:

Example 7


0 0 0 0

Problem:

Causes:

Resolution:

Thanks

hemovigilance & blood banking

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