1. NEONATAL THROMBOCYTOPAENIA

2. History Baby DP, white male Born 30 March 2011 (now 5 weeks old)
Normal vaginal delivery
Birth weight 2830g
Length 45cm
Head circumference 33cm
APGARS 8/10, 9/10, 10/10
Given oxygen via face mask at delivery, but no need for resuscitation

3. Mother
21 years old
Apparently 32 weeks pregnant by dates, but unsure of dates, birthweight suggests closer to term
Primigravida
Rhesus positive, RPR negative, HIV negative
Had a ?pustular rash throughout pregnancy, otherwise well during pregnancy
No medications taken other than routine vitamins, folate

4. Intern called to see baby on Day 1 of life for a ‘rash’ on the body
On examination
Baby looked like a term neonate by Ballard score
Pink in room air
No distress
Not obviously dysmorphic
Petechial rash over trunk, arms, legs
Bruising on trunk (right upper quadrant), palms and soles
Rest of examination noted to be normal
Assessment
Term male neonate with a suspected clotting abnormality/ bleeding tendency presenting on Day 1 of life
Mother not known to be hypertensive or diabetic, no history of perinatal asphyxia

5. Investigations FBC INR, PTT White cell count 22.35 Haemoglobin 18.2
Platelet count 10 (not clumped)
MCV 112.4
Platelet volume normal
INR, PTT
1.26, 39.9 (normal)

6. Mother’s results
FBC (31/03/2011)
WCC 20.8
Hb 12.0
Plts 293 (normal)

7. Management and course Admitted to neonatal ward Antibiotics, Vitamin K
Platelet (random donor) infusion ordered immediately
Cranial sonar (1 April 2011) showed possible intracerebral haemorrhage
CT brain confirmed a haemorrhage in right frontal lobe (suspected to be about a week old already)

9. Baby developed jaundice on day 3, phototherapy started, resolved quickly
No seizures in ward
Blood cultures remained negative
CRP, U&E day 3 unremarkable
Fed well
Gained weight (latest 3.36kg)
Much later noted to have abnormal ‘webbed’ toes on left foot – Xrays taken – showed polydactyly of middle and terminal phalanx 5th and 6th toes

10. Platelet count trend 21/04 24/04 25/04 26/04 28/04 04/05 12.6 10.1 137 6 44 22
Platelets given
Platelets + IVIG
Platelets + steroids

11. Expert advice sought Oncologists (08/04)
Considered alloimmune thrombocytopenia
Suggested testing mother for HPA-1a antibodies
Advised IVIG (intravenous immunoglobulin) if platelets not improving
Plasma serum screen for antibodies to platelet glycoproteins GPIIb/IIIa, GPIa/IIa and GPIb/IX and platelet antigens HPA 1a, -Ib, -3a, -3b, -4a, -5a and -5b:
NEGATIVE
Plasma serum screen for HLA Class I antibodies:

12. Head of SA National Blood Transfusion Service
Oncologist (21/04)
May be false negative results
Advised to test father’s blood also
Give normal platelets again, but would need specific platelets in near future
Consider steroids
lood
Head of SA National Blood Transfusion Service
Unusual to have no antibodies on screen performed if alloimmune thrombocytopenia
AIT unlikely
Not for HPA-1a negative platelets as diagnosis not confirmed
Head of Neonatal ICU
Try steroids
HPA-1a negative platelets/ mother’s platelets

13. Other results
Urine CMV Shell vial culture – negative
HIV negative
Rubella serology – IgG positive, IgM negative – suggestive of maternal antibodies
Xrays long bones – no periosteal reactions or features of congenital infections (rubella, syphilis)
Xray skull – no calcifications noted suggestive of CMV, Toxoplasmosis

19. Platelet production Process similar in neonates and adults
Important developmental differences
Tpo concentrations higher in neonate
But neonates with thrombocytopenia have lower Tpo concentrations than adults with similar degrees of thrombocytopenia
Megakaryocyte progenitors have higher proliferative potential, more sensitive to Tpo, and are present in BM and blood
Each megakaryocyte produces fewer platelets (smaller)
Can increase number of megakaryocytes

20. Neonatal Thrombocytopenia (NT)
Relatively common haematological problem in neonates
Defined as count < 150 x 10⁹/L
Occurs in 1-5% of all newborns
Depends on population – sick prems, ICU – more common (22-35% of all admissions)
Most have mild-moderate thrombocytopenia
5-10% - severe (<30 x10⁹/L)
urgent investigation and management needed
List of causes
Until recently many called “idiopathic”
Reduced platelet production main underlying mechanism
Newer classifications based on timing of onset

21. Classification based on timing of onset (more common causes)
Early (< 72 hours)
Late (> 72 hours)
Placental insufficiency (IUGR, PIH)
Perinatal asphyxia
Perinatal infection (GBS, E coli)
DIC
Alloimmune (severe NT)
Autoimmune (ITP, SLE)
Late-onset sepsis
NEC
Account for >80% cases

22. Less common causes Late Early Congenital infection
Kasabach-Merritt syndrome
Metabolic disease
Congenital/ inherited
Autoimmune
Congenital infection (TORCH)
Kasabach-Merritt syndrome
Metabolic disease
Congenital/ inherited (TAR, CAMT)
Thrombosis
Bone marrow replacement

23. Fetal causes
Alloimmune
Autoimmune
Congenital infection
Aneuploidy
Severe Rhesus disease
Congenital/ inherited

24. Classifying causes helps guide towards investigations and management
Often a dilemma – when to transfuse platelets?
Variation in platelet transfusion practice between centres
No study has yet shown benefit of platelet transfusion in NT
Possibly even adverse effects – increased mortality, increased risk of short-bowel syndrome in transfused neonates surviving NEC

25. Chronic fetal hypoxia
Occurs in infants of mothers with PIH (pregnancy-induced hypertension), diabetes
Manifest in fetus by IUGR
Mild-moderate thrombocytopenia
Self-limiting, usually resolves within 10 days
Mechanism
Reduced megakaryopoeisis
Associated neutropenia, increased circulating nucleated red cells, +/- polycythaemia
Tpo normal/ only minimally elevated – inadequate up-regulation of Tpo production
If severe/ not resolving after 2 weeks, look for other causes of thrombocytopenia

26. Thrombocytopenia develops very rapidly over 1-2 days
Sepsis, NEC
Thrombocytopenia develops very rapidly over 1-2 days
Often very severe (< 30 x 10⁹/L)
May recover over weeks
Uncommon causes of NT in first few days of life
Mechanism – peripheral consumption
Elevated levels of Tpo
Increased numbers of circulating megakaryocyte progenitors
Actually up-regulation of thrombopoiesis

27. Neonatal Autoimmune Thrombocytopenia
Transplacental passage maternal platelet auto-antibodies
Maternal platelets low
Immune thrombocytopenic purpura, SLE
1-2:1000 pregnancies
Much less clinically problematic than NAIT
thrombocytopenia only occurs in 10% of those with auto-antibodies
Incidence of ICH only 1%
Caesarean section not indicated
Maternal disease severity, thrombocytopenia, previous severe NT most useful indicators of likelihood of significant NT in current pregnancy

28. Infant of mother with AI disease
Platelet count checked at birth (cord/peripheral blood – NOT heel-prick)
If >150 x 10⁹/L – no further action
If platelet count low – repeat after 2-3 days – drop to lowest at this time, then rise spontaneously by day 7
Thrombocytopenia may persist for several weeks in few cases – if severe (< 30)/ prolonged, treat with IVIG – most respond promptly

29. Congenital infections
HIV, CMV, enterovirus, Rubella can cause NT
HIV
Immune-mediated destruction
Sequestration
Suppression of production, though increased megakaryocytes (ineffective thrombopoiesis)
CMV
Quite common
Thrombocytopenia 50-75% of cases
Massive splenomegaly causes sequestration
Decreased production

30. Inherited thrombocytopenia
Numerous, rare
Present in fetus/neonate
Unexplained, persistent thrombocytopenia
due to reduced platelet production
Due to abnormal haemopoeitic stem cell development
Often associated congenital abnormalities > guide investigations, diagnosis
Advances in molecular techniques aid in diagnosis and management

31. Bernard-Soulier Syndrome
May present in neonatal period though bleeding not usually severe
Mild-moderate thrombocytopenia
Giant platelets
AR pattern of inheritance
Defect in glycoprotein Ib-IX-V complex
Platelet transfusion effective, but reserve for life-threatening haemorrhage
Transfused patients may form allo-antibodies to GPIb, GPIX, GPV
Offspring of mothers with BSS may present as NAIT with severe fetal ICH
due to formation of such allo-antibodies

32. Blood film showing giant platelets

33. Wiskott-Aldrich Syndrome
WAS, X-linked thrombocytopenia spectrum of disorders
Mutation in WAS protein gene, short arm X chromosome
Over 100 different mutations
Microthrombocytopenia, eczema, recurrent bacterial, viral infections, propensity to autoimmune conditions
Present in first year, rarely neonatal period unless known family history
Bleeding due to abnormal platelet function and reduced platelet survival, number
X-linked thrombocytopenia – other clinical features absent, thrombocytopenia milder

34. Fanconi Anaemia
Usually presents after infancy, but thrombocytopenia reported in neonates
Result of a genetic defect in a cluster of proteins responsible for DNA repair
Consider diagnosis
Unexplained thrombocytopenia
Especially if typical dysmorphic features – malformations of skin, thumb, face, eyes
If parental consanguinity
Diepioxybutane test diagnostic
Treatment rarely necessary in neonatal period

36. Thrombocytopenia absent radii (TAR) syndrome
Bilateral absence of radii
Thrombocytopenia – present at birth/ within first 4 months
Both thumbs present and normal
Platelets usually < 50 x 10⁹/L
WCC elevated (sometimes > 100 x 10⁹/L) in > 90% of patients, may mimic congenital leukaemia
Associated abnormalities
Cow’s milk protein intolerance
Lower limb abnormalities
Renal, cardiac abnormalities
Platelet count seems to improve spontaneously
AR inheritance
Tpo increased, megakaryocytes decreased > presumed defect in Tpo signalling pathway

38. Some other rare inherited conditions
Amegakaryocytic thrombocytopenia with radio-ulnar synostosis
Severe thrombocytopenia at birth, absent megakaryocytes in BM
Congenital amegakaryocytic thrombocytopenia (CAMT)
Nearly always in neonatal period
Platelet count < 20 x 10⁹/L
Evidence of bleeding
Physical anomalies present in 50%
50% later develop aplastic anaemia, leukaemia, myelodysplasia
AR inheritance – mutations in Tpo receptor (c-mpl)
Stem cell transplant curative

39. Giant platelet syndromes
Many, rare
Present in neonatal period/fetus
May-Hegglin anomaly
Giant platelets, thrombocytopenia, leucocyte Dohle-like inclusion bodies
Rare cause of fetal/neonatal intracerebral haemorrhage

40. Kasabach-Merritt Syndrome
Neonatal period
50% vascular tumour diagnosed at birth, 90% diagnosed by 1 year of age
Life-threatening consumptive coagulopathy
Profound thrombocytopenia PLUS microangiopathic anaemia, disseminated intravascular coagulation
Due to enlarging vascular lesion – usually obvious – cutaneous, involving face, neck, trunk or extremities
20% visceral/retroperitoneal involvement (liver) without cutaneous signs; abdominal distension, organ dysfunction, high-output cardiac failure

41. Previously thought to be haemangioma, actually histological features of kaposiform haemangioendothelioma or tufted angioma
Locally aggressive vascular tumours
Trapping of platelets on endothelium, exacerbated occasionally by DIC
Diagnosis – MRI most frequently used modality
Diffusely enhancing
Significant morbidity and mortality
Haemorrhage
Invasion/compression of vital structures
10-30% mortality

42. Maintain haemostasis
Platelets for active bleeding/ prior to surgery only, as can increase size of tumour or even exacerbate KMP
Aminocaproic acid
Antiplatelet agents (Aspirin, diprridamole) may reduce platelet aggregation
Fresh frozen plasma if clinically indicated
Curative therapy – treat underlying tumour
No treatment uniformly effective
Surgery not usually possible
Tumour embolization with medical/ surgical therapy
Corticosteroids, alpha interferon, vincristine – alone/in combination

44. Thrombotic disorders
Seen in adults, older children, but reported in neonates
TTP, HUS, Heparin-induced thrombocytopenia
Inherited deficiency of von Willebrand factor cleaving protease
Thrombocytopenia, hyperbilirubinaemia, anaemia
Diagnosis delayed as condition rare, signs common in sick neonates
HUS reported due to B pertussis
NT may occur after thrombosis of major vessel (renal vein thrombosis)
Consider in neonate with thrombocytopenia and renal failure

45. Metabolic disorders
Thrombocytopenia common presenting feature in certain inborn errors of metabolism
Methylmalonic acidaemia, propionic acidaemia, Gaucher disease
Can also complicate induced hypothermia used to treat HIE

46. Aneuploidies
Trisomy 18, 13, 21, Turner syndrome and triploidy can be associated with thrombocytopenia
Down syndrome frequently associated with mild thrombocytopenia
Mechanisms
Similar to that seen in IUGR infants – chronic fetal hypoxia
10% neonates develop a pre-leukaemic disorder (Transient Abnormal Myelopoiesis); increased myeloblasts, variable degrees of thrombocytopenia
Usually resolves spontaneously but 20-30% develop AMKL (acute megakaryoblastic leukaemia)

47. Neonatal Alloimmune Thrombocytopenia (NAIT)
Also referred to as AIT, FM(fetomaternal)AIT
Life-threatening bleeding disorder
Represents <5% cases of early NT, most common cause of severe thrombocytopenia in well, term neonates
Due to maternal platelet antibodies produced in response to fetal platelet antigens inherited from the father
Antibodies cross placenta
Destroy fetal platelets
Cause severe thrombocytopenia, bleeding, ICH

48. Incompatibility between maternal and fetal platelets for HPA 1a accounts for most cases of NAIT

49. Clinical presentation
Clinical picture varies from mild thrombocytopenia at birth to intracerebral haemorrhage (ICH) in utero/ at delivery/ in first days life
Characterised by low platelets and bleeding in an otherwise healthy neonate
Severe thrombocytopenia
(often < 20x10⁹/L)

50. Incidence NAIT affects approximately 1:2000 newborns
Approximately 2% of women are at risk (HPA-1a negative)
10% of these have detectable antibodies
30% of these have affected fetus
20% of these have intracerebral haemorrhage

51. .
Fig 1. Pyramid model of NAT. Each tier shows the number of affected pregnancies and infants expected out of a sample of white women. Each tier represents a proportion of the tier below, shown in parentheses along the right side of the pyramid

52. Pathophysiology
3 major platelet antigen systems on surface of platelets
HLA class uncertain role in NAIT
ABH blood group antigens
Specific platelet antigens (HPA’s)
HPA system assigns a name to platelet antigens based on recognition by specific antisera, in chronological order in which they were identified; high- and low-frequency alleles designated a and b respectively
16 unique polymorphisms been described
Human platelet antigens are epitopes on platelet glycoproteins
Most polymorphisms expressed on GPIIIa
Polymorphisms readily detected by PCR techniques

53. Table 3. Platelet Antigens Implicated in NAT by Frequency
Major platelet antigens (high frequency of NAIT) HPA-1a HPA-5a HPA-5b HPA-15a HPA-15b
Platelet antigens accounting for
up to 2% of NAT collectively
HPA-3a HPA-2a HPA-2b
Platelet antigens implicated in NAT
but found on very few individuals
in the general population HPA-6b HPA-7b HPA-8b HPA-9b HPA-10b HPA-11b HPA-12b HPA-13b HPA-14b HPA-16b
Platelet antigens rarely associated
with NAT
HPA-1b HPA-3b
Platelet antigens commonly
implicated in NAT in the
Asian population HPA-4a

54. HPA-1a implicated in +/- 80% of serologically confirmed NAIT – white population
Japanese – HPA-4a alloimmunization far more common
HPA-5b, HPA-15a implicated in up to 20% of cases of NAIT
All other specificities account for remaining 2%
Because of relatively high expression of both alleles in population, genetic incompatibilities are common
However not sufficient for NAIT – observed syndrome far less common than would be expected
Other factors involved (HLA haplotypes)

55. Many antibodies exist to low-frequency antigens (6b-14b, 16b) – antigen expressed in very few individuals
Antibodies to these will recognize the GP target on paternal platelets, but not on most random donor target platelets
Implication – diagnostic testing – platelets from FATHER optimal platelet targets for detection of NAIT antibodies
Fetal platelets/ platelet antigens enter maternal circulation early in pregnancy (unknown mechanism)
First pregnancy usually affected

56. Complications High risk of bleeding
?not only due to low platelet numbers but also defect in platelet function, antibodies to endothelial cells > loss of vessel integrity
Generalised petechiae, mucocutaneous purpura common
ICH most feared complication
20% of those with anti HPA-1a antibodies
Risk not same for all antigen incompatilities
1/3 of ICH events fatal

57. Up to 80% occur in utero, as early as 16 weeks
Non-fatal often associated with serious neurological sequelae, mental retardation, cerebral palsy, seizures
Up to 80% occur in utero, as early as 16 weeks
Lead to migrational disorders, porencephalic cysts, hydrocephalus
Bleeding elsewhere unusual
Thrombocytopenia may last weeks – antibody-mediated destruction of megakaryocytes also

58. Predictors of severity
Most NB predictor history of NAIT in sibling
Severity worsens with each subsequent pregnancy and increasing gestational age
Depends on antigen incompatibility – HPA-1a severe, but HPA-5a/5b and HPA-15a/15b rarely cause severe disease
Maternal antibody titre not consistently reliable as predictor of severity
NAIT CAN OCCUR IN ABSENCE OF DETECTABLE ANTIBODIES

59. Lab testing for NAIT
Glycoprotein assays – monoclonal antibody immobilisation of platelet antigen (MAIPA)
More sensitive than previous tests
Enzyme immunoassay
Uses GP-specific monoclonal antibodies to identify antigenic target of maternal alloantibodies
Goals of investigations
Determine if maternal-fetal platelet antigen incompatibilty exists
Detect platelet alloantibodies in maternal serum
Risk to fetus then determined

60. What do you need to send? Whole blood samples from mother and father
Genotyping and platelet phenotyping
Genotyping by PCR techniques
Use known ‘anti-sera’ for all relevant alloantigens
ELISA kits for common platelet antigens – BUT limited in ability to detect new antigen targets, and high cost
Maternal serum
For antibody investigations (MAIPA, RIP)
Carried out with paternal platelets as ‘target platelets’ where possible, to allow for detection of antibodies against rare / new antigens

61. Management of NAIT
Multidisciplinary – haematologists, obstetricians, neonatologists, fetal medicine specialists, transfusion services, specialised platelet testing lab
Antenatal
Previously affected infant
Start treatment by as early as 12 weeks’ gestation – if previous intrauterine ICH
Serial fetal sonars – ICH, signs of distress – need for treatment escalation
IVIG weekly
2g/kg/week if previous fetus had ICH in 2nd trimester
1-2g/kg/week if previous fetus had ICH in 3rd trimester/perinatally
Start at weeks if no history of ICH

62. Headaches, fever most common Steroids Not dexamethasone
Side effects
Headaches, fever most common
Steroids
Not dexamethasone
Used in combination with IVIG in high-risk mothers/ when treatment escalation required
Mood alterations, diabetes, fluid retention
Fetal blood sampling, intrauterine platelet transfusion
Complicated by exsanguination, worsening of alloimmunisation, induction of labour
Risks outweigh benefits
Not advised

63. Mode of delivery Summary Screening
Caesarian section often used, but evidence to support this lacking
Use of fetal platelet count impractical
Advantage is planning ahead – have platelets ready, personnel
Summary
Weekly IVIG 1g/kg/week from 24 weeks
Benefit of steroids not proven, but considered
FBS, intrauterine procedures too risky
Screening
Shown to be cost effective
Lack of well-defined pre-clinical phase, insufficient specificity of diagnostic tests, high risk and cost of treatment

64. Treatment of newborn with NAIT

65. Once suspected, treat before having results of workup
Need to prevent ICH – ‘safe’ platelet count not really defined
IVIG (2g/kg over 2-5 days) effective in 75%
Platelet transfusions - for severe thrombocytopenia and/or bleeding
Random donor platelets if antigen-negative (HPA-1a and 5b negative) or maternal platelets not available
Latter platelets ideal
Platelet count response higher, lasts longer
Maternal platelets must be washed to get rid of antibodies
Generally threshold to transfuse platelets – 30 x 10⁹/L (some say 50 x 10⁹/L), higher if ICH or other bleeding
No proven role for steroids

67. ??? TERM OTHERWISE WELL SEVERE THROMBOCYTOPENIA DAY 1 OF LIFE (EARLY)
1ST BABY
HIGHLY SUGGESTIVE OF NAIT
BUT
NO RESPONSE TO IVIG
NOT IMPROVING AFTER 5 WEEKS
MATERNAL ANTIBODY TESTS NEG
?THUMB/ TOE ABNORMALITIES > ?INHERITED THROMBOCYTOPENIA - but plt count should rise after transfusion

68. WHAT’S NEXT?
BMAT today
Finally to receive HPA-1a neg plts (?will they help)
Mother’s platelets ideal