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

2. Outline Pre-test Major blood group basics Trauma ABO incompatibility
Bag o’blood
Massive blood transfusion protocol (MBTP)
ABO incompatibility
Acute hemolytic transfusion reaction
Hemolytic disease of the fetus/newborn (HDFN)
Post-test

3. What is blood
Blood has an interesting medical and philosophical history, it permeates our lives and culture
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
Bram Stroker’s Dracula centralizes around a character who can drink the blood of his victims and attain immortality (with huge pop culture implications)
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
Egyptian hieroglyphics show treatment of patients by bleeding from the neck and ankle – circa BC
“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
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
French physician Jean-Baptiste Denis transfuses a teenage boy suffering from a persistent fever with nine ounces of lamb's blood – 1667
Shakespeare’s Macbeth plays at the Global theater with the theme of blood representing a character’s masculinity and remorse – circa 1600
Clara Barton, “Angel of the battelfield” during American civil War, founded the American Red Cross
Christianity teaches the blood shed by Jesus Christ formed a new covenant and is the sacramental symbol of the Eucharist AD to present

4. Entire process: Blood transfusion safety
What the Blood Blanker does for the clinicians and patients Transfusion therapy is a set of processes, not just a product
Recruit donor
Medical reason to TX
Screen donor
Pre-TX testing
Collect unit
Issue unit
Prepare components
Administer at bedside
Infectious diseases
testing
Monitor & evaluate
Product: Blood safety
Entire process: Blood transfusion safety
After S. Dzik, MD Blood Transfusion Service, MGH, Boston

5. Some blood bank basics Basically, there are 4 major blood groupsAB 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

6. A gene
If you have the A gene (N-acetyl galactosyltransferase), you make the A antigen
Thus you are blood Type A
A Blood type
A gene
N-acetyl GalT

7. B gene
If you have the B gene (galactosyltransferase), you make the B antigen
Thus you are blood Type B
B Blood type
B gene
GalT

8. Both A and B genes AB Blood Type
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
AB Blood Type
GalT
A gene
B gene
N-acetyl GalT

9. Neither A or B genes O Blood Type
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

10. 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)

11. Major antigens and antibodies
Type A
Type AB B B B B B B A A
A,B B A A A A A B
A,B A A B B A A
A,B A
Type B
Type O

12. 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

13. So what happens when… We mix type A whole blood with…
Type AB whole blood B B B B B B B B

14. When transfusing whole blood the blood types must be identical.
So what happens when…
We mix type A whole blood with…
Type AB whole blood? B B B B B B B B
When transfusing whole blood the blood types must be identical.

15. But if we just… Mix type A RBCs… with type AB whole blood… B B B B B B

16. And if we apply this to patients…
Donor
Recipient
It is the basis for blood component therapy

17. Blood Component Therapy
For RBC units
For plasma based units
Donor blood type
Donor blood type
Recipient blood type
Recipient blood type
Who is the universal RBC donor?
Type O
Who it the universal RBC recipient?
Type AB
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.

18. 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….

19. Olympus Has Fallen!

20. Yes, this could happen to you!
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!
Calling in all available resources including clinicians is a part of IU Health’s emergency response plan – this will be mimicked at several institutions.

21. 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”

22. 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
These are typically done all at once with priority given in alphabetical order – a patient who cannot protect his/her airway will generally die before they exsanguinate. When an issue is identified the remaining steps are paused until the issue is resolved.

23. 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

24. 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
This is a normal Babinski’s reflex, plantar reflex, or negative Babinski’s test
A positive Babinski’s sign is dorsiflexion of the big toe with splaying of the smaller digits – which would indicate nerve damage.

25. 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

26. 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?
Will review questions on next slide

27. 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
Will differentiate between a type & screen and a type & cross in the next case
Why use NS if patient is bleeding – 2 reasons
– Volume resuscitation is essential
– dilution of the blood means less true blood volume is lost
Direct pressure to reduce bleeding
Arterial line for real time monitoring of BP
Warm blankets… bleeding not only causes hypovolemia but blood maintains body temperature. Massive loss of blood = massive drop in body temp.
Elevate extremities to get blood back to the central circulation
Foley catheter to monitor urine output – urine production will indicate correction of hypovolemia.
Can you manage this patient on your own?
To whom do you need to communicate this patient?

28. 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.
Get them to internalize that the patient will not live twenty minutes in his current state without blood products
Blood flow through the femoral artery in a healthy male is generally at least 500ml/min
– Blood volume is approximately 70ml/kg, in a 70kg male that’s ~5L (70x70 = 4900mL)
– Hemorrhagic shock occurs when blood volume drops to 30% or below.
– This patient has approximately 3.5 minutes before they develop shock/organ failure (70% is ~3500ml at a rate of loss of 1000ml/min) if no blood loss had
occurred at the scene
Give them a moment to respond to what products the patient needs – bleeding whole blood so they need either whole blood or a combination that recapitulates whole blood.

29. Irradiated or not irradiated?
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?

30. Whole Blood Description: Storage: Indications:
500+/- 50 ml mixed with 70 ml CPD
Storage:
21 days stored at 1-6º
Indications:
Recently used in military hospitals in combat areas settings
Proposed clinical trials to examine feasibility and efficacy in civilian setting

31. Packed Red Blood cells Description: Storage = 42 days Indications:
200 ml of RBC with 111 ml of additive solution
Packed cell volume = 60%
Storage = 42 days
Indications:
Acute blood loss exceeding 15-20% of blood volume (pediatric patients 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

32. Packed Red Blood cells Indications: Contraindications:
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
Contraindications:
For volume replacement
In place of a hematinic
To enhance wound healing
To improve general “well-being”

33. Leukocyte reduced pRBC
Description:
Packed red cells with leukocytes reduced (residual leukocyte count less than 5x106)
Processing of Product:
Product made during transfusion with filter attached to unit
Pre-storage leukocyte reduction at blood center
Indications:
Prevention of HLA/WBC alloimmunization
Prevention of recurrent non-hemolytic febrile reactions
Prevention of CMV transmission in select groups of patients

34. Saline washed pRBC 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
Processing: manual and automatic methods
Storage: once washed, 24-hour outdate
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

35. Platelet Concentrates
Description:
Random donor unit contains 5.5 X 1010 platelets suspended in ml of plasma
Apheresis donor unit contains
3.0 X 1011 platelets suspended in ml of plasma
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*
Storage:
Stored at 20-24º C on a rotator
5-day outdate

36. 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)

37. Plasma Description: Storage: 1 year at -18ºC
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
Storage: 1 year at -18ºC
Outdate once thawed (1-6ºC)
24 hours for FFP or 24FP
hours if relabeled as Thawed Plasma

38. Plasma Indications: Contraindications:
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)
Contraindications:
Volume expansion
Treatment of nutritional deficiencies

39. Cryoprecipitate
Description: each unit consists of ml residual plasma
80 units of factor VIII
250 mg of fibrinogen
Storage: 1 year at -18ºC
Indications:
Hypofibrinogenemia (≤ 100 mg/dl)
Dysfibrinogenemia
Factor XIII deficiency - rare
MTP

40. Irradiated Units Products irradiated:
Whole blood, packed red cells, platelets, and granulocyte concentrates
Indications: preventing graft versus host disease
Immunocompromised patients
Directed donations from blood relatives
Premature infants ≤ 1200 gms
Fetuses receiving intrauterine transfusions
Neonatal exchange transfusions
Processing and final product:
Irradiate with 2500cGy
Mitotic capacity of lymphocytes is reduced or eliminated without significant functional damage to other cellular elements
Storage:
Red cells outdate 28 days from irradiation (or original expiration if less than 28 days)

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

42. 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

43. 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

44. Back to the case You initiate that massive blood transfusion protocol
You order rapid infusion as units arrive
Lab results come back

45. Labs/Studies
Venous blood
Total 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 = gm / dl)
Sodium (Na+) = 138 mEq / L
(normal = mEq / L)
Potassium (K+) = 5.1 mEq / L
(normal = mEq / L)
Chloride (Cl-) = 104 mEq / L
(normal = mEq / L)
BUN = 27 mg / dl
(normal = mg / dl)
Creatinine = 1.9 mg / dl
(normal = mg / dl)
Glucose = 165 mg / dl
(normal = 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 = mmol/L)
Arterial blood
Blood pH = 7.28
(normal = )
pCO2 = 31 mm Hg
(normal = 40 mm Hg)
pO2 = 78 mm Hg
(normal = mm Hg)
Hemoglobin - O2 saturation = 88%
(normal = %)
[HCO3-] = 14 mEq / L
(normal = mEq / L)
Urinary output in first 60 minutes in ER was 20 ml (color was dark yellow). Urine specific gravity = (normal = ). 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

46. 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.

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

48. Case Presentation #2 HPI PMHx
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

49. Case Presentation #2 Medication: FmHx/SocHx/RoS: unremarkable
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

50. 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)
5.2
9.8
220
29.4
4.9
140
102 25
0.80 19 80
Ca: 9 mg/dL
Alk: 100 U/L
Bili: 0.4mg/dL
AST: 5 U/L
ALT: 2 U/L
Protein: 7g/dL
Albumin: 4g/dL

51. 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

52. 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

53. Patient’s T&C 4+ A+ A Forward Reverse Anti-A Anti-B Anti-Rh Type?
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
Forward
Reverse
Anti-A
Anti-B
Anti-Rh
Type?
A Cell
B Cell 4+ A+ A

54. 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

55. 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?

56. Suspected Transfusion Reaction
STOP THE TRANSFUSION

57. 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

58. 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

59. Transfusion reaction work-up
Forward
Reverse
Anti-A
Anti-B
Anti-Rh
Type?
A Cell
B Cell O- 4+ O
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?

60. 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

61. Acute hemolytic transfusion reaction
Signs and symptoms
Laboratory findings
Triad
Fever/Chills (>2˚F or >1 ˚C)
Flank pain
Dark urine
Additional
Tachycardia
Dyspnea
N/V
Bleeding
Hypotension
Feeling of impending doom
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

62. 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

63. Risk of transfusion reaction
Carson J L et al. Ann Intern Med doi: /

64. 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!!

65. 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.

66. Case presentation #3 PMHx Medication FmHx/SocHx/RoS Birth history
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

67. Case presentation #3 Physical exam Routine labs and exams ordered
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

68. 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

69. Type and Screen Results
Forward
Reverse
Anti-A
Anti-B
Anti-Rh
Type?
A Cell
B Cell O- 4+ O
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?

70. 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 identified
Other Minor blood group antibodies are implicated as well – however Anti-D alloantibodies are the most immunogenic/potent
Walk through of mechanism/pathophysiology of disease

71. 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
Walk through of mechanism/pathophysiology of disease

72. A B A B Minor blood groups e k D Jka Fya E c K Fyb Jkb C 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 e k D
Jka B
Fya E c K
Fyb A B
Jkb C

73. Development of fetal allo-antibodies
Mother must be antigen negative
Antigen positive individuals will not form antibodies to self
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
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
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
Self explanatory

74. Development of fetal alloantibodies
Rh+
Rh - Rh
Unlike A and B antibodies,
many minor antibodies are
not naturally occurring
D d
d d
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…
Second Child
D d
First Child
D d
d d
d d
Rh +
Rh +
Rh -
Rh -

75. 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 )

76. 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
More detailed explanation

77. Hydrops and Erythroblastosis Fetalis
Two pics on top – hydrops
Lower left – precursor RBCs in peripheral blood showing erythroblastosis fetalis
Lower right – extramedullary hematopoesis

78. 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

79. Acute Bilirubin Encephalopathy and Kernicterus

80. 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
Usual approach to diagnosis – did not apply to patient due to third world health care during first pregnancy

81. 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
Self explanatory

82. Algorithm for HDFN

83. 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
More than one product… have options
Some IM only
Some IV or IM

84. 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
Can tell anecdotal story about the 72hr rule and how it originated in Singapore by testing inmates  investigators only allowed re-admittance 72hrs after administration of Rh+ cells for administration of RhIg
Remind them that you can give RhIg two weeks out or later if neglected to administer within 72 hrs

85. 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
Previous US looking for blood in trauma setting – may miss details of fetus

86. …You decide to go into psych.
Questions ?
All of these blood bank situations and complications have driven you so crazy......
…You decide to go into psych.