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VEXAS Syndrome: Pathology Lab Approach to Bone Marrow Aspirate

Background

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Step 1 - Gross/Pre-Analytical Triage

• Middle-aged/elderly male
• History of: relapsing polychondritis, Sweet syndrome, neutrophilic dermatosis, polyarteritis nodosa, MDS (30-50% of VEXAS), MGUS (10%)
• Unexplained macrocytic anemia, cytopenias, elevated inflammatory markers (CRP, ferritin)
• Recurrent fevers unresponsive to antibiotics
• Venous thromboembolism (40% incidence)

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Step 2 - Morphology (The Core Pathology Finding)

Hallmark Finding: Cytoplasmic Vacuolation

Vacuolation in >10% of neutrophilic (myeloid) precursors, with >1 vacuole per cell = diagnostic threshold for VEXAS (Reumatologia Clinica, 2024).

Other BMA Morphologic Features

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Step 3 - Differential Diagnosis of Vacuolated Myeloid/Erythroid Precursors

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Step 4 - Pathology Report Language

• Presence, degree, and lineage(s) of vacuolation (myeloid, erythroid, or both)
• Estimated percentage of affected precursors
• Presence or absence of concurrent dysplasia/MDS features
• A specific comment recommending UBA1 molecular testing if the clinical picture is compatible

"Prominent cytoplasmic vacuolation is identified in myeloid and erythroid precursors affecting approximately X% of neutrophilic precursors. In the appropriate clinical context (systemic inflammation, cytopenias, elderly male), this morphology is consistent with VEXAS syndrome. Correlation with UBA1 molecular sequencing of peripheral blood is strongly recommended."

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Step 5 - Triaging Molecular Testing (Done on Peripheral Blood, Not Just BMA)

• Peripheral blood (whole blood) - preferred, sufficient in most cases
• Bone marrow aspirate specimen itself

UBA1 Mutation Hotspots

• p.Met41Val - most common (~60%)
• p.Met41Thr - second most common
• p.Met41Leu - less common
• Rare variants outside this region exist

Recommended Molecular Test

• Targeted amplicon sequencing / Sanger sequencing of UBA1 exon 3 (fast, cost-effective first step)
• Or a myeloid mutation NGS panel that includes UBA1 (if ordering a panel for concurrent MDS workup anyway - efficient parallel testing)

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Step 6 - Concurrent Hematologic Malignancy Workup

• Complete BMA morphologic assessment for MDS (WHO 2022 criteria)
• Cytogenetics (conventional karyotype + FISH panel)
• Flow cytometry (myeloid phenotyping, plasma cell quantification)
• Full myeloid NGS panel (DNMT3A, TET2, ASXL1, SF3B1, etc.) - these co-occur with UBA1 mutations

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Step 7 - Summary Diagnostic Algorithm (Pathology Perspective)

BMA received → examine smear
         |
         ↓
Cytoplasmic vacuoles in myeloid/erythroid precursors?
         |
    YES  |
         ↓
>10% neutrophil precursors affected?
         |
    YES  |
         ↓
Assess clinical context: elderly male + systemic inflammation?
         |
    YES  |
         ↓
Comment in report → recommend UBA1 sequencing (peripheral blood)
         + concurrent MDS workup (karyotype, flow, myeloid NGS panel)
         |
         ↓
UBA1 p.Met41 mutation confirmed → DIAGNOSIS: VEXAS syndrome

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Key Takeaways for Pathologists

1. Vacuolation of myeloid AND erythroid precursors in a middle-aged/elderly man with inflammation = always consider VEXAS first.
2. The >10% myeloid precursor vacuolation threshold achieves 100% sensitivity and specificity in validated series.
3. Standardize your report language - explicitly mention VEXAS and recommend UBA1 testing rather than attributing vacuolation to "non-specific dysplasia."
4. MDS and VEXAS coexist frequently - do not let an MDS diagnosis satisfy the workup without excluding VEXAS.
5. UBA1 testing is most practically done on peripheral blood - the pathologist can request this without waiting for a repeat BMA.
6. Skin biopsy in VEXAS shows neutrophilic dermatosis (similar to Sweet syndrome) with immature myeloid infiltrates carrying the same UBA1 mutation - if skin biopsy is also available, this further supports the diagnosis.

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• Goldman-Cecil Medicine (2024), Chapter on Autoinflammatory Diseases
• Harrison's Principles of Internal Medicine 22E (2025), FUO chapter
• Cherniawsky H et al. "VEXAS syndrome: A review of bone marrow aspirate and biopsies reporting myeloid and erythroid precursor vacuolation." Eur J Haematol 2023. PMID 36788756
• Hagiya A et al. "How I diagnose and manage VEXAS syndrome." Am J Clin Pathol 2024. PMID 38511841
• Mekinian A et al. "ACR Guidance Statement for Diagnosis and Management of VEXAS - International VEXAS Working Group." Arthritis Rheumatol 2026. PMID 40787890 - first formal international consensus guidance

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Haemoglobinised Red Cells on Peripheral Smear - Differential Diagnosis

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Category 1 - HYPOCHROMIC Red Cells (Reduced Hb Content)

Differential Diagnosis of Hypochromic Cells

Pearl: In thalassemia trait, the microcytosis is disproportionately severe for the degree of anaemia (Mentzer index <13), while in IDA the anaemia is worse relative to MCV drop (Mentzer index >13). However, overlap exists and Hb electrophoresis is definitive.

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Category 2 - HYPERCHROMIC Red Cells (Increased Hb Density)

Differential Diagnosis of Hyperchromic Cells

Critical note: True hyperchromia (elevated MCHC) is essentially pathognomonic of spherocytes. When the analyzer flags high MCHC, always look for spherocytes on the smear. Any MCHC >36 g/dL should prompt a manual PS review.

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Category 3 - POLYCHROMATIC (Polychromatophilic) Red Cells

A polychromatic cell on Wright's stain is larger than a mature RBC and lacks central pallor. Marked polychromasia with anemia = hemolysis until proven otherwise.

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Category 4 - DIMORPHIC Red Cell Population (Mixed Haemoglobinisation)

Differential Diagnosis of Dimorphic Film

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Category 5 - ABNORMAL Hb DISTRIBUTION WITHIN THE CELL (Special Patterns)

5a. Target Cells (Codocytes)

• Haemoglobin C disease (most target cells per field)
• Anemia - thalassemia (β > α)
• Liver disease (obstructive jaundice, cirrhosis)
• Thalassemia
• Chromic - post-splenectomy / asplenia

5b. Sickle Cells (Drepanocytes)

5c. Heinz Bodies / Bite Cells

5d. HbH Inclusion Bodies

5e. Hemoglobin C Crystals

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Summary Algorithm: Approach to Haemoglobinisation Abnormality on PS

Observe RBC colour/Hb pattern
            |
   ┌─────────────────────────────────────┐
   │                                     │
Hypochromic                        Hyperchromic/No pallor
(large central pallor)             ─────────────────────
   │                                Macrocytes → B12/folate, liver disease
   ↓                                Spherocytes → HS, AIHA
IDA / Thalassemia /                 (confirm with osmotic fragility / DAT)
ACD / Sideroblastic /
Lead poisoning
   │
   ↓
Check: Is the film DIMORPHIC?
→ YES → Sideroblastic anemia / iron therapy / post-transfusion / dual deficiency
→ NO → Iron studies + Hb electrophoresis
            |
   ┌─────────────────────────┐
   │                         │
Low ferritin             Normal/High iron
→ IDA                    + HbA2 ↑ → β-thal trait
                         + HbA2 normal, microcytosis → α-thal
                         + Ring sideroblasts → Sideroblastic anemia

Check also:
Polychromasia? → Reticulocytosis → Hemolysis / blood loss / response to Rx
Target cells? → HALT C causes
Bite cells? → G6PD/oxidant hemolysis (supravital stain)
Sickle cells? → Sickling disorders

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• Henry's Clinical Diagnosis and Management by Laboratory Methods, Chapter 31 (Hemoglobin Content, Color, Polychromatophilia)
• Henry's Clinical Diagnosis and Management, Chapter 33 (Microcytic/Hypochromic anemias, Dimorphic anemias)
• Goldman-Cecil Medicine (Microcytic Anemias, Sideroblastic Anemia)
• Tietz Textbook of Laboratory Medicine 7E (Red Blood Cell Parameters)
• Robbins Pathologic Basis of Disease (Iron Deficiency Anemia, peripheral smear)

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Advanced Coulter Hematology Parameters: IPF, RHe, LFR, MFR, HFR

Important note on nomenclature: These parameters exist on multiple platforms with different names for the same concept:

• Beckman Coulter (DxH): uses RHe (Reticulocyte Haemoglobin equivalent), LFR/MFR/HFR, IPF
• Sysmex: uses RET-He (same concept as RHe), IRF (= MFR + HFR sum), IPF
• Siemens ADVIA: uses CHr (same concept as RHe)

RHe/RET-He/CHr are functionally equivalent and the literature uses them interchangeably.

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PART 1 - LFR, MFR, HFR (Reticulocyte Maturity Fractions)

What They Measure

Biological Principle

• High HFR/MFR = reticulocytes just exited the bone marrow = intense erythropoietic drive
• High LFR = predominantly mature reticulocytes = steady-state or recovering erythropoiesis
• The IRF rises before the total reticulocyte count increases - it is the earliest indicator of marrow erythroid activation

Clinical Applications of LFR/MFR/HFR

1. Bone Marrow Transplant (BMT) / Stem Cell Transplant Engraftment Monitoring

• Earlier cessation of G-CSF and prophylactic antibiotics
• Earlier discharge planning
• Cost savings

2. Monitoring Response to Treatment in Nutritional Anemias

• In iron deficiency anemia on iron therapy: IRF rises within days of effective treatment, well before reticulocyte count or Hb changes - useful for early confirmation of response
• In megaloblastic anemia on B12/folate: same early IRF rise signals effective marrow stimulation
• In EPO therapy for CKD: rising IRF confirms pharmacological erythropoietic response

3. Differential Diagnosis of Anemia

Key clinical rule: A dissociation between low reticulocyte count and high IRF = ineffective erythropoiesis (MDS, megaloblastic, thalassemia).

4. Neonatal Assessment

• Used to assess transfusion needs in premature neonates
• IRF higher in premature infants; rising IRF indicates erythropoietic recovery

5. Aplastic Crisis Detection

• In compensated hemolytic anemia (e.g., hereditary spherocytosis), a sudden fall in IRF precedes the reticulocyte drop during a parvovirus B19-induced aplastic crisis - gives earlier warning

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PART 2 - RHe (Reticulocyte Haemoglobin Equivalent)

What It Measures

• Units: pg/cell (same as MCH)
• Normal range: approximately 28-35 pg (exact range varies; most use >28 pg as normal)
• Key cut-off for iron deficiency: RHe/RET-He < 28 pg = iron-deficient erythropoiesis

Why It Is Superior to Conventional Iron Markers

RHe is a real-time mirror of iron availability for erythropoiesis - it reflects the iron status at the moment the reticulocyte was being produced, not weeks ago.

Clinical Applications of RHe

1. Diagnosis of Iron Deficiency Anemia (IDA)

2. Diagnosis of Functional Iron Deficiency (FID)

• CKD patients on EPO therapy - EPO drives erythropoiesis faster than iron stores can release iron
• Post-surgical/ICU patients on IV iron + EPO
• Inflammation states - hepcidin blocks iron release from macrophages

• Serum ferritin is NORMAL or HIGH (misleading)
• TSAT may be borderline
• RHe is LOW (<28 pg) → reveals that despite adequate stores, iron is not reaching the erythroblast

3. Monitoring Response to Iron Therapy

• Iron deficiency without anemia
• Functional iron deficiency
• True iron deficiency anemia
• Iron replete erythropoiesis

4. Preoperative Assessment

5. Screening Blood Donors

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PART 3 - IPF (Immature Platelet Fraction)

What It Measures

• IPF % (percentage of total platelets)
• Absolute IPF (IPF × platelet count = immature platelet count in /μL)

Biological Principle

• High IPF = bone marrow megakaryopoiesis is active, producing/releasing many new platelets → seen when platelet destruction/consumption is high (marrow compensating)
• Low IPF = megakaryocyte production is suppressed or absent → hypoproductive thrombocytopenia
• IPF rises before the platelet count recovers - it is the earliest indicator of thrombopoietic recovery

Clinical Applications of IPF

1. Differential Diagnosis of Thrombocytopenia

Clinical pearl: In a patient with thrombocytopenia of unknown cause, high IPF (>10%) + low platelet count = peripheral destruction (ITP, TTP, DIC, drug-induced) = NO bone marrow biopsy needed in most cases. Low IPF + low platelet count = marrow failure = bone marrow biopsy indicated.

2. BMT/SCT Engraftment Monitoring

• Earlier platelet transfusion cessation
• Earlier confirmation of graft function
• Earlier hospital discharge trigger

3. Guiding Platelet Transfusion Timing

• High IPF in a thrombocytopenic patient = marrow is producing platelets → own platelets are coming → transfusion may not be urgently needed (especially in ITP)
• Low IPF = marrow is not making new platelets → transfusion support needed sooner

4. Sepsis Management

5. Other Applications

• COVID-19: Elevated IPF was observed in thrombocytopenic COVID-19 patients, reflecting immune-mediated platelet destruction
• DIC: High IPF confirms peripheral consumption
• Essential thrombocythemia / thrombocytosis: IPF may help distinguish reactive from clonal thrombocytosis
• Plateletpheresis quality control: IPF of the product reflects the proportion of metabolically active young platelets

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Summary Integration: Using All Parameters Together

Thrombocytopenia
    ├── IPF ↑ → Peripheral destruction (ITP, TTP, DIC, sepsis)
    └── IPF ↓ → Marrow failure (aplasia, chemo, MDS)

Anemia with reticulocytopenia
    ├── IRF (MFR+HFR) ↑ but retics ↓ → Ineffective erythropoiesis (MDS, megaloblastic)
    └── IRF ↓ + retics ↓ → Marrow failure / EPO deficiency

Anemia with reticulocytosis
    └── IRF ↑ + retics ↑ → Hemolysis / blood loss / treatment response

Iron status (regardless of ferritin)
    ├── RHe < 28 pg → Iron deficient erythropoiesis (true OR functional)
    └── RHe > 28 pg + low ferritin → Pre-latent iron deficiency

Post-transplant monitoring
    HFR rises first → MFR rises → Absolute retics rise → Hb rises (erythroid)
    IPF rises first → Platelet count rises (platelet lineage)

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• Tietz Textbook of Laboratory Medicine 7E, Sections on IRF, Reticulocyte Hb Content, IPF (Chapter 74/77)
• Henry's Clinical Diagnosis and Management by Laboratory Methods, Chapter 31 (Reticulated Platelets)
• Zhang Y et al. "From reticulated platelets to immature platelet fraction." Platelets 2025. PMID 40035091
• Poventud-Fuentes I et al. RET-He in pediatric IDA. Int J Lab Hematol 2023
• PMC Reference range study: PMC5119661