Transfusion associated graft versus host disease - case history, pathophysiology, clinical features, investigations, management and prevention.

1. Journal club presentation
G.D.A. Samaranayaka

2. Case history
• 63 year old male – Heavy smoker (90 pack year)
• Past history of minimally invasive squamous cell
carcinoma of the larynx – curative radiotherapy
completed 1 year back.
• Known patient with COAD and peptic ulcer disease
• Presented to emergency medical department with acute
onset chest pain
• Diagnosed to have NSTEMI with coronary artery disease
– (TVD with LMCA >90%)

3. Case history
• On admission
– Hb – 8.5mg/dl
– WBC – 8900/μL
– Lymphocytes - 630/μL
– Renal and liver profile - normal
• Bone marrow biopsy – Normocellular marrow
• Received 4 random units of RBC

4. Case history
• Underwent CABG 1 week following admission
• During surgery – 3 units of random RCC
• Post operative period – Uncomplicated
• Discharged 4 days following the surgery
• Readmitted 5 days after surgery – with fever nausea, vomiting
and abdominal pain
• O/E
– Febrile, icteric
– Erythematous petechial rash-trunk + extremities
– Cardiac and respiratory –NL
– No lymphadenopathy

5. Case history
• Biochemical Investigations
– S.Cr – 3.7mg/dL
– ALP – 200U/L
– Hb – 8g/dL
– SGOT – 1231U/L
– SGPT – 3241U/L
– LDH – 4360U/L
– Total bilirubin – 6.3g/dL direct – 5.8g/dL
– Amylase – 477U/L Lipase - 2,145 mIU/mL

6. Case history
• Serological test - Hepatitis A,B,C & Parvovirus B19 –
Negative
• HIV- negative
• All cultures – negative
• ANA, RF & Anti-mitochondrial antibody - negative
• Started on broad spectrum antibiotics
• In the next few days
• Deterioration of renal functions
• Continuing fever
• Diarrhoea
• Worsening coagulopathy
• Encephalopathy

7. Case history
• Admitted to ICU and started on CRRT
• Pancytopaenia
• WBC - 200/μL
• Plt - 3 x103/μL
• Hb – needed multiple transfusions
• DIC and HIT were excluded
• A bone marrow biopsy - severe hypoplasia with
less than 5% overall cellularity.
• Skin biopsy of rash - vacuolar dermatitis with a
sparse lymphocytic infiltrate and abundant
necrotic keratinocytes.

8. Case history
• Continued deterioration despite aggressive medical
mx with broad-spec antibiotics, antifungal treatment,
ET-intubation with mechanical ventilation and IABP
and CVP monitoring.
• Died on 26th day following first transfusion due to
rapid declining of Hb 9.5 -> 5g/dL
• Working clinical diagnosis at the time of death
• Viral-induced haemophagocytic syndrome
• Severe drug reaction

9. However
• A pathologist had suspected TA GvHD
• First 4 RBC donors – consented for HLA typing
• DNA fingerprinting - Multiplex PCR
• Patient’s myocardium, lymph nodes, and spleen -for DNA
extraction.
• Myocardium as control – Histologically no lymphocyte
infiltration

10. Autopsy findings
• Skin biopsy – vacuolar dermatitis with a mild lymphocytic
infiltrate and abundant necrotic keratinocytes.
• Liver - portal triads - scattered lymphocytes invading bile duct
epithelium with atypia
• GI tract - complete mucosal denudation with prominent
mucosal hemorrhage, reactive epithelial changes, and focal
apoptosis

11. Autopsy findings
• Bone marrow - overall cellularity of less than 5%. The marrow
spaces were replaced by numerous macrophages
• Diffuse alveolar damage and early Aspergillus flavus
pneumonia of the lungs.
• Immunohistochemical stains for cytomegalovirus and
herpesvirus in the skin, liver, gastrointestinal tract - negative

12. • DNA fingerprinting - absence of foreign DNA
HLA Typing

13. Transfusion Associated Graft
versus Host Disease

14. Introduction
• Rare complication due to transfusion of lymphocyte
containing blood products.
• Clinically similar to BMT & HSCT associated GvHD
• Usually arises 4 to 30 days after transfusion
• Onset of symptoms occur early with signs and
symptoms of bone marrow apalasia

15. Pathophysiology
• Immunocompetent host - When
donor is Homozygous and recipient is
heterozygous for HLA haplotype (sp
Class I) – Host does not recognize
donor lymphocytes as foreign
• Immunocompromised host –
congenital/acquired- lack the ability
to reject the donor T cells
• Not removed from the recepient – T
cell engraftment

16. • Host APCs present host antigens to Donor CD4+ (HLA Class II) and
cytotoxic CD8+ cells (HLA Class I ) – recognized as foreign
• Activation of T lymphocytes, proliferation and migration (migration
by chemokines like CCL2-5, CXCL2, CXCL9-11)
• Massive release of TNF-alpha IL-1 and 2 (by Th1 TLC)
• TNF alpha – key inflammatory cytokine
• Activate dendritic cells
• Recruitment and activation of T lymphocytes (effectors) – cause target cell
apoptosis
• Directly cause tissue necrosis
• Causes a cytokine storm – wide spread
tissue damage – manifest as skin rash,
abnormal liver functions ect
• Target organ damage by both soluble
inflammatory mediators and cell
mediated cytolysis

18. Factors for developing TA GvHD
1) HLA haplotype sharing
• Donor who is HLA homologous to the recipient.
• Ex - donor is a family member, HLA matched products
• Degree of population’s genetic diversity
– The estimated risk of developing TA-GvHD
• In Japan - 1:874
• In France – 1:16,835

19. Factors for developing TA GvHD
2) Degree of recipient immunodeficiency
– Inability to reject the donor T cells
3) No. of viable T cells in transfusion
• Minimum no. of leukocytes required to cause the disease is unknown.
• Some studies <1 to 5x10^5 T cells/kg recipient body weight are safe.
• Can also occur after transfusion of even 4x10^3 T cells/kg recipient
body weight.

20. Factors for developing TA GvHD
3) CABG
– reduced mitogenic lymphocyte transformation and reduced
interleukin 2 production
– Usage of fresh blood
4) Usage of fresh products
– Contains maximal numbers of viable lymphocytes. (Blood less
than 4 days old)
– Lymphocyte viability is retained in stored red cells for at least 3
weeks
– During storage the number of viable lymphocytes declines as
does the expression of cell-surface lymphocyte activation
antigens.

21. Patients at risk of developing TA GvHD
• Recipients of donations from first or second degree relatives.
• Patients receiving HLA-matched components.
• All T lymphocyte immunodeficiency syndromes - SCID
• Intra-uterine transfusion of red cells or platelets.
• Red cell or platelet transfusions – who had in-vitro transfusions -
up to the age of 6 months after the EDD.
• Hodgkins disease
• Patients treated with purine analogues, e.g. fludarabine,
cladribine or deoxycoformycin.
• Following alemtuzumab (anti-CD52) therapy.
• Aplastic anaemia patients receiving immunosuppressive therapy
with ATG.

22. • Recipients of allogeneic SCT from the time of initiation of
conditioning chemoradiotherapy until GVHD prophylaxis is
completed and/or lymphocyte count >1 x109/L.
• Following SCT, if chronic GvHD or if continued
immunosuppressive treatment is required, irradiated blood
components should be given indefinitely.
• Patients undergoing bone marrow or peripheral blood stem cell
'harvesting' for future autologous transplant, should receive
irradiated cellular components during and for 7 days before the
bone marrow/stem cell harvest - prevent the collection of viable
allogeneic T lymphocytes - potentially withstand
cryopreservation
• All patients undergoing autologous bone marrow transplant or
peripheral blood stem cell transplant, from initiation of
conditioning chemo/radiotherapy until 3 months post-
transplant (6 months if total body irradiation)

23. Patients not at risk
• Solid organ transplant
• HIV
• Solid tumours
• Routine surgery
• Non-Hodgkins lymphoma
• Premature or term infants (unless previous IUT)
• Acute leukemia

24. Clinical Presentation
• Signs and symptoms usually begin 4-30 days after
transfusion.
• Initially fever with skin manifestations
• GI manifestations
• Hepatic dysfunction
• BM failure with pancytopenia
• Death often occurs with infection or bleeding
manifestations

25. Skin manifestations
• Erythematous Maculopapular
rash
• Pruritic
• Involves palms and soles and
spreads throughout the body
• Blisters and ulcers - in severe
cases.

26. GIT - manifestations
• Diarrhoea – secretory, voluminous (>2L/day)
• Bleeding - life threatening intestinal hemorrhage.
• Nausea, vomiting.
• Anorexia
• Abdominal pain

27. Liver
• Jaundice and hepatomegaly
• Mainly cholestatic hepatitis
– lymphocytic infiltration of portal tracts
– damage to bile duct epithelium
– consequent destruction of bile ducts.
• Increased liver enzymes
• Increased serum billirubin

28. Diagnosis
• TA-GVHD is probably underdiagnosed since it may be wrongly
attributed to
 Intercurrent infection
 Severe drug reaction
 Auto immune diseases
• Histopathological/hematological features and detection of
donor lymphocytes or DNA (mixed chimerism)

29. Histopathology of skin
• Epidermal mononuclear
infiltrates
• Basal membrane degeneration
• Necrotic keratinocytes
• Bullae formation in the
absence of eosinophils
• Eosinophils would be more
supportive of a drug reaction.

30. Histopathology of GIT
• Apoptosis of crypt epithelial
cells
• Lymphocytic infiltration
• Patchy ulceration
• Loss and flattening of
surface epithelium

31. Histopathology Liver
• Subendothelial lymphocyte
infiltration
• Portal and lobular inflammation
• Bile duct damage
• Canalicular cholestasis

32. Histopathology of Bone marrow
• Marked hypoplasia with numerous macrophages and
erythroid precursors.
• Haemophagocytosis
• Lymphohistiocytic infiltrate

33. Detection of donor lymphocytes
Techniques to establish donor lymphocyte engraftment are based
on genetic differences between the donor and the host and include
• HLA Typing
• Chromosome differences – DNA fingerprinting - Variable number
tandem repeat & short tandem repeat profiles
– Pre and post transfusion blood samples, bone marrow, affected and non
affected skin
– Hair follicle or nail clipping samples – alternative source of pre-tarsnfusion
DNA

34. Differential diagnoses
• Acute seroconversion illness due to HIV infection - fever, skin
rash, diarrhea, abdominal discomfort, jaundice and
lymphadenopathy,
• Acute viral hepatitis can present with similar illness and is
associated with progressively rising AST and ALT levels and
positive serologic tests.
• Severe drug reaction
• Dengue fever and leptospirosis

35. Management

36. Management of Suspected/proven
disease
• Poor prognosis with >90% mortality rate
• Must be treated in a specialized unit
• High dose steroids –First line - antilymphocyte and anti-
inflammatory activity
• Methotrexate & Cyclosporine-A – to prevent the disease
• Steroid refractory GvHD
– Anti-thymocyte globulin (ATG)
– Azathioprine
– Intravenous immunoglobulins
• Supportive therapy – Antibiotics

37. Management of at-risk patients received
non-irradiated blood components
• Establish which blood components are involved.
• HLA type patient and store mononuclear cell and
DNA samples.
• Careful clinical observation of the transfusion
recipient – for features of TA-GVHD

38. Prevention
• Prevention is better than cure
• Irradiation and non irradiation methods

39. Prevention
γ-Irradiation
• 25Gy all parts of the pack. – Max 50Gy
• Prevent TA GvHD by lymphocyte DNA cross linkage.
• Does not damage platelets or granulocytes
• But shortens the shelf life of red cell components -
increased plasma membrane permeability, potassium
leakage, hemolysis
– IUT / ET RCC - 24 hours.
– Other RCC - irradiate within 14days of collection and store for
not more than 14 days post-irradiation.
– Platelets. Irradiate at any stage in shelf life - no effect on total
shelf life of 5 days except 24 hours for IUT platelets.
– Granulocytes. Transfuse as soon as possible after preparation

40. Prevention
γ-Irradiation
• Controversy exists regarding the optimal prevention strategies
due to rarity of the disease
• Irradiating all units - not practicle
• Targeted irradiation – does not prevent in immune competent
patients
• Japan – genetically homogenous population
– Universal cellular product irradiation.

41. Prevention
Non-irradiation Prevention Strategies
• Leukocyte reduction has been shown to reduce the risk of TA-
GVHD, especially in a genetically diverse population, but is not
a substitute for irradiation in at-risk populations.
• Psoralen (S59) + ultra-violet A – used for pathogen
inactivation

42. Case Discussion
• Mild lymphocytopaenia and the 2 doses of
methylprednisolone 80mg for COAD
• Shared class I HLA haplotypes - critical for the development
of TA-GVHD.
• Negative DNA fingerprinting result - Number of foreign
lymphocytes in the tissue sections was very small
• The negative DNA fingerprint supports the degree of
difficulty in diagnosing

43. References
• Transfusion-Associated Graft-vs-Host Disease. A Fatal Case Caused by
Blood From an Unrelated HLA Homozygous Donor. Timothy E. Gorman,
DO, Carmen J. Julius, Rolf F. Barth, A. Ng, Melanie S.Kennedy, Thomas W.
Prior, James Allen, Larry C. Lasky;Am J Clin Pathol2000;113:732-737
• Transfusion-Associated Graft-VersusHost Disease in Severe Combined
Immunodeficiency; S Sebnem Kilic, S Kavurt,S Balaban Adim: J Investig
Allergol Clin Immunol2010; Vol. 20(2): 153-156
• Transfusion-associated graft-versus-host disease; D. M. Dwyre & P. V.
Holland :Vox Sanguinis, 2008 95;85–93
• Review - Transfusion-associated graft-versus-host disease, BJH
2002;117:275–287
• Rossi's Principles of Transfusion Medicine
• TA-GvHD management guidelines -NHS