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Laboratory Approach to Red Cell Disorders

Ralph Green MD PhD

Department of Pathology and Laboratory Medicine

ISLH May 2017

Pre-Meeting Educational WorkshopHonolulu

Anemia Classification

• Morphologic– Red cell size (micro-, macro- or normocytic)

– Hemoglobinization (hypo-, hyper- or normochromic)

– Red cell shape

• Mechanistic– Blood loss

– Increased destruction (hemolysis)

– Impaired production

Morphologic classification of anemiaa partial list…

Microcytic Normocytic Macrocytic

Iron deficiency Acute blood loss Megaloblastic anemia

Hemoglobinopathies Hemoglobinopathies Reticulocytosis

Anemia of inflammation (chronic disease)

Anemia of inflammation (chronic disease)

Liver disease

Sideroblasticanemia

Hemolytic anemia Myelodysplasia

Anemia – an algorithm

MCV-BASED ALGORITHMIC APPROACH TO ANEMIA DIAGNOSIS

RBC, Hct, Hb

Microcytic

Anemia

MCV

80-100 fl

Normocytic Macrocytic

Ferritin and iron status tests

Reticulocyte Count Blood Smear Morphology

NormalLow High

<80 fl >100 fl

Iron deficiency

anemia

Thalassemia Hemoglobin-

opathy

Anemia of chronic disease

Sideroblastic Anemia

Bone marrow ringed sideroblasts

Hemoglobin studies

NormalLow High

Aplasia Anemia of

chronic disease

Acuteblood loss

Hemolysis Chronic

blood loss

LDH BilirubinHaptoglobin

Direct anti-globulin test

No hypersegmented neutrophils

Hypersegmented neutrophils

Polychromasia Target cells

Reticulocytosis Liver disease

Liver enzymes

Vit. B12

deficiency

Folate deficiency

B12 or folate levels MMA

CRP and other cytokines

Causes of Macrocytic Anemia

Green R, Dwyre DM. Evaluation of Macrocytic Anemias. Semin Hematol. 2015 Oct;52(4):279-86

Megaloblastic Anemia

▪ Megaloblastic anemia results from an abnormality of DNA synthesis

▪ Commonest causes are deficiencies of vitamin B12 or folic acid

▪ Features include raised MCV, oval macrocytes and hypersegmented PMNs in the blood smear and nuclear/cytoplasmic dyssynchrony in the BM

Green R & Datta Mitra A Med Clin North Am. 2017 Mar;101(2):297-317

Blood Smear in Megaloblastic Anemia

Blood Smear in Megaloblastic Anemia

Bone Marrow in Megaloblastic Anemia

Laboratory Features of Megaloblastic Anemia

▪ Macrocytic anemia with raised MCV, MCH and RDW

▪ Increased SI, ferritin Tf saturation

▪ Increased LDH, indirect bilirubin

▪ Low plasma B12 or folate (and RBC folate)

▪ Raised plasma homocysteine

▪ Raised plasma methyl malonate (in B12 deficiency)

Green R & Datta Mitra A Med Clin North Am. 2017 Mar;101(2):297-317

Green R. Vitamin B12 deficiency from the perspective of a practicing hematologist Blood May 11 2017 (in press)

Vitamin B12 absorption and metabolism

Vitamin B12-Dependent Reactions

Homocysteine* + MethylTHF Methionine + THFMethyl-B12

Methylmalonyl-CoA Succinyl-CoAAdenosyl-B12

Methylmalonic Acid*

*Levels rise in B12 deficiency

Diagnosis of Vitamin B12 Deficiency

• Macrocytic megaloblastic anemia with or without neurological involvement.

• Atypical presentations (neurological syndrome without anemia or macrocytosis

• Low plasma B12 as an isolated lab finding

• Raised plasma metabolites (methylmalonic acid and homocysteine

• Low transcobalamin B12 (HoloTC) = “Active B12”

Differentiating B12 from Folate Deficiency

Condition Serum B12 Serum folate

Red Cell folate

B12 deficiency

Low

Nl or high

Low

Folate deficiency

Normal

Low

Low

Note:Plasma homocysteine is raised in B12 and folate

deficiencies. Methylmalonic acid rises in B12 deficiency

alone

Macrocytic Anemia Diagnostic Algorithm

Green R, Dwyre DM. Evaluation of Macrocytic Anemias. Semin Hematol. 2015 Oct;52(4):279-86

MCV-BASED ALGORITHMIC APPROACH TO ANEMIA DIAGNOSIS

RBC, Hct, Hb

Microcytic

Anemia

MCV

80-100 fl

Normocytic Macrocytic

Ferritin and iron status tests

Reticulocyte Count Blood Smear Morphology

NormalLow High

<80 fl >100 fl

Iron deficiency

anemia

Thalassemia Hemoglobin-

opathy

Anemia of chronic disease

Sideroblastic Anemia

Bone marrow ringed sideroblasts

Hemoglobin studies

NormalLow High

Aplasia Anemia of

chronic disease

Acuteblood loss

Hemolysis Chronic

blood loss

LDH BilirubinHaptoglobin

Direct anti-globulin test

No hypersegmented neutrophils

Hypersegmented neutrophils

Polychromasia Target cells

Reticulocytosis Liver disease

Liver enzymes

Vit. B12

deficiency

Folate deficiency

B12 or folate levels MMA

CRP and other cytokines

Differential Diagnosis of Microcytic Hypochromic Anemia

▪ Iron Deficiency Anemia (IDA)

▪ Anemia of Chronic Disease (ACD)

▪ Thalassemias

▪ Sideroblastic Anemia

Body iron fluxes

Hepcidin – “the master” regulator of iron metabolism

Anemia of inflammation (“chronic disease”)

Bunn F HemOnc Today 2013

Differentiation of IDA from ACD

Differentiation of IDA from Thalassemia minor

Discriminant Function (DF) to distinguish IDA from Thalassemia minor*

DF = MCV2 X RDW100 X Hgb

If > 65 ……….Iron DeficiencyIf < 65…………Thalassemia minor

* Green R, King R. Blood Cells. 1989;15(3):481-91

MCV-BASED ALGORITHMIC APPROACH TO ANEMIA DIAGNOSIS

RBC, Hct, Hb

Microcytic

Anemia

MCV

80-100 fl

Normocytic Macrocytic

Ferritin and iron status tests

Reticulocyte Count Blood Smear Morphology

NormalLow High

<80 fl >100 fl

Iron deficiency

anemia

Thalassemia Hemoglobin-

opathy

Anemia of chronic disease

Sideroblastic Anemia

Bone marrow ringed

sideroblasts

Hemoglobin studies

NormalLow High

Aplasia Anemia of

chronic disease

Acuteblood loss

Hemolysis Chronic

blood loss

LDH BilirubinHaptoglobin

Direct anti-globulin test

No hypersegmented neutrophils

Hypersegmented neutrophils

Polychromasia Target cells

Reticulocytosis Liver disease

Liver enzymes

Vit. B12

deficiency

Folate deficiency

B12 or folate levels MMA

CRP and other cytokines

Microcytic Anemia Diagnostic Algorithm

Blood count including red indices/smear Presumptive

diagnosis

Serum ferritin- iron- iron binding capacity- transferrin saturation

ANEMIA OF

CHRONIC DISEASEIRON DEFICIENCY

ANEMIA

THALASSEMIA

MINOR

SIDEROBLASTIC

ANEMIA

Hemoglobin studiesFor Hgb A2, F, E(hplc, genetic studies)

Definitive

diagnosis

bone marrow sideroblasts

- Infection- collagen vascular

disease- cancer

-blood/iron lossGIgynecologicalurinarypulmonaryintegumentary

- β thalassemia- α thalassemia- Hgb E disease

- congenitalX-linkedautosomalrecessive

- acquiredlead

causes

Laboratory approach to hemolytic anemia

Laboratory approach to hemolytic anemia

Anemia Classification

• Morphologic– Red cell size (micro-, macro- or normocytic)

– Hemoglobinization (hypo-, hyper- or normochromic)

– Red cell shape

• Mechanistic– Blood loss

– Increased destruction (hemolysis)

– Impaired production

Drawings of common red cell abnormalities

Don’t forget morphology! Shape and size!

Thank you! - Any questions?

Thank you! or Any questions?