What is the diagnostic approach for Bernard Soulier syndrome and Glanzmann thrombasthenia?

Diagnostic Approach for Bernard-Soulier Syndrome and Glanzmann Thrombasthenia

Diagnose Bernard-Soulier syndrome (BSS) by demonstrating deficient GPIb/IX expression on flow cytometry combined with absent ristocetin-induced platelet aggregation, and diagnose Glanzmann thrombasthenia (GT) by showing deficient GPIIb/IIIa expression on flow cytometry with absent aggregation to all agonists except ristocetin. 1

Initial Clinical Assessment

Begin by documenting specific bleeding manifestations that suggest inherited platelet disorders:

• Unexplained or extensive bruising from early childhood 1
• Severe epistaxis requiring medical intervention 1
• Menorrhagia in females 1
• Bleeding following dental extractions or invasive procedures 1
• Family history of similar bleeding, particularly in consanguineous marriages 2

Use a validated bleeding assessment tool (BAT) to quantify bleeding severity, as this has proven validity in identifying patients requiring platelet function testing. 1

First-Line Laboratory Testing

Complete Blood Count with Peripheral Smear

• BSS characteristically shows mild thrombocytopenia (platelet count marginally reduced) with giant platelets visible on peripheral smear 3, 4
• GT typically presents with normal platelet count and normal platelet size 3, 2
• Exclude pseudothrombocytopenia by repeating counts in sodium citrate or heparin tubes if platelet clumping is suspected 5

Light Transmission Aggregometry (LTA)

Perform LTA as the gold standard first-line functional test using standard agonists: ADP, collagen, ristocetin, and arachidonic acid. 6

For BSS:

• Normal aggregation to ADP, collagen, and arachidonic acid 3, 4
• Absent aggregation to ristocetin (diagnostic hallmark) 1, 3
• Absent aggregation to bovine plasma 4

For GT:

• Absent or severely reduced aggregation (<30%) to ADP, collagen, and arachidonic acid 1, 3
• Normal aggregation to ristocetin (distinguishes from BSS) 1

Critical caveat: If initial aggregation studies show atypical abnormalities, repeat testing after at least 1 month, as 20% of initially abnormal results normalize on repetition. 3, 6

Flow Cytometry (Confirmatory and Diagnostic)

Flow cytometry is essential for definitive diagnosis and can be performed on small blood volumes, making it particularly suitable for pediatric patients. 7

For BSS diagnosis, perform flow cytometry on resting platelets:

• Use antibodies against GPIb (CD42b) and GPIb/IX (CD42a) 1, 6
• BSS shows markedly reduced or absent GPIb/IX expression (typically <5% of normal mean) 4, 7
• Normal expression of GPIIb/IIIa (CD41/CD61) distinguishes BSS from GT 4, 7

For GT diagnosis, perform flow cytometry on resting platelets:

• Use antibodies against GPIIb/IIIa (CD41), GPIIIa (CD61) 1, 6
• GT shows absent or severely reduced GPIIb/IIIa expression 7
• Normal GPIb expression distinguishes GT from BSS 7
• Also test activated platelets with PAC-1 antibody (anti-GPIIb/IIIa activation epitope), which shows defective activation in GT 1

Diagnostic Algorithm

Step 1: Obtain 21-28 mL of blood for combined LTA and flow cytometry 6

Step 2: If peripheral smear shows giant platelets with thrombocytopenia:

• Perform ristocetin aggregation (absent = suspect BSS) 1
• Confirm with flow cytometry showing absent GPIb/IX 1, 7

Step 3: If peripheral smear shows normal-sized platelets with normal count:

• Perform full LTA panel (absent aggregation to all agonists except ristocetin = suspect GT) 1
• Confirm with flow cytometry showing absent GPIIb/IIIa 1, 7

Step 4: Confirm diagnosis in all cases with flow cytometry using multiple monoclonal antibodies, as this provides rapid, definitive diagnosis even in pediatric patients 3, 7

Important Diagnostic Pitfalls

• Ensure patients avoid all medications affecting platelet function (aspirin, NSAIDs) for 7-10 days before testing 3
• Do not rely on bleeding time testing, as it lacks sufficient specificity and sensitivity 6
• Standardize time between blood collection and analysis, as pre-analytical variables significantly affect results 8
• In pediatric patients, flow cytometry is superior to aggregation studies at initial presentation due to technical difficulties with aggregometry in small children 7
• Whole blood impedance aggregometry can be used as an alternative to platelet-rich plasma methods when separating giant platelets is technically difficult 4

Molecular Confirmation

After establishing diagnosis by flow cytometry and aggregometry, genetic testing can identify specific mutations:

• BSS: mutations in GP9, GP1BA, or GP1BB genes 1
• GT: mutations in ITGA2B or ITGB3 genes 1

Molecular genetics is particularly valuable for genetic counseling in populations with high consanguinity rates 2