Second Year Medical Student Fall Pathology Lab and Indiana Blood Center Tour: Transfusion Medicine and Blood Banking October, 2014.

Second Year Medical Student Fall Pathology Lab

and Indiana Blood Center Tour:

Transfusion Medicine and Blood Banking

October, 2014

Outline

• Pre-test• Major blood group basics• Trauma

– Bag o’blood– Massive blood transfusion protocol (MBTP)

• ABO incompatibility – Acute hemolytic transfusion reaction

• Hemolytic disease of the fetus/newborn (HDFN)• Post-test

What is bloodBlood has an interesting medical and philosophical

history, it permeates our lives and culture Egyptian hieroglyphics show treatment

of patients by bleeding from the neck and ankle – circa 2500 BC

Hippocrates postulates that similar to the four elements, the body is comprised of four humors -- blood, phlegm, black bile, and yellow bile -- and their imbalance causes disease – circa 400 BC

Christianity teaches the blood shed by Jesus Christ formed a new covenant and is the sacramental symbol of the Eucharist - 0 AD to present

Shakespeare’s Macbeth plays at the Global theater with the theme of blood representing a character’s masculinity and remorse – circa 1600

French physician Jean-Baptiste Denis transfuses a teenage boy suffering from a persistent fever with nine ounces of lamb's blood – 1667

“I was a queen, and you took away my crown; a wife, and you killed my husband; a mother, and you deprived me of my children. My blood alone remains: take it, but do not make me suffer long.” Marie Antoinette 1793

Clara Barton, “Angel of the battelfield” during American civil War, founded the American Red Cross - 1881

Bram Stroker’s Dracula centralizes around a character who can drink the blood of his victims and attain immortality - 1897 (with huge pop culture implications)

Surgeon General of the U.S. Army and Navy, the American Red Cross agrees to organize a civilian blood donor service to collect blood plasma for the war effort -1941

I was born with music inside me. Music was one of my parts. Like my blood. It was a force already within me when I arrived on the scene. It was a necessity for me - like food or water. Ray Charles 1930-2004

Product: Blood safety

Entire process: Blood transfusion safety

Recruit donorScreen donor

Collect unitPrepare components

Infectious diseasestesting

Pre-TX testing

Medical reason to TX

Issue unitAdminister at bedside

Monitor & evaluate

After S. Dzik, MD Blood Transfusion Service, MGH, Boston

What the Blood Blanker does for the clinicians and patients Transfusion therapy is a set of processes, not just a product

Some blood bank basics

• Basically, there are 4 major blood groups – A– B– AB – O

• These groups are defined by surface antigens present on the Red Blood Cell (RBC)

• Which antigens are present is determined by the expression of genes which code for enzymes found on the long arm of chromosome 9 (9q34)

• These genes are expressed co-dominantly

A gene

• If you have the A gene (N-acetyl galactosyltransferase), you make the A antigen – Thus you are blood Type A

N-acetyl GalTA gene A gene

A Blood type

B gene

• If you have the B gene (galactosyltransferase), you make the B antigen – Thus you are blood Type B

GalTB gene B gene

B Blood type

Both A and B genes

• If you have the A and B genes on separate chromosomes, you make both the A and B antigens – Thus you are blood group AB

GalTB geneA gene

N-acetyl GalT

AB Blood Type

Neither A or B genes

• If neither A or B genes are present, you will make neither A no B antigens– Thus you are blood group O

O Blood Type

Why do we care?

• Try to stay awake because it has to do with immunology – Recognition of self and non-self

• A and B like antigens are found constitutively in nature– We are exposed to them early in life – As a result the body will make antibodies against

the antigens it doesn’t have (non-self) and not make antibodies against the antigens it does (self)

Major antigens and antibodies Type A

B

A

B

A,B

B

B

B

B

B

B

Type B

A

AA

AAA

A

Type AB

Type O

AB

A

A

B

BB

A,B

A,B

A

A, B, and A,B allo-antibodies

• Allo-antibody is an antibody against an antigen of the same species

• All potent activators of complement and the membrane attack complex – Cause massive intravascular hemolysis of RBCs

So what happens when…

We mix type A whole blood with… Type AB whole blood

B

B

B

BB B

B

B

So what happens when…

We mix type A whole blood with… Type AB whole blood?

B

B

BB

BB

BB

When transfusing whole blood the blood types must be identical.

But if we just…

Mix type A RBCs…

B

B

B

BB B

B

B

with type AB whole blood…

And if we apply this to patients…

Donor Recipient

It is the basis for blood component therapy

Blood Component Therapy

For RBC units For plasma based units

Donor blood type

Reci

pien

t blo

od ty

pe

• Who is the universal RBC donor?• Type O

• Who it the universal RBC recipient?• Type AB

Donor blood type

Reci

pien

t blo

od ty

pe

• Who is the universal plasma donor?• Type AB

• Who it the universal plasma recipient?• Type O

Approximately 45% of the population is Type O, 40% type A, 11% type B, and 4% AB.

Case presentation #1

• You have been accepted to Emergency Medicine residency in Washington DC and are enjoying a nice day off of sight seeing following three days on 16 hour shifts when you walk past a TV wall and see this….

Olympus Has Fallen!

https://www.youtube.com/watch?feature=player_detailpage&v=kWPNediW79I

Case presentation

• Within a minute your pager goes off. • You return the page and you are informed that

Emergency Response Plan has been initiated and you are to report to your training hospital for assignment.

Yes, this could happen to you!

Case presentation

• You are assigned to trauma management as a team leader

• You meet the EMS at the ED entrance to accept your first patient – As you take the patient the driver quickly states “this is a

27 year old male with proximal lower limb injuries secondary to large caliber gunshot, GCS 15/15, vitals are 98.0F, 135, 32, 108/62, O2 sats 95% NC. Approximately 2 liters of blood loss at the scene. Intraosseous lines placed – patient has received approximately 1000ccs NS”

What do you Do?

• ABCDEs

–Airway assessment and protection possible cervical spine stabilization

–Breathing and Ventilation assessment for oxygenation

–Circulation assessment to control hemorrhage and maintain end organ perfusion

–Disability assessment by basic neurologic evaluation

–Exposure by undressing patient and searching for additional injury while preventing hypothermia

A & B

• As you move the patient into the ED you ask the patient his name – He states “Channing Tatum” between rapid breaths – Besides realizing that’s the wrong movie, you just

covered your A and B (patient’s ability to “mentate” & phonate generally indicates an ability to breath and protect his/her airway)

– You note the tachypnea and maintain a low threshold for intubation

C &D

• Pulse in upper extremities is 1+ weak and thready with regular tachycardic, approximately 130, while the posterior tibia and dorsus pedis pulses are absent bilaterally. – An abnormality is identified in the lower extremities (C), now you

must look for a cause while assessing for disability (D) of the injury which may change treatment

• Blood soaked gauze is present at the upper thigh bilaterally • You remove the patient’s socks to reveal two pale, grey feet

and 10 pale toes. – You run the sharp end of you reflex hammer up the plantar aspect

of the patient’s feet – the toes curl in and the patient groans – you just assessed for disability

E

• You order your staff to start undressing the patient as you arrive in Triage and demand a trauma room. – To which they respond “All of the trauma rooms are full!”

• You turn your attention to the blood soaked gauze at the thighs– Removing the gauze reveals two entrance and exit

puncture wounds with pulsatile blood– A scan of the rest of the body including back reveals no

other injuries but the patient’s skin is cold and clammy

Diagnosis and additional management

• Diagnosis: Traumatic laceration of bilateral femoral arteries complicated by probable hemorrhagic shock

• What can you do for this patient?– What studies or labs should you order?– Intravenous access?– Environment?

Traumatic laceration of bilateral femoral arteries

• Studies– EKG with telemetry – Continuous pulse ox

• Labs – CBC stat– CMP stat– Blood gas stat– Lactate stat– Type and Cross stat– Thromboelastography (TEG)?

• Interventions – Oxygen therapy– Two large-bore 16 gauge catheters

with continuous NS push– Direct pressure/turniquet/BP

cuff/upstream occlusion– Arterial line – Warm blankets to prevent

hypothermia – Elevate extremities/ Trendelenburg– Foley catheter

Can you manage this patient on your own?To whom do you need to communicate this patient?

Transition of care and support

• You page the trauma/vascular surgery team– Nurse returns the page and says that due to the volume the

soonest they can evaluate the patient is in 20 minutes• You call the blood bank and they haven’t received the

specimen for type and cross.– They say, due to the volume, it will take 5-10 minutes before a

type can be done.– Still, they ask what products you want? – You try to think back to medical school and recall if anyone ever taught you anything about blood therapy.

Options

• Whole blood • Packed red blood cells

– Leukocyte reduced – Washed RBC– CMV negative

• Platelets – Pooled random donor – Apheresis

• Plasma – Fresh Frozen Plasma– Plasma frozen <24 hrs – Thawed Plasma – Liquid plasma – Plasma cryoprecipitate

reduced

• Cryoprecipitate

Irradiated or not irradiated?

Whole Blood

A. Description:– 500+/- 50 ml mixed with 70 ml

CPD

B. Storage:– 21 days stored at 1-6º

C. Indications:– Recently used in military

hospitals in combat areas settings

– Proposed clinical trials to examine feasibility and efficacy in civilian setting

Packed Red Blood cells A. Description:

– 200 ml of RBC with 111 ml of additive solution

– Packed cell volume = 60%

B. Storage = 42 daysC. Indications:

– Acute blood loss exceeding 15-20% of blood volume (pediatric patients - 10-15 ml/Kg) and failure to obtain hemodynamic stability with reasonable volume of crystalloid and/or colloid solutions

– Acute blood loss of any amount if there is clinical evidence of inadequate oxygen carrying capacity

Packed Red Blood cells

C. Indications:– Hemoglobin of ≤ 7 gm/dl

(hematocrit ≤ 21%), if not due to a treatable cause (treatment of underlying case is preferable if patient is not symptomatic)

– Symptomatic anemia regardless of hemoglobin level

– Hemoglobin ≤ 8 gm/dl (hematocrit ≤ 24% ) and acute cardiac disease / or shock

D. Contraindications:– For volume replacement– In place of a hematinic– To enhance wound healing– To improve general “well-being”

Leukocyte reduced pRBCA. Description:

– Packed red cells with leukocytes reduced (residual leukocyte count less than 5x106)

B. Processing of Product:– Product made during transfusion

with filter attached to unit– Pre-storage leukocyte reduction at

blood center

C. Indications:– Prevention of HLA/WBC

alloimmunization– Prevention of recurrent non-

hemolytic febrile reactions– Prevention of CMV transmission in

select groups of patients

Saline washed pRBCA. Description: packed red cells washed

with saline – 99% of plasma proteins are removed– 85% of leukocytes are removed– Post-wash K + is 0.2 meq/L

B. Processing: manual and automatic methods

C. Storage: once washed, 24-hour outdateD. Indications:

– History of allergic or febrile reactions secondary to plasma proteins not prevented by pre-transfusion administration of antihistamines and leukocyte reduction

– IgA deficiency with documented IgA antibodies

– History of anaphylactic reaction to blood components

Platelet Concentrates A. Description:

– Random donor unit contains 5.5 X 1010 platelets suspended in 30-50 ml of plasma

– Apheresis donor unit contains – 3.0 X 1011 platelets suspended in 300-

400 ml of plasma

B. Dosage:– 4-6 platelet concentrates– 1 apheresis unit– Platelet count should increase 25,000 –

30,000/cc3– Each dose has equivalent of one unit of

fresh plasma*

C. Storage:– Stored at 20-24º C on a rotator– 5-day outdate

Platelet Concentrates D. Indications: prevention and cessation of bleeding

– Severely thrombocytopenic (less than 10,000 or 20,000 depending on institution)

– Moderately thrombocytopenic and bleeding (less than 50,000)

– Surgery or invasive procedure (less than 50,000)

– Diffuse microvascular bleeding following cardiopulmonary bypass or with intra-aortic balloon pump (less than 100,000)

– Bleeding with qualitative platelet defect– Massive Transfusion Protocols (MTP)

E. Contraindications:– Idiopathic Thrombotic

Thrombocytopenic Purpura (ITP)– Thrombotic Thrombocytopenic Purpura

(TTP)

Plasma A. Description:

– 225-275 ml of plasma and CPDA-1, including 25 meq of citric acid

– Jumbo plasma 400 cc or greater

– Frozen within 8 hrs = FFP– Frozen within 24 hrs = 24FP

B. Storage: 1 year at -18ºC C. Outdate once thawed (1-6ºC)

– 24 hours for FFP or 24FP– 72-120 hours if relabeled as

Thawed Plasma

Plasma D. Indications:

– Treatment of coagulopathy due to clotting factor deficiencies

– Patient is bleeding actively with PT and/or PTT greater than 1.5 normal (INR > 1.8) and platelet count above 50,000

– Coumadin overdose with major bleeding or impending surgery

– Treatment of TTP– Massive Transfusion Protocol

(MTP)E. Contraindications:

– Volume expansion– Treatment of nutritional

deficiencies

Cryoprecipitate

A. Description: each unit consists of 10-30 ml residual plasma – 80 units of factor VIII– 250 mg of fibrinogen

B. Storage: 1 year at -18ºCC. Indications:

1. Hypofibrinogenemia (≤ 100 mg/dl)

2. Dysfibrinogenemia3. Factor XIII deficiency - rare4. MTP

Irradiated UnitsA. Products irradiated:

– Whole blood, packed red cells, platelets, and granulocyte concentrates

B. Indications: preventing graft versus host disease

– Immunocompromised patients– Directed donations from blood relatives– Premature infants ≤ 1200 gms– Fetuses receiving intrauterine transfusions– Neonatal exchange transfusions

C. Processing and final product:– Irradiate with 2500cGy– Mitotic capacity of lymphocytes is reduced

or eliminated without significant functional damage to other cellular elements

D. Storage:– Red cells outdate 28 days from irradiation

(or original expiration if less than 28 days)

Confused?

• As you are running through the options in your head…. you hear the technician, still on the line, ask…

“Would you like to initiate a Massive Blood Transfusion Protocol?”

Massive Blood Transfusion Protocol (MBTP)

• Massive blood transfusion – transfusion of 10 or more blood components within a 24 hour window

• Blood bank maintains an adequate inventory of type-specific and type compatible units for emergent situations – When the patient’s blood type is unknown O Rh positive red

blood cells and AB plasma products will be released– Special component processing cannot be provided due to

urgency – Once blood type has been identified type specific units will be

released; if incompatible blood has been released the clinician will be notified retrospectively

Massive Blood Transfusion Protocol (MBTP)

• Since the patient is bleeding whole blood each component of the blood must be replaced– At IU Health components are released in the

following ratios• 8 units packed, leukoreduced red blood cells• 4 units plasma • 1 unit leukoreduced, apheresis platelets

Back to the case

• You initiate that massive blood transfusion protocol– You order rapid infusion as units arrive

• Lab results come back

Labs/Studies

Urinary output in first 60 minutes in ER was 20 ml (color was dark yellow). Urine specific gravity = 1.029 (normal = 1.003 - 1.030). Central venous pressure ranged from 1 to 3 cm H2O throughout the cardiac cycle (normal = range = 5.5 to 13 cm H2O).

ECG revealed normal sinus rhythm with slight ST-depression in most leads

Venous bloodTotal white blood cell (WBC) count = 7,400 WBCs /mm3 (normal = 4,000 to 11,000)Differential WBC count revealed 59% neutrophils (normal = 55-70%)Hematocrit = 46% (normal = 42-54%)Hemoglobin = 15.0 gm / dl (normal = 14-18 gm / dl)Sodium (Na+) = 138 mEq / L (normal = 136-145 mEq / L)Potassium (K+) = 5.1 mEq / L (normal = 3.5-5.1 mEq / L)Chloride (Cl-) = 104 mEq / L (normal = 96-106 mEq / L) BUN = 27 mg / dl (normal = 6 - 23 mg / dl)Creatinine = 1.9 mg / dl (normal = 0.7 - 1.5 mg / dl)Glucose = 165 mg / dl (normal = 70 - 160 mg / dl)SGPT = 41 IU / L (normal = 0-33 IU / L)SGOT = 48 IU / L (normal = 0 41 IU / L)Lactate = 4.2 mmol/L (normal = 0.6-2.3 mmol/L)

Arterial blood Blood pH = 7.28 (normal = 7.35-7.45)pCO2 = 31 mm Hg (normal = 40 mm Hg)pO2 = 78 mm Hg (normal = 90-100 mm Hg)Hemoglobin - O2 saturation = 88% (normal = 94-100%)[HCO3-] = 14 mEq / L (normal = 22-26 mEq / L)

Final Diagnosis and Treatment

• Hemorrhagic shock– Anion gap secondary to lactate acidosis from glycolytic pathway,

indicating poor oxygenation and end organ dysfunction. • As units are infused and new labs are ordered the trauma

surgery team arrives to evaluate the patient.– They commend you for your excellent work and indicate that they

are taking the patient to surgery now.

Case presentation #2

• You decided Emergency medicine just wasn’t for you so you transfer into General Surgery at a small community hospital outside Seattle, WA that didn’t fill.

• You are asked to admit a 35 year old female for non-urgent cholecystectomy for symptomatic cholelithiasis confirmed by ultrasound.

You’re thinking “Now this is more like it!”

Case Presentation #2

• HPI– Started feeling intermittent RUQ pain with fatty meals

during her third trimester. Pain brief and resolves spontaneously. Last attack was 3 weeks ago. No fevers, N/V, change in bowel habits. Wants the surgery preformed while on maternity leave.

• PMHx– Just gave birth to her second child 1 month ago, she claims

to have lost some blood during birth but felt fine, did not get transfused, and was discharged at 48hrs

– No other significant history

Case Presentation #2

• Medication: – multivitamin

• FmHx/SocHx/RoS: unremarkable • Physical exam: unremarkable

– Vitals within normal limits– Pertinent positives/negatives

• Abd: Soft non-distended, normal bowel sounds; No guarding or rebound tenderness

• You order standard pre-op labs– CBC, CMP, Type & screen

Labs

• CBC

• CMP

• Normal Hemoglobin levels – Men: 13.8 – 18.0 g/dL– Female 12.1 – 15.1 g/dL

• Hematocrit is the proportion of whole blood occupied by RBC, calculated %– HCT = RBC volume/total blood volume (or Hgb x 3)

5.29.8

22029.4

4.9140 102

25 0.8019

80

Ca: 9 mg/dLAlk: 100 U/LBili: 0.4mg/dLAST: 5 U/LALT: 2 U/LProtein: 7g/dLAlbumin: 4g/dL

Diagnosis

• Mild anemia, otherwise ready for surgery

• You discuss the patient with your staff on rounds.– He tells you to change the Type & Screen to a Type

and Cross – He says transfuse two units when available… He

never operates on a patient with a Hgb < 10 g/dL• A single unit of packed RBC will increase a normal adults

hemoglobin 1gm/dL

T&C vs T&S

• Type and Screen – used in patients without and immediate need for transfusion (pregnant and pre-surgical patients)

– Identify blood and Rh type (with RBC and serum)– Screen serum for minor blood group antibodies which could

cause hemolysis• Type and Cross – used in patients with an imminent need for transfusion

(trauma patients, sickle cell crisis, invasive surgical patients)

– Perform a Type and screen– Identify compatible units for transfusion and mix patient

serum with RBC product to prove there is no reaction/hemolysis (CROSS-match)

– Units are then reserved for that patient if needed

Patient’s T&C

Forward Reverse

Anti-A Anti-B Anti-Rh Type? A Cell B Cell Type?

4+ 0 4+ A+ 0 4+ A

• Red blood cell typing – done two ways, remember ABO antigen and antibodies correlate

• Forward – Patient’s red blood cells with known anti-sera• Reverse – Patient’s serum with known red blood cell types

Transfusion

• Screen is negative and serum sample cross-matched with two units – Units arrive in the OR as the patient receives anesthesia prior

to intubation• The nurse checks the patient labels and everything

matches, confirms that they are A+ units which is witnessed by the anesthesia tech, and takes the patient’s vitals which are normal

• She starts the transfusion and goes about her pre-op routine– As she places the foley catheter she notes dark urine

Transfusion

• There is no one besides the anesthesia tech in the room, so the nurse pages you– On the phone the nurse tells you about the urine

• You quickly request an update on her vitals– The patient is now intubates but otherwise vitals show

increased temperature of 1.9˚C, increased heart rate, and decreased blood pressure

– She includes that the patient is now oozing from all IV sites• What is the next, MOST important step you need to

take to decrease morbidity and mortality for your patient?

Suspected Transfusion Reaction

STOP THE TRANSFUSION

Suspected Transfusion Reaction

• The nurse stops the transfusion • You tell her to call a code, start a transfusion

reaction work-up, and you will be there as soon as possible

• You arrive in the midst of aggressive resuscitation efforts– Ultimately the team is unsuccessful and the

patient is pronounced dead

Transfusion reaction work-up

• Post-mortem labs are drawn – Direct anti-globulin test– Plasma free hemoglobin– Urine for hemoglobin testing– Blood bank receives a specimen for retyping and

cross match • Body bank notes the serum is pink that does not

change with centrifugation

• Clerical paper work review revealed no errors

Transfusion reaction work-up

• What is the patient's blood type?– Original pretransfusion specimen retyped A+

• DAT is positive• Repeat cross-match strongly positive (4+)• Plasma free hemoglobin elevated• Urine hemoglobin elevated

What is the diagnosis and how did this happen?

Forward Reverse

Anti-A Anti-B Anti-Rh Type? A Cell B Cell Type?

0 0 0 O- 4+ 4+ O

Acute hemolytic transfusion reaction

• Rapid destruction of donor RBC by recipient Ab• Medical emergency

– Most severe transfusion reaction – Can be fatal, thus treat all transfusion reactions as possible AHTR (Stop

the transfusion for all possible reations)• Pathophysiology

– Ab bind donor RBCs Complement activation formation of MAC RBC lysis

– Ag-Ab complexes coagulation cascade activation intravascular thrombi and consumption of coag factors DIC and schistocytes

– Both pathways result in the release of numerous cytokines causing symptoms

– All dose dependant

Acute hemolytic transfusion reaction

Signs and symptoms• Triad

– Fever/Chills (>2˚F or >1 ˚C)– Flank pain– Dark urine

• Additional – Tachycardia– Dyspnea – N/V– Bleeding – Hypotension– Feeling of impending doom

Laboratory findings • Hemoglobinemia (pink or red

serum/plasma)• Hemoglobinuria (NOT hematuria)• Usually positive direct

antiglobulin test (DAT) but can be negative (all Ab coated cells already lysed)

• Elevated indirect bilirubin and LDH

• Decreased haptoglobin • Hyperkalemia• Peripheral smear: Schistocytes

Acute hemolytic transfusion reaction

• Treatment is supportive

• Case resolution– Patient in a two person room– Patient asked to switch beds with roommate because she liked to

be by the window– When nurse came to draw Type and Cross specimen, she drew the

specimen from the patient in the door bed (where the patient was assigned) without checking patient ID

– The patients roommate typed A+ while our patient was actually O-– Anti-A, Anti-B, and Anti-A,B antibodies in patient destroyed A+

transfused cells

Risk of transfusion reaction

Carson J L et al. Ann Intern Med doi:10.1059/0003-4819-156-12-201206190-00429

Case Presentation #3

• You decide that General Surgery isn’t for you either. You love babies, so you figure Ob/Gyn is the place for you.

• You transfer into a residency program in a large metropolitan city with a diverse population.

You think, its just catching babies – They’re about as big as a blood bag and inside someone else. I’m

finally done with these pesky transfusions!!

Case presentation #3

• A 25 year old pregnant Chinese female presents to your clinic for her first obstetric appointment. She just immigrated from China and is late in her second trimester. She received her prenatal care in rural China.

Case presentation #3

• PMHx– None, reports always being healthy

• Medication– None

• FmHx/SocHx/RoS– Unremarkable

• Birth history – G3 P2002

• First pregnancy uncomplicated • Second child born “golden” which resolved after several weeks• Her husband is the father of all children

Case presentation #3

• Physical exam– Vitals within normal limits – Pertinent physical exam findings: Fundal height 26cm (correlates

with weeks of gestation)

• Routine labs and exams ordered – CBC, CMP– Infectious serology (ToRCHeS)– Group B strep screen– Type and Screen– Fetal heart rate by doppler – Fetal ultrasound

Lab and Exam results

• Fetal heart rate: 170 bpm (normal 120-150 bpm)

• Awaiting ultrasound findings• Laboratory work up shows a mild anemia but

is otherwise unremarkable – Remember pregnant patients will develop a

physiologic anemia due to a disproportionate increase in blood volume over red blood cell volume

Type and Screen Results

• Antibody screen is positive– Reflex red blood cell panel is performed and an Anti-D

antibody was identified• What diagnosis do these findings suggest?

– What does the Rh +/- mean?– Does it have anything to do with the Anti-D antibody?

Forward Reverse

Anti-A Anti-B Anti-Rh Type? A Cell B Cell Type?

0 0 0 O- 4+ 4+ O

Hemolytic Disease of the Fetus/Newborn (HDFN)

Defined as the destruction of fetal and neonatal red blood cells by maternal antibodies

Historically IgG anti-Rh (anti-D) minor blood group alloantibodies were first identifiedOther Minor blood group antibodies are implicated as well – however Anti-D

alloantibodies are the most immunogenic/potent

Hemolytic Disease of the Fetus/Newborn (HDFN)

The Rh Minor blood group antigen system contains multiple antigens

However during typing the Rh +/- demarcation refers only to the Rh(D) antigen

Thus Anti-Rh allo-Ab are often incorrectly used interchangeably with Anti-D allo-Ab

Minor blood groups

• Genetics – Similar to Major ABO genes– Minor blood group genes code for surface

proteins (Rh system), enzymes, or nothing (silent/amorph)

– Minor genes are also inherited in a Co-dominant pattern

• Each parent contributes half of the inheritance

• Individual traits are inherited independent of each other

• Serology– Allo-antibodies can be formed against any

minor blood group antigen– Requires exposure which is often by unnatural

means, i.e. exposure to blood

A

BA

BD

C

cE

e

K

kFya

Fyb

Jka

Jkb

Development of fetal allo-antibodies

1. Mother must be antigen negative– Antigen positive individuals will not form antibodies to self

2. Mother must be exposed to minor antigen– Feto-maternal hemorrhage – Transfusion with ABO compatible, minor group incompatible blood – Injection with needles contaminated with minor group antigen positive blood– Minor blood group mismatch allogeneic stem cell transplant

3. Minor antigen exposure induces antibody formation– Exact volume unknown (varies between individuals) but as little as 0.1 mL of Rh+ RBC have been

shown to stimulate antibody production – Larger volume of exposure tends to produce a more robust response

4. Following antibody production mother must become pregnant with an antigen positive fetus

– The initial response will be IgM which will not cross the placenta • In most cases, the first minor group incompatibility between mother and fetus will not be affected, with

exceptions

– Subsequent exposure will induce memory cells to produce IgG antibodies which will in turn cross the placenta and cause hemolysis or clearance

Development of fetal alloantibodies

Rh+ Rh Rh -

D d d d

D d D d d d d d

Rh + Rh + Rh - Rh -

Firs

t Ch

ild

Seco

nd

Child

First Rh + child sensitizes Mother

Mother develops Anti-D antibodies - First round IgM antibodies

Second child conceived

IgG Alloantibodies have developed which cross placenta and…

Unlike A and B antibodies, many minor antibodies are not naturally occurring

IgG antibodies

• Only IgG type antibodies can cross the placenta• Actively transported via a

receptor specific to IgG Fc region

• Starts in the second trimester and continues until birth (passive immunization )

Pathophysiology of HDFN• Maternal IgG antibodies cross the placenta and attach to fetal red blood

cells• Red blood cells hemolyzed or removed via reticulo-endothelial system • Resultant anemia causes accelerated production of RBCs by bone

marrow, termed erythroblastosis fetalis • In severe disease, bone morrow inevitably falls short of necessary RBC

production• Body responds with extramedullary hematopoesis in the spleen and liver• Hepato-splenomegaly causes portal hypertension and hepatocellular

damage • Anemia coupled with hypoproteinemia (decreased osmotic pressure)

leads to massive, diffuse edema (hydrops fetalis) and high cardiac output heart failure

Hydrops and Erythroblastosis Fetalis

Sequelae of HDFN

• Hyperbilirubinemia– RBC destruction does not cease with delivery

• IgG due to its small size as a monomer distributes throughout tissues (intravascular and extravascular)

• IgG has a half life of 25 days

– Prior to delivery bilirubin is transported across placenta and conjugated by maternal liver preventing hyperbilirubinemia in utero

– Bilirubin conjugation system in a neonate is immature • Without therapy unconjugated/indirect bilirubin can reach

toxic levels (18-20 mg/dL) and diffuse into the brain causing kernicterus and acute bilirubin encephalopathy

Acute Bilirubin Encephalopathy and Kernicterus

Risk Stratification and Management for Anti-D HDFN

• Type and screen at first prenatal visit

• If Rh(D) positive there is no risk of alloimmunization

• If Rh(D) negative, fetus may be at risk• Determine biological fathers Rh(D) status and zygocity

– If dd homozygote there is no risk of allommunization – fetus is Rh(D) negative– If DD homozygote fetus is Rh positive – fetus may be at risk depending on screen results – If Dd heterozygote risk of fetus being Rh(D) positive is 50% - fetal DNA studies can be performed

to confirm status – fetus may still be at risk depending on screen results

• Fetus Rh(D) positive and screen is negative – no antibodies have developed, but may yet

– Repeat screen at 28 weeks and following any incident in which there is increased risk of feto-maternal hemorrhage

• Fetus Rh(D) positive and screen is positive, fetus is at risk– Perform titer of Anti-D antibodies (>1:32 is clinically significant)– Perform diagnostic techniques followed by treatment if indicated

Diagnosis and Treatment of Intrauterine fetal anemia

• Diagnostic techniques:– Ultrasound– Doppler assessment of MCA peak velocity – Percutaneous umbilical cord sampling for fetal hematocrit– Allele-specific PCR on fetal cells in amniotic fluid

• Treatment: – Intrauterine fetal transfusion

• Transfuse when fetal hematocrit falls below 2 standard deviations of mean hematocrit for gestational age

• Can be performed between 18 and 35 weeks• Intraperitoneal transfusions not as effective as intravascular transfusions in

hydropic fetus due to congested lymphatics • ABO type O, Rh- packed RBC utilized• Fetal loss 1-2% with overall survival of 85% after transfusion

Algorithm for HDFN

Prevention of Rh Alloimmunization

• Anti-D immune globulin/RhIG– IgG anti-D manufactured from human plasma (primarily

male who undergo repeat injections of Rh+ blood)– Preparation methods

• HyperRho S/D®, RhoGAM®– Cohn cold ethanol fractionation followed by viral-clearance

ultrafiltration – intramuscular only as IgA and other plasma proteins have the potential to produce anaphlyaxis

• Rhophylac®, WinRho SDF®– Ion-exchange chromatography isolation – intramuscular or IV

Prevention of Rh Alloimmunization

• Anti-D immune globulin/RhIG– Mechanism of action: not well understood, epitope

masking?, rapid clearance of fetal RBC? – Guidelines

• Weak D positive managed as Rh-• Mother Rh-, fetus confirmed or suspected Rh+

– 300 micrograms early in third trimester– 300 micrograms if there is increased risk of feto-maternal

hemorrhage » Repeat doses if risk is ongoing, guided by titers or Kleinhauer-

Bethke test– 300 micrograms within 72 hrs after delivery

» If inadvertently not administered, give ASAP – partial protection has been seen up to 13 days after birth

Case Presentation• Finalizing the case

– What do you order?• Maternal Anti-D titer

– 1:64

• US of fetus and MCA peak velocity

– Slightly hydroptic fetus – MCA peak velocity elevated

• Fetal CBC – Moderate anemia

• Intrauterine fetal transfusion performed by IR

– Mom transferred to high risk OB service where last you heard she was doing very well and expecting to be discharged

Questions

?All of these blood bank situations and complications

have driven you so crazy......

…You decide to go into psych.