Daily Image Based Questions – IBQs

Answer = [B] Gray platelet syndrome (GPS)

Bleeding phenotype (mucosal) is s/o PLT disorders and the smear shows large platelets (macrothrombocytopenia). If you notice the platelets are not normally stained (lightly stained) = These features are s/o Gray PLT syndrome.

Signs of GPS typically arise at birth or in childhood, these signs and symptoms include thrombocytopenia, bruising susceptibility, and epistaxis.

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by macrothrombocytopenia and absence of platelet α-granules resulting in typical gray platelets on peripheral smears. GPS is associated with a bleeding tendency, myelofibrosis, and splenomegaly. (Myelofibrosis is because of contents of alpha granules are abnormaly released into marrow) Marrow shows normal number of megakaryocytes and thrombocytopania (mild to moderate) is due to reduced PLT survival.

GPS is diagnosed based on clinical findings and requires demonstration of absence or marked reduction of alpha-granules by electron microscopy (EM).

NOTE – Giant platelets are associated with inherited diseases like Bernard–Soulier syndrome, gray platelet syndrome and May–Hegglin anomaly.

NOTE –  Wiskott–Aldrich syndrome is associated with small platelets.

Answer = [B]

Platelet-type von Willebrand disease (PT-VWD) is an autosomal dominant bleeding disorder and is unique among platelet disorders because it is characterized by platelet hyperresponsiveness rather than decreased function. The disease is caused by gain-of-function mutations in the platelet GP1BA gene, which codes for the platelet von Willebrand factor (VWF) receptor, GPIbα.

Patients with PT-VWD present with mild to moderate mucocutaneous bleeding, which becomes more pronounced during pregnancy and following aspirin ingestion or drugs that have antiplatelet activity.

Laboratory testing shows low VWF:ristocetin cofactor and low or normal VWF:antigen and characteristically an enhanced ristocetin-induced platelet agglutination (RIPA). These laboratory features are also indicators of the closely similar and more common bleeding disorder type 2B VWD and hence patients with PT-VWD are often misdiagnosed as Type 2 vWD.

Treatment is based on making a correct diagnosis of PT-VWD where platelet concentrates instead of VWF/factor VIII preparations should be administered.

Simplified RIPA mixing assays and flow cytometry can differentiate between the two disorders. However, the gold standard is to identify mutations within the VWF gene (indicating type 2B VWD) or the platelet GP1BA gene (confirming PT-VWD).

RIPA Mixing studies involves:

MIX A) Patient plasma and health control platelets

MIX B) Donor plasma and patient platelets

RIPA is performed using low doses of Ristocetin and the pattern of results observed. If the abnormality lies within the VWF protein ie Type 2B VWD then Mix A will demonstrate agglutination whereas Mix B will not whereas if the mutation is in the GpIB receptor then Mix A will show no agglutination but Mix B will.

So here answer would be [B] as diagnosis is PT-VWD so mutation will be in GP1BA gene

Answer = [D] AML M3 = APML

Blasts in APML can be initially identified using an SSC vs CD45 plot. Correlation to morphology is important as hypergranular APL blasts generally fall in the same region as normal granulocytes (AS IN PLOT) because of their large size and highly granular content as compared to typical blasts which have low side scatter also.

NOTE: Hypogranular APL blasts have a lower SSC and fall in blast region only.

Now the flow given for the atypical cells is = CD34-DR-CD11b-CD117+CD33+cMPO+

The triple-negative (CD34-/HLA DR-/CD11b-) profile rapidly and specifically identifies an acute promyelocytic leukemia.

Answer = [D] AML M3 = APML

There are two subtypes of APML based on morphologic features; hypergranular and microgranular (hypogranular).

[1] Hypergranular APL blasts vary greatly in size. The cytoplasm is densely packed with large granules occasionally obscuring the nucleus. Characteristic Auer rods or bundles of Auer rods are frequently present.

[2] Microgranular/Hypogranular APL blasts have distinct morphologic features such as the bilobed nucleus and paucity or absence of granules. (as in image)

The flow given in Q is – The atypical cells are CD34+DR-CD117+CD33+cMPO+ ie s/o Microgranular APML

Answer = [B] Shows paracortical plasmacytosis.

The image A shows vascular, atrophic germinal center with surrounding concentric “onion skin” layers of lymphocytes.
Immunohistochemical staining for CD23 (panel B) highlights several follicular dendritic cell clusters within an expanded mantle zone of CD23-positive cells. CD34 (panel C) identifies a positively staining blood vessel within an atrophic follicle.

So this is typical of hyaline vascular type of Castleman disease.

Plasma cell accumulation in the interfollicular areas is a feature of plasma cell variant of Castleman disease

Answer = [A] Band Marked 1

Different types of hemoglobinopathies and diseases can be detected by hemoglobin electrophoresis. Alkaline gel electrophoresis doesn’t allow for separation of HbC, HbE, and HbO-Arab as these are on the same band along with A2.

The blood sample is placed in the middle of this gel in this test and some hemoglobin will flow toward the anode (+) and some will flow toward the cathode (-).

So in this situation Acid electrophoresis allows confirmation of variant hemoglobins observed in the Cellulose Acetate Electrophoresis procedure and allows good separation of HbC from HbE and HbO-Arab. HbC migrates maximum towards + Anode hence band 1 is HbC.

Similarly, HbS can be separated from HbD and HbG which appear on the same band in Alkaline gel electrophoresis but S forms a different band on Acid electrophoresis.

Answer = [B] Hb Barts

Migration of Hemoglobin in Alkaline Electrophoresis
Of the hemoglobins normally present in an adult, Hb A migrates the fastest, followed by Hb F. Hb A2 moves only slightly from the point of origin near the cathode. (See upper panel in the image for normal migration)

Abnormal hemoglobins show the following migration patterns:
Hb C, Hb E and Hb O migrates with Hb A2 near the cathode.
Hb S lies between Hb A2 and Hb F and Hb D and HbG migrate with S making accurate diagnosis difficult.
Hb H and Bart’s hemoglobin are unstable and very fast moving, with Hb H being the faster of the two. They are located nearer the anode past Hb A.

So in Q [2 = Hb Bart and 3 = HbH]

Answer = [B] VWD Type II B

The test shown here is Ristocetin-Induced Platelet Agglutination [RIPA]

The aggregation curve shows that – In normal plasma, no aggregation curve occurs when the ristocetin concentration is below 0.8 mg/mL. In patient – aggregation occurs at much lower ristocetin concentrations ie < 0.8 mg/ml.

FACT: In type 2B vWD (or platelet type vWD), aggregation occurs at much lower ristocetin concentrations.

So Answer he is [B] VWD Type II B

Type 2B vWD also has an AD inheritance and is characterized by loss of large-molecular-weight multimers, but the mechanisms for such change are quite distinct from type 2A. The gain of function mutations in or around the A1 domain (exon 28) results in an increased affinity of GpIb binding site on vWF for the platelet GpIb receptor, leading to spontaneous binding in circulation. The large multimers have a higher number of GpIb binding sites and are thought to be rapidly cleared from plasma, along with bound platelets, thus resulting in characteristic thrombocytopenia and lack of Large multimers in the patient’s plasma.

This characteristic is also demonstrated in vitro when the increased binding is seen with low concentrations of ristocetin on ristocetin-induced platelet aggregation (RIPA) testing ie Low Dose RIPA (with ristocetin dose <0.8 mg/ml). Clinical phenotype usually is of moderate to severe mucocutaneous bleeding.

ALSO NOTE: In cases in which there is enhanced agglutination with Ristocetin at low concentrations, it is important to establish whether this is a case of Type 2B VWD or Platelet-type von Willebrands disease.

Answer = [C] Column C

Von Willebrand disease (VWD) is considered the most common congenital bleeding abnormality in the world. Accurate diagnosis of von Willebrand disease (VWD) can often be very challenging for clinicians. In addition to patient and family history, clinicians utilize a panel of assays provided by the clinical laboratory to aid in diagnosis. These most often include an assessment of von Willebrand factor (VWF) activity and antigen levels, along with coagulation FVIII activity levels.

VWF multimeric analysis is a method for analysing the concentration and distribution of Von Willebrand Factor [VWF] multimers that are present in plasma. In normal plasma, VWF multimers appear as a series of bands separated by the mass of 2 subunits as can be seen in Lane/column A.

In the image above – Lane A shows normal plasma as a control. Lane B shows type 1 VWD with all multimers sizes present, but reduced concentration (as shown by sparse lines). Lane C shows type 2A VWD with a loss of the high and intermediate multimers. Lane D shows type 2B with a loss of only the large multimers. Lane E show type 3 VWD with no detectable multimers.

Results of multimer analysis is shown below:-

Answer = [D]

The MRI image shows cerebellar atrophy (Arrow) and ocular telangiectasia hence s/o Ataxia telangiectasia

Ataxia telangiectasia (A-T) is an autosomal recessive disorder primarily characterized by cerebellar degeneration, telangiectasia, immunodeficiency, cancer susceptibility, and radiation sensitivity. A-T is often referred to as genome instability or DNA damage response syndrome.

A-T is caused by mutations in the ATM (Ataxia Telangiectasia, Mutated) gene which encodes a protein of the same name. The primary role of the ATM protein is the coordination of cellular signaling pathways in response to DNA double-strand breaks, oxidative stress, and other genotoxic stress.

The diagnosis of A-T is usually suspected by the combination of neurologic clinical features (ataxia, abnormal control of eye movement, and postural instability) with one or more of the following which may vary in their appearance: telangiectasia, frequent sinopulmonary infections, and specific laboratory abnormalities (e.g. IgA deficiency, lymphopenia especially affecting T lymphocytes and increased alpha-fetoprotein levels).

Answer = [A]

Here in the aggregometry plot notice on the y axis, ie % aggregation – With ADP aggregation response is there as % aggregation increasing up to 75% whereas there is no aggregation with other agonists ie Epinephrine, collagen, and Arachidonic acid. Note that the Ristocetin plot is not given.

Principle: Platelet aggregation testing measures the ability of various agonists to platelets to induce in vitro activation and platelet-to-platelet activation. Platelet Rich Plasma [PRP] is stirred in a cuvette at 37°C and the cuvette sits between a light source and a photocell. When an agonist is added the platelets aggregate and absorb less light and so the light transmission increases and this is detected by the photocell and the curve can be plotted in two ways i.e. y-axis can be increasing % aggregation or decreasing optical density (OD). In this question % aggregation has been plotted (so always notice what is written on the y-axis in aggregometry plots)

Summary of the ‘commonly’ described abnormalities seen with light transmission aggregometry [LTA]

Answer = [A]

Patients of inherited disorders of platelets are characterized by a prolonged clinical history of mucocutaneous bleeding. Glanzmann thrombasthenia (GT) is a rare, inherited autosomal recessive disorder characterized by qualitative or quantitative deficiency of integrin αIIbβ3 [glycoprotein IIa (GPIIb)/IIIa, CD41/CD61] diagnosed by absent or reduced platelet aggregation to physiological agonists, namely, collagen, adenosine diphosphate (ADP), epinephrine and arachidonic acid (AA).

The role of flow cytometry in the analysis of Glanzmann’s Thrombasthenia and Bernard Soulier Syndrome has evolved based on following markers – CD41, CD61 and CD42b directed against specific platelet membrane glycoproteins.

In Images 5 the gated platelets are negative for both CD41 and 61 which together forms the GpIIb/IIIa complex hence the patient lacks GpIIb/IIIa complex and so diagnosis is – Glanzmann’s Thrombasthenia.

In image 6 – the gated platelets are negative for CD 61 but positive for CD42b (GPIb) hence  Bernard Soulier Syndrome is excluded.

Answer = [A]

TEST PRINCIPLE: Cells lacking CD55 (DAF) or CD59 (MIRL) do not agglutinate and form pellet at the bottom of the microtube

In this gel card test Positive reactions of 3+ to 4+ (agglutinated cells forming a red line on the surface of the gel) indicate a normal red cell population with the presence of the corresponding DAF and MIRL antigens. If DAF and MIRL are absent on red cell population then there would be no antigen-antibody reaction on the addition of anti-DAF and anti MIRL antibodies and so agglutinate will not be formed and the red cell pellet will settle at the bottom on centrifugation.

As we can see that in the patient’s sample (the right side of the card) the red cell pellet is settled on the bottom in both DAF and MIRL tube thereby indicating the absence of DAF and MIRL on patients RBCs so the patient has PNH clone. The normal control sample result is shown in the left side of the card where the agglutination reaction is visible.

NOTE: Gel test is a useful screening test for PNH in resource-constrained countries because of its simplicity and increased sensitivity to diagnose PNH.

Answer = [B]

For initial detection of PNH-type populations, screening of granulocyte and monocyte populations is overall more sensitive than RBC screening, since active hemolysis or transfusion may markedly reduce the number of circulating RBCs that lack GPI-linked proteins, potentially resulting in false-negative PNHnscreens or in the underestimating of PNH clone size.

FLAER-based PNH screen commonly employs a 5-color, single-tube flow cytometry approach that uses antibodies to three markers (CD15, CD45, CD64) for selection (gating) of granulocyte and monocyte populations, and two reagents (FLAER and anti-CD157) for detecting GPI-deficient (PNH-type) populations.

• [1] The assay takes advantage of the action of proaerolysin which binds to GPI anchor in the plasma membrane of cells. Cells affected by PNH lack GPI anchoring proteins, and thus are not bound by proaerolysin.
• [2] CD157 is a GPI-linked protein that is consistently expressed at relatively high levels by both granulocytes and monocytes, eliminating the need for additional lineage specific GPI-linked protein targets, and avoiding the lower sensitivity of markers that show lower or more variable levels of expression in normal populations.
• PNH Granulocytes and Monocytes will be negative for both CD157 and FLAER.

Left plot shows CD15+ granulocytes which are positive for both CD157 and FLAER and the right plot shows CD64+ monocytes which are positive for both CD157 and FLAER. As CD157 and FLAER negative population is not identified hence the patient doesn’t have PNH.

Answer = [C]

The aspirate image shows infiltration by lymphoid cells (smaller ones with dense blue chromatin) along with well-formed plasma cells.

The flow cytometry also is suggestive of a dual population of cells – Lymphoid cells [strong CD19&20+] and Plasma cells [CD138+]. These dual population of clonal cells is characteristic of Waldenstrom’s macroglobulinemia.

Lymphoplasmacytic lymphoma/Waldenstrom’s macroglobulinemia.

Lymphoplasmacytic lymphoma involving either the bone marrow or the extramedullary sites typically exhibits a cytologic spectrum ranging from small lymphocytes with clumped chromatin, inconspicuous nucleoli, and sparse cytoplasm to well-formed plasma cells. Frequently present are “plasmacytoid lymphocytes” having cytologic features intermediate between these 2 extremes, although the cytologic composition and the degree of plasmacytic differentiation vary from case to case.

The cytologic spectrum of lymphoplasmacytic lymphoma in Waldenström macroglobulinemia is reflected in the immunophenotypic attributes of the neoplastic cells. A monotypic lymphocytic component is almost always detected, typically with high levels of surface CD19, CD20, and immunoglobulin light chain expression. By comparison, the plasmacytic component expresses the same immunoglobulin light chain as the lymphocytic component, is positive for CD138 (particularly when assessed by immunohistochemistry), and shows diminished expression of B-cell–associated antigens such as CD19, CD20, and PAX5.

Answer = [B]

HPFH is a disorder in which Hb F is increased above the normal adult level and there are no morphological changes to the red cells.

HPFH heterozygotes differ from thalassemia heterozygotes in that they have no imbalance between the synthesis of α and non-α-chains (i.e. γ and β-chains) and thus are characterized by an asymptomatic heterozygous state without microcytosis (As in this question the patient has normal Hb, he is asymptomatic and MCV is also within normal range). The elevated Hb F ranges from 3 to 35%, depending on the type of the mutation.

Heterozygous HPFH is usually found incidentally through family studies. Patients present with a slightly elevated erythrocyte count with the corresponding elevation of the hematocrit and a slightly decreased MCH (27 pg) with near-normal MCV. HbF is 10–30% of the total hemoglobin. HbA2 is decreased to 1–2% and the remainder is HbA.

Homozygous HPFH: Erythrocytes are microcytic and slightly hypochromic with a mean MCV of 75 fL and a mean MCH of 25.0 pg.
There is a mild degree of anisocytosis and poikilocytosis. The reticulocyte count range is 1–2%. It is doubtful that this disorder has any
significant degree of hemolysis because the reticulocyte count, bilirubin, and haptoglobin levels are normal. Electrophoresis demonstrates
100% HbF.

Note – In this question, a normal MCV essentially rules out δβ-Thalassemia heterozygotes and β-Thalassemia heterozygotes as these both will have low MCVs. (Usually < 70 fl).

The hematological picture in δβ-Thalassemia heterozygotes is of microcytic, hypochromic erythrocytes without anemia. HbA2 is normal or slightly decreased, whereas HbF is increased to 5–20%. HbA is usually < 90%.

β-Thalassemia heterozygotes (minor) have mild anemia microcytic, hypochromic erythrocytes with increased HbA2 (<5-7%) and normal or mildly increased HbF (less than 5%) with the rest being HbA. In summary, b thalassemia minor is indicated when the MCH is < 27 pg and the HbA2 is > 3.5%.

Answer = [A]

Image: Electron microscope picture showing the characteristic nuclear ‘Swiss cheese like’ appearance in congenital deyserythropoietic anaemia type 1.

CDA type I (CDAI) is characterized by severe or moderate anemia, which is generally macrocytic, and relative reticulocytopenia and congenital anomalies, such as skeletal abnormalities, chest deformity, and short stature. Distal limb anomalies are well documented for ∼10% of these patients (As seen in this patient), although they have been described also in other CDA subtypes.

In the bone marrow, 2.4% to 10% of late erythroblasts are binucleate, and most of these have nuclei at different stages of erythroid differentiation. However, the typical morphological feature of CDAI is the presence of thin internuclear chromatin bridges between the nuclei pairs of intermediate erythroblasts (1% to 8% of cells examined) as seen in the image above. Internuclear chromatin and cytoplasmic bridges have been observed in 79% of patients with CDAI.

Under electron microscopy, their heterochromatin is denser than normal and forms demarcated clumps with small translucent vacuoles, which gives rise to the metaphor of the classical “spongy” or “Swiss cheese” appearance of the nucleus.

CDAI is inherited as an autosomal recessive disorder that is caused by biallelic mutations in 2 different loci that account for 90% of CDAI cases: CDAN1 and C15orf41.

Answer = [C]

The image below shows the thin internuclear bridge in erythroblasts (in the center) along with erythroid prominence and in view of the clinical scenario it is s/o CDA type I

CDA type I (CDAI) is characterized by severe or moderate anemia, which is generally macrocytic, and relative reticulocytopenia and congenital anomalies, such as skeletal abnormalities, chest deformity, and short stature. Distal limb anomalies are well documented for ∼10% of these patients, although they have been described also in other CDA subtypes.

In the bone marrow, 2.4% to 10% of late erythroblasts are binucleate, and most of these have nuclei at different stages of erythroid differentiation. However, the typical morphological feature of CDAI is the presence of thin internuclear chromatin bridges between the nuclei pairs of intermediate erythroblasts (1% to 8% of cells examined) as seen in the image above. Internuclear chromatin and cytoplasmic bridges have been observed in 79% of patients with CDAI.

Under electron microscopy, their heterochromatin is denser than normal and forms demarcated clumps with small translucent vacuoles, which gives rise to the metaphor of the classical “spongy” or “Swiss cheese” appearance of the nucleus.

CDAI is inherited as an autosomal recessive disorder that is caused by biallelic mutations in 2 different loci that account for 90% of CDAI cases: CDAN1 and C15orf41.

Answer = [B]

Cold agglutinin disease accounts for about 10-20% of cases of AIHA. Idiopathic (primary) chronic cold agglutinin disease has its peak incidence after age 50 years. This disorder is characterized by monoclonal IgM cold agglutinins.

Most patients with idiopathic cold agglutinin disease have chronic hemolytic anemia. Other patients exhibit episodic, acute hemolysis with hemoglobinuria induced by chilling.

Acrocyanosis involving the fingers, toes, nose, and ears is caused by sludging of RBCs in the cutaneous microvasculature as seen in this image (left). Livido reticularis is also commonly seen.

Patients with chronic cold agglutinin disease exhibit mild to moderate anemia, hematocrit levels ranging sometimes as low as 15-20%.  The blood film exhibits several features common to all types of AIHA ie polychromasia (reticulocytosis), spherocytes, nucleated RBCs, and erythrophagocytosis by monocytes may be seen.

RBC autoagglutination may be seen in the blood film and in chilled anticoagulated blood from patients with cold-antibody AIHA as seen in image (right) of peripheral blood smear (This finding is not seen in warm AIHA).

So the case is likely of COLD AIHA

HENCE ANSWER HERE WOULD BE [B] DAT + AGAINST C3D (COMPLEMENT) ONLY

Answer = [D] Southeast Asian ovalocytosis (SAO)

Peripheral blood film shows macro-ovalocytes and stomatocytes some of which has off center, transverse or less frequently, longitudinal slits. Several red cells have more than one stoma. The Y-shaped stoma can also be seen in the smears.

These findings are suggestive of Southeast Asian ovalocytosis (SAO).

Southeast Asian ovalocytosis (SAO) is a very common condition in the aboriginal peoples from Papua New Guinea, Indonesia, Malaysia, the Philippines, and southern Thailand, in areas where malaria is endemic, with prevalence varying between 5% and 25%. SAO is now known to be caused by a 27 base-pair deletion in SLC4A1, which codes for band 3, a 911 amino acid protein that is both a structural component of the red cell membrane cytoskeleton and the chloride-bicarbonate anion-exchanger in this membrane.

Individuals with SAO are characterized as having oval-shaped red blood cells with increased membrane rigidity and decreased anion transport, but no clinical symptoms beyond sporadic associations with anemia in both adults and neonates. The diagnosis is made accidentally as a result of a peripheral blood smear examination, showing the characteristic rounded elliptocytes (ovalocytes).

NOTE

Spherocytic elliptocytosis is characterized morphologically by two populations of cells: red cells that are more rounded than typical hereditary elliptocytes and a variable number of microspherocytes.

Stomatocytic Xerocytosis is characterized morphologically by the presence of stomatocytes.

Sitosterolemia, also known as phytosterolemia, is a recessively inherited disorder associated with elevated plasma levels of plant sterols. Affected patients exhibit early-onset xanthomatosis and premature coronary artery disease. Hematologic manifestations include macrothrombocytopenia and stomatocytic hemolytic anemia