Anaemias. Blood groups and transfusion

Dr. Istenes Ildikó

SE, I.sz. Belklinika, Budapest

What are we going to talk about?

• Symptoms of hematological disorders

• Normal haemopoesis

• Anaemias

• Blood groups

• Transfusion

Normal haemopoesis

• Where: bone marrow• process:

– Stem cells proliferate, differentiate– Differentiation:

– Myeloid cells: in the bone marrow– lymphoid cells: in the lymph nodes and

thymus

• Regulation: cytokins– Bind to specific receptors of cell

progenitors, which activate intracellularmessenger mechanisms that lead toproliferation and differentiation.

– E.g.:– colony stimulating factors (EPO,

thrombopoetin, G-CSF etc), interleukins, tumor necrosis factors, interferons, chemokins)

Red blood cells

Platelets

white blood cellsNormal

Bone

marrow

Location of hematopoesis

Differentiation: –Myeloid cells: in the bone marrow

–lymphoid cells: in the lymph nodes and thymus

White blood cells

Peripheral blood count

Leukaem

ia, infectio

n

Hem

olysis

Normal peripheral

blood smear

Multiple myeloma-

Rouloux formation due to paraproteins

Acute myeloid leukaemia

Chronic myeloid leukaemia

Normal

Haematogical disorders• Non oncological disorders:

– Quantitative disorders• (anaemia-polyglobulia, thrombocytopenia- thrombocytosis, leukopenia- leukocytosis:

in many cases not caused by hematological, but other diseases)

– Disorders of the hemostasis• (hemophilia, trombophilia)

• Oncohaematological disorders– Myeloid cell line:

• Myeloproliferative diseases

• Myelodysplasia syndrome

• Acut myeloid leukaemia

– Lymphoid cell line:

• Lymphoproliferative disorders

• lymphoma: if malignant cells are mainly in the lymph nodes (Hodgkin/Non-Hodgkin lymphoma)

• Leukaemia: if malignant cells are mainly in the peripheral blood. (chronic lymphoid leukaemia, acut lymphoid leukaemia)

Peripheral blood count

Parameter Normal Parameter Normal

RBC count (T/L) male: 4,3-5,5female: 3,6-4,7

WBC (G/l) 3,7-10,0

Hemoglobin (g/l) male:135-160female: 120-150

Neutrophil (%) 40-80

Hematocrit male: 0,41-0,53female: 0,37-0,44

Lymphocyte(%)

10-45

MCV (mean cellvolume) (fl)

80-95 Monocyte(%) 3-12

MCH (mean cellhemoglobin, pg)

28-35 Eosinophil(%) 0-7

MCHC (mean cellhb conc, g/l)

315-355 Basophil(%) 0-2,5

RBC distributionwidth(RDW, %)

11-14 Thrombocyte(G/l)

150-450

Reticulocyte (%) 0,4-1,7

Quantitative abnormalitiesRed blood cells-Anaemia- polycytaemia

White blood cells-Leukocytosis-Leukopenia

Platelets-Thrombocytosis-thrombocytopenia

When to callfor a hematologist?

Symptoms suspicious for haematological disorders!

Complaint Reason Symptoms at physical

examinationFever, weight loss, night sweat Activity sign in

malignancy (B symptoms)

Signs of weight loss

Weakness, dizziness, decreased

ability to exercise, effort

dyspnoe, chest pain, ankle

oedema

Anaemia

Paleness, icterus, ankle

oedema, tachyardia, systolic

murmur

Recurrent oral infections, skin

infections, other diseases with

fever

Decreased immun response

(neutropenia)

Eg.: soor oris, aphta, other

signs for infection

Long lasting nose

bleeding/menses, spontaneous

hematoma, bleeding

Impaired hemostasis Petechia, hematoma, wet

purpura in the oral cavity

Abdominyal dyscomfort, pain,

early fullness

Liver/spleen/lymph node

enlargement

Hepato/splenomegaly,

peripheral and retroperitoneal

lymph nodes

Double vision, headache,

dizzyness

Hyperviscosity Vessel alteration in the retina

Weakness , paraesthaesia in the

limbs, segmental pain

Central nervous system

involvement (root

compression)

Decreased/absent reflexes,

paresthesia, paresis

Plummer Vinson syndrome: iron/vitamin deficiencyGlossitis, tongue burningCheilitis, difficulty in swallowing

Stomatitis aphtosa

Gothic palate-

could be present in sphaerocytosis e.g.

Hyperplastic gingiva in monocytic/myelomonocytic

leukaemia

Pale limbs

Oral bleedings:

Wet purpuras in thrombocytopenia

Disorders of red blood cell count

• Anaemia:

Hemoglobin, hematocrit or RBC count falls below normal level

Hgb <13,5 g/dl (male) Htk <40% (male)

<12 g/dl (female) <37% (female)

• Polycytaemia

Hemoglobin, hematocrit or RBC count rises above normal level

Prevalence of anaemiaApprox. 1/3 of the population (approx. 2 000 million)

Population prevalence (%)

All 32,9

Men (15-60years) 12,7

Children (0-5 years) 43

Children (5-15years) 25,4

Elderly (over 60 years) 23,9

Non pregnant women (15-49years) 29

Pregnant women (15-49 years) 38

Anaemia

• Not a disease, it is a SYMPTOM

• It is not enough to normalize hemoglobin levels, the cause of anaemia has to be found.

RBC destruction

-reticulo-endothelial system (RES)

-monocyta-macrophag system of the spleen

Red blood cellproduction needs:

Elements:

• Iron, Vitamin B12, Folicacid

Factors:

• Erythropoetin

- produced in the kidney

- to hypoxia

- binds to the receptor of the pluripotent stem cellwhich then differentiatesinto red blood cell

Background Etiology Type of anaemia

I. Impairedproduction

1. Defect of the erythropoeticstem cell

2. Impaired DNA synthesis

3. Impaired Hgb synthesis4. Decreased erythropoetin5. Decreased hemopoesis (due

to lack of space)

6. Multifactorial

Aplastic anaemiaMyelodysplastic syMegaloblastid anaemia due to decreasedvitamin B12 or folic acidIron deficiency anaemiaRenal anaemia, Bone metastasis of tumors, malignanthematological diseasesAnaemia of chronic diseases (tumor, infection, rheumatic diseases)

II. Increaseddistruction

1. Red blood cell defect

2. Eytraerythrocyter causes

Corpuscular haemolytic anaemias:-membrane defect-enzyme defect-haemoglobinopathy

Extracorpuscular haemolytic anaemias-iso-/autoantibodies-drugs-infections-physical/chemical noxa-metabolic disorders

III. Blood loss bleedings Anaemia due to bleeding

IV. Impaireddistribution

Red blood cells are pooled in theenlearged spleen

Hypersplenic syndrome

Iron deficiency anaemia• 80% of anaemias (80% female: menses, pregnancy...)

• Iron:

– Absorbed in the proximal small intestine, transported by transferrin and stored as ferritin

– Needed for the synthesis of hem

– Iron deficiency causes: hypochrom, microcytaer, hyporegenerative (reticulocyte count is low) anaemia

• Etiology of iron deficiency

– Decreased intake (children vegetarians)

– Decreased absorption (after gastric resection, bowel diseases)

– Increased need (growth, pregnancy)

– Iron loss due to bleeding (GI bleeding, surgery, blood donation, too often blood sampling, haemophilia)

• Symptoms

– Fragile hair, hair loss, dry skin, itching

– Pale skin and mucosa, weakness, effor dyspnoe, systolic murmur

• Therapy: – Treat the cause

– Iron supplementation (oral, iv. if needed): effect: 3-4 weeks: hb rise of 20g/l, normal ferritin 3-6 months

Diagnose?

Which parameters will help?

Anaemia: RBC count, MCV, MCH, reticulocyte,

iron parameters: ferritin↓, transferrin↑, transferrin

saturation ↓, total iron bindig capacity ↑Is it enogh?

Stages of iron deficiency

Prelatent latent manifest(iron storage is deficient) (hemopoesis is iron deficient) (iron deficiency anaemia)

Serum ferritin and bone marrow iron is low

Iron absorption is increased

Serum iron ↓, transferrin↑

Number of bone marrow sideroblasts ↓

Hb, Ht, RBC ↓

As time goes by….

• Mild, microcyter, hypochrom anaemia, which does not improve to oral iron supplementation….

Anaemia of chronic disease

ACD: anaemia of chronic disease

1. Increased RBC destruction: due to mild extracorpuscular hemolysis

2. Decreased bone marrow RBC production

a) Iron is scarcely available to hemopoesis:

Hepcidin (produced by the liver, increased levels in inflammation)

- inhibits iron absorption from the gastrointestinum → decreased ironabsorption in inflammation (oral iron suppl is less effective)

- inhibits mobilisation from hemosiderin (iron from RBC-s is stored in the

RES and in the macrophages of the inflammated tissue as hemosiderin) → iron storageincreases, reuse of iron decreases

- transferrin is not increased in proportion to the need A → irontransport does not increse paralelly to the need

b) Erythropoetin production is not increased despite tissue hypoxy, and theeffect of EPO decreases as well.

c) Direct inhibition of bone marrow RBC production : due to elevated IL- 1 and TNF alfa

Differential diagnose of ACD and iron deficiency anaemia

Iron deficiency ACD

Serum iron Very low Normal or low

Serum transferrin elevated low

Serum iron bindingcapacity

elevated low

Serum ferritin low elevated

Solubile transferrinreceptor

elevated Normal or low

Transferrinreceptor/ferritin ratio

Highly increased >4 low ˂1

Bone marrow ironstorage

missing increased

Ferritin should be evaluated with CRP!

Megaloblastic anaemiasVitamin B12 and/or folic acid deficiency

→DNA- synthesis, maturation of the nucleus is impaired

Vitamin B12 storage of the liver is enough for 3 years

Absorption of Folic acid: Jejunum: as a monoglutamate, after deconjugation

from polyglutamate.

Drugs (oral anticoncipients) may inhibit deconjugation,

thus causing folic acid deficiency)

Folic acid storage of the liver is enough for 3 months.

What symptoms would you expect?

Megaloblastic anaemiasSymptoms of B12 and/or folic acid deficiency: • Symptoms of chronic anaemia

very pale (elderly) patient with very low hb, which is tolerated relatively well(mouth: atrophic glossitis (burning tongue), angular cheilosis)

• Neurological complaints due to neuropathy: (in case of B12 deficiency)– Tingling, weakness, pain…

• Gastrointestinal symptoms

Diagnose: macrocytic anaemia (RBC↓, MCV↑, MCH↑), haemolysis: LDH, Indirect bi↑,

B12, folic acid measurement (Schilling test is no longer available- confirmsintrinsic factor deficiency in anaemia perniciosa)

Therapy:treat the cause, if possiblevitamin supplementation (usually both):

B12 (oral, parenteral)folic acid (oral)

Cause B12 deficiency Folic acid deficiency

1. Decreased intake Vegetarian/vegan dietB12 source: meat, fish, egg, milk etc.Elderly: decreased intake and absorption

undernutrition (alkoholism, „tea and toast” diet of theelderly)Source: liver, yeast, spinach, green leaf vegetables, nuts

2. Increased need - Hemolysis- pregnancy

3. Decreasedabsorption

Intrisic factor is missing: - anaemia perniciosa- After gastrectomy

Drugs interfering withdeconjugation (someantiepileptics, oralanticoncipients, metformin)

Severe malabsorption Malabsorption

Intestinal helminthiasis (helmints use it)

CoeliakiaIleitis terminalis

Ileum resesctionGastrointestinal bypass surgery

Lymphoma infiltrating the intestine

4. Egyéb Long term antacid and PPI treatment,Metformin

Folic antagonist therapy

74-year-old female• Examination because of recurrent gastrointestinal complaints for

years (cramps), – virtual colonoscopy negative in 2011,

– Maldigestion

– H. pylori eradication

2014

• B12 deficiency, anaemia- B12 supplementation- Anaemia is notimproved

• ???

• Bone marrow problem? – Peripheral flow cytometry: lymphoproliferative disease, mantle cell

lymphoma?

• Repeated colonoscopy (not virtual):– colon polyp histology: mantle cell lymphoma

• Bone marrow biopsy: lymphoma infiltrates bone marrow.

Renal anaemia

chronic renal failure causes

Erythropoetin (EPO) deficiency

Symptoms:

Anaemia…

Renal failure (fluid retention…)

-reticulocyta↓

-Dg.: anamnesis+symptoms

-Th.: recombinant human EPO

(rule out iron deficiencyanaemia)

Aplastic anaemia• Gross reduction or absence of haemopoietic precursors in all 3 cell

lineages resulting in pancytopenia in peripheral blood• Rare 5/million/year• Types

– Hereditary• Fanconi anaemia

– Aquired: • Idiopathic• Secondary (post viral, drug induced, irradiation, chemotherapy)

• Clinical features reflect pancytopenia: – Mucosal bleeding, infections, anaemia– Normochrom, normocyter anaemia, reticulocyte low

• Treatment– Supportation (transfusion, antibiotics)– Allogenous bone marrow transplantation– Immune suppressive treatment

Differential diagnose of anaemias based on mean cell volume (MCV) and mean cell hemoglobin (MCH)

Hypochrom microcyticanaemia

Normochrom normocyticanaemia

Hyperchrom macrocyticanaemia

MCH↓+MCV↓

MCH˂28pg/l

MCH+MCV normalMCH 28-35pg/l

MCH↑+MCV↑

MCH˃35pg/l

Ferritin norm or ↑:Hemoglobinopathy

Iron and ferritin↓:Iron deficiency anaemia

Reticulocyte↑:Haemolytic anaemiaAnaemia due to bleeding

Reticulocyte↓: AplasticanaemiaRenal anaemia

Reticulocyte normal:Megaloblastic anaemia (B12, folic acid deficiency)

Reticulocyte ↓:- myelodysplasia syndrome- drug (eg. hydroxiureatreatment) - pregnancy, hypothyreosis

Iron↓, ferritin↑, ret ↓: anaemia due to inflammation, infection, tumor

Etiology? : decreased RBC, Hb, MCH, (MCV), reticulocyte, ferritin

Ferritin should be evaluated with CRP!Reticulocyte: reflects bone marrow function:

Hyporegenerative: low, hyperregenerative: high

How long do they live?

Giant tortoise:

Up to 150-170 years

African elephant:

Up to 60-70 years

Tiszavirág: Ephemeroptera

1 day

Ant: 1 year

Fly: 3 months

rabbit

8 years120days

Haemolytic anaemias

The life of RBCs shortens to couple of weeks/days (normal: 120 days)

Clinical course:

chronic:Signs of anaemiaicterus, splenomegaly

acute: Fever, shivering, collapse, icterus, headache, abdominal pain, backpain, hemoglobinuriaa

Pl: hemolytic transfusion reactions Pl: hemoglobinopathies

Where are RBCs broken down?

Normally: ExtravascularRBC destruction extravascularly in theRES (reticuloendothelial system) of thespleen

In case of hemolysis: in the liver, bonemarrow as well, and if their capacity is fulfilled

Intravascular hemolysis:RBCs are destroyed within the blood vessel

↓free hgb binds to haptoglobin

↓free haptoglobin↓ (measurable value)

↓free hgb will be present in the plasma, which

transforms to haematin, which is transferred to RES by haemopexin → finally haemopexin level↓

↓Free hgb is filtrated and reabsorbed in the kidney

If reabsorption capacity is overrun↓

hemoglobinuria (red urine)

Laboratory alterations in hemolytic anaemia!

Laboratory parameter reason

Hb↓, Ht ↓ Anaemia

LDH ↑, serum iron↑ cell destruction

Indirect bilirubin ↑ (icterus), urobilinogenuria

Hem destruction is increased → non-conjugated, albumin-bound (indirect) bilirubin is elevated

Haptoglobin ↓ Hgb coming out of intravascularly destroyed RBC binds to haptoglobin

Hemoglobinuria (brownishurine)

In case of massive intravascular hemolysis, whenthe tubular reabsorption capacity of the kidney is overrun

Reddish serum Free haptoglobin in the serum in case of intravascular hemolysis

Retikulocyte number↑ In case of intact bone marrow function: erythropoesis is increased, ratio of prematureRBC forms is increased

Etiology- hemolytic anaemia example

RBC defect=corpuscularHemolyticanémia

-membrane defect inherited: Spherocytosis, elliptocytosisacquired: paroxysmal nocturnal hemoglobinuria

-enzyme defect Glucose- 6-phosphate dehydgrogenase defectPiruvate kinase defect

-hemoglobinopathy Qualitiative problem: sickle cell anaemiaQuantitative problem: Thalassaemia (alfa, beta)

Eytra-Erythrocytercauses=Extra-CorpuscularHaemolyticanaemia

-alloantibodies hemolytic transfusion reactionMorbus hemolyticus neonatorum

-autoantibodies Autoimmune hemolytic anaemia- cold-warm- Idiopathic- secundary

-drugs Immune-mediated: Penicillin, kinidin, metildopanon immun-mediated= oxidative stress: salazopyrin

-infections Malaria, clostr. Perfringens sepsis

-mechanic: RBC fragmentationsyndrome

Cardiac origin: artificial valve, graftMicroangiopathic: TTP, HUS, DIC, HELLP, vasculitisArteriovenosus malformations

-physical- chemicaleffects

Metabolic disorder: Wilson disease (elevated copper)drugschemicals: lead, arsene, chlorineSevere burning

TTP. Trombotic trombocytopeniic purpura, HUS: hemolytic uraemic syndrome, DIC: disseminated intravascular coagulation)

Corpuscular haemolytic anaemia

Mentzner index: MCV (fl)

RBC (million/l)

Thalassaemia: < 13Iron deficiency anaemia: > 13

Spaerocytosis- membrane defect

Gothic palateG6PD deficiency

Heinz body: dense bodies composed

of precipitated hb

Hemolytic shubs to oxidative stress

Bite cells:

Heinz bodies removed by the spleen

Sickle cell anaemia

Sickle cells obstruct vessels,

causing organ infarctions-

hepatoplenomagly

Acantocytes in

piruvate kinase deficiency

Immunmediated hemolytic anaemia

• Hemolytic anaemia caused by antibodies• Allo-antibody

• Auto- antibody

• Antibodies against RBC:

- IgM- complete antibody: it can agglutinate (big enough to cover the distance between 2

RBC-s), Cold antibody (optimum temperature is below 22C)

- IgG -incomplete antibody: it can’t agglutinate, just cover the cell warm antibody (optimum temperature is at 37C)

How can we show the presence of incomplete antibodies?

•Coombs test= antiglobulin test-direct Coombs test: direct antiglobulin test (DAT)

Shows antibodies that cover RBCs

-indirect Coombs test:

shows free antibodies in the serum

•Coombs’ serum:

- serum that contains antihuman globulin:

antibodies against IgG and complement (retreived from a rabbit or other animal previously immunized

with purified human globulin)

=Antihuman

globulin

Coombs tests

Hemolysis caused by allo-antibodies:… see later

- incompatibile transfusion: e.g. ABO incompatibile transfusion

- morbus hemolyticus neonatorum…

Hemolysis caused by auto-antibodies= autoimmune hemolytic anaemia

Warm AIHA: caused by incomplete IgG autoantibodies

- 45% idiopathic, 55% secondary (NHL, M.Hodgkin, SLE, drug, virus)

-IgG binds at body temperature to RBCs, that are destroyed in the liver and in thespleen → erythropoesis compensates for a while, then→ symptoms

Cold AIHA: caused by complete IgM autoantibodies

- Cold temperature causes acrocyanosis, hemolysis

- Antibodies activate complements (DAT test: complement coverage)

- acut: usually preceded by infection

- chronic: idiopathic or secondary (B cell lymphoma)

Treatment of AIHA: treat the cause (e.g. leave the drug):

-warm AIHA-steroid, Iv Ig, immunsuppressants, splenectomy

-cold AIHA: avoid cold, immunsuppr., plasmapheresis

Search for a primary cause!!!

Case report 55-year-old male

• Chronic lymphoid leukaemia, comes to regularcheck-up

• Anaemia

• AIHA? – common in CLL

• LDH, Tbi, haptoglobin norm

• ???

• RDV: Weber positive

• Gastro-colono:source of bleeding is not found

• Capsule endoscopy: small intestine: angiodysplasia

Summary

• Anaemia is a symptom, find the cause!

• Differential diagnose:

– Hypochrom- normochrom- hyperchrom (MCH)

– Reticulocytes

– Iron parameters (ferritin + CRP)

– Other: observe parameters for hemolysis

• Treat the cause

BLOOD GROUPS

ABO Basics

• It is the most important blood group system

• Blood group antigens are actually sugarsattached to the red blood cell.

• Antigens are “built” onto the red cell.

• Individuals inherit a gene which codes for specific sugar(s) to be added to the red cell.

• The type of sugar added determines the blood group.

Group 0: nothing

H antigen: codes a fucosyl transferase

(responsible for fucosylation)

Group B: Galactose

Group A: N-acetyl galactosamin

Fucose is important:

if the H antigen is missing-

fucose is absent:

Bombay fenotype

with anti H, anti A, anti B

antibodies.

transfusion only with Bombay

fenotype

H

9. chromosome: the genes encode

a certain glucosyl transferase

Inheritance of ABO blood groups

• Follows the mendelian rule

• On the ABO locus there are 3 allels: A, B, 0

• A and B are cododominant

• A and B dominates over 0 gene

• The „O” gene is non-functional, it does not produce a detectable antigene (no sugar transfer)

• Genes are expressed gradually: – 5-6 week embryo: detectable

– Newborn: weaker compared to later

series…

Who can be the father?

At the science lesson, they check

AB0 as an exercise…

Child Mother Man 1 Man 2

A A 0 AB

AB B A B

0 A AB A

0 AB A 0She is not the biological mother- assuming that the child is not having any

Hematological diseases that cause Ag weakening…

ABO Type Frequencies In Europe and Hungary.

ABO Type Europe Hungary

O 35-42% 31%

A 38-42% 42%

B 5-15% 18,5%

AB 3-6% 8,5%

Jakab Judit dr

A, B, H antigenes- outside RBCs• Diluted in different body fluids (depending on the individual’s

secreting feature)– Saliva, serum, breast milk, urine, amnion fluid etc.

• Not found on the surface of granulocytes, but– Granulocyte transfusion also has to be matched to AB0 group due to RBC

contamination.

• Lymphocytes– Adheres to the surface from the plasma

• Thrombocytes– They synthetize it, present on megacariocytes as well

– Passive adsorption on the surface

• Vascular endothel, epithel, primary sensory neuron – Independent of secreting feature

• In cells as wellDr. Jakab Judit

(! Helps to determine the ABO blood

groups, if it is uncertain from the

serum due to eg. ABO

mistransfusion, stem cell

transplantation, antigen weakening,

hereditary weak antigens

ABO antigens and regular antibodies

•Individual’s will form immune antibodies to ABO blood group antigens they do not

possess: Landsteiner’s rule

•Substances are present in nature which are so similar to blood group antigens

which result in the constant production of antibodies to blood group antigens they

do not possess.

•Critical for understanding compatibility between ABO blood groups.

BOMBAY?

Anti A, Anti B

Anti H

Regular/natural antibody

• Regular= if it is missing, the reason has to be found

• Natural: it is not produced as a response to a RBC antigen– It is produced as a response to the antigens of the individual’s gut flora

• The production – starts at 3-6 months of age

– The amount varies depending of age, infection, vaccination

• It can be found in the plasma, saliva, colostrum

• Usually IgM type (can be IgG, IgA as well)– They bind complements and cause immediate hemolytic transfusion

reaction

– In vitro cold type, but they react very well on room temperature and cause direct agglutination

Rh blood groupName comes from Rhesus monkey:

The RBC-s of the Rhesus monkey was injected in rabbits

and guineapigs →

they produced antibodies against these RBCs →

these antibodies reacted 85% of human RBCs:

these are called Rh positive, the remainings are negative

Inheritance of Rh blood groups

• Genetics:

– D, C, c, E, e antigens (1. chromosome)

– Inherited in triplets: CDE, cde

– Independent from ABO blood group system

– Dominant inheritance

Characteristics of Rh antigens• Rh antigens are proteins

• ONLY RBCs carry Rh antigen

• Present in 5-6 week-old embryo

• The D antigen is the most immunogenic: anti-D is produced against it

• There are variants: variant D antigens give weaker agglutination to anti Dthan expected, meaning:

RBC with variant D antigen mixed with standard anti-D will give a negative or uncertain result, but

the direct Coombs test will be positive. – Changes in DNA: insertion, deletion, missense mutation- the protein is modificated on

the surface which is responsable for the weaker reaction

– Antigen expression is weakened, or changed- standard anti D test will be negative

Clinical consequences of D variant features:

- If it is a donor/ fetus RhDvar :

- it is considered positive, because it can immunise an Rh negative

individual

- If it is a recipient/pregnant woman RhD var:

- it is considered negativ, because it can produce anti-D antibody to Rh

positive RBC

Antibodies of the Rh system

• Usually immun-antibodies, irregular antibodies (produced to an antigen- eg. Transfusion, pregnancy)

• Can be autoantibodies (anti-e, anti-C)

• Transfusion: – RhD matching is important and sometimes the other (C, c,

E, e) antigens have to match as well

– Cause transfusion reaction

• Ususally type IgG but can be IgM or IgA

– Warm antibodies: optimal temperature is 37C

– Usually don’t bind complements

– IgG crosses the placenta- causing morbus hemolyticus neonatorum

• CAVE: pregnant women and D variants!

Rh negative pregnant women with Rh positive fetus:

Morbus hemolyticus neonatorum:

Fetal hemolysis: fetal RBCs are destroyed

Mild case: Anaemia, icterus

Severe case: Kernicterus, CNS damage, hydrops fetalis

(heart works harder due to anaemia, heart will be insufficient, causing oedema…)

What can be done?

Give the Rh negative mother human anti D globulin:

it binds the fetal Rh positive RBCs that went into the mother’s blood

the Rh positive fetal RBCs will not provoke immunisation in the mother

When?

28-32. gestational weeks,

within 72 hours after giving births or abortion

Main blood groups: ABO, Rh

can cause transfusion reaction or morbus

Hemolyticus neonatorum:

Kell (K),

Duffy (Fy),

Kidd (Jk)

others:

MNS, Lewis, P , I etc…

Other blood group systems:

Thrombocyte and granulocyte antigens and antibodies

Thrombocyte antigens

- RBC antigens (A, B, Lewis, I, P) (Rh is not present but an Rh negative female can

only receive Rh negative thrombocyte due to RBC

contamination of the trombocyte pack)

- HLA 1 (human leukocyte antigen)

- HPA1a, 1b (human platelet antigen)

Clinical significance of anti-platelet

antibodies:

- Thrombocyte refractory stage (no

platelet count rise after thr transfusion

- Neonatal alloimmun thrombocytopenia

- Immunthrombocytopenia

- Drug induced thrombocytopenia,

- Post transfusion purpura,

- Non hemolytic transfusion reaction

with fever

Granulocyta antigens

- HNA 1, 2, 3, 4, 5

Clinical significance of anti-granulocyte

antibodies:

- Autoimmun neutropenia

- Neonatal alloimmun neutropenia

- TRALI: transfusion associated lung

injury

- Non hemolytic transfusion reaction

with fever

Determining blood groupIn the laboratory:

1.AB0, RhD

2.Direct Coombs teszt (DAT)

3.Antibody screening and identification

Autoagglutination control

Examined RBCs mixed together with the

examined serum without reagent

The whole examination is valuable if:

autocontrol is negative.

Autocontrol is positive if:

• RBCs are covered with:

autoantibody

alloantibody

anti-drug antibody

- Serum is making sympexis

„Bed-side” test

A Rh positive

TRANSFUSION

What are we going to discuss?

• Definition

• products

• Compatibility

• Indications of transfusion

• Complications of transfusion

– Hemolytic

– Non-hemolytic

Transfusion- definition

• The transfer of blood or blood components from one person (the donor) into the bloodstream of another person (the recipient).

What can be transfered?

• Red blood cells

• Thrombocyte

• Fresh frozen plasm

• (granulocyte)- rarely used, in severe infections of neutropenic patients

Products

Packed red cells

• Stored at 2-6 ºC for up to 35

days

– 1 unit -> Hb rise by

1g/dl in adult

• compatibility:

– ABO, Rh, other

Platelets

• Stored at room temperature for

up to 5 days, kept agitated

• Thr rise by 12-14000/ul

• Pooled (from more donors)

• Apheretic (from 1 donor)

• compatibility: – ABO (in Rh negative females Rh as

well)

Fresh frozen plasm

• Stored at -30 ºC for up to 1

year

• Clotting factors, Protein C, S,

fibrinogen,

• ABO compatibility:

• Indications:– Massive blood transfusion

– DIC

– Coagulation defect with no

available factor concentrate

Other plasma products

Blood products derived

by fractionation of

plasma:

• albumin,

• Factor VIII

concentrate

• Factor IX concentrate

• Human Ig

• Cryoprecipitate

• Separated by freezing FFP, allowing it to thaw to 4-8ºC

• Re-frozen & stored at –30ºC for up to 1 yr

• Enriched with FVIII, vWF and fibrinogen

• Indications:– Fibrinogen deficiency

– DIC

Preparation of Blood Components

Donor:

Age: between 18-65

Weight: almost 50kg

healthy individual.

Male: max 5x/year

Female max 4x/year

Blood samples are examined for:

Hepatitis B antigen

HIV 1-2

Hepatitis C antibody

Lues serology

PREOPERATIVE AUTOLOGOUS BLOOD DONATION (PABD)

• convenient, predictable, safe and widely practised form of transfusion support.

• It is not available to all patients, it requires– time to pre-donate– a starting hemoglobin >110 g/lwhich effectively excludes most emergency surgery.

• hospital admission and operative dates must be guaranteed, as donated blood has a limited storage life of 35 days.

• Required hb:

– Men 110-145 g/l

– Women 130-145 g/l.

CompatibilityCompatible: AB0, Rh

Same group: eg. „A” Rh+ gets „A” Rh+ RBC

Not the same groupEg. „A” Rh+ gets „0” Rh- RBC

Plasm has to be removed („washed”) to

Get rid of the antibodies in the blood product

Eg. Anti A and B in 0 RBC product)

chosen:

Other antigens are matched as welleg: recipient: „A” Rh+ with anti-C, anti-e antibodies will

receive blood that does not contain C or e antigen

• If the antibody screening of the recipient is positive (either allo or auto

antibody) �

• Positive direct coombs test �

• If the recipient has anamnestic data for antibody; �

• Immunisation within 3 months

• Surgery performed in hypothermia; �

• Transfusion of infants �

• every situation where the possibility of immunisation is very high (eg.. MDS,

thalassaemia, sickle cell anaemia)

Not chosen:

Other antigens are not checkedYou go to the fridge and get an A+ blood to

an A+ patient,

When can you do it?

- If the direct coombs test and the antibody

screening of the recipient is negative

-No anamnestic data for positive Coombs

test and antibody screening

-- No immunisation within 3 month

(transfusion, pregnancy, vaccination,

transplantation)

Only ABO, Rh compatible

blood products can be transfused:

Recipient does not have AB to donor RBCEmergency situation

This is usually the policy

How long is it valid?

Transfusion within 3 month: 72 hours

Transfusion within 3 month: ˂72 hours

Compatible products

Recipient blood group Compatible RBC/platelet compatible FFP

0 0 0, A, B, AB

A A, washed 0 A, AB

B B, washed 0 B, AB

AB AB, washed: B, A, 0 AB

RhD positive Rh pozitive, negative RhD does not have to be consideredRhD negative Rh negative

RhD variant Based on consultation: + or -

unknown 0 Rh Dnegative, maximum 2 units

AB

Rh variant female of child

bearing potential can get

only Rh negative

In case of FFP:

Think of the plasm content!

In Hungary FFPs are derived from male donors

(to avoid antibodies of previously pregnant

women)

Rh does not have to be considered

Transfusion reactions

• Acute- within 24 hours

• Delayed- from 1-14days

– Due to previous RBC Ag exposure

• recipient was previously sensitized to an Ag- antibody titers are low- when the recipient meets this Ag again-anamnestic response takes place

– Destruction of donor RBC with the antigen

– Usually mild, self limited

– Mild fever, gradual decline of Hb, jaundice

A: Hemolytic transfusion reactions

B: Non hemolytic transfusion reactions

• Symtoms

– Fever and chills: 80%

– Back or infusion site pain

– Hypotension/shock

– Hemoglobinuria

– DIC/increased bleeding

• Lab:

– See previously

– Hemoglobinaemia (pink or red serum/plasma)

– Hemoglobinuria

– Positive DAT (direct antiglobulin test= Direct Coombs test)

Due to incompatible blood transfusion: the

recipient has a great amount of Ab-s

against donor RBC

Transfusion reactions

B/1: due to WBC contamination

– FNHTR: febrile non hemolytic transfusion reaction (citokins)

– Thrombocyte refracterity (HLA antibody)

– Transfection of WBC associated virus (CMV, EBV)

– Transfusion associated graft versus host disease (fatal)

– Immunmodulation (leukocytes)

– Posttransfusion purpura (anti HPA1a antib)

Prevention:

- getting rid of the WBC (filtration)

- irradiation (blocks immun active WBCs-in case of TA GVHD

B/2: due to plasm contamination

– Urticaria (allergy to transmitted solubile

substances)

– Anafilaxia

– Passive transmission of ABO Antibodies (in case of compatible but not

identical transfusion)

– TRALI: transfusion associated lunginjury (passive transmission of antileukocyte

antibodies)

Prevention:

• getting rid of the plasm (washing)

A: Hemolytic transfusion reactions- due to incompatibility

B: Non hemolytic transfusion reactions

B/3. Transfusion associated circularory

overload

TA-GVHD

Occurs 4-30 days after transfusion

Symptoms: fever, skin rash, liver dysfunction, diarrhoea

Bone marrow dysplasia

˃90% fatal

Transfused T lyRecipient T ly

TA-GVHDIf Child1 gives blood to the mother (or the father)

Child 1’s „yellow” WBCs

will attack mother’s „blue”

cells.

Mother cannot counter

attack, because she also

carries „yellow” HLA, so

child1’s yellow cells are not

foreign to her

transfusion TA-GVHD

No GVHD

Mother’s „blue” WBCs will not

attack child1’s yellow cells,

because she also has yellow

cells

Prevention: irradiation

HLA: human leukocyte antigen

Transfusion reactions• Acute (<24 hours)

– Immunologic:

• hemolytic, FNHTR, allergic, anaphylactic, TRALI

– Non-immunologic:

• volume overload, hemolytic (physical or chemical destruction of RBC), air embolus, hypocalcaemia, hypothermia

• Delayed (>24 hours)

– Immunologic:

• hemolytic, GVHD, PTP

– Nonimmunologic:

• iron overload

– Infectious complications

Transfusion Reactions- what to do?

• STOP the transfusion

• keep line open with normal saline, using a new set,

• Check I.D of patient, bag and cross-match form

• Refer to handbook for further management

Indication for RBC transfusion

• Absolute: life

threatening anaemia

– Circulatory insufficiency

due to blood loss

(hypovolaemia, shock)

– Impaired oxygenization

• Chest pain, dyspnoe

• Relative: all other cases

– Consider „pro”s and „con”-s.

– Things to consider:

• Lung, heart, circulation, Hb, Htk, Hb-O2 affinity, metabolism affecting O2 uptakeand release

• Transfusion

• According to international consensus:

• Hb: 70 g/l (21 % Htk), usually makes transfusion necessary

depends on: age (> 65 years), concomitant diseases(heart, lung), activity of the patient

• Over 100 g/l Hb transfusion is usually not needed.

• Aim of transfusion: stop hypoxia• 1 unit RBC concentrate produces: 10 g/l Hb or 2-3 % Htk rise)

↓↓

2 units of RBC cc will get the patient out from hypoxia.

- speed of transfusion: 2-4 ml/kg/hour.

- 2 units a day is recommended

•Therapeutic support:•Bleeding due to thrombopenia or thrombopathy („wet purpura”, inner bleeding)

•Preventive support:•At critical platelet numbers (without bleeding)

Indication for platelet transfusion

Preventive support Plt count

Stable, afebrile patient < 5-10G/l

Patient with fever, sepsis, DIC, severe anaemia, extreme

leukocytosis, deepening thrombopenia

< 20G/l

Lumbal puncture, intrathecal chemotherapy < 30G/l

Surgery, invasive diagnostics (except for sternal punction

and crista ilei biopsy)

< 50G/l

Neurosurgery, ophtalmological surgery, patient with

polytraumatization

< 100G/l

Platelet transfusion

• 4E/70kg (pooled product)

• Compatibility:

- ABO identical or compatible product.

- Rh neg female of gestational age have to get Rh negative platelet

Preventive support is contraindicated usually in: – TTP, HUS, HIT (heparin induced thrombocytopenia)

Preventive support is ususally not indicated in: – ITP (except for splenectomy if plt 10 xG/l thr)

– PTP (post-transfusion purpura)

– Thrombocyte functional problem

– Thrombocyte transfusion refractory state

• But: it can be done in case of life threatening bleeding.

Take home messages!

• Anaemia is a symptom, find (and treat) the CAUSE

• Differential diagnose of anaemia

• Transfusion:

– Blood groups and compatibility

– Double check everything to avoid incompatible transfusion!

Thank you!