11 Hematology by Urvi Trivedi

SECTION 11

Haematology 58.

Approach To Leucocytosis Madhuchanda Kar, S Kartthik, Prantar Chakrabarti

319

59.

Practical Approach to Lymphadenopathy Niranjan Rathod

324

60.

ABC of CBC Pankhi Dutta

327

61.

A Practical Approach to Anaemia MB Agarwal

334

62.

An Approach to a Patient with Bleeding Disorder Nidhi Sharma, Sandhya Gulati, Sudhir Mehta, Shaurya Mehta

339

Approach To Leucocytosis

C H A P T E R

Madhuchanda Kar, S Kartthik, Prantar Chakrabarti

ABSTRACT

Leukocytosis means elevation of WBC count for the patient’s age. Leukocytosis might be because of lymphoid or myeloid series of cells. There is usually wide spectrum of causes starting from benign diseases including infections and chronic inflammation or it might indicate an underlying malignancy. So the approach in diagnosis should be very systematic starting from meticulous peripheral smear examination and then proceed to further tests for clinching the diagnosis. So we therefore discuss the approach to various types of leukocytosis in this article.

INTRODUCTION

Leukocytosis is defined as an elevation of the 9WBC count for the patient’s age. WBC count of 30 X 10 /L is considered elevated in an adult, but this value is normal in the early neonatal period, so an appropriate reference value is critical. So reference intervals for WBC counts and relative percentages and absolute cell counts vary by patient age and hospital population. Leukocytosis is a common finding with a broad differential diagnosis, encompassing both benign and malignant entities. The

Table 1: Causes of Leucocytosis Primary hematologic etiology : Hereditary neutrophilia Chronic idiopathic neutrophilia Myeloproliferative disorders (eg, CML, PV,ET) Familial myeloproliferative disease Congenital anomalies and leukemoid reaction Down syndrome Leukocyte adhesion factor deficiency Familial cold urticaria and leukocytosis 2. Secondary to other disease entities: Infection Chronic Inflammation Cigarette smoking and Stress Drug induced (Corticosteroids, beta agonists, lithium, G-CSF etc) Heat Stroke Marrow stimulation and Post splenectomy status 3. Spurious Platelet clumping Mixed cryoglobulinemia

newer generation of hematology analyzers can examine thousands of leukocytes using flow cytometry–based methodology, some in combination with cytochemistry or fluorescence or conductivity, so they could elucidate different types of WBCs, including neutrophils, lymphocytes, monocytes, basophils, and eosinophils. Spurious elevations of the WBC count can also be seen, including platelet clumps, nucleated RBCs, incomplete lysis of RBCs, cryoglobulins, and cryofibrinogen. Hyperleukocytosis refers to a WBC count greater than 100,000/mL, and is seen almost exclusively in leukemias and myeloproliferative disorders. Leukostasis, or sludging of WBC in small vessels of the brain, lungs, and kidneys, is an oncologic emergency that may cause life-threatening cerebral infarcts, cerebral hemorrhage, or pulmonary insufficiency caused by impaired blood flow. Leukostasis is more common in acute myelogenous leukemia than in acute lymphoblastic leukemia, because myeloblasts are larger and more adhesive than lymphoblasts; it is rarely seen in chronic leukemias, even with extremely high WBC counts (Table 1)

WHAT MIGHT BE THE CAUSES OF LEUKOCYTOSIS? Leucocytosis Blasts

Neutrophilia

Eosinophilia

Lymphocytosis

Monocytosis

Basophilia

Blasts

Blasts can be morphological identified by its atypical morphology, but its not always reliable to distinguish between myeloid and lymphoid blasts. There are some blast equivalents described by WHO for hematololymphoid malignancies 2008 which includes promonocytes, megakaryoblasts and atypical promyelocytes. The further characterization of the blast is done using immunophenotyping by flow cytometry or immunohistochemistry. Blast count more than or equal to 20 % is defined as acute leukemia. There are lower than 20% circulating blast count seen in chronic myeloid neoplasms, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and overlap MDS/ MPN. Iatrogenic or endogenous excess granulocyte colony-stimulating factor (G-CSF) stimulation can cause a left shift of the myeloid lineage to the blast stage. Bone marrow infiltration by fibrosis, malignancy, or infection can be associated with circulating immature cells (leukoerythroblastosis), including blasts and nucleated red blood cells.

Myeloid Leukocytosis

Myeloid leukocytosis may represent granulocytosis (ie, neutrophilia, eosinophilia, and basophilia) or monocytosis

320

Neutrophilia (Figure 1)

conditions. A leukoerythroblastic reaction caused by myelophthisis is similar (but the total WBC does not need to be high) and also includes nucleated RBCs.

Leukemoid reactions represent exaggerated leukocytosis (typically 50,000– 100,000/mL) and may include in the peripheral blood all recognizable stages of neutrophil maturation, that is, from myeloblasts to mature granulocytes. Leukemoid reactions typically last hours to days and may be caused by either benign or malignant

Causes of Neutrophilia

HAEMATOLOGY

Neutrophilia is defined as an elevated circulating neutrophil count (>7.7 X 109/L in adults).

Features supporting reactive WBC <50 x 10 /L Predominantly mature Toxic granulation and vacuoles DÖhle bodies Thrombocytosis Neutrophilia

Features supporting neoplastic WBC >50 x 10 /L Pronounced left shift Basophilia or Eosinophilia Dacrocytes Dysplasia

Fig. 1: Causes of Nuetrophilia

A 65 yr old female presented with low back pain and pallor for last 6 months, on examination she had no other significant findings apart from mild pallor. Peripheral blood examination showed Hb – 7 g/dl, total leukocyte count – 62 X 109cells/L and platelet count – 94 X 109 cells/L, Peripheral smear examination shows Rouleaux formation with neutrophilic leukocytosis (ANC – 41 X 109cells/L) and occasional nRBCs with myelocytes and metamyelocytes. Biochemistry evaluation showed S.Urea – 64 mg/dl and S.Cr – 2.6 mg/dl, Liver function showed normal bilirubin and enzymes, but Total protein – 7.2 g/dl, S. Albumin – 3.2 g/dl and S. Globulin- 4g/dl. How to proceed? (Figure 2) 1.

Digital rectal examination

Stool for occult blood

3. LDH

Neutrophilia

Repeat counts

Yes

Peripheral smear examination Leukoerthroblastic

Normalised

Bone marrow examination Morphology, cytogenetics, culture

Tumor/ Granulomatous disease

No Further Investigation

Reactive causes more likely

Ph or BCR ABL positive CML

JAK 2/CaIR mutation (Non CML MPN)

Fig. 2: Approach to Neutrophilia

• Parasitic (helminths, ectoparasites, isospora, sarcocystis) • Viral (HIV, HTLV) • Fungal (coccidiomycosis) • Bacterial (tuberculosis)

Rule out reactive eosinophilia Peripheral blood screening with FISH (for CHIC2) or RT-PCR (for FIP1L1-PDGFRA fusion gene)

Fig. 3: Cases of Eosinophilia Urine R/E and microscopic examination

Serum protein electrophoresis

USG whole abdomen and pelvis

S. PSA and CEA

Upper GI endoscopy and colonoscopy

S. Calcium

10.

Bone marrow aspiration and biopsy

GENERAL APPROACH TO NEUTROPHILIA

Yes

Positive?

Other PDGFRA or PDGFRB rearrangements by cytogenetics or RT-PCR

Myeloproliferative neoplasm

Underlying disorder?

Yes

Present? No

Other WHO-deﬁned myeloid (AML, MDS, classic MPN, MPN/MDS overlap disorders, 5M) or lymphoid (B- or T-cell lymphoblastic lymphoma) entities

Imatinib 100 mg/d

Symptoms AND/OR End-organ damage AND/OR Marked eosinophilia

Present?

Neutrophilia in Childhood

CHAPTER 58

321

Finding of eosinophilia

Yes

During the first few days of life the upper limit of the normal neutrophil count ranges from 7000 to 13,000 cells/ μL for neonates born prematurely and at term gestation, respectively, and is followed by a decrease to adult levels within the first few weeks of life. Congenital primary neutrophilia is rare. Autosomal dominant neutrophilia kindred of 12 patients in three generations had an activating mutation in the CSF3R gene leading to constitutive activation of the G-CSF receptor and increased proliferation and differentiation of neutrophil precursors. One of the patients progressed to MDS.

- Escalate imatinib (up to 400 mg/d); - nilotinib; - dasatinib; - sorafenib; - midostaurin

Treat according to disease-speciﬁc guidelines AND/OR imatinib 400 mg/d

- Corticosteroids; - FNo; - Hydroxyurea; - Cytotoxic agents; - Imatinib 400 mg/d; - Alemtazumab; - Clinical trial

Observe

Fig. 4: Approach to Eosinophilia

Marked neutrophilia is a hallmark of functional disorders of neutrophils that are caused by impaired adhesion or motility, such as in patients with leukocyte adhesion deficiencies or actin dysfunction.

examination mild pallor, scratch marks over the body and enlarged bilateral cervical, axillary and inguinal lymphnodes with maximum size of 3X3 cms . There was mild hepatomegaly and mild splenomegaly. Peripheral blood examination revealed Hb – 9g/dl, Total leucocyte count of 45 X 109 cells/L with prominent eosinophilia (Absolute eosinophil count – 13 X 109 cells/L) and platelet count of 164 X 109 cells/L. Biochemistry evaluation showed mildly elevated liver enzymes with normal bilirubin and renal parameters were within normal limits. How to proceed? (Figure 4)

Eosinophilia

Lactate dehydrogenase

S. Uric acid

Stool for parasite

Chest X ray and USG whole abdomen and pelvis

Lymph node biopsy including histopathological examination by H&E and Immunohistochemistry

CECT whole abdomen & pelvis and CECT thorax including neck

Bone marrow aspiration and biopsy

Molecular studies for PDGFR alpha and beta & FGFR1 mutation

The normal absolute eosinophil count (AEC) is 350 to 500/mm3. The severity of eosinophilia has been arbitrarily divided into mild (AEC 500–1500/mm3), moderate (AEC 1500–5000/mm3), and severe (AEC >5000/mm3). Features supporting reactive Transient Clinical presence of drugs, allergy or infection Eosinophilia

Features supporting neoplastic Persistent Immature cells present Cytopenias and dysplasia in other lineages

Causes of Eosinophilia

AD familial eosinophilia has been reported in several families in which individuals displayed marked eosinophilia, but few had pulmonary, cardiac, or neurologic involvement.The disorder has been mapped to chromosome 5q31-q33, a region that contains a cytokine cluster including genes encoding IL-3, IL-5, and GM-CSF. A 15 yr old boy presented with low-grade fever, pruritus and multiple swelling over the neck for last 2 months. On

9. Echocardiography

Monocytosis

An absolute monocytosis is defined as >1 X 109/L monocytes. A reactive monocytosis may be seen with malignancy (ie, carcinoma, plasma cell myeloma, or lymphoma), subacute bacterial endocarditis, chronic infections like tuberculosis, syphilis, Rocky mountain spotted fever, and kala-azar, autoimmune disorders, and splenectomy.

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The persistence of an absolute monocytosis should prompt examination of the BM with flow cytometric immunophenotyping and cytogenetic studies, because persistent monocytosis raises a differential diagnosis, including chronic myelomonocytic leukemia (CMML), acute monoblastic/monocytic leukemia, CML, juvenile myelomonocytic leukemia, atypical (BCR-ABL negative) CML, and myelodysplastic/myeloproliferative neoplasms, unclassifiable.

HAEMATOLOGY

Basophilia (Figure 6)

Basophilia, defined as > 0.3 X 109/L in adults, is extremely rare. Isolated basophilia is extremely uncommon. Reactive basophilia has been linked to hypersensitivity disorders, iron deficiency, chronic inflammation, and rarely infection, including influenza and chicken pox. Features supporting reactive Transient Clinical presence of drugs, allergy or infection Monocytosis

Features supporting neoplastic Transient Predominantly mature Reactive changes

Fig. 5: Differentiating features of Reactive Vs Neoplastic Monocytosis Features supporting reactive Very rare

Basophilia

Features supporting neoplastic Other lineage abnormalities

Lymphocytosis

In adults, an absolute lymphocyte count of > 3.5 X 109/L can be considered lymphocytosis. Absolute lymphocyte counts are higher in children and infants compared with adults, so the appropriate reference intervals must be used.

Causes of Lymphocytosis

Reactive lymphocytosis - Viral infections, some bacterial infections, toxoplasma, malaria, Babesiosis, Drug hypersensitivity, autoimmune disease, cytokines, vaccination, smoking, stress, endocrine disorders and secondary to malignancy ( lymphoma, leukemia). A 80 yr old male presented with swellings all over the body including neck, axilla and inguinal region for last 2 yrs. On examination he had generalised lymphadenopathy and moderate hepatosplenomegaly. Peripheral 9 smear examination showed Hb â&#x20AC;&#x201C; 8g/dl, TLC â&#x20AC;&#x201C; 124 X 10 /L, There are 90% small mature lymphocytes with small round nuclei and plenty of smudge cells and platelet count of 123 X 109/L. No comorbidities and his biochemistry reports were unremarkable. How to proceed?

Fig. 6: Basophilia

Reticulocyte count

Direct Coombs Test

3. LDH

Features supporting reactive Usually Pleomorphic Lymphocytosis

Myeloproliferative neoplasms like chronic myeloid leukemia and polycythemia vera can have basophilia. In CML, quantification of eosinophilia is very important to distinguish chronic phase from accelerated phase (>20%). Increased numbers of marrow basophils may occur in MDS and sideroblastic anemia. Peripheral blood or bone marrow basophilia may also accompany acute myeloid leukemias, usually in association with 6p or 12p chromosomal abnormalities, or juvenile myelomonocytic leukemia.

Features supporting neoplastic Usually monomorphic

S. Uric acid

Chest X ray and USG whole abdomen and pelvis

Immunophenotyping from peripheral blood

Neoplastic lymphocytosis based on peripheral blood morphology can be as follows: (Figure 8)

To summarize

In conclusion, leukocytosis in a patient should prompt

Fig. 7: Lymphocytosis

Neoplastic Lymphocytosis

Small round nuclei

Folded or cleaved nuclei

Convoluted nuclei

Villous cytoplasm

Plasmacytoid

Large cells

C.I.I. monoclonal B cell lymphocytosis

Follicular/mantin cell/lymphoma atypical; C.I.I.

Sezary syndrome Adult t cell leukemia

Hairy cell leukemia and its variant/ Splenic marginal zone lymphoma

Lymphoplasmacytic lymphoma

Burkitt/DLHCL

Fig. 8: Lymphocytosis

APPROACH IN A CASE OF LEUKOCYTOSIS:

Leukocytosis

Peripheral smear examination

Blast

Lymphoid

Monocytosis Basophilia

Pleomorphic reactive

Neutrophilia

Eosinophilia

Reactive causes including infection, malignancy, splenectomy and autoimmune disorders

Favouring malignancy then BMA and ancillary test

Rule out MPN

Rule out reactive causes and then proceed with evaluation of hematolymphoid malignancy

Fig. 9: Approach in a Case of Leukocytosis

323

REFERENCES

Chabot-Richards. D. S, George. T. I. Leukocytosis. Int Jnl Lab Hem 2014; 36:279–88.

2. George. T. I. Malignant or Benign Leukocytosis. ASH Education Book 2012; 475-84. 3. Cerny J, Rosmarin A.G. Why does my patient have Leukocytosis? Hematol Oncol Clin N Am 2012; 26:303–19. 4.

Falchi L, Verstovsek S. Eosinophilia in hematological disorders. Immunol Allergy Clin N Am 2015; 35:439–52.

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BM examination with Flow cytometry, cytogenetics and molecular studies

Myeloid

Monomorphic rule out lymphoproliferative disorders

confirmation of the CBC and WBC differential. Examination of the blood smear should be performed to establish a manual differential or to confirm the automated differential. This will allow the distinction of myeloid from lymphoid disorders. Distinguishing myeloid leukemoid reactions from myeloid malignancies is difficult, with features such as dysplasia, basophilia, WBC count >50 X 109/L, a pronounced left shift, and increased blasts favoring a myeloid malignancy with recommended BM examination and appropriate ancillary testing.

Practical Approach to Lymphadenopathy

C H A P T E R

Enlarged lymph node always put dilemmas in mind of physicians due to its association with certain conditions like malignancy. It is important for all physicians to have systemic but practical approach to these patients. Understanding of anatomical locations of lymph nodes and their drainage areas is essential (Table 1). Evaluation of lymphadenopathy begins with good history which focuses on possible aetiologies of lymphadenopathy (Table 1). Physical examination directed towards finding out if it is localized or generalized lymphadenopathy and

Niranjan Rathod

characteristics of enlarged nodes like size, site, mobility & consistency. In one large retrospective study from India in tertiary referral hospital studying diagnosis in 1724 patients who have under lymph node excision biopsy showed tuberculous adenitis in 31%, , malignancy in 26% and had non-specific lymphadenitis in 31%1. However incidence of malignancy is likely to be much lower in primary care situations.

Table 1: Lymph node drainage areas and causes of localised & generalised lymphadenopathy Lymph node

Areas of drainage

Common cause of enlargement

Cervical

Neck and scalp region

Infections- Infectious mononucleosis, Tuberculosis. Ear nose throat & dental infections Malignancy Lymphoma Head and neck cancer Thyroid cancer

Supraclavicular

Breast & mediastinum on right side

Infections- e.g TB

Abdomen on left side

Hodgkinâ&#x20AC;&#x2122;s lymphoma Non-Hodgkinâ&#x20AC;&#x2122;s lymphoma Bronchogenic carcinoma Breast carcinoma

Epitrochlear

Upper limb-ulna, forearm and hand

Sarcoidosis Lymphoma Infectious mononucleosis Local infections in upper extremity Secondary syphilis HIV

Mediastinal

Mediastinum

Tuberculosis, histoplasmosis, Sarcoidosis, silicosis Lymphoma, other malignancies

Axillary

Upper extremities, breast and thorax

Skin infection with Streptococcal or staphylococcal Malignancies like breast carcinoma, metastatic melanoma etc

Abdominal

Mesenteric or retroperitoneal space

Tuberculosis, Lymphoma, Gastric malignancies etc

Generalised

> 3 lymph node or groups

Infectious mononucleosis, HIV infection, Tuberculosis infection, Medications, SLE

Table 2: Drugs causing lymphadenopathy Hydralazine Phenytoin Primidone Allopurinol Atenolol Carbamazepine Cephalosporins Quinidine Practical approach to lymphadenopathy

HISTORY

Obtaining information about age of patient- older (higher probability of malignancy) or younger, possible infection or malignancy in drainage of particular lymph node enlarged, travel history and drug history (Table 2), eating habits like undercooked meat (for toxoplasmosis), high risk behaviour (multiple sexual partners, injection drug use) 2. Duration and evolution of lymphadenopathy can also give clue about aetiologies and severity. Longer duration of symptoms and lymph nodes enlargement would suggest possible chronic infections like TB or indolent cancers like follicular lymphoma. Malignancy possibilities suggested by symptoms related mechanical compression like dysphagia, hoarse voice, cough, haemoptysis and systemic systems like weight loss, anorexia, fatigue & fever.

PHYSICAL EXAMINATION

All lymph node groups should be examined with attention to their characteristics, associated splenomegaly or signs of systemic illness. Size, site, mobility, consistency & location should be noted. Size: Normal lymph nodes are usually less than 1 cm in size. Generally greater than 1 cm nodes are considered abnormal except one in inguinal region where size greater than 2 cm is considered abnormal3. Larger the size more is the possibility of pathological enlargement of lymph nodes. Consistency: Softer nodes are seen commonly in acute leukaemia but chronic leukaemias and lymphomas have firm and rubbery nodes. Harder nodes are seen in malignancies and chronic infections leading to fibrosis. Mobility: Fixation to underlying tissues can make nodes immobile. This can occur due to inflammation and cancers leading to matted or fixed nodes. This is generally a bad sign and should alert physician for possibilities of malignancy or chronic infections with fibrosis. Tenderness over lymph node is generally seen in nodes with infections or rapidly growing malignancy. Location: Localised enlargement occurs due to infection or malignancy in drainage area of lymph nodes. Generalised lymphadenopathy generally manifest with

325

Associated splenomegaly is seen more commonly in patients with leukaemia, lymphoma.

DIAGNOSTIC INVESTIGATIONS

Routine tests like CBC, ESR along with viral markers like HIV, EBV, CMV and x ray chest is indicated in most of patients with lymphadenopathy. Specific tests like Mantoux test, ANA, tests for syphilis etc are required in further evaluation in specific suspected situations. Patients having localized lymphadenopathy could be observed for duration up to four weeks if there is no suspicion for malignancy. This time will not lead to missed opportunity even if later turns out to be tuberculosis or lymphoma, as one will be able to still manage patient without adverse impact on treatment outcome. If lymph nodes remain enlarged even after 3-4 weeks or if there is high suspicion for malignancy right in beginning, further testing like lymph node biopsy is indicated. Definitive diagnosis is generally made on lymph node biopsy of most representative enlarged node. It allows view of complete architecture of node and histopathological confirmation of pathology4. Excision biopsy is best for this purpose; however imaging guided core needle biopsy is next best alternative in cases where excision biopsy is not possible. Supraclavicular, cervical, mediastinal, axillary, inguinal are the choices for biopsy chosen in descending order. It is very important to convey likely diagnosis and samples required to surgical team, so that samples are taken in formalin for histopathology with biopsy and in saline for molecular tests for malignancies or tuberculosis. Fine needle aspiration cytology (FNAC) allows quick and lesser invasive option for evaluation. However it is of limited value in diagnosis of certain conditions like malignancies as tissue architecture cannot be studied and there is a substantial false-negative rate. It may used for screening of cytology in selective patients with suspected infections like TB or relapse of malignancies before planning for biopsy5. Occasionally incision and drainage of fluctuating painful lymph node may be done to offer symptomatic relief to patient, but it is hardly useful for diagnosis purpose. Imaging studies like computed tomography (CT), ultrasound, Doppler, or magnetic resonance imaging (MRI) play important role in evaluation of lymphadenopathy6. Imaging helps distinguish nodes from other lesions and also assess extent of lymphadenopathy for staging of cancers7. Imaging guided core needle biopsy is helpful in patient with internal lymphadenopathy, aborting need for laparatomy for diagnosis purpose. There is no role for empirical antibiotics as it is very challenging to pinpoint bacterial cause from non bacterial causes of lymphadenopathy. And there is no clinical evidence to support its use in clinical practice. Treatment will depend upon aetiology identified in diagnostic work up. Wait and watch is enough for

CHAPTER 59

Sulfonamides

systemic disease like constitution symptoms etc. (Table1).

HAEMATOLOGY

326

conditions like infectious mononucleosis and early stage follicular lymphoma or chronic lymphocytic leukaemia. Anti tubercular treatment for documented diagnosis or occasionally empirically when suspicion is strong but no diagnostic evidence could be achieved. However due rising number of resistant tubercular strains, it would better to go for detailed work up like sensitivity and susceptibility tests along with histopathological diagnosis. Corticosteroids and other immunosuppressant are used for autoimmune conditions like SLE or sarcoidosis. Chemotherapy, radiation or surgical removal may be necessary for malignancy. Lymphoma is highly treatable cancer with high response rates.

TAKE HOME MESSAGE

Systematic approach to lymphadenopathy is highly useful due to divergent aetiologies. It is important to do step wise evaluation based on history & physician examination followed by blood investigation, biopsy in necessary patients along with imaging to come conclusion about diagnosis. Selection of appropriate node for biopsy is equally important to avoid false negatives. Treatment is guided by diagnosis.

REFERENCES

1. Mohan A, Reddy MK, Phaneendra BV, Chandra A. Aetiology of peripheral lymphadenopathy in adults: analysis of 1724 cases seen at a tertiary care teaching hospital in southern India. Natl Med J India 2007; 20:78 2. Ferrer R. Lymphadenopathy: differential diagnosis and evaluation. Am Fam Physician 1998; 58:1313. 3. Pangalis GA, Vassilakopoulos TP, Boussiotis VA, Fessas P. Clinical approach to lymphadenopathy. Semin Oncol 1993; 20:570. 4. Chau I, Kelleher MT, Cunningham D, et al. Rapid access multidisciplinary lymph node diagnostic clinic: analysis of 550 patients. Br J Cancer 2003; 88:354. 5. Steel BL, Schwartz MR, Ramzy I. Fine needle aspiration biopsy in the diagnosis of lymphadenopathy in 1,103 patients. Role, limitations and analysis of diagnostic pitfalls. Acta Cytol 1995; 39:76. 6.

van den Brekel MW, Castelijns JA. Imaging of lymph nodes in the neck. Semin Roentgenol 2000; 35:42.

Sumi M, Ohki M, Nakamura T. Comparison of sonography and CT for differentiating benign from malignant cervical lymph nodes in patients with squamous cell carcinoma of the head and neck. AJR Am J Roentgenol 2001; 176:1019.

ABC of CBC

C H A P T E R

Pankhi Dutta

INTRODUCTION

The complete blood count (CBC) is the most commonly ordered blood test in a healthcare set up. The fully automated sophisticated cell counters give us very accurate and precise blood counts rapidly alongwith a number of additional newer parameters which give valuable extra information added to the routine CBC.The origin of the modern cell counters date back to 1953, when Wallace H Coulter, an American engineer by training, obtained a patent for a principle for counting and sizing microparticles suspended in a fluid.1 The principle was named the â&#x20AC;&#x2DC;coulter principleâ&#x20AC;&#x2122; and is the basis of the technology of most cell counters even today.

White cell count differential (DC)- 3 parts /complete 5 part differential count. Absolute white cell counts

Platelets

Platelet count (PC) Mean platelet volume (MPV) Platelet distribution width (PDW)

Haematocrit (Hct)

Over and above all these , few new parameters available on some analyzers have found clinically utility and are being incorporated into the routine CBC. Two new US FDA approved parameters are the Reticulocyte Haemoglobin Content (CHr) or Reticulocyte Haemoglobin Equivalent (Ret-He) and Immature Platelet Fraction (IPF). Promising research parameters include Leucocyte Positional Parameters(LPP).2

Red blood cells(RBCs) and indices

The Hb and the Hct

A basic CBC comprises of the following parameters Haemoglobin (Hb)

Total Red Cell count (TRBC)

Mean corpuscular volume (MCV) Mean corpuscular haemoglobin (MCH) Mean corpuscular haemoglobin concentration (MCHC)

The Hct can be used synonymously with the Hb to classify a sample as anaemic or polycythemic. The Hct is the given volume of packed RBCs as compared to the plasma and can be measured by a microhaematocrit centrifuge (as in Sysmex analyzers) or calculated (as in Beckman Coulter instruments) by taking into account the Hb and the MCV. Due to differences in the methodologies there can be considerable variation in the Hct between analyzers while the Hb value is more reproducible. Hence, it is better to use the Hb to make clinical decisions.

Red cell distribution width (RDW)

White blood cells (WBCs)

Total white cell count (WBC)

a pe rtu re

e xt ern a l e le ct ro d e

The haemoglobin is measured after the RBCs are lysed and the Hb converted to a stable coloured compound whch is measured spectrophotometrically. Hence, like lipemia and hyperbilirubinemia can interfere with the results.

in tern al e lect ro d e

TRBC and MCV va cu um U=RxI

I m p u ls e 3-p ar t D iff te ch no lo gy

V 0 70 6

Fig. 1: Impedance based cell counter. https://www.sysmex.com Source-https://www.sysmex.com

The most common technology for counting and sizing the blood cells is the impedance technology based on the coulter principle. The basic counting chamber comprises of two electrodes with a partition between them. The partition has an aperture through which electrolyte flows and maintains flow of electrical current between the electrodes (Figure 1). The cells are suspended in the electrolyte and when an individual cell passes through the aperture, there is a break in the flow of current as blood cells are poor conductors of electricity. Each break is counted as a pulse. The number of pulses generated give the cell count. The TRBC and the platelet counts are obtained in this manner.

328

Incubation 30 min

RBC-Histogram

100

200

RBC HGB HCT MCV MCH MCHC (fL) RDW

Results

RBC-Histogram

Results

RU 2.23x10 /L 14.4g/dl 24.9% RU± 111.7ﬂ RU± 64.6pg RU± RU± 57.8g/dl ± 25.4ﬂ ±

100

200 (fL)

12 RBC 4.35x10 /L 14.5g/dl HGB 43.5% HCT 100.0ﬂ MCV 33.3pg MCH MCHC 33.3g/dl 14.7ﬂ RDW

HAEMATOLOGY

Fig. 2: CBC printout

Fig. 4: Algorithmic approach to Anaemia

Fig. 3: RBC Histogram The height/size of a pulse generated depends on the size of the cell causing it. In the case of RBCs, the average height of the pulses gives us the MCV. The MCV is expressed in femtolitres (fl) and red cells with MCVs in the range of 80-100fl are considered normocytic. Cells with MCV less than 80fl are called microcytic. An MCV above 100fl would classify the cells as macrocytic. Besides numerical values, the instruments also give RBC volume distribution histograms.

MCH AND MCHC

Having obtained the Hb, Hct , TRBC and MCV, the instruments give us the calculated parameters of MCH (Hb x 1/TRBC) and MCHC (Hb x 1/Hct) which give information about the concentration of Hb in the RBCs. Values below the reference range are used to classify the RBCs as hypochromic. Samples with higher concentrations of MCH indicate dense RBCs as may be seen in megaloblastic anaemias or in the presence of spherocytes. Very high (non-physiological) values of MCV, MCH and MCHC indicate presence of red cell clumping, often due to cold agglutinins. The abnormalities are reversed on reanalyzing the samples after incubation at 37 deg C. The CBC printout with the characteristic RBC indices is diagnostic of red cell agglutination. The following RBC histogram illustrates the same (Figure 2).

RDW

From the RBC volume distribution histogram, the analyzers calculate an index of variation of RBC size (anisocytosis) called the RDW. Anisocytosis is appreciated on the peripheral smear (PS) but it is not possible to

quantify anisocytosis and give a numerical value. On the other hand, on the analyzers, a numerical value of the RDW is obtained as a percentage coefficient of variation (CV%) or as the standard deviation of the mean size (SD). A raised RDW indicates a heterogenous population of RBCs from the size point of view.3 In nutritional anaemias, this parameter is often the first to be altered and a raised RDW even with a normal MCV warrants a PS examination to look for a small population of microcytic or macrocytic RBCs. It is important to realise that the MCV is just an average value and even if a population of microcytes /macrocytes are present in a sample, the MCV may fall in the normal range if majority of the cells are normocytic. Moreover, an admixture of microcytes and macrocytes as may be seen in a case of nutritional anaemia may average out the MCV, causing it to fall in the normal range. However, the RDW would be much raised. Thus, the RDW is an useful parameter to differentiate between nutritional and other causes of anaemias. The following RBC histogram shows marked anisocytosis and a raised RDW (Figure 3).

RETICULOCYTE COUNTS

An underutilised but useful parameter in the work up of anaemias is the reticulocyte count. As reticulocytes are the youngest forms of RBCs, their presence in the blood establishes the fact that the bone marrow is producing RBCs. Increased bone marrow production of RBCs is reflected in a raised reticulocyte count. Unlike mature RBCs, the reticulocytes contain remnant RNA which is stained by a supravital dye and which appears as a ‘blue reticulum’ under the microscope.4 Thousand RBCs have to be counted and reticulocytes enumerated and expressed as a percentage of RBCs which can be difficult and unreliable. Today’s cell counters provide automated

characterised by macrocytic anaemia and thrombocytosis. Increased reticulocytes seen in haemolytic anaemias may lead to slight macrocytosis. Normocytic indices may be seen in ACD, haemolytic anaemias and even combined nutritional anaemias.

reticulocyte counts on the basis of their larger volumes and presence of RNA. For ease and familiarity, the reticulocytes continue to be expressed as a percentage of the RBCs which may lead to falsely raised values when the absolute number of RBCs is low as seen following haemolysis or blood loss. A better way is to ‘correct’ the reticulocyte count for the patient’s Hb or Hct. A simple formula is- Corrected reticulocyte count= % of reticulocyte x patient’s Hct/45.5 Getting used to absolute reticulocyte numbers provided by the automated analyzers may be a better solution but limited by the limited access to automated reticulocyte counts.

CBC IN THE WORK UP OF ANAEMIAS (FIGURE 4)

The following is a simple algorithm for the start up of the work up of a case of anaemia using the Hb, red cell indices and reticulocyte count.5 If the reticulocyte count is raised and blood loss is ruled out, few routine biochemical tests like serum bilirubin and fractions, haptoglobin, lactate dehydrogenase (LDH), etc, may establish ongoing haemolysis. Added to these, a peripheral smear examination may help to clinch the exact diagnosis/cause of haemolysis. For example, finding a spherocyte would suggest an autoimmune haemolytic anaemia or hereditary spehrocytosis while a ‘bite cell’ or ‘blister cell’ would suggest oxidant damage as seen in G6PD enzyme deficiency. Alternatively, one may look at the RBC indices. Macrocytic anaemias may be due to B12 /folate deficiencies but may be also seen with use of anti retroviral drugs, liver disease, hypothyroidism.6 Macrocytic anaemia as a part of generalised pancytopenia may be seen in megaloblastic anaemia or aplastic anaemia. A unique type of myelodysplastic syndrome, associated with loss of the long arm of chromosome 5 (5q- syndrome) is

CHr/ Ret-He

A close differential diagnosis of iron deficiency anaemia (IDA) is anaemia of chronic disease (ACD), characterised by functional iron deficiency (FID) where iron is not incorporated into developing erythroid cells in spite of adequate bone marrow iron stores. It is now well established that release of hepcidin from liver cells under the influence of inflammatory cytokines like interleukin -6 is responsible for blockage iron iron movement from iron storing macrophages into the developing red cells.8 This block also occurs at the level of the intestinal cells wherein absorbed iron cannot enter the blood stream. Over weeks, this lead to formation of RBCs with low MCV and low MCH. The traditional biochemical tests like serum iron, transferrin and ferritin levels fail to convincingly distinguish between IDA and ACD. An improvement over these tests is the soluble transferrin receptor level (sTfr) which is raised only in IDA and remains low in ACD.9 This parameter is limited by its complexity of testing and its relative unavailability. Changes in the RBC parameters as described occur over the life span of RBCs (120 days) while changes occurring in reticulocytes occur quickly, i.e., within 2-3 days (the life span of the reticulocytes). CHr provided by instruments manufactured by Siemens and Ret-He provided by Sysmex analyzers measure reticulocyte haemoglobin content or its equivalent directly. Both are helpful parameters to detect iron insufficient erythropoiesis,10 either due to pure and simple iron deficiency or due to FID at a very early phase. Effective treatment of these conditions bring about a rapid change or increase in the CHr or Ret-He as these are measurements on the reticulocytes. Moreover, this information is obtained at the cost and at turn around time of a routine CBC. However, it should be noted that the Ret-He is a size based assay and will be useless in the presence of an underlying beta thalassemia or in the presence of concomitant megaloblastic anaemia. It has been shown that using a combination of sTfr, serum

CHAPTER 60

Fig. 5: Scattergram from a sysmex x class analyzer. Sourcehttps://www.sysmex.com

In the presence of microcytosis, the TRBC (raised in thalassemia) and RDW (increased in IDA) can help differentiate between iron deficiency and beta thalassemia trait. Indices like the Mentzer’s index7 have been put forward to make this differentiation more objective. If the quotient of the MCV (in fl) divided by the TRBC (in millions/cubic mm) is less than 13, thalassemia is more likely. If the result is greater than 13, iron deficiency is more likely. Co-existing iron deficiency and beta thalassemia trait make these values redundant. Beta thalassemia carriers have microcytic hypochromic RBC indices with a normal RDW as the red cells are uniformly small , i.e., homogenously microcytic. On the other hand, iron deficiency shows microcytosis along with an altered RDW.

329

are characteristic. Any deviation from these as given by the mean value and standard deviation of each of these parameters reflect changes in size and complexity for a particular cell type which in turn may be reflective of specific diseases. These positional parameters have been used to differentiate between chronic lymphoproliferative disorders (CLPD) like chronic lymphocytic leukaemia (CLL) vis a vis reactive lymphocytosis as in the former, the cells are uniformly small.14 Changes in the parameters of neutrophils have been found to indicate sepsis and bacterial infections.15 Changes involving lymphocytes and monocytes have also been used to flag for presence of malaria.16 As of now, these are still research parameters and yet to be incorporated into routine clinical practice.

HAEMATOLOGY

330

Fig. 6: Platelet clumps in EDTA ferritin and the Ret-He, it is possible to accurately diagnose and provide guidelines for appropriate management of all microcytic hypochromic anaemias (Thomas plot, 11). Both Retâ&#x20AC;&#x201C;He and CHr have been incorporated into guidelines for management of anaemia in chronic renal disease patients and FID.12 A high haematocrit which is inexplicable (no history of living at high altitude, no smoking , no chronic obstructive airway disease, etc) should prompt one to work up for a primary haematological disorder like polycythaemia vera (PV). A the Jak2 mutation performed on peripheral blood is warranted.13

WBCS

Popular technologies for enumeration and differentiation of WBCs include the Volume Conductivity and Scatter (VCS) used by the Beckman Coulter instruments (from Beckman Coulter Inc, USA) and the Fluorescence Flowcytometry (FFC) principle used by the Sysmex analyzers (from Sysmex Corporation, Japan). The VCS principle differentiates cells based on their volume measured by impedance , conductivity of radio frequencies which give information about the internal structure of cells and and scatter of light which tells us about surface structure and granularity. In the FFC technology, cells are hit by a laser beam which gets scattered in various directions. The forward scattered light is a measure of the cell volume while the side scattered light is a measure of cell granularity. When a fluorescent dye is used, side fluorescence gives information about the nucleic acids in the cells. Thus, monocytes have the highest forward scatter owing to their large size while eosinophils have the highest side scatter owing to the presence of the prominent granules. immature granulocytes have high fluorescence. Thus, various scatter plots are generated and the total WBC and the differential counts obtained. Leucocyte positional parameters- Numerical data coordinates generated by VCS technology are available as research parameters on LH Beckman Coulter analysers. Volume, conductivity and light scatter for each WBC type

The CBC will indicate if there is leucocytosis or leucopenia. The type of cell involved suggests the possible differential diagnoses. For example, increase in neutrophils could suggest a bacterial infection, an inflammatory state, steroids use, etc. An extremely high count with left shift suggests a chronic myeloid leukaemia (CML) or a leukamoid reaction. A bimodal peak of myelocytes and neutrophils alongwith presence of basophils point towards CML and the molecular test for the diagnostic fusion gene, BCR:ABL1 should be asked for. Similarly, finding sustained absolute lymphocytosis without any chronic infection would warrant a haematological work up for a CLPD like CLL. Lymphoma spillover into blood may also cause a lymphocytosis. Peripheral smear may reveal characteristic morphology but immunophenotyping using flowcytometry is the main stay of diagnosis.17 Eosinophilia is most often secondary to allergic conditions, parasitic infestations, drugs, vasculitides. If no such cause is found after reasonable investigations and if eosinophilia is sutained, a primary eosinophilia (clonal or hypereosinophilic syndrome) needs to be considered. Molecular studies for FIP1L1-PDGFR mutation are important to determine response to a tyrosine kinase inhibitor like Imatinib.18 Monocytosis may be seen in some chronic infections or during recovery of counts following chemotherapy or drugs. However, sustained absolute monocytosis of at least 1000/ul in absence of chronic infections warrant investigating for a haematological disorder like chronic myelomonocytic leukaemia in an adult and juvenile myelomonocytic leukaemia in children. In the presence of leucopenia , it is prudent to look at the absolute cell counts to see if any particular cell line is predominantly suppressed. Neutropenia is most commonly encountered due to drugs and severe infections which would be apparent from the patientâ&#x20AC;&#x2122;s clinical condition. When neutropenia occurs alongwith anaemia and thrombocytopenia, a serious bone marrow disorder like aplastic anaemia should be thought of. Inherited conditions of neutropenia are rare but known (e.g., Kostman syndrome). Acquired lymphopenia may be seen following use of monoclonal anti CD20 antibodies

331

CHAPTER 60

Fig. 7: RBC & Platelet Histogram like Rituximab.19 Absolute monocytopenia is a feature of Hairy cell leukaemia while eosinopenia is a feature of sepsis.

PLATELETS

A small percentage of people develop platelet clumping when blood is collected in EDTA (the preferred anticoagulant for CBC analysis) because they carry antibodies against certain epitopes of platelets unmasked in the presence of EDTA (Figure 6).20 In such cases, a repeat count after a repeat collection in citrate is indicated. It should be noted that in the presence of platelet clumps it is not possible to give an accurate platelet count , whatever be the technology used. The most common method of counting platelets is impedance as described earlier in the section on RBCs. The difference in size between the RBCs and platelets makes it possible to separate out the the platelets and RBCs. Problems occur when there are very large platelets which may be falsely picked up as RBCs leading to a falsely lowered platelet count. Pathological conditions associated with large platelets like Bernard Soulier syndrome and familial platelet disorders with myosin heavy chain9 (MYH9) gene mutation are well documented but are rare.21 More commonly, in the eastern part of our country , a large chunk of the population have mild thrombocytopenia and very large platelets (constitutional macrothrombocytopenia) which cause further artefactual lowering of the platelet count on impedance cell counters.22 There is no risk of bleeding but the low platelet count leads to panic and unnecessary investigations. Extremely microcytic or fragmented RBCs may be wrongly counted as platelets causing an inaccurate higher platelet count. This becomes more significant when the platelet counts are low. In these situations, the platelet and the RBC histograms overlap with each other and the instrument gives a flag which warrants a peripheral smear check. A manual platelet count is useful in such situations.

Fig. 8: Measurement of IPF using platelet-F technology. Source-https://www.sysmex.com The following figure shows overlapping RBC and platelet histograms (Figure 7). As manual platelet counts are difficult and time consuming, there have been attempts to innovate technology to overcome the problems with impedance counts like optical counts based on light scatter, use of monoclonal anti platelet antibodies (Abbott), fluorescent platelet counts (platelet-F, Sysmex),23 etc. Platelet â&#x20AC;&#x201C;F involves use of a fluorescent dye which is highly specific for platelet membrane and counts obtained by this method have been found to be very accurate.24 Besides the counts, platelet indices like the MPV and PDW have also been useful reflecting the average platelet size and platelet anisocytosis respectively. A PS confirmation of thombocytopenia is a must to rule out EDTA induced agglutination, presence of large platelets, fibrin strands, etc. Infections and sepsis alongwith drugs are the commonest causes of thrombocytopenia encountered in a hospital set up wherein the clinical history is indicative of the cause. Thrombocytopenia associated with abnormalities in other cell lines may indicate a bone marrow disorder like an acute leukaemia. Isolated thombocytopenia in the absence of any other cytopenia (except perhaps for an IDA secondary to bleeding) may be due to an immune thrombocytopenia (ITP) which may be primary or secondary to an associated immunological disorder.25 ITP is a diagnosis of exclusion. It is crucial to rule out a thrombotic thrombocytopenia purpura (TTP) in the appropriate clinical setting by looking for fragmented cells (schistocytes) in the PS and analysing the serum LDH as TTP has a 90% mortality if plasmapheresis is not started early. Another life threatening situation is Heparin induced thrombocytopenia which needs to be considered in patients exposed to unfractionated heparin.26 Thrombocytosis(>450,000/ul) is seen in inflammatory

332

states, infections and IDA. However, persistent thrombocytosis in the absence of these conditions may indicate an underlying chronic myeloproliferative neoplasm like CML, essential thrombocytosis , idiopathic myelofibrosis, etc. In such situations, bone marrow trephine biopsy and molecular tests for BCR;ABL1, JAK2 mutation, CALR mutations are warranted.27

HAEMATOLOGY

IMMATURE PLATELET FRACTION (IPF)

Akin to the reticulocyte count in RBCs is the IPF in the platelets (Figure 8). In the higher end Sysmex analyzers it is possible to reliably quantify the fraction of the most immature platelets containing RNA (reticulated platelets) which is expressed as the IPF % (reference range-1.1 to 6.1%).28 In a case of thrombocytopenia, a high IPF indicates adequate bone marrow production with peripheral loss/ destruction of platelets. It is seen that with recovering platelet counts, the IPF value falls. Thus, it is a very useful parameter in the diagnosis of various thrombocytopenias associated with increased peripheral destruction, eg., ITP/ TTP.28 IPF has been found useful in the management of Dengue. In a small study of 32 patients, 93.75% of the patients showed platelet recovery within 24–48 h if the IPF was more than 10% and 100% patients showed a recovery within 24 h of the fall of the IPF compared with the previous days.29

SUMMARY

The CBC is often the starting point in the investigation of a sick patient and important clinical decisions are made based on the findings. Today, the CBC is obtained from automated analyzers which give fast, accurate and reproducible results. Parameters like the RDW are obtained only from the automated counters. Newer parameters like Ret-He have been incorporated into international guidelines for investigation of FID. A basic understanding of the technologies makes one aware of the pitfalls and possible problems. While interpreting an abnormal CBC, it is important to take the patient’s clinical status into account and to review previous reports if any. For eg., neutrophilia in a hospitalized patient occurring over 2-3 days is likely to be due to an acute stress/infection while neutrophilia sustained over months in the absence of any apparent illness may be due to a myeloproliferative neoplasm like chronic neutrophilic leukaemia. Platelets are the most difficult cells to count due to the inherent problems associated with impedance technology. Methods like platelet-F give highly accurate counts. Newer parameters like IPF have been found to be in management of dengue and are being incorporated into the routine CBC.

REFERENCES

1. 2016 Wallace H. Coulter Foundation. http.//www.whcf.org 2. Briggs C. Quality counts:new parameters in blood cell counting. Int J Lab Hem 2009; 31:277-297. 3. Lin CK, Lin JS, Chen SY et al. Comparision of haemoglobin

and red blood cell distribution width in the differential diagnosis of microcytic anaemias. Arch Pathol Lab Med 1992; 116:1030-32. 4. Bain B J, Lewis M S, Bates I 2006. Basic haematological techniques in Lewis, Bain and Bates (ed) Dacie and Lewis Practical Haematology. Elseiver Ltd. 5. Glader B. 2004. Anaemia:general consideration in Greer JP, Foerster J, Rodgers G M, et al (ed) Wintrobe’s clinical haematology. Lippincott Williams and Wilkins. 6. Aslinia F, Mazza JJ, Yale SH. Megaloblastic anaemia and other causes of macrocytosis. Clin Med Res 2006; 4:236-241. 7. Mentzer WC. Differentiation of iron deficiency from thalassemia trait. Lancet 1973; 1:449-52. 8. Weiss G, Goodnough L T. Anaemia of chronic disease. NEJM 2005; 352:1011-1023. 9. Punnonen K, Irjala K, Rajamaki A. Serum transferrin receptor and its ratio to serum ferritin in the diagnosis of iron deficiency. Blood 1997; 3:1052-1057. 10. Brugnara C, Schiller B, Moran J. Reticulocyte haemoglobin equivalent (Ret He) and assessment of iron deficient states. Clin Lab Haem 2006; 28:303-308. 11. Thomas C and Thomas L. Biochemical markers and haematologic indices in the diagnosis of functional iron deficiency. Clin Chem 2002; 48:1066-1076. 12. Thomas Wayne D, Hinchliffe Rod F, Briggs C et al. Guideline for the diagnosis of functional iron deficiency. British Journal of Haematology 2013; 161:639-648. 13. Tefferi A, Gilliland DG. The JAK 2V617F tyrosine kinase mutation in myeloproliferative disorders:status report and immediate implications for disease classification and diagnosis. Mayo Clin Proc 2005; 80:947-958. 14. Silva M, Fourcade C, Fartoukh C et al. Lymphocyte volume and conductivity indices of the haematology analyzers Coulter GEN.STM in lymphoproliferative disorders and viral diseases. Clin Lab Hem 2006; 28:1-28. 15. Chaves F, Tierno B and Xu D. Quantitative determination of neutrophil VCS parameters by the Coulter automated hematology analyzer:new and reliable indicators for acute bacterial infection,. Am J Clin Path 2005; 124:440-444. 16. Briggs C, Da Costa A, freeman L, et al. Development of an automated malaria discriminant factor using VCS technology. Am J Clin Path 2006; 126:691-698. 17. Hallel M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukaemia. Blood 2008; 111:5446-5456. 18. Pardanani A, Brockman SR, Paternoster SF et al. FIP1L1PDGFRA fusion:prevalece and clinicopathologic correlates in 89 consecutive patients with moderate to severe eosinophilia. Blood 2004; 104:3038-3045. 19. Plosker GL, Figgit DP. Rituximab:a review of its use in non Hodgkin’s lymphoma and chronic lymphocytic leukaemia. Drugs 2003; 63:803-843. 20. Bizzaro, N., et al. EDTA-Dependent Pseudothrombocytopenia: A Clinical and Epidemiological Study of 112 Cases, With 10-Year Follow-Up. American Journal of Hematology 1995; 50:103-109. 21. Drachman J G. Inherited thrombocytopenia:when a low platelet count does not mean ITP. Blood 2004; 103:390-398. 22. Harris V. K. N and Harris S. Harris Platelet Syndrome—

Underdiagnosed and unrecognized. Archives of Pathol Lab Med 2008; 10:1546.

molecular weight heparin prophylaxis:a meta analysis. Blood 2005; 106:2710-2715.

23. Briggs C, Harrison P, Machin SJ. Continuing developments with the automated platelet count. Int J Lab Hem 2007; 29:77-91.

27. Arber DA, Orazi A, Hasserjian R, Thiele T, et al. The 2016 World health Organisation classification of myeloid neoplasms and acute leukaemias. Blood 2016; 127:23912405.

24. Schoori M, schoori M, Oomes J, van Pelt J. New fluorescent method (PLT-F) on Sysmex XN2000 hematology analyzer achieved higher accuracy in low platelet counting. Am J Clin Pathol 2013; 140:495-9. 25. Stasi R. How to approach thrombocytopenia. Hematology Am Soc Hematol 2012; 2012:191-197.

28. Briggs C, Kunka S, Hart D, Ogumi S, et al. assessment of immature platelet fraction in peripheral thrombocytopenia. British Journal of Haematology 2004; 126:93-99. 29. Dadu T, Sehgal K, Joshi M, Khodaiji S. Evaluation of the immature platelet fraction as an indicator of platelet recovery in dengue patients. Int J Lab Hematol 2014; 36:499504.

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26. Martel N, Lee J, Wells PS. Risk for heparin induced thrombocytopenia with unfractionated heparin and low

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C H A P T E R

A Practical Approach to Anaemia

Anaemia is defined as a fall in hemoglobin (Hb) below once individual baseline value. Often, such baseline value is unavailable. In this situation, physician utilizes age-specific, sex-specific and race-specific reference ranges. There are large number of different diseases that are included in the World Health Organization (WHO) list regarding causes of anaemia. Our todayâ&#x20AC;&#x2122;s goal is to provide a simple approach to anaemia which is based on clinical and simple laboratory tests. In clinical practice, the standard method of taking a good history followed by proper examination supported by laboratory parameters still forms the best approach1. We have tried to propose a hierarchical and logical way to reach a diagnosis as quickly as possible by properly managing the medical interview, physical examination, appropriate laboratory tests including bone marrow examination and other complementary tests. There are various ways of classifying anaemia. None is perfect. The approach to find the cause of anaemia differs between men and women, children and adults and also between different ethnic groups. Various algorithms exist. It is important to approach the problem carefully. One should not miss treatable disorders like nutritional deficiencies, GI bleeding, hemolysis and anaemia of renal origin. The traditional approach of sub categorizing anaemia in to microcytic, normocytic and macrocytic subtypes is still probably the best. The basis of this subtyping is Mean Corpuscular Volume (MCV). At the same time, one must remember that this is just a starting point. Subsequently, each category has to be deciphered using a stepwise approach which utilizes various laboratory tests.

CLASSIFICATION

One can approach the problem of anaemia from three angles i.e. based on : 1.

Pathogenesis

Clinical presentation

Red cell morphology and indices

MB Agarwal Pathogenic mechanisms involved in the production of anemia include 1.

Inadequate production

Excessive destruction (hemolysis)

Blood loss (bleeding)

CLASSIFICATIONS BASED ON PATHOGENESIS

Hypo-regenerative: Here blood production is decreased. This could be due to lack of nutrients (iron, vitamin B12 or folic acid), paucity of stem cells, defective marrow function or marrow infiltration

Regenerative: Here marrow is normal and it responds appropriately to anaemia by increasing production of erythrocytes.

CLASSIFICATIONS BASED ON CLINICAL PRESENTATION

Acute (bleeding or hemolysis)

Chronic (primary marrow disorders and various chronic diseases)

CLASSIFICATION BASED ON RED CELL MORPHOLOGY AND INDICES

In practice, classification based on red cell morphology & indices chiefly mean corpuscular volume (MCV) is very useful. From this angle, anaemia is classified as microcytic, normocytic or macrocytic. Red cell morphology as assessed by microscopic examination of peripheral blood film, MCV as analysed by electronic cell counter together with reticulocyte count which differentiates between regenerative and hyporegenerative anaemias can give an accurate diagnosis in most of the patients We will now look at various causes of anaemias based on MCV

MICROCYTIC ANAEMIA

MCV below 80 fl is considered as microcytosis. Common causes of microcytic anaemia are listed in Table 1.

Iron deficiency anaemia (IDA) Table 1 : Causes of microcytic anaemia (MCV < 80 fl) Iron deficiency anaemia (IDA) Thalassaemia & Hemoglobinopathies Anaemia of chronic disease (ACD) Sideroblastic anaemia Lead intoxication

Iron deficiency is the commonest cause of microcytic anaemia. It has one specific symptoms i.e. pica. Pica relates to persistent and compulsive craving to eat non-food items. It comes from a Latin word for a bird known for its indiscriminate appetite (Figure 1). Pica includes eating of dirt, clay, paint chips, plaster, chalk, cigarette ashes, soap, toothpaste, burnt match heads, cigarette butts, ice, glue, hair, buttons, paper & sand etc. IDA has a specific sign

Table 2 : Investigations to differentiate IDA (iron deficiency anaemia) from ACD (Anaemia of chronic disease) Test

ACD

S. Iron



Total Iron Binding Capacity (TIBC)



 or N

Transferrin saturation



S. Ferritin (SF)



N or 

Soluble transferrin receptor (STfR)



Ratio of STfR / SF Cytokine levels N : Normal;  Increase ;  Decrease

Fig. 1 : Pica (A bird with indiscriminate appetite)

Another condition that needs to be differentiated from IDA is thalassaemia minor. Increased red cell distribution width (RDW) is associated with IDA. In thalassaemia minor, RDW is normal. For a given level of Hb, RBC count is relatively higher in thalassaemia minor. Peripheral smear examination in IDA shows pencil cells which are quit characteristic. As against this, in thalassaemia, one has basophilic stippling and target cells. Also, thrombocytosis is often associated with iron deficiency. Lastly, a therapeutic trial with iron supplementation is both cost-effective and definitive way to diagnose IDA. In each case of IDA, underlying cause must be detected. Nutritional deficiency is the commonest while bleeding from GI tract or menorrhagia accounts for most important underlying disorders. Other conditions include celiac disease (gluten enteropathy) etc. Although, Hepcidin is important in iron metabolism, it’s assay is neither clinically available or required.

Thalassaemia and hemoglobinopathies

Fig. 2 : Koilonychia i.e. Koilonychia (Figure 2). Differentiating iron deficiency anaemia (IDA) from anaemia of chronic disease (ACD) is important. The tests used for differentiating these are listed in Table 2. A diagnostic test for IDA is low S. ferritin (SF). This finding is diagnostic and specific of iron deficiency state.2 Raised S. ferritin, specially above 100 ng/ ml is very unusual in iron deficiency, however, ferritin is a acute phase reactant and hence it may be normal or high despite iron deficiency if person has infection, inflammation or malignancy. Serum Iron (SI) is low in both IDA and Anaemia of Chronic Disease (ACD). Total iron binding capacity (TIBC) is high only in iron deficiency and hence TIBC also helps. Soluble Transferrin Receptor (STfR) is elevated in IDA and hence the ratio of STfR to SF is high in IDA as against ACD. A gold standard to diagnose iron deficiency state is to document deficiency of iron in bone marrow, however, this is not necessary in day-to-day practice. Also, this may also be inaccurate due to certain technical problems3.

In India, in community practice, thalassaemia minor (heterozygous state or carrier state or trait) is the second most common cause of microcytic anaemia. It is β-thalassaemia minor which is common and more important then relatively rare and clinically insignificant α-thalassaemia trait. The definitive test is documenting raised Hb-A2 fraction which is usually above 3.8%. Normal individuals have Hb-A2 between 2.0 and 3.5 % while in IDA, it decreases. This tests can be carried out by HPLC (High Pressure Liquid Chromatography) or Hb Electrophoresis, the first being more reliable. In case of difficulty, molecular techniques are required. As stated above, thalassaemia trait will have normal RDW and RBC count is much higher for a given level of Hb while peripheral smear shows morphological changes in red cells as mentioned above. Common hemoglobinopathies in India include Hb-S, HbE, Hb-D, Hb-Q etc. Detailed description of diagnosing these is beyond the scope of this write-up

Anaemia of chronic disease (ACD)

Third important cause of microcytic anaemia is ACD. It is not uncommon and probably the most common cause in hospitalised patient. Usually, ACD is normocytic, however, various chronic disorders e.g. tuberculosis,

CHAPTER 61

IDA

335

HAEMATOLOGY

336

Fig. 3 : Bone marrow showing ring sideroblasts

Fig. 4 : Hypersegmented neutrophils

Table 3 : Causes of macrocytic anaemia (MCV > 100 fl) Anaemia with reticulocytosis Megaloblastic anaemia Drug induced disorders Alcohol related anaemia Primary marrow disorders Liver disorders Spurious macrocytosis rheumatoid arthritis, other collagen vascular diseases & even malignancies are accompanied by microcytosis. The microcytosis of ACD is mild. RDW is usually normal. Peripheral blood smear is unremarkable. Diagnosis is often suspected on clinical grounds. Bone marrow examination is hardly ever required. Many patients are febrile or have other constitutional symptoms due to raised cytokines (Table 2).

Acquired sideroblastic anaemia

This is a rare disorder which is often drug induced. An important cause is low grade myelodysplastic syndrome (MDS). Here, peripheral smear shows dimorphic morphology, RDW is high and marrow shows ring sideroblasts (Figure 3).

Lead intoxication

This is not an unusual cause of microcytic anaemia, specially in India. Unlike the causes mentioned in hematological text books, ayurvedic medicines containing â&#x20AC;&#x153;Bhasmasâ&#x20AC;? is a common etiology. Clinically, patients may have characteristic blue line over the gums, peripheral smear shows punctate stippling and blood lead levels are high

MACROCYTIC ANAEMIA

MCV above 100 fl is required to consider anaemia as macrocytic. Common causes of macrocytic anaemia are listed in Table 3. One should first look for reticulocytosis

Fig. 5 : Bone marrow showing megaloblasts as the cause of mild macrocytosis.4 This is reflected as polychromasia in peripheral blood film. If present, it will suggest a regenerative anaemia i.e. hemolysis or hemorrhage. If there is no reticulocytosis, the next thing is to look for red cell morphology to differentiate between macro-ovalocytosis which is seeing in megaloblastic anaemia vs round macrocytosis which is common with remaining disorders. In patients with round macrocytosis, it is important to ask for history of alcohol and drugs e.g. hydroxyurea, methotrexate, trimethoprim, zidovudine etc. In India, nutritional vitamin B12 and/or folic deficiency is common.5 Often, these patients have pancytopenia as vitamin B12 and folic acid are required for formation of all nucleated cells. Neutrophils are often hypersegmented

Table 4 : Causes of normocytic anaemia (MCV : 80-100 fl) Dimorphic anaemia Anaemia of renal insufficiency

NORMOCYTIC ANAEMIA

337

Anaemia with MCV between 80-100 fl are called normocytic. Table 4 enlists the common causes. Dimorphic anaemia (combined deficiency of iron and vitamin B12 / folic acid) is a common condition. Anaemia of renal origin has a unremarkable hemogram with normal looking peripheral blood smear. Here, anaemia is proportional to the degree of renal failure. Marked anaemia will invariably be associated with high S. Creatinine level. Once again, both ACD & anaemia secondary to marrow disorders are often normocytic. Lastly, haemolytic anaemias are usually normocytic (Figure 6). These disorders will have clinical features of hemolysis i.e. icterus and splenomegaly. Laboratory evidence will include indirect hyperbilirubinemia, raised LDH, decreased haptoglobin & erythroid hyperplasia in the marrow. There is reticulocytosis and polychromasia (Figure 6). Once haemolytic anaemia is suspected, itâ&#x20AC;&#x2122;s cause has to be found. Peripheral smear is an important test in assessing the etiology of hemolysis (Figure 7). In addition, doing coombs test differentiates between immune hemolysis and others (Figure 8). In patients without hemolysis, a large number of bone marrow disorders feature and hence marrow examination become a must. This will give diagnosis of important primary marrow disorders e.g. Aplastic anaemia,

Haemolytic anaemia Anaemia of chronic disease (ACD) Primary marrow disorders

Indirect hyperbilirubinemia

Anemia

Reticulocytosis

Evaluate for hemolysis: CBC, reticulocyte count, LDH, indirect bilirubin, haptoglobin, peripheral blood smear

Negative

Consider alternative diagnoses, including other causes of normocytic anemia (e.g., hemorrhage, chronic disease, chronic kidney disease)

Positive

Spherocytes, positive DAT

Spherocytes, negative DAT, family history

Immune hemolysis: Hereditary lymphoproliferative spherocytosis disorder/cancer, autoimmune diseases, drugs, infections, transfusion

Schistocytes

Microangiopathic hemolytic anemia

Hypochromic, microcytic anemia

Thalassemia

PT/PTT, renal and liver function, blood pressure

Sickle cells

Sickle cell anemia

Hemoglobin electrophoresis

TTP, HUS, DIC, eclampsia, preeclampsia, malignant hypertension, prosthetic valve

Fig. 6 : An approach to haemolytic anaemia

Infection/drug exposure

Fever/recent travel

G6PD activity

Thick and thin blood smears, Babesia serologies, bacterial cultures

CHAPTER 61

(Figure 4). Platelet count may be low. Homocystein levels are high. S. Vitamin B12 or RBC folic acid level should be ordered. Although not common in practice, S. methylmalonic acid level is high in vitamin B12 deficiency.5 Often, a bone marrow examination may be needed to differentiate between non megaloblastic macrocytic anaemia vs megaloblastic anaemia (Figure 5). This is also important as many primary bone marrow disorders produce mild to moderate macrocytic anaemia. These include aplastic anaemia, myelodysplastic syndrome and others. If vitamin B12 deficiency is confirmed, one has to search for its etiology. In India, many vegetarians have nutritional vitamin B12 deficiency, specially if they do not take milk which is an important dietary source of vitamin B12 for vegetarians. It is also important to look for pernicious anaemia by testing for intrinsic factor antibodies. Schilling test is not so widely available. Lastly, various primary intestinal malabsorption disorders e.g. tropical sprue, celiac disease, inflammatory bowel diseases etc. should be considered and if required, investigated.

HAEMATOLOGY

338

Fig. 7 : Importance of peripheral blood smear examinations in assessing the etiology of haemolytic anaemia. DIC = disseminated intravascular coagulation; HUS = hemolytic uremic syndrome; MAHA = microangiopathic hemolytic anemia; PCH = paroxysmal cold hemoglobinuria; PNH = paroxysmal nocturnal hemoglobinuria; TTP = thrombotic thrombocytopenic purpura acute leukaemia, myelodysplastic syndrome, multiple myeloma, infiltrative disorders etc.

CONCLUSION

The human mind is a marvelous albeit complex tool. It is difficult to understand how it works. Clinicians are no exception in their varied approach to any clinical problem. The guidelines outlined above have to be used in the context of good history and clinical examinations. Over 100 diseases cause anaemia. It is important to look at various points in history and examination which cannot be dissociated from laboratory investigations. A simple example is a patient with clinical frank features of myxoedema requiring no other investigations except TSH. Similarly, history of recent blood transfusions can vitiate many results. Laboratory tests such as complete blood count obtained from an electronic cell counter, peripheral smear examination & reticulocyte count are the first line investigations. These give a guideline for selecting further tests. Serum iron studies and S. ferritin assay are sufficient

Fig. 8: An approach to hemolytic anaemia based on coombs tests and other investigations. G6PD - Glucose-6-Phosphate dehydrogenase to in arriving at the diagnosis of IDA. For macrocytic anaemia, smear examination and reticulocyte count form the first line of investigation. Based on the results, further laboratory tests should be ordered i.e. S. Vitamin B12, RBC folic acid, hemolytic profile or marrow examination. If ACD is suspected, various tests e.g. S. Creatinine, TSH, ANA, Anti CCP Antibody & tumor markers may be needed.

REFERENCES

1. Gjorup T, Bugge PM, Hendriksen C, Jensen AM. A critical evaluation of the clinical diagnosis of anemia. Am J Epidemiol 1986; 124:657-665. 2. Guyatt GH, Oxman AD, Ali M, Willan A, McIlroy W, Patterson C. Laboratory diagnosis of iron-deficiency anemia: an overview [published correction appears in J Gen Intern Med 1992; 7:423]. J Gen Intern Med 1992; 7:145-153. 3. Barron BA, Hoyer JD, Tefferi A. A bone marrow report of absent stainable iron is not diagnostic of iron deficiency. Ann Hematol 2001; 80:166-169. 4. Colon-Otero G, Menke D, Hook CC. A practical approach to the differential diagnosis and evaluation of the adult patient with macrocytic anemia. Med Clin North Am 1992; 76:581-597. 5. Beck WS. Diagnosis of megaloblastic anemia. Annu Rev Med 1991; 42:311-322.

An Approach to a Patient with Bleeding Disorder

C H A P T E R

Nidhi Sharma, Sandhya Gulati, Sudhir Mehta, Shaurya Mehta

ABSTRACT

Hemorrhagic diathesis can be caused by disorders in primary hemostasis (von Willebrand disease, inherited platelet function disorders), secondary hemostasis (hemophilia A, B, other coagulant factor deficiencies) and fibrinolysis, and in connective tissue or vascular formation. This review summarizes the approach to a bleeding patient starting from structured patient history, to applying bleeding assessment tools (BATs), the utilization of currently available diagnostic methods for bleeding disorders and finally investigating with highly specialized tests. A comprehensive framework for a genetic diagnostic work up to inherited bleeding disorders is the need of the hour. The discovery of a wide spectrum of mutations in the various genes associated with coagulation and their association with different severity of the disease has allowed for the development of a rational strategy for mutation detection in clinical settings. Characterization of the genetic defects is also required for carrier detection, antenatal testing, and predicting risk of factor inhibitor development.

KEYWORDS

bleeding disorder, approach, investigations, genetics

INTRODUCTION

Hemostasis involves interactions between the blood vessels, platelets and coagulation factors1. A defect in any of these phases of coagulation can result in a bleeding problem which may be inherited or acquired. The aim of hemostasis is to prevent blood loss following injury by the formation of a stable blood clot. Blood clots are eventually dissolved by the fibrinolytic system. A delicate balance between formation and dissolution of a blood clot is maintained2,3. A disruption of this unique balance results in bleeding or thrombosis. The objectives of this review is to provide primary care physicians with a systematic diagnostic approach in dealing with patients suffering from bleeding disorders, and demonstrate the importance of clinical and laboratory evaluation as highlighted in Table 1.

Table 1: Evaluation of a patient with bleeding disorder Clinical evaluation

History Family history Bleeding assessment tools (BATs) Physical examination

Laboratory First line investigations (screening tests) evaluation Second line investigations (confirmatory test) Third line investigations (genetic workup)

History taking: A good detailed comprehensive history is the best predictor of a bleeding problem.

NATURE OF BLEEDING

If the bleeding involves the skin (cutaneous) and mucous membranes i.e. petechiae, purpura bruises, epistaxis, gingival bleeding, menorrhagia and/or hematuria, it would suggest a platelet and/or vascular abnormality. Bleeding into deep tissue, joints and muscles suggest a coagulation factor defect4.

HISTORY SUGGESTING CONGENITAL/ACQUIRED BLEEDING

A history of easy bruising or exaggerated bleeding after injury suggests an inherited bleeding problem. There should be questions on any childhood history of epistaxis, umbilical stump bleeding, bleeding after circumcision hinting an inherited bleeding disorder. Any history of blood transfusion or other blood components is very important. Information on all surgeries including tooth extractions and any history of abnormal bleeding during or after surgery should be evaluated. The response to trauma is an excellent screening test. Any significant injury without abnormal bleeding is good evidence against the presence of an inherited haemorrhagic disorder. A very careful family history is critical; any family history of abnormal bleeding in parents, maternal grandparents, aunts, uncles, and siblings as well as any history of consanguineous marriage (or among relatives) should be taken. However, a negative family history does not exclude an inherited coagulation disorder. As an example, up to 30-40% of patients with hemophilia A have a negative family history. A comprehensive history will guide the direction and extent of the laboratory evaluation and also help in determining management.

MEDICATION USE

A careful history of medication use is important, including prescribed medications, over-the-counter medications and herbal products. Drugs cause bleeding due to thrombocytopenia or platelet dysfunction, aplastic anemia or vascular purpura. Bleeding scores (BSs) have been proposed for obtaining standardized quantitative histories5. Because symptoms might be subtle, especially in younger children, a detailed clinical history is crucial. In order to standardize this, questionnaires have been drafted and are known as bleeding assessment tools (BATs). The main advantages of using BATs are: 1.

As a screening tool, particularly for mild bleeding disorders.

340

To assess disease severity.

3. To address phenotypic heterogeneity and correlations between clinical and laboratory phenotype, and between genotype and phenotype 4.

To improve communication in a clinical setting.

HAEMATOLOGY

PHYSICAL EXAMINATION

From the clinical assessment, one should be able to assess whether: (1) the bleeding is the result of a local anatomic defect or part of a systemic defect in hemostasis, (2) the bleeding is due to a vascular defect, platelet abnormality or coagulation disorder, or (3) the haemostatic defect is inherited or acquired.

DISORDERS OF PLATELETS OR BLOOD VESSELS

They are characteristically associated with mucosal (epistaxis, gingival) and cutaneous bleeding (petechiae, ecchymoses). Patients with platelet abnormalities tend to bleed immediately after vascular trauma and rarely experience delayed bleeding, which is more common in the coagulation disorders.

COAGULATION DISORDERS

The typical manifestations of bleeding in the coagulation disorders are large palpable ecchymoses and large spreading deep soft tissue hematomas. Haemorrhage into synovial joints most often indicates a severe inherited coagulation disorder such as hemophilia.

LABORATORY INVESTIGATIONS

For each phase of hemostasis, screening tests which help in distinguishing a platelet disorder from a coagulation defect are available. The use of these tests are never a substitute for clinical assessment, as there is enough evidence that screening tests are unhelpful in the prediction of a bleeding disorder especially when applied indiscriminately. The quality of the report issued from the laboratory will depend on the quality of the sample. Clotted, hemolyzed and inappropriately collected sample will lead to erroneous results. Sodium citrate is the anticoagulant of choice. First line investigations (screening tests) include a complete blood count (CBC) to assess platelet count, peripheral blood smear examination, prothrombin time (PT), activated partial thromboplastin time (APTT), a thrombin time (TT) and a bleeding time (BT) or platelet function analysis (PFA)6,7,8. Second line investigations include mixing studies, factor levels, platelet aggregation studies and von Willebrand factor level. Platelet counting and the peripheral smear — Platelets may be counted directly or with the use of fully automated electronic methods. While some automated methods may flag for the presence of unusually small or extremely large platelets, there is no substitute for direct examination of the peripheral blood smear for detection of quantitative as well as qualitative (ie, abnormalities of platelet size) platelet abnormalities. Examination of the peripheral blood smear is essential in patients with low platelet counts to exclude the presence of pseudothrombocytopenia due to in vitro platelet agglutination in the presence of EDTA. This phenomenon is thought to result from a “naturally

occurring” platelet autoantibody directed against a normally concealed epitope on the platelet membrane, which becomes exposed by EDTA. Use of alternative anticoagulants (eg, citrate or heparin), may circumvent this technical problem. Bleeding time — The bleeding time (BT) is a measure of the interaction of platelets with the blood vessel wall. A prolonged bleeding time may occur in thrombocytopenia (platelet count usually below 50,000/microL), qualitative platelet abnormalities (eg, uremia), von Willebrand disease (VWD), some cases of vascular purpura, and severe fibrinogen deficiency, in which it is probably the result of platelet dysfunction. Among patients with a normal platelet count who are not taking aspirin, the bleeding time is used primarily to screen patients for inherited disorders of platelet function. An abnormal test in a patient with mucocutaneous bleeding would justify further testing for platelet dysfunction or specific tests for von Willebrand disease (VWD). However, a normal value for the BT should not preclude testing for VWD. Platelet Function Analyzer is more sensitive for detection of VWD than is the BT. A normal BT does not predict the safety of surgical procedures, nor does an abnormal BT predict for excessive bleeding. Since assessment of the BT is subject to considerable variation due to technical factors in executing the test, a normal range for the test varies from laboratory to laboratory, and cannot be generalized here. Of importance, the BT is not recommended as a preoperative screening test. Because of considerable variation due to technical factors in executing the test, the BT plays a limited role, if any, in evaluating hemostatic defects. The Platelet Function Analyzer — The commerciallyavailable Platelet Function Analyzer (PFA-100) is an alternative technology that assesses platelet function with greater sensitivity and reproducibility than the bleeding time (BT). Because the BT is insensitive, invasive, time consuming, and subject to variation due to technical factors, many centers have adopted the PFA-100 in place of the BT as their screening test of platelet function. This test may be performed on citrated samples of whole blood that have been stored at room temperature, and is considerably faster to perform than platelet aggregation studies. Normal PFA-100 test results may obviate the need for further expensive platelet function testing. Unlike the in vivo BT, the PFA-100 test does not provide a measure of vascular function. Prothrombin time — The production of fibrin via the extrinsic pathway and the final common pathway (common to both extrinsic and intrinsic cascades) requires tissue thromboplastin (tissue factor), factor VII (extrinsic pathway), and factors X, V, prothrombin (factor II), and fibrinogen. The functioning of these pathways is measured by the plasma prothrombin time. The test bypasses the intrinsic pathway and uses thromboplastins to substitute for platelets. Within this combined pathway, factors VII, X, and prothrombin are vitamin-K dependent and are altered by warfarin. For this reason, the PT is used

Table 2: Screening coagulogram for a patient with bleeding disorder Normal range

Platelet count

150-450 x 103 /cmm

Platelet morphology

Size and clumps

Clot retraction study

Good

Prothrombin time

10-14 seconds

Activated partial thromboplastin time

25-38 seconds

Thrombin time

15-19 seconds

Clot solubility test

Insoluble

as a measure of the anticoagulant activity of warfarin and other vitamin K antagonists. Activated partial thromboplastin time — The activated partial thromboplastin time (aPTT) measures the intrinsic and common pathways of coagulation. It is called partial since platelet substitutes are used which are only partial thromboplastins; they are incapable of activating the extrinsic pathway, which requires complete tissue thromboplastin (tissue factor). In the original method, a glass test tube provided contact activation. However, the addition of activators such as ellagic acid or particulate silicates provided better and more standardized contact activation. This activated version of the PTT (aPTT) is now the routine assay used to evaluate intrinsic coagulation and the degree of heparin anticoagulation. The aPTT is sensitive to inhibitors such as heparin and to deficiencies of all coagulation factors except factors VII and XIII. It is less sensitive than the PT to deficiencies of the common pathway (factors X and V, prothrombin, and fibrinogen). High levels of a single factor (eg, factor VIII) can shorten the aPTT. However, an association between a short aPTT and a hypercoagulable state remains controversial. Thrombin time and reptilase time — The thrombin time (TT) and reptilase time (RT) measure conversion of fibrinogen to fibrin monomers and the formation of initial clot by thrombin and reptilase, respectively. Reptilase, a thrombin-like snake enzyme, differs from thrombin by generating fibrinopeptide A but not fibrinopeptide B from fibrinogen and by resisting inhibition by heparin via antithrombin. Fibrin strand cross-linking, which is mediated by factor XIII, is not measured by these assays. Prolonged thrombin times and reptilase times may be due to hypofibrinogenemia, structurally abnormal fibrinogens (dysfibrinogens), or increased fibrin split products. Since heparin prolongs the TT but not the RT, the RT is useful for determining if heparin is the cause of a prolonged TT. Alternatively, one can test for heparin activity via its anti-factor Xa activity, or with the use of a commercial heparinase. Factor deficiencies and inhibitors — A prolonged aPTT can be due to a deficiency (or absence) of a coagulation factor or the presence of a coagulation factor inhibitor. A factor deficiency should be correctable by addition of

The presence of a factor inhibitor is suspected when the abnormal test does not correct, or only partially corrects, following an immediate assay of a 1:1 mixture of patient and normal plasma. In some cases, such as acquired factor VIII antibodies, the aPTT may correct immediately after mixing, but becomes prolonged after 60 to 120 minutes of incubation at 37º. In addition to factor inhibitors, lupus anticoagulants can result in a prolonged aPTT that is not correctable by the addition of normal plasma. The effect of these antibodies on the aPTT can be overcome by adding excess platelet phospholipid (particularly a hexagonal phase phospholipid) or by assessing the diluted Russell’s viper venom time. Paradoxically, the antiphospholipid syndrome is usually associated with a tendency to thrombosis rather than bleeding; the prolonged aPTT is an artifact of the antiphospholipid phenomenon. Tests for fibrinolysis — Fibrin and fibrinogen degradation products (FDP) are protein fragments resulting from the action of plasmin on fibrin or fibrinogen, respectively. Elevated levels are seen in states of fibrinolysis such as disseminated intravascular coagulation (DIC). FDP assays do not differentiate between fibrin degradation products and fibrinogen degradation products. It is possible to accurately measure the concentration of fibrin D-dimers, which are degradation products of cross-linked fibrin. The method of choice is the enzyme-linked immunosorbent assay (ELISA). When fibrinolysis exceeds thrombin generation, thereby increasing the risk of hemorrhage rather than thrombosis (eg, disseminated intravascular coagulation associated with acute promyelocytic leukemia), quantitative FDP levels may be more sensitive than D-dimer levels as an indication of the degree of fibrinolytic activity. Because D-dimers specifically reflect fibrinolysis of cross-linked fibrin (ie, the fibrin clot), assessment of D-dimer levels suggests thrombosis more reliably. As an example, in patients who have a low pretest probability of deep vein thrombosis, the negative predictive value of D-dimers is high. The euglobulin lysis time, which assesses overall fibrinolysis is less useful, since results from this test may vary significantly in relation to calcium ion concentrations as well as plasma levels of tissue plasminogen activator and plasminogen activator inhibitor-1. Alpha-2 antiplasmin, an inhibitor of fibrinolysis, is not measured in this test.

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Test

normal plasma to the test reaction tube. This is normally done by performing a PT or aPTT on a 1:1 mixture of patient and normal plasma (mixing study). Specific factor deficiencies are then determined by assessing the PT or aPTT in mixes of test plasma with commercially available plasmas deficient in known factors. Factor levels can be functionally assessed by comparing test results to standard curves generated by mixtures of serially diluted normal plasma and factor-deficient plasma. Immunologic assays can also be used to measure factor levels. Immunologic and functional assays should give equivalent results when a factor deficiency is present. On the other hand, a low functional assay but normal immunologic assay indicates the presence of a functionally abnormal factor.

HAEMATOLOGY

342

Table 3: Screening test results in various bleeding disorders Platelet count

Prothrombin time

Activated partial Differential diagnosis thromboplastin time

Normal

Increased

Normal

Factor VII deficiency(early liver disease, early vitamin K deficiency, early warfarin therapy) Dysfibrinogenemia Factor VII inhibitor Some cases of DIC

Normal

Increased

Factor VIII, IX, XI deficiency Inhibitors to Factor VIII, IX, XI VWD Heparin Lupus inhibitor

Normal

Increased

Vitamin K deficiency Liver disease Warfarin Heparin Factor V,X, deficiency

Decreased

Increased

DIC Liver disease HIT

Decreased

Normal

Increased platelet destruction Decreased platelet production Hypersplenism

Increased

Normal

Myeloproliferative disorders

Normal

Mild VWD Qualitative platelet defects Factor XIII deficiency

More specific tests of the fibrinolytic system include assays for plasminogen, tissue plasminogen activator (t-PA), alpha-2 antiplasmin, plasminogen activator inhibitor-1 (PAI-1), and thrombin-activatable fibrinolysis inhibitor (TAFI). Assays for alpha-2 antiplasmin are used clinically to identify patients with alpha-2 antiplasmin deficiency, an inherited disorder associated with delayed bleeding. However, specific assays for t-PA, PAI-1 and TAFI are of uncertain use clinically. The screening coagulogram is tabulated in Table 2. The aim of the screening tests is thus, to reveal broadly the source of problem, and accordingly request further investigations.Â Interpretation of screening tests is tabulated in Table 3. The second line investigations include mixing studies in the case of abnormal PT or aPTT for further information on the nature of the defect; coagulation factor assays to confirm and assess the severity of the coagulation factor deficiency such as in Hemophilia A or B; platelet aggregation studies to confirm platelet qualitative defects and investigations for Von Willebrand disease. If all baseline-screening tests are normal then investigations for factor XIII deficiency and alpha 2-antiplasmin deficiency which are not detectable by the routine screening tests are warranted. Factor XIII deficiency may be diagnosed by a clot solubility test, and the alpha 2-antiplasmin activity can be measured by a chromogenic assay. If all investigations are found normal, the patient should be investigated for

blood vessel wall abnormalities. A vessel wall defect can result in abnormal bleeding despite an otherwise normal coagulation system. Since there are no reliable clinical tests of vascular integrity, diagnosis depends on a high level of suspicion, when all laboratory tests are normal.

BLEEDING DISORDERS WITH NORMAL SCREENING COAGULOGRAM

There can be scenarios in which the coagulation disorders leading to a bleeding diathesis may be associated with normal screening coagulation tests (i) if the factor is not involved in the steps in coagulation measured by in vitro tests or (ii) if the degree of deficiency is mild. The most common amongst these rare clinical disorders is factor XIII deficiency9. Factor XIII stabilizes and cross-links fibrin strands. Factor XIII deficiency may present with delayed bleeding, usually 24 to 36 hours after surgery or trauma. Coagulation testing shows normal values for the PT, aPTT and TT. The diagnosis is made by measurement of reduced plasma factor XIII activity; an immunoassay for factor XIII or demonstration of clot dissolution in 5 molar urea or monochloroacetic acid. Rare abnormalities in regulators of plasminogen activation or plasmin degradation have been reported as causes of familial bleeding disorders (eg, alpha-2 antiplasmin deficiency, plasminogen activator inhibitor-1 deficiency). Mild hemophiliac patients with factor activity levels above approximately 15 to 20 percent are often sufficient to prevent spontaneous bleeding and to produce a normal PT and aPTT. However, patients with mild deficiency of

Table 4: Genetics of common bleeding disorder Disorder

Genetics

Hemophilia A (Factor VIII deficiency

Sex-linked recessive Xq28

Hemophilia B Christmas Disease Sex-linked recessive (Factor IX deficiency) Xq27.1-27.2

Thrombasthenia (Impaired platelet function) Glanzmann, Bernard-Soulier disease

Autosomal recessive

Owren’s disease / Parahemophilia (Factor-V deficiency)

Autosomal recessive 1q21-25

Factor-VII (Proconvertin

Autosomal recessive 13q34

(Factor I deficiency) Afibrinogenemia/ Hypofibrinogenemia/ Dysfibrinogenemia

Autosomal recessive 4q23-34

a coagulation factor may have increased bleeding with hemostatic challenges (eg, excessive surgical bleeding, menorrhagia). This may be seen in an individual who is heterozygous for a coagulation factor defect, such as a hemophilia carrier. Lastly, hemostatic disorders resulting due to structural abnormalities (eg, hereditary hemorrhagic telangiectasia), hereditary disorders of connective tissue (eg, EhlersDanlos disease, osteogenesis imperfecta), acquired connective tissue disorders (eg, scurvy, steroid-induced purpura), small vessel vasculitis, and purpura associated with the presence of paraproteins will also have a normal screening coagulogram.

A number of mutation screening methods have been used for detecting mutations, which include denaturing gradient gel electrophoresis (DGGE), single strand conformational polymorphism (SSCP), conformational sensitive gel electrophoresis (CSGE) and chemical cleavage mismatch (CMC). These result in a mutational detection rate up to 90%. All 1st degree female relatives of severe and moderate hemophilia must get factor assays done because some of them may be vulnerable to postprocedural or post-traumatic bleeding.

CONCLUSION

The approach to a patient with a bleeding disorder needs a comprehensive detailed history and thorough physical examination. There must be a logical systematic approach and a discriminate use of laboratory investigations to reach the diagnosis and assess severity. Particular emphasis should be placed on family and drug history. A simple approach to detect the cause is to look at the hemostatic system as three compartments- blood vessels, platelets and coagulation proteins. Genetic workup should be done whenever feasible to personalize the management of the patient.

REFERENCES

2. Greaves M, Watson HG. Approach to the diagnosis and management of mild bleeding disorders. Journal of Thrombosis and Haemostasis 2007; 5:167-74. 3.

Hayward CP, Moffat KA, Plumhoff E, Van Cott EM. Approaches to investigating common bleeding disorders: an evaluation of North American coagulation laboratory practices. American journal of hematology 2012; 87: S45-50.

Gopinath R, Sreekanth Y, Yadav M. Approach to bleeding patient. Indian journal of anaesthesia 2014; 58:596.

Rodeghiero F, Tosetto A, Abshire T, Arnold DM, Coller B, James P, Neunert C, Lillicrap D. ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders. J Thromb Haemost 2010; 8:2063–5.

Some patients are encountered with a significant bleeding history for which there is no explanation. Abuse, occasionally self-inflicted, should be considered.

GENETICS OF BLEEDING DISORDERS

Ever since the cloning and characterization of the first coagulation factor gene in 1982, considerable progress has been made in the use of molecular genetic strategies to assist in diagnosis of bleeding disorders. Working up on the mutational data base also helps in knowing the genetic basis of the coagulation disorders within a region and helps in determining management and prevention strategies10,11. A study from western part of India reported on 630 patients, diagnosed to have hereditary bleeding diathesis12. Amongst these, 598 (95%) patients had a coagulation disorder and 32 (5%) patients had a platelet function abnormality. In a group of coagulation disorders, hemophilia A (70.5%) was the most common disorder followed by hemophilia B (14%) and von Willebrand disease (10.8%). However Glanzman’s thrombasthenia (84.3%) was found to be the most common platelet function

Dahlbäck, Björn. “Blood coagulation.” The Lancet 355.9215 (2000): 1627-32.

6. Wintrobe’s Clinical Hematology thirteenth edition Page 1168-70. 7.

McPherson, Richard A., and Matthew R. Pincus. Henry’s clinical diagnosis and management by laboratory methods. Elsevier Health Sciences, 2016.

Triplett, Douglas A. “Coagulation and bleeding disorders: review and update.” Clinical Chemistry 2000; 46:1260-9.

9. Peyvandi F, Kaufman RJ, Seligsohn U, Salomon O, BOLTON‐MAGGS PH, Spreafico M, Menegatti M, Palla R, Siboni S, Mannucci PM. Rare bleeding disorders. Haemophilia 2006; 12:137-42. 10. Dalal A, Pradhan M, Agarwal S. Genetics of bleeding disorders. International journal of human genetics 2006; 6:27. 11. Manisha M, Ghosh K, Shetty S, Nair S, Khare A, Kulkarni B, Pathare AV, Baindur S, Mohanty D. Spectrum of inherited bleeding disorders from Western India. Haematologia 2002; 32:39-47.

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Von Willebrand Disease (Most Autosomal common disorder of platelet dominant, function, many variants (Type recessive, X -linked I-III, Pseudo, etc.) manifestations mild to severe

disorder followed by Bernard-Soulier syndrome (12.5%). Table 4 summarises the common bleeding disorders and their genetic basis.