Journal club presentation

G.D.A. Samaranayaka

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%)

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

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

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

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

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.

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

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

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

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

• DNA fingerprinting - absence of foreign DNA

HLA Typing

Transfusion Associated Graft versus Host Disease

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

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

• 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

Factors for developing TA GvHD1) 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

Factors for developing TA GvHD2) 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.

Factors for developing TA GvHD3) 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.

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.

• 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)

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

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

Skin manifestations • Erythematous Maculopapular

rash• Pruritic• Involves palms and soles and

spreads throughout the body• Blisters and ulcers - in severe

cases.

GIT - manifestations • Diarrhoea – secretory, voluminous (>2L/day)

• Bleeding - life threatening intestinal hemorrhage.

• Nausea, vomiting.

• Anorexia

• Abdominal pain

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

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)

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.

Histopathology of GIT• Apoptosis of crypt epithelial

cells• Lymphocytic infiltration• Patchy ulceration• Loss and flattening of

surface epithelium

Histopathology Liver

• Subendothelial lymphocyte infiltration

• Portal and lobular inflammation• Bile duct damage• Canalicular cholestasis

Histopathology of Bone marrow

• Marked hypoplasia with numerous macrophages and erythroid precursors.

• Haemophagocytosis• Lymphohistiocytic infiltrate

Detection of donor lymphocytesTechniques 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

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

Management

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

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

Prevention

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

Preventionγ-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

Preventionγ-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.

PreventionNon-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

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

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