HEME

WBCs

Basophils, CDs, Cytokines, Eosinophils, Monocytosis, Neutrophils

RBCs

Causes of Anemia, AIHA, DAT, Iron Deficiency, Macrocytosis, Methemoglobinemia, Polycythemia, Peripheral Smear, Renal Failure,

Platelets

Peripheral Smear
Thrombocytopenia (Drugs, ITP, Pregnancy, TTP, HIT, APLA Syndrome)
Thrombocytosis
Dysfunction (Acquired, Inherited)

Other

Amyloidosis, Blood Group Systems, Blood Transfusion Reactions, Castleman’s, Hemochromatosis, Hyperviscosity, Heme Syndromes, Myelodysplasia, Myeloma, PBSCT Indications, Waldenstrom’s

 

ACQUIRED PLATELET DYSFUNCTION

MEDICATIONS

- Aspirin: blocks cyclo-oxygenase
- NSAIDs: blocks cyclo-oxygenase
- Cephalosporins: Interferes with agonist-receptor interactions
- Penicillins: Interfere with agonist-receptor interactions
- Thrombolytic agents: proteolysis of membrane glycoproteins
- Dextran
- Prostacyclin
(Iloprost): decreased aggregation, Increased cAMP
- Beta blockers
- Ca-channel blockers (nifedipine, verapamil, diltiazem): interfere with Ca flux
- Nitroglycerin: increases in nitric oxide
- Nitroprusside: increases in nitric oxide
- Quinidine
- Tricyclic antidepressants
- Antihistamines
- Eicosapentanoic acid
(omega-3 fatty acids): Compete with arachidonate
- Ticlopidine: interferes with GPIIb-IIIa receptors
- Herbs: cat's claw, dong quai, evening primrose, feverfew, red clover, horse chestnut, garlic, green tea, ginseng, ginkgo

 

MEDICAL CONDITIONS

- CRF: abnormal fibrinogen & vWF binding
- Cardiopulmonary bypass: abnormal fibrinogen & vWF binding, in vivo activation --> acquired storage pool disease
- DIC: in vivo activation -> acquired storage pool disease
- Chronic liver disease: abnormal fibrinogen & vWF binding
- Myeloma: adsorption of vWF by tumor cells or increased clearance of vWF by monoclonal gammopathy
- Collagen vascular, especially SLE: antiplatelet Ab binding membrane glycoproteins and agonist-binding sites, or preventing uptake of substances into granules
- Myeloproliferative disease: decreased a-adrenergic receptors, decreased GPIb and GP-IX, decreased PGD2 receptors, Increased GPIV receptors, Increased Fc receptors
- MDS

 

AIHA: Classification, Therapy, DAT

Classification Schemes

On Basis of Serologic characteristics of involved antibody process:

- Warm-autoantibody type: autoAb maximally active at body temp (37C)
- Cold-autoantibody type: autoAb active at temps below 37C
- Mixed cold and warm autoAbs

On Basis of Presence or Absence of underlying or significantly associated disorder

- Primary or idiopathic AHA
- Secondary AHA: Associated with lymphoproliferative disorders (HD, NHL), rheumatic disorders (especially SLE), infections, certain nonlymphoid neoplasms (ovarian), certain chronic inflammatory disease (UC), certain drugs (aldomet)

The retic count is usually elevated altho early on up to 30% may have transient reticulocytopenia despite a normal or hyperplastic marrow -- maybe because the autoAb also attacks RBC precursors.

Patients with a positive IAT (indirect antiglobulin test) due to a warm autoAb should also have a positive DAT. A patient with a serum anti-RBC Ab (positive IAT) and a negative DAT probably has, not an autoimmune process, but, rather, an alloAb stimulated by prior txfusion or pregnancy.

Negative DAT may be caused by titer of IgG autoAb too low to give a positive DAT reaction (subthreshold IgG). You can concentrate the eluates or use ELISA to detect these. These patients usually have mild hemolysis and respond well to steroids. This phenomenon can also be the case sometimes in the "complement alone" pattern of DAT in the absence of drug sensitivity or cold agglutinins.

Recent organ transplant recipients can develop an ALLOimmune HA that mimics AHA.

AIHA -- Therapy

Transfusions: Problem due to trouble cross-matching and shortened RBC survival. Use "least incompatible" blood.

STEROIDS: Prednisone 60-100 mg qd x 10-14 days. (20% CR, 10% no response). When Hct stabilizes, can decrease in rapid steps to 30 mg qd. Then decrease at rate of 5 mg/day every week to a dose of 15-20 mg qd. These doses should be administered for 2-3 months after the acute episode, then wean pt over 1-2 months or switch to qod (eg 20-40 mg qod). Therapy should not be stopped until the DAT has become negative. They then need to be followed for several years due to risk of relapse.

Steroids probably work by suppressing RBC sequestration by the spleen (short term effect), and also by decreasing Ab production (long term).

SPLENECTOMY -- 30% of patients require splenectomy. Candidates include:

- No response to steroids within 3 wks

- Still requiring > 15 mg/d prednisone after 1-2 months.

Splenectomy RR = 65%. Relapse rate still high, but can often be maintained on lower dose of steroids than before, or even qod dosing.

IMMUNOSUPPRESSION -- Reserved primarily for patients who fail to respond to steroids and/or splenectomy or who are poor surg risks.

CTX 60 mg/m2 qd

Azathio 80 mg/m2 qd

If response, can try for 6 months.

 

AMYLOIDOSIS

Diagnosis: based on the identification of amyloid in a tissue biopsy obtained from an involved organ. In systemic amyloid, a sample of subcutaneous abdominal fat (obtained by needle aspiration or surgical biopsy) stained with Congo red will be + in 85%. A gingival or rectal biopsy is diagnostic in about 80%. Biopsies performed on involved tissues, such as liver, kidneys, or nerves, are almost always positive. In B2M amyloid, abdominal fat, rectal, skin and joint biopsies are of questionable value. Biopsy must be obtained from affected tissue.

IHC techniques using monoclonal Abs specific for proteins AA, AL, B2M, TTR and others are used to identify specific types of amyloid with the greatest precision. (But less sensitive in AL because light chains derive from the N-terminal variable domain, much of which is too unique. In AL amyloid, bone marrow b may be stained for AL in 50%.

If there is no family history, then patient should be worked up for plasma cell dyscrasia if amyloid tissue found.

Therapy: Generally aimed at preventing further deposition of the amyloid protein and treating the organs involved and complications.

Specific interventions:

- AL amyloid -- chemo: treat like myeloma, including transplant.

- AA amyloid -- treat underlying inflammatory process. Colchicine may help. Immunosuppressive drugs (chlorambucil, cyclophosphamide, steroids).

- ATTR (Familial amyloid polyneuropathy) -- liver transplant helps since liver produces most of the ATTR found in the serum.

- B2M amyloid -- renal transplant - which reduces the need for dialysis.

[Amyloid protein, Protein Precursors, Clinical]

AA -- Serum amyloid A (SAA) -- Reactive (secondary) amyloid, familial fever, familial amyloid nephropathy with urticaria and deafness (Muckle-Well syndrome)

AL -- lambda>kappa Ig light chains -- idiopathic (primary) amyloid, myeloma- or macroglobulinemia-associated

AH -- Ig heavy chain -- idiopathic (primary)

ATTR -- transthyretin (TTR) and its variants -- familial amyloid polyneuropathy, cardiomyopathy, systemic senile amyloidosis

AB2M -- B2 microglobulin -- associated with chronic dialysis

 

ANEMIA DDX (Low retic, High retic)

LOW RETIC (AbsRetic <75K or Index < 2%)

Hypoproliferative:

H/M: Iron deficiency, Sideroblastic (increased Fe/ferritin, think Pb intoxication if see basophilic stippling), ACD (decreased Fe/TIBC, increased ferritin, normal STRs), HbE

N/N: ACD, Renal disease (SCr >2), Endocrine (hypopituitary, hypoT4, adrenal, testic), Aplastic, Pure RBC Aplasia (vacuolated normoblasts, B19), Marrow replacement (leukemia, tumor, fibrosis, granuloma), Marrow damage (radiation, chemicals, vacuoles), Dyserythropoiesis (MDS, PNH), Myeloma (check serum/urine IFE)

Abnormal Maturation:

Macrocytic (B12, folate, Schilling Test)
Dimorphic (Sideroblastic - increased LDH, sideroblasts in BM)

 

HIGH RETIC (AbsRetic >100K or Index >2%)

Normal erythrocytic response: Blood loss, partially treated deficiency
Hemolytic Anemia

 

ANEMIA IN RENAL FAILURE

Degree of anemia does NOT correlate with degree of renal failure. (Treatment)

Causes of anemia in CRF:

1. Decreased Epo due to loss of nephrons and interstitial cells
2. Decreased folate (lost in dialysate)
3. Hematuria (from nephron damage)
4. Hemolysis (extrinsic due to metabolic defects, especially patients on hemodialysis)
5. ACD and cytokine suppression of erythropoiesis (IL-1, TNF, IFN-g)
6. Hyperparathyroidism --> osteitis fibrosa (marrow fibrosis). NOTE: These patients may be resistant to Epo.
7. Rare hypersplenism
8. Hemolysis due to: Dialysis, ACD, Uremia, Increased sensitivity of RBCs to oxidant damage due to decreased glutathione and decreased methemoglobin reductase

Treatment:

Folate if on hemodialysis

Epo 50-150 mcg/kg TIW (will correct anemia in 80%). See response in 3 days. Max response in 1-2 months.

If no response to Epo, rule out: Fe deficiency, Alum toxicity, Hyperparathyroidism, Infection, Occult blood loss

Target Hct = 35%. Once there, decrease Epo to maintain Hct. May be able to get as low as 1000u BIW.

Epo (Epogen, Procrit) Supplied in 2000, 3000, & 10,000 units/cc vials in boxes of 10.

 

APLASTIC ANEMIA (Acquired, Inherited, Treatment)

Progressively rising incidence from age 2-20, then after 60.

Clinically see bleeding (low platelets), weakness (anemia), and infection (low WBCs).

Presence of lymphadenopathy, splenomegaly, or hepatomegaly should prompt search for other disease.

Anemia usually N/N with mild macrocytosis.

ANC usually < 1500 with monocytopenia.

Presence of NRBCs on smear should raise suspicion of other disease.

Serum Fe usually elevated.

BM usually --> scant material with few spicules.

"Severe": ANC<500 Plt<20 Retic<1

<30% hematopoietic cells in BM

Overall mortality is 70%, and median survival is 1 yr.

25% live < 4 mon
25% live 4-12 mon
35% live > 1yr
10-20% spontaneously recover partially or completely.

Aplastic anemia associated with hepatitis: 60% mortality within 2 mon

Increased risk of other malignancies, MDS, and leukemia, especially patients treated with immunotherapy (compared with BMT).

Differential Diagnosis

ACQUIRED

Idiopathic (50%)

Secondary

Radiation

Drugs (2nd most common cause) (Chemotherapy, Benzene, Chloramphenicol, NSAIDs, Anticonvulsants, Gold, Arsenic, Sulfa)

Viruses: EBV, HCV, HIV, CMV, Parvo (Viral hepatitis most common virus associated with aplastic anemia. Usually develops within a few weeks or months of the hepatitis.)

Immune: Eosinophilic fasciitis, Hypoimmunoglobulinemia, Thymoma & Thymic cancer (most common hematologic disorder associated with thymoma is pure RBC aplasia, but aplastic anemia has been described.), GVHD in immunodeficiency

Clonal disorder: PNH

- Pregnancy

INHERITED

Fanconi's anemia (congenital aplastic anemia)

Schwachman-Diamond syndrome (aplas anemia+pancreatic insuf)

Reticular dysgenesis

Amegakaryocytic thrombocytopenia

Preleukemia (monosomy 7)

Non-hematologic syndromes: Downs, Dubowitz, Seckel

Treatment

Evaluate for BMT, if possible: Any patients with identical twin or Patients 20-50yo with HLA-identical sib

Support with blood products (unless BMT candidate), antibiotics.

Growth factors sometimes help.

Androgens-try if not BMT candidate and immunotx failed: Oxymethalone, 3-5 mg/kg/d x 3-6 months.

Immunosuppression:

ATG 10-20 mg/kg/d x 8-14 days. Give over 4-6hrs in NS. (To prevent serum sickness: Prednisone 50 mg/m2/d x 14 d.)

Tox: fever, chills, rash, arthralgia, anaphylaxis, platelets

Contra: horse serum allergy

Survival at 1 yr: 40-50%

Patients < 20yo with ANC <200 less likely to respond. Female<male.

1/3 relapse.

Cyclosporine (CsA)

3-7 mg/kg/d in 2 doses with weekly dose adjustments to maintain whole blood CsA drug level at 400-800 ng/ml.

Continue x 3 months unless tox. If responds, cont until max response, then taper over several months.

Tox: azotemia,liver,HBP,gum hypertrophy,psychiatric,infx

ATG plus CsA

Greater RR, but no diff in survival.

 

BASOPHILS (Basophilia, Basopenia)

Basophilia (Abs > 150/uL or >3%)

Hypersens reaction: Drugs, food, urticaria

Infection: Influenza,zoster,TB

Inflammation:    UC, RA

Lung ca

Myeloproliferative Dz: AML, CML, MDS

CRF

Basopenia

Acute infection
Steroids
Epi
Thyroxicosis
T4 replacement

 

CASTLEMAN'S DISEASE

(Giant LN hyperplasia, angiofollicular LN hyperplasia)

Young adults with mediastinal mass (70%) or localized lymphadenopathy. High ESR.

Two histologic subtypes:

1.   Hyaline-vascular (80-90%) -- Usually benign course. Amenable to surgery. +/- radioresistant, but some success with RT

2.   Plasma cell variant -- Freq associated with systemic symptoms, anemia chronic disease, hypergammaglobulinemia, & paraneoplastic syndromes (myasthenia, nephrotic syn, periph neuropathy, amyloid, temporal arteritis)

Two forms:

1.   Localized - younger patients. Treat with local treatment.

2.   Multicentric - older adult with diffuse adenopathy & variable but often bad clinical course. Most likely to transform to frank lymphoma. Med survival: 26-30 months. 20-30% -> Kaposi's or B cell NHL

Two clinical syndromes:

1.   Localized (usually hyaline type).

2.   Multicentric (usually plasmacytic, but not always). Cells often CD5+. Unpredictable course with high mortality. Increased risk of KS or lymphoma (usually unresponsive to chemo).

Nodal lesions indistinguishable from plasma cell variant have been observed in LNs from patients with AIDS, WAS, autoimmune disease, & POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein, skin changes).

IL-6 may be responsible for lymphadenopathy as well as constitutional symptoms.

There is an association with the human Herpes 8 virus, which is responsible for KS both in HIV and non-HIV patients.

TREATMENT:         

Debulking surgery

RT

Steroids

Anti-IL6 MoAb

Hyper-CVAD (used at MDACC)

 

COMMON CDs (Click here for Patterns)

T-cells

CD1 Common thymocyte

CD2 Sheep RBC receptor. Pan T-cell and some NK cells

CD3 Pan T-cell. Signal transduction proteins for Ag receptor. Immunocompetent T-cell.

CD4 Helper T-cells

CD5 Mature T-cells. Pan T-cell and a subset of B-cells

CD8 Suppressor or cytotoxic T-cells

CD9 Monocytes & pre-B cells. NOT seen on mature B-cells

B-cells

CD10           CALLA (common acute lymphoblastic ag); on pre-B cells

CD19           Pan-B. Expressed on earliest recognizable B-cell

CD20           On all peripheral B-cells & some late progenitors

CD22           Resting B-cell

CD23           Activated B-cell

CD25           IL2 receptor or T-cell growth factor receptor. Present on activated T- and B-cells

CD37           Mature B-cell. Function unknown. See in B-CLL, HCL, B-cell NHL, and B-ALL.

CD38           Plasma Cell marker

Myelomonocytic

CD11           One of earliest T-cell markers that is expressed & retained

CD11C         Monos/MPs/NK cells -- increased in Hairy Cell Leukemia

CD13           Monocytes, grans

CD15           Grans. LeuM1: + in Hodgkins Disease

NK cells

CD16           NK cells and Large granular lymphocytes

CD56           NK cells. + in LGL leukemia/lymphoma

CD57           NK cells, T&B cells

Miscellaneous

CD30           Ki-1 antigen

CD34           Stem cells or very undifferentiated leukemias

CD45           LCA. On all leukocytes and many T&B cell lymphomas. Negative in HD (except LP HD)

CD55           DAF (decay accelerating factor). Absent in PNH. Normally helps reduce the activity of C3. Absence increased C3 activity

CD58           LFA-3 (Lymphocyte function-associated Ag). Absent in PNH.

CD59           MIRL (Membrane inhibitor of Reactive Lysis). More impt than DAF in regulating complement action. Prevents interaction between C8 & C9. Absent in PNH. Absence on platelets may have something to do with venous thrombosis.

CD61           GP IIIa (platelet marker)

CD103         Strongly expressed in Hairy Cell Leukemia.

CD117 – Marker for the KIT protein (tyrosine kinase) expressed in GISTs. Malignant GISTs also commonly express CD34.

HLA-DR found on Monocytes, B-cells, activated T-cells

Glycophorin - RBC marker

 

CYTOKINES

Erythropoietin - Synthesized by peritubular cells of kidney. Chrom 7 (C7, q11-22). ~10% of endogenous Epo is made by liver, so still have some in CRF. Plasma 1/2 life (6-9hrs) shortens with continued treatment

IL-3 --             Made by T-cells. Chrom 5 (C5, q23-31). Stimulates production of pluripotent stem cells, causing them to diff into myeloid line. Synergistic with GM-CSF

GM-CSF -- Made by bone marrow stroma. --> grans, monos, RBCs, and Eos.

G-CSF -- Potent, LMW glycoprotein. --> prolif & maturation of G precursors. Chrom 17 (C17, q11-21). Immediate (48h) effect.

M-CSF -- Stromal cells. Heavily glycosylated glycoprotein (is a dimer). Chrom 5. Stim macrophages --> other cytokines

IL-2 (Tcell stim factor) -- Syn by T4 cells. --> clonal expansion of antigen-specific T-cells and induces expression of IL-2 (CD25) receptors on T cells. Activates T cell cytotoxic responses. Sl stim prolif of NK and B cells.

IL-4 (Bcell stim factor) -- Fr T cells & mast cells. --> prolif & diff of B cells.

IL-5 (Bcell stim factor-2) -- Eos activation/stim factor. Stim B cell diff & ab production.

IL-6 --             Fr lymphoid & non-lymphoid cells. Identical to IFN-B2. Major role in mediation of inflammation & immune response. --> prod of acute phase proteins. Stimulates hematopoietic cells. Important growth factor for myeloma cells. Co-stimulant of IL-2 and IL-2 receptors.

IL-7 --> growth/diff T cells

IL-8 --> chemotactic activating factor for PMNs

IL-9 --> synergistic with IL4 to --> abs

IL-10 -> suppresses macrophage function

IL-11 -> inflammatory mediator

 

DAT

DAT detects IgG and C3d (complement) on RBC membrane.

In Coombs negative hemolytic anemia:

- Can use more sensitive assay (ELISA or RIA)

- Can try specific assay for RBC-bound IgA

The DAT detects cold agglutinins because of C3d on RBC membrane, not bec of the IgM. (Most commercial sera only detect monospecific IgG and C3). In cold-agglutinin syndrome, get less agglutination as warm the blood from 4ψ-30ψC.

Occ see +DAT in normal people.

See +DAT in up to 40% HIV+ patients.

 

DEFEROXAMINE (Desferal)
(MKSAP)

Standard:

40 mg/kg continuous SQ infusion over 8-12 hrs

(Add 2cc sterile water to each vial 250mg/cc concentration)

For life threatening complications (CHF, arrhythmia):

5-12 g/day IV (given thru central line). Rate should not exceed 15 mg/kg/hr.

CONTRA: Severe renal disease (excreted by kidney)

SUPPL : 500mg vials.

COMPLICATIONS:

Skin: Pain/swelling/itching at injection site. Can minimize this by rotating inj sites or by adding 0.1mg HCT/ml of deferoxamine. Use cold compresses and antihistamines for established rxs.

Hypersensitivity Rxs: have been reported, so must give slowly.

Eye & Ear: Need to get annual audiologic (hearing loss/tinnitus) and ophthalmologic evaluations. (blurred vision/cataracts/visual loss)

 

DRUG INDUCED THROMBOCYTOPENIA

Decreased Production: Thiazides, Amrinone, EtOH, Estrogens, TMP/SMX, Chemo

Increased Destruction: Quinine, Quinidine, Heparin, Gold, Rifampin, Sulfas, PCNs, Valproate (induces vWD in kids)

 

EOSINOPHILS (Eosinophilia, Eosinopenia

Eosinophilia

(Abs > 700/uL or >10%)

Allergy: Asthma, hay fever, drugs
Dermatitis
GI: IBD,CAH
Hereditary: Fam eosinophilia, thrombocytopenia with absent radii
Hypereos Syn
Eosinophilic leukemia
Idiopathic: Loeffler's syn
Other pulm syndromes
Neoplasia: NHL, HD, mucin-secreting ca
Parasites: Trichinosis, echinococcus, schisto, filaria
Misc: Angiitis, GVHD, radiation, RA, Sarcoid, Wegener's
Pancreatitis
Drugs: ASA, sulfa, iodides, chlorpromazine
Fungal: Cocci, aspergillus

 Eosinopenia

Acute infection
Acute inflam
Acute MI
Stress
Thymoma (spindle cell variety with hypogammaglobulinemia)
Dysgammaglobulinemia with recurrent infx

 

HB ELECTROPHORESIS

Cellulose Acetate (Origin at -)

Disease---------           -__ A2/C__S__F__A__ +

Normal -| | | ||  

Sickle trait - | | | ||

Sickle disease | ||| ||

Sickle C || || |

Sickle Thal | | | |

Thal major | || |

Thal minor || | ||

Hbs that migrate together:

D migrates with S but doesn't sickle

C migrates with A2 on gel, but separates on acid

E migrates with A2 and A. Clue is that you get inordinantly high (15%) A2 levels, higher than expect for thalassemia, which it mimics.

On Acid, note that:

E migrates with A2 on cellulose & A on acid citrate.

D migrates W S on cellulose

C migrates W A2 on cellulose

 

Hb electrophoresis is usually a poor test to "rule out" unstable (Heinz body) hemoglobins. CONSIDER INSTEAD:

Supravital stain (1% methyl violet) - demonstrates Heinz bodies

Isopropanol stability test - 37 degrees in a 17% solution --> precipitation of abnormal Hb

Heat stability test - expose up to 50C

 

HEME SYNDROMES

ALPOT'S S. - deafness, nephritis, thrombocytopenia (but large platelets)

BLACKFAN-DIAMOND - congenital RBC aplasia

BERNARD-SOULIER - giant platelets, GPIb deficiency--> decreased platelet adhesion

CHEDIAK-HIGASHI S. - rare congenital disorder associated with partial oculocutaneous albinism, recurrent pyogenic infections, neuropathy, and giant lysosomal granules in the PMNs. Can also have dense granule deficiency of platelets.

DiGUGLIELMO - Erythroleukemia (M6). Rheumatic signs may occur, including synovitis, serositis, and effusions. Can also see +Coombs, +ANA, elevated RA, & hypergammaglobulinemia.

EPSTEIN'S - hereditary macrothrombocytopenia, nephritis, and deafness

EVANS - combined immune thrombocytopenia & hemolytic anemia

FANCONI - congenital aplastic anemia

HERMANSKI-PUDLAK S. - oculocutaneous albinism & deficiency of dense granules in the platelets.

JOB S. - Hyper-IgE - abnormal neutrophil function associated with recurrent cutaneous & deep cold abscesses, pulmonary infections, very high IgE levels, eczema, mucocutaneous candidiasis, abnormal T-cell dysfunction.

KASABACH-MERRITT S. - hemangiomas that give rise to DIC

KOSTMAN - congenital agranulocytosis (neutropenia)

LGL - (large granular lymphocytes) associated with neutropenia and recurrent infections

MARCHIAFAVA-MICHELI - other name for PNH

MAY-HEGGLIN ANOMALY - Large but functionally abnormal platelets. Decreased production of platelets. Also see Dohle bodies in PMNs.

OSLER-WEBER-RENDU - hereditary hemorrhagic telangiectasia

PEARSON - sideroblastic anemia + pancreatic insufficiency

SCHWACHMAN - agranulocytosis + pancreatic insufficiency

TAR (thrombocytopenia & absent radii) - congenital amegakaryocytic thrombocytopenia

T gamma - see LGL syndrome

TROUSSEAU'S - migratory venous thrombosis associated with solid tumors of visceral origin

WISKOTT-ALDRICH S. - sex-linked. Thrombocytopenia with increased megas in the marrow. Platelets appear normal, but have decreased survival.

 

HEMOCHROMATOSIS

HEREDITARY FORMS:

Hereditary hemochromotosis is, in most cases, caused by a mutation in HFE, a protein that is abundantly expressed in the duodenal mucosal crypt cells. Its function in iron metabolism is not entirely known. It usually results from missense mutations in C282Y, H63D, or S56C genes. However, in 10-15% of US patients, none of the above mutations are found.

HFE is structurally similar to major histocompatibility complex (MHC) class I proteins It seems to be tightly linked with the HLA gene on chromosome 6. Therefore, family members with identical HLA type may be at risk for HLA-linked hemochromotosis. (homozygotes, since Auto R). Heterozygotes may show biochemical evidence of Fe overload, but usually do not have end-organ damage.

Non HLA-linked - sub Saharan Africa - natives who have "African Hemochromatosis gene" and who make beer in iron pots.

ACQUIRED FORMS:

Due to Increased Erythropoiesis with Intramedullary Hemolysis: Thalassemia, megaloblastic anemia, sideroblastic anemia. The problem is increased Fe release PLUS increased Fe absorption due to increased Epo

Due to iatrogenic Fe overload in Low Erythropoiesis states: Renal disease, aplastic anemia, MDS, sickle cell. Problem not usually as bad because don't have increased Fe absorption.

TREATMENT

Approach: If transferrin saturation > 45% (especially > 60% in men and 50% in women) --> look for C282Y mutation. If positive, then patient has HFE-related hemochromotosis. If liver size normal, AST normal, and ferritin < 1000, then treat with phlebotomy weekly until ferritin < 50 mcg/L and transferrin saturation is < 20%. If liver is enlarged or AST elevated or ferritin > 1000, then need to rule out cirrhosis. If cirrhosis present, then need to screen for hepatocellular carcinoma. (Good discussion in ASH 2000 Education Book p. 39 ff)

Phlebotomy - can do this if not also anemic. Fe burden can be as high as 20-50 grams. Phlebotomy 1-2 times/wk can lead to removal of 200-500 mg/wk. Can phlebotomize until ferritin falls.

Chelation - if patient is anemic, can't do phlebotomy. So must use chelation. Usual - deferoxamine. Usually given SQ, but in life-threatening situations can give IV through central line.

 

HEMOGLOBIN C

GAG->AAG ==> glutamic acid-->lysine at 6th position of B-globin chain.

Positively charged lysine interacts with -charged RBC membrane resulting in intracellular K+ and H2O loss --> cellular dehydration --> decreased deformability --> hemolysis --> enlarged spleen and anemia.

Incidence of HbC trait in American blacks is 2%. Heterozygotes (AC) are clinically normal, but have 20-50% target cells on smear. Homozygotes (CC) have mild-mod hemolytic anemia with Hct 25-37% and 4-8% retics. Homozygotes have >80% target cells on smear.

Suspect diagnosis if see excessive target cells and microspherocytes. Diagnosis: Band that migrates with A2, E, and O-arab on cellulose acetate gel, but separates out on acid agar gel electrophoresis.

Treatment: Not needed. May give folate.

 

HEMOGLOBIN D

Think: India

D Punjab (most common)

D Los Angeles

Caused by glutamic acid-->glutamine at 121st position of B-chain.

Does not cause either anemia or hemolysis. Does --> decreased osmotic fragility and increased O2 affinity.

Important because:

1. HbD migrates with S on cellulose EP (must do acid gel to distinguish)

2. D and S are coinherited (since different positions on globin chain molecule) which could --> varying degrees of severity of sickle syndrome.

 

HEMOGLOBIN E (SE Asia, Thailand)

Think: "Far East"

2nd most prevalent Hb variant.

Caused by glutamic acid --> lysine at 26th position of B-globin chain.

Substituted lysine is involved at critical a1b1 contact site --> oxidatively unstable --> "thalassemic hemoglobinopathy".

Homozygotes: usually asymptomatic, but microcytosis (MCV 65-69) and target cells.

Heterozygotes: No anemia and less microcytosis (MCV 71-75).

Concomitant thalassemia, which is also common in Asia, will exacerbate the anemia and microcytosis. Indeed, HbE/B thal patients may appear clinically more like B-thal major.

DIAGNOSIS: E migrates with C and A2 on cellulose, and with A on acid.

Because HbE unstable, patients should avoid oxidant drugs.

 

HEMOGLOBIN H

Causes Alpha thalassemia syndrome due to 3 gene deletions (a-/--). Presents as moderately severe anemia with splenomegaly and hypochromic, microcytic indices. Hb H is demonstrable by special stains or Hb electrophoresis (other thalassemias not detectable on electrophoresis). Generally the anemia is partially compensated with average Hb levels 8-10 g/dL.

Chronic transfusion treatment is usually not needed. Splenectomy may be indicated for progressive anemia.

 

HEMOLYTIC ANEMIA

(Abnormal Hb, Abnormal Membrane, Abnormal Metabolism, Immune destruction, Drugs, Mechanical, Infections)

HEMOGLOBINOPATHY (Qualitative, Quantitative)

Qualitative:

HbS

HbC - target cells, microspherocytes

HbD - subtypes Punjab (India) & Los Angeles. No anemia or hemolysis. See increased O2 affinity. Migrates with HbS on cellulose. Do acid gel.

HbE - think "Far East". HbE, a mutation of the beta globin chain, which is also associated with reduced expression, causes few abnormalities other than microcytosis and hypochromia when present in a heterozygous or homozygous state. A variable and occasionally much more severe phenotype is seen when this mutation is combined with a sickle cell or beta thalassemia mutation. This mutation is very frequent in southeast Asia. Hemoglobin E is also mildly unstable to oxidative damage. Clinically mild with low MCV, target cells. Migrates with C & A2 on cellulose, and A on citrate. Can look like thalassemia, especially if co-inherited.

Unstable Hbs - usually child/teen with congenital HA. See abnormal Heinz-body stain (crystal violet) and heat denaturation test. Avoid oxidants

Quantitative (Thalassemia): Alpha, Beta

Alpha Thalassemia (chrom 16)

aa/a- = heterozygous thal-2 (a+) = Silent carrier

a-/a- = homozygous thal-2 (a+) = silent carrier

aa/-- = heterozygous thal-1 (a0) = silent carrier, Asians, but increased risk for hydrops fetalis (Hb Barts)

a-/-- = Hb H disease. Moderate H/M anemia, splenomegaly

--/-- = Hydrops fetalis

Hb Constant Spring = elongated a-globin chain: aCS

Divisions of Genetic Determinants:

a0 thal (formerly called a thal 1): no functioning genes on the affected chromosome

a+ thal (formerly called a thal 2): one gene functioning on the affected chromosome

Thalassemic Hemoglobinopathies

Hb Indianapolis, Quong Sze = decreased synthesis due to mRNA instability

Hb E, Knossos = decreased synthesis due to abnormal mRNA splicing

Hb Vicksburg, N Shore = decreased synthesis due to unknown cause

 

B Thalassemia (Chromosome 11) [Therapy]

Suspect if very low MCV with normal RBC.

B+ = suboptimal B-globin synthesis
Bo = total absence of B-globin synthesis
dB = total absence of both d and B globin synthesis
Hb Lepore = absence of d & B with synthesis of fusion globin that migrates with Hb S on cellulose acetate
HPFH = reduced or absent d & B synthesis and increased Hb F

"Major/Minor" are clinical classifications:

B thal trait - heterozygote - asymptomatic

B thal intermedia - homozygote with some A/A2 production. Does not usually need transfusion.

B thal major - homozygote - transfusion dependent.

Note: Diagnosis is based on demonstration of increased Hb A2 (4-6%) and F (5-20%) levels. Can get false negative quantitative Hb electrophoresis if also have concomitant Fe deficiency (ie, levels don't elevate). Can be hard to differentiate from Fe deficiency. RDW is high in Fe deficiency, but normal in B-thal. Can use radiolabeled leucine to demonstrate B-globin production.

dB Thal will have high HbF, but normal or low A2.

B-thal is frequently seen in combination with HbE, which migrates with A2 but in greater amounts than usually seen for A2 alone.

Therapy of Beta Thalassemia

Transfusion therapy has extended life expectancy from 2yrs to 20s. This is even longer if institute chelation treatment.

1. Begin transfusion therapy when Hb <7 and no other cause.

2. Goal: Maintain Hb >= 10.

3. Use leukopoor blood to avoid reaction and in case BMT candidate.

4. HBV vaccinate

5. Chelation (SQ deferoxamine)

6. Splenectomy if transfusion requirement increases 1.5 x baseline

7. BMT in selected patients

8. Hydroxyurea or 5-azacytidine -> increased gamma chains which bind to excess alpha chains, also -> a Hb (F) that functions.

- AlloBMT has been used in Europe with >75% long-term DFS. Better if done early before lot of Fe overload and cirrhosis.

- Chelation also needs to started early to prevent cardiac disease.

 

MEMBRANE ABNORMALITIES

Inherited:

Spherocytosis - decreased or abnormal ankyrin/spectrin/band 4.2, increased osmotic fragility
Elliptocytosis - Common HE - most common - bad 4.1
Pyropoikilocytosis - mutant spectrin
SE Asian ovalocytosis - asymptomatic, spoon-shaped cells
Spherocytic HE - hybrid

Acquired:

PNH
Liver disease, Uremia
Hypophosphatemia

 

METABOLIC ABNORMALITIES
(cause decreased ATP/2,3DPG)

Normal RBC metabolism involves 2 pathways of glucose metabolism:

  1. Glycolytic pathway: Purpose is to generate ATP. ATP needed to maintain Na/K and Ca membrane pumps
  2. Rapaport-Luebering Shunt: Controlled by diphosphoglyceromutase (which converts 1,3-DPG to 2,3-DPG). 2,3-DPG facilitates transfer of O2 from Hb to tissue binding sites.

In addition to glucose metabolism, also have:

HMP Shunt - G6PD (G6PD assay/EP) "bite cells". Preserves & regulates glutathione to protect cell against oxidant injury.

Glycolytic pathway - PK deficiency (spiculed cells), PFK deficiency causes Autohemolysis test

Acquired - Wilson Disease - increased Cu --> inhibits hexokinase --> intermittent hemolysis

 

IMMUNE HEMOLYSIS

            Autoimmune, Alloimmune, Drugs, Mechanical, Infection

Autoimmune

Idiopathic (AIHA), collagen disease, lymphoma

Cold Agglutinin: Post infectious (mycoplasma, EBV), Diagnosis: cold agglutinins, Anti-I, anti-I. (The finding of anti-i without viral infection should prompt a search for lymphoma.)

Alloimmune: Transfusion reactions, hemolytic disease of newborn

Drug related

Drug-induced autoimmune Abs (procainamide)
Hapten reaction (PCN)
Innocent Bystander (stibophen reaction - quinidine)

Mechanical

Microangiopathic: (DIC, TTP, Ca, vasculitis) --> intravascular hemolysis
Heart valve
"March hemoglobinuria"
Heat (burns)

Infection

Erythroparacytosis - malaria, babesiosis, bartonellosis
Sepsis (staph, pneumoc, neisseria)

Clostridium perfringens infection - Clostridium perfringens releases enzymes that acutely degrade the phospholipids of the red cell membrane bilayer and the structural membrane proteins. Phospholipase C is the toxin thought most likely to be implicated in the hemolysis associated with clostridial infection. In one case report, the serum phospholipase C concentration increased five-fold over four hours, the period of time from presentation to death. Other enzymes elaborated by C. perfringens including neuraminidase and T-lectin were not elevated in a similar manner, suggesting the dominant role of phospholipase C.

The signs of clostridial infection may be obvious, but fever may be unimpressive. Any site of infection can be associated with septicemia and massive hemolysis but a local source of infection may not be apparent.

The diagnosis of clostridial sepsis should be entertained in patients who rapidly become gravely ill with evidence of intravascular hemolysis. Examination of the peripheral smear may reveal a spherocytosis that develops within hours. A clue to the severity of the process may be the inability of the laboratory to perform chemical assays or to type and cross-match the blood because the sample is hemolyzed. The massive hemolysis may be accompanied by the development of acute renal failure.

With even the slightest suspicion, one should institute appropriate antibiotic therapy with full doses of intravenous penicillin plus either clindamycin or tetracycline. The patient should also be evaluated for disseminated intravascular coagulation.

 

HYPERVISCOSITY SYNDROME

2-4 symptoms rare
5-8 symptoms common
8-10 most patients symptomatic
>10 all patients symptomatic

Symptoms:

- Weakness, fatigue, anorexia.

- Bleeding (caused by effects of macroglobulin on the clotting system)

- Polyneuropathies, headache, dizziness, vertigo, stupor (due to intracerebral occlusions)

- Visual impairment, blindness.

- Cardiac failure (due to increased plasma volume)

Treatment:

-  Plasmapheresis - try to reduce viscosity by 50% or more. Measure viscosity before & after. Usually have to do maintenance because effects are temporary. Usually 2-4 units of exchange every week or so.

 

IRON FACTS (Iron Deficit, Therapy)

The body has ~3 gm of Fe. 2/3 is in Hb. 1/3 is non-Hg iron. < 1% is bound to ferritin or transferrin in blood

Normal menstrual period --> loss of 25mg Fe

Pregnancy --> 250 mg loss (ie, same as 10 periods)

1 ml PRBCs contains 1mg Fe

1 ml whole blood with Hct 50% contains 0.5mg Fe

1/2 life of transferrin: 10 days

Soluble transferrin receptors:

The soluble transferrin receptor is a soluble portion of the transferrin receptor that has probably been proteolytically released from the cell membrane. The soluble form reflects the total body mass of cellular transferrin receptor. In normal subjects, over 80% of the cellular transferrin receptor is in the erythroid marrow, and the soluble portion is determined by erythroid marrow activity. Thus STR levels are decreased in erythroid hypoplasia (eg, aplastic anemia, chronic renal failure) and increased with erythroid hyperplasia (hemolytic anemias). STRs are also increased in iron deficiency because the cells increase the manufacture of the transferrin receptor which eventually gets shed. Therefore, in the absence of other conditions causing erythroid hyperplasia, an increase in STRs is a sensitive, quantitative measure of iron deficiency.

It is also important to note that the STR level is not increased by inflammation or infection as is ferritin, so this helps to differentiate iron deficiency and anemia of chronic disorders (STR levels are normal in ACD).

The most sensitive means to distinguish between ACD and iron deficiency is the "transferrin receptor-ferritin index" which is the STR level divided by the log of the ferritin level. Index levels < 1 are most consistent with ACD. Levels > 4 are consistent with iron deficiency.

  1. Help distinguish Fe deficiency from ACD (N in ACD, increased in Fe deficiency)
  2. Best test in pregnancy
  3. Earliest indicator of tissue iron depletion.

                         |-- TFRs --|

Stem cell |BFUe|CFUe|Retic|RBC

|- EpoR --|

 

Integrin: Transports Fe through luminal cell wall

Mobilferrin: Transports Fe within intestinal cell

Ferritin: Stores Fe within cells

Transferrin: Transports iron in blood

Fe Therapy: (Oral, Parenteral)

Only 5% of FeSO4 tablet is actually absorbed (20% bioavailable and then 25% of that absorbed). If not anemic, absorb 10%.

Two 300mg tabs/day --> production of 9gm Hg/dl/week = increase in Hct by 3/wk. Chk H/H in 3-4 wks.

Retic count peaks in 5-10 days but may not be that noticeable if anemia is mild.

Start with one 300mg/day with food (less absorption, but better tolerated at first. Do this 3 days, then bid with meals.

Continue therapy for 6 months after Hg normalized.

Parenteral Fe Therapy: (Ferrlecit, InFeD, Calculation of iron deficit)

Ferrlecit [ferric gluconate] – shorter half-life and less immunogenic because it does not contain dextran (which is a larger molecule).

There is no experience giving this as a total dose replacement. European data suggest there is less risk of an allergic reaction than using iron dextran (3.3 versus 8.7 allergic events per million doses per year. Also, if an allergic reaction occurs, the risk of death is near zero with Ferrlecit compared to 16% for iron dextran).

Experience in the US is limited, and it is only approved for use in renal failure or if other forms of iron cannot be tolerated. It is the preferred product except for cost. Some reserve it only for those who have had a reaction to iron dextran. It can be used in those patients, but the risk of reaction is increased seven-fold.

Supplied as 62.5 mg in 5 cc vials (12.5 mg/ml).

No test dose is necessary except in patients who are intolerant to iron dextran, but it isn’t a bad idea for the first dose.

Administration: May be diluted and administered at a rate of 2.1 mg/minute (which equals 126 mg/hr). Repeat daily or weekly until iron stores are repleted.

People usually give 125 mg per weekly until iron stores are repleted. 250 mg over 2 hours is the maximum dose that has been given.

The main side effects are hypotension, flushing, and GI symptoms - which are usually related to the total dose given at a time.

InFeD [iron dextran]

Supplied 50 mg/ml vials

Dose in ml = Iron deficit in mg divided by 50 mg/ml

Administration: Total dose replacement with dilution is saline is no longer recommended and is not approved in the USA. (UpToDate: Treatment of Iron Deficiency Anemia topic last updated Feb 2001) It can be given in repeated doses similar to Ferrlecit as follows:

Premed: Benadryl + Tylenol if desired

Give a test dose of 0.5 ml (25 mg) IV over 1 minute. If no reaction after 1 hour, give the remaining dose at a rate no greater than 1 ml/minute. No more than a total of 2 ml (100 mg) should be given per day.

So, the order should be something such as:

-     100 mg iron dextran IV daily x 7 days (or however many days needed) as follows:

-     Each day give 0.5 mg IV over 1 minute. If no reaction in 1 hour, then give the remaining dose over 2-5 minutes.

This is no longer recommended, if you really want to replace the total dose all at once, mix it in 500 ml NS. Give 25-50 ml IV over 5 minutes, then stop the infusion for 1 hour. If no reaction, then give the remainder over two hours.

 

Calculation of Iron Deficit

Iron deficit (mg) = hemoglobin deficit * blood volume * 3.3 mg Fe/g Hb,

So

Iron deficit (mg) = (14 – Hb) * (0.65 * Kg) * 3.3

 

Assumptions:

- Weight is LEAN body weight

- Blood volume is 65 ml/kg which equals 0.65 dL/kg (There are 110 ml/dL.)

- Hemoglobin is corrected to 14 gm/dl

- There is 3.3 mg Fe per gram Hb

- No additional iron given for repletion of body stores

ITP IN PREGNANCY (ITP, Pre-eclampsia, HELLP, Therapy, Fetus)

PREGNANCY

Incidence: 8% of women without a history of thrombocytopenia will normally have platelet counts 100-150K. This is an incidental finding without clinical significance.

ITP is the most common cause of thrombocytopenia in first two trimesters of pregnancy. It occurs in at least 1-2 out of every 10,000 pregnancies.

(In women with a history of ITP, it seems to worsen during pregnancy and to get better after delivery.)

Cause: Caused by the development of IgG antibodies that recognize distinct epitopes on platelet surface glycoproteins, including GP IIb/IIIa and Ib/IX complexes. Once coated with antibody, target platelets are removed from the circulation by binding to Fc receptors expressed by macrophages within the RE system, primarily the spleen.

Making the Diagnosis: 90% of patients with ITP have platelet-associated IgG, but the fact that 10% do not makes measurement of platelet antibodies somewhat debatable in proving the diagnosis of ITP.

At present, the diagnosis of ITP may be presumed if you have:

- an otherwise healthy patient
- isolated thrombocytopenia
- normal-appearing or mildly enlarged platelets on the peripheral smear
- a bone marrow containing normal to increased numbers of megakaryocytes
- no history of drug ingestion.

Fetal Thrombocytopenia: The aspect of ITP that is unique to the pregnant patient is that the fetus, as well as the mother, may also be affected by this disorder.

Maternal platelet-reactive IgG appears to be actively transported to the fetal circulation.

10-30% of women with ITP have an infant with platelet count < 50. However, CNS bleeding is rare. Some docs give prednisone during last month of pregnancy to decrease the risk of severe thrombocytopenia in fetus.

Correlation with Mother's Platelet Count

The level of Platelet-associated Ab Does not correlate with the mother's platelet count at all. Neither does the degree of fetal thrombocytopenia necessarily correlate directly the amount of platelet-reactive IgG in cord blood.

There are several factors that interact to determine the degree of fetal thrombocytopenia:

- Amount of PAIgG
- Affinity of the Ab for fetal platelets
- Maturity of the fetal RE system
- Ability of the fetal bone marrow to compensate for increased platelet destruction

Statistical Attempts to Predict Fetal Thrombocytopenia

Since the degree of fetal thrombocytopenia may have a profound impact upon the baby's health, especially near delivery, we would like to be able to know how much the baby is affected, if at all.

In one important study (NEJM 323:229, 1990) Philip Samuels from the Univ of Penn found that there are two maternal characteristics that predict a low risk of severe neonatal thrombocytopenia:

- The absence of a history of ITP before pregnancy
- The absence of circulating Ab's in women who do have a history of ITP.

They concluded:

Mother with previous history of ITP = 20% risk of severe fetal thrombocytopenia. However, if there are no circulating Ab's detected in her serum, then the risk falls to 0.

If the mother has no previous history of ITP and her platelet count is >75K, then there is virtually no risk of the baby having severe thrombocytopenia.

If the mother has no previous history of ITP but has platelet count <75K, insufficient evidence to predict, but probably increased risk.

Measuring the Baby's Platelet Count

Fetal scalp vein sampling:

- Remove blood via capillary tube from small laceration made in fetal scalp

- Unsatisfactory because:

- Have to rupture mother's membranes
- Have to fully dilate mother's cervix
- Have to engage the fetal head firmly in the mother's pelvis (applies pressure)
- Specimens usually contaminated with maternal blood and amniotic fluid.
- In experienced hands, can be useful, especially if obtain a normal platelet count.

Percutaneous Umbilical Blood Sampling (PUBS):

- Technically difficult - done using ultrasound guidance
- Postpuncture bleeding in 38% - usually contained in Wharton's jelly
- Could be even worse outcome if the test confirms severe fetal thrombocytopenia
(resulting in emergency C-Section or fetal death)
- Nevertheless, preferred test because more accurate results

MANAGEMENT OF PREGNANT WOMAN WITH THROMBOCYTOPENIA

Make the Correct Diagnosis !!

ITP must be distinguished from:

Gestational Thrombocytopenia or "Pseudo-ITP" - very similar to ITP and can manifest platelet associated Ab's thus making it almost impossible to distinguish from ITP. However, maternal platelet counts usually remain above 75K, and there is no associated fetal thrombocytopenia.

Pre-eclampsia - most common medical disorder of pregnancy.

- Associated with thrombocytopenia 15-50% of cases.

- Postulated that pre-eclampsia is caused by production of prostacyclin (PGI2) and PGE2, and production of thromboxane (TXA2) and PGF2 leading to placental ischemia and platelet activation.

Distinguishing features of pre-eclampsia:

- Occurs during third trimester
- Nearly always women <20 or >30 years old
- BP > 140/90
- Proteinuria > 0.3 g/24h

HELLP Syndrome - appears to be a variant of preeclampsia since many patients with this also manifest hypertension and proteinuria.

Distinguishing features of HELLP:

- 80-90% present with malaise, RUQ and/or epigastric pain, and nausea
- Microangiopathic hemolytic anemia
- Elevated liver tests (Bili, LDH, SGOT)
- Thrombocytopenia - typically < 100K and often <50K
- Lack of HBP and proteinuria may help distinguish from preeclampsia

TTP - characterized by a pentad of:

1. Microangiopathic hemolytic anemia
2. Thrombocytopenia
3. CNS symptoms
4. Fever
5. Renal dysfunction

Less than half will have complete pentad, but 75% will have first three.

DIC - various causes, including amniotic fluid embolism, placental abruption, or uterine rupture

Type II vWD - production of the abnormal vWF protein is increased due to the hyper-estrogenic milieu of pregnancy. The protein binds to platelets increasing their clearance rate.

Drugs - especially cocaine, quinines, thiazides, and hydralazine (latter two used to control HBP and/or edema in pregnancy)

HIV

Malignancy

SLE - 15-20% have antiphospholipid Ab's which can induce thrombocytopenia and thrombosis.

Therapy for the Mother

Your patient is the Mother. Treat her, then worry about the baby. Therapy does not differ significantly from that of a nonpregnant person with ITP.

Treat only if platelet count <30K or if bleeding.

If Need Immediate Response:

IVIG

Most immediate response
Response may last up to 2 weeks
Dose: 2 gm/kg in divided doses over 2-5 days.

Splenectomy

Use if emergency bleeding develops
Optimal time is in early second trimester because:

- Uterus still small enough to be out of surgical field
- Surgery at this time associated with relatively low incidence of premature labor

Less Urgent Cases

Prednisone

Drug of choice - 1 mg/kg
Response rate: 60-70%
May not get response for up to two weeks.
Keep dose as low as possible to avoid HBP, eclampsia, and adrenal suppression of the fetus.

 

Therapy for the Baby

Again, must have correct maternal diagnosis - worry about fetal thrombocytopenia if ITP.

If Mother's platelet count is >75K:

Baby's risk is low
No need for further testing
Can deliver vaginally

If Mother has history of ITP or platelet count <75K:

Baby may be at risk

Consider PUBS when mother better

Baby's platelet count:

> 50K - vaginal delivery
< 50K - C-section

Factors That Determine Degree Of Fetal Thrombocytopenia

- Amount of platelet-associated IgG that crosses placenta
- Affinity of the Ab for fetal platelets
- Maturity of the fetal RE system
- Ability of fetal bone marrow to compensate for increased platelet destruction

 

ITP (Acute, Chronic, Splenectomy, Pregnancy)

(See ASH Guidelines in Blood 88:1, 7/96)

 

ACUTE ITP

Risk of intracranial bleeding due to platelet count <20K is highest during first 2 wks after onset of thrombocytopenia.

Treatment options:

Prednisone 2-4 mg/kg po

Methylprednisolone 30 mg/kg IV

IVIG 1 g/kg x 2 days. (Adverse effects: flu-like symptoms, aseptic meningitis, rarely alloimmune hemolysis, HepC, renal failure, thrombosis)

In Rh+ patients who still have a spleen: Anti-D (WinRho) immune globulin or RhoGAM. (Thought to block the phagocytic system with the anti-Rh-coated RBCs). As effective as IVIG, easier to give, and costs less.

 

CHRONIC ITP

Due to IgG Abs against GP IIb, IIIa, or Ib-IX. Removed when IgG binds to Fc receptors on macrophages in spleen. In some patients may be due to C3 on platelets --> removed by macrophages.

Estrogen and progesterone in pregnant women may increase MP receptor expression --> increased destruction.

May also have Abs against megakaryocytes --> decreased production.

Treatment options:

Prednisone

- 0.25-1 mg/kg po
- 50% response rate by 4-6 weeks
- As tapered, most will redevelop thrombocytopenia.

Splenectomy:

- Indicated if requires high steroid dose OR if no response to steroids.
- 70% response rate
- Accessory spleens in 10-20%

Azathioprine

Cytoxan

Vincas

Danazol

Anti-D Abs (WinRho)

IVIG

Note: Patients who relapse after splenectomy and who are not bleeding may not need further treatment.

 

WinRho (Anti-D)

As effective as IVIG
Cheaper and easier to give.

CONFIRM THAT PATIENT IS Rh+ AND STILL HAS A SPLEEN !!

Mechanism: Anti-D binds to RBCs, blocking phagocytic system. This may cause a transient hemolytic anemia which usually mild. Hemolytic anemia is NOT dose dependent, even up to 100 mcg/kg.

CAUTION: Patients who are already anemic (Hb < 10) or who have underlying hemolytic anemia.

Goal is to keep platelet count 30-50 range.

DOSE:

Initial: 50 mcg/kg (250 IU/kg) given as a single injection or divided over two days. If Hb <10, reduce dose to 25-40 mcg/kg (125-200 IU/kg).

Subsequent: If patient responds with satisfactory increase in platelet count, then decrease to maintenance dose of 25-60mcg/kg q3-4 weeks.

If patient did not respond, then dose according to Hb --

Hb < 8: Use with caution
Hb 8-10 -- 25-40 mcg/kg
Hb > 10 -- 50-60 mcg/kg

Monitor for signs of intravascular hemolysis, anemia, and renal insufficiency.

If necessary to give platelets or blood, avoid Rh+ !!

 

MACROCYTOSIS
(Wintrobe)

NONMEGALOBLASTIC

Accelerated erythropoiesis: Hemolytic anemia, Post-hemorrhagic anemia

Increased membrane surface area: Liver disease, Obstructive jaundice, Post-splenectomy

Myelodysplasia: Aplastic anemia, 5q- syndrome, Acquired sideroblastic anemia, Idiopathic, Alcoholism, Drugs (INH,chloramphenicol), Cu deficiency (nutritional, zinc toxicity), Hypothermia, Hereditary dyserythropoietic anemia, type I (Splenomegaly common, Cabot rings, basophilic stippling)

Alcoholism

Hypothyroidism

Myelophthisic anemia

COPD

                       

METHEMOGLOBINEMIAS

Hbs containing Fe in the ferric (Fe3+) state. They can arise from mutations that impair the ability of the globin chain to maintain the Fe in the reduced ferrous form (eg, Hb MIwate), or they can arise as an inherited deficiency of methemoglobin reductase.

Acquired methemoglobinemia is the most common form, arising from exposure to substances that oxidize the heme iron. Drugs that induce methemoglobin:

Nitrites
Acetanilid
Phenacetin
Acetaminophen
Sulfonamides
Nitroprusside
Local anesthetics (benzocaine, lidocaine)

The color of methemoglobin causes the complexion of patients to resemble cyanosis, yet the arterial O2 sat is normal, a key diagnostic feature.

 Treatment: Methylene Blue

METHYLENE BLUE

For methemoglobinemia (especially with Hb M levels > 40%):

1 mg/kg IV/5 min. May repeat in 1 hr, then every 4-6h

Should not exceed 7 mg/kg

CONTRA: G6PD deficiency because acts as an electron receptor for NADH, thereby reducing the pt's already low levels.

The drug acts as an electron acceptor for NADPH in the transfer from methemoglobin to form hemoglobin. (In high doses may have opposite effect.)

The intrinsic RBC reduction system is NADH. NADPH is a latent system that requires an exogenous electron carrier such as VitC or methylene blue.

Oxidation: make element more + (eg, Fe2 Fe3)

Reduction: make element more - (eg, 2H+ H2)

 

MONOCYTOSIS
(Abs mono count > 900/uL)

Chronic bacterial infections:

TB, SBE, Syphilis, Brucellosis, Protozoa and Rickettsial Infection, Malaria, Typhus, RMSF, Trypanosomiasis, Kala-azar

Autoimmune Disease: SLE, RA

Granulomatous dzs: Sarcoid, Crohn's, UC

Hematologic malignancies

Monocytic leukemias (AMoL, CMML)
Lymphomas (NHL and HD)
CML
Myeloma

Carcinomas: Breast, Gastric, Ovary

Lipid storage dzs

Recovery from acute bacterial infections

Recovery from chemotx

 

MYELOMA (Diagnosis, Staging, Indolent, Smoldering, Treatment, POEMS)

MYELOMA STAGING SYSTEM
(Durie-Salmon)

STAGE CRITERIA

I - All of the following:

Hb >= 10
Ca <= 12
X-ray: normal bone structure or soloitary bone plasmacytoma.
Low M component production:

(IgG< 5gm/dl
IgA< 3gm/dl
Urine light chain < 4 gm/24hrs)

II Fitting neither Stage I nor Stage III

III - One or more of the following:

Hgb < 8.5
Ca > 12
Advanced lytic bone lesions
High M-component production rates:
(IgG > 7gm/dl, IgA > 5gm/dl, Urine light chain > 12gm/24 hrs)

SUBCLASSIFICATION

A-Relatively normal renal function (SCr < 2mg/dl)
B-Abnormal renal function (SCr >=2mg/dl)

POOR PROGNOSTIC FACTORS

10% wt loss or more
Fever not associated with infection
BUN > 50mg/dl

 

MYELOMA: CRITERIA FOR DIAGNOSIS

MAJOR

Plasmacytoma on biopsy
BM plasmacytosis > 30%
Monoclonal M protein
> 3.5 g/dl for IgG
> 2.0 g/dl for IgA
1 gm or more/24h of k or l light chain in urine in absence of amyloidosis

MINOR

BM plasmacytosis 10-30%
M-protein, but less than above
Lytic bone lesions
Normal IgM < 0.05
IgA < 0.1
IgG < 0.6

DIAGNOSIS: 1 major + 1 minor OR 3 minor in symptomatic patients with progressive disease.

Support diagnosis (but not specific):

Anemia
Hypercalcemia
Azotemia
Bone demineralization
Hypoalbuminemia

-------------------------------------

INDOLENT MYELOMA

Like myeloma except:

< 4 bone lesions & no compression fxs
M-protein
IgG < 7 g/dl
IgA < 5 g/dl
No symptoms or associated disease features:

KPS > 70%
Hb > 10
Ca normal
Creat < 2.0
No infections

--------------------------------------

SMOLDERING MYELOMA

Same as Indolent, except:

No bone lesions
BM plasma cells 10-30%

--------------------------------------

MGUS

Monoclonal gammopathy
M-protein

IgG < 3.5
IgA < 2.0
BJ < 1 g/24h
BM plasma cells < 10%
No bone lesions
No symptoms

 

MYELOMA: INDICATIONS FOR TREATMENT

(Chemotherapy)

Stage I / Indolent / Smoldering: Observe unless symptoms or high risk for complications (such as renal failure)

Stage II/III:

Bone pain
Hypercalcemia
Renal failure
Marrow failure
Cord compression

 

NEUTROPHILS (Neutropenia (Drugs), Neutrophilia

Neutropenia

*=ANC usually <500

After chemo/radiation*

Primary Hematologic Disease

Autoimmune neutropenia
Cyclic neutropenia*
Kostmann's Syndrome (congenital hypoplastic neutropenia)
Schwachman's Syndrome (neutropenia & pancreatic insufficency)
Pure WBC aplasia*
Agranulocytosis
MDS
LGL Syndrome (T-gamma lymphocytosis)

Secondary Neutropenia

Infection (most common cause of neutropenia)
Viruses (including HBV, EBV, HIV)
Bacteria (including Salmonella typhi)
Rickettsia
Protozoa
Overwhelming Infection
Sepsis
Disseminated mycobacteria infection
Collagen Vasc Disease (SLE, Felty's syndrome)
Starvation and kwashiorkor
Hypothermia
Alcoholism
Malignancy (leukoerythroblastic smear)

Drugs (second most common cause of neutropenia)

(1-4+ indicates approx frequency encountered.)

Heavy metals (Au,As)
Analgesics
NSAIDs (including ibuprofen) in clinical practice
Acetominophen
Gold salts
Aminopyrine
Dipyrone
Antipsychotics: Phenothiazines (4+)
Antileptics (1+)
Phenytoin
Carbamazepine
Antithyroid (3+): (PTU, Methimazole)
Cardiovascular (1+): (Procainamide, Captopril, Aprindine)
Sulfa drugs
Thiazides
Oral hypoglycemics
Sulfasalazine
Dapsone
Sulfa abxs
Spironolactone
Acetazolamide
Antibiotics
Sulfas (4+)
Macrolides
PCNs (3+, usually high dose IV)
Cephalosporins
Chloramphenicol (1+)
Vanc
Clinda
Anti-TB: (Levamisole)
Antimalarials
Antiviral,especially zidovudine
Antihistamines
Cimetidine,ranitidine

Normal Variation

Ethnic (Black) & familial neutropenia
Benign chronic neutropenia

 

NEUTROPHILIA

Shift from marginated pool to circulating pool: Exercise, Epinephrine, Hypoxia, Acute stress

Shift from marrow storage pool to circulating pool: Corticosteroids, Endotoxin, Chemical intoxication (heavy metals), Chronic stress, Sickle cell anemia

Increased Production: Infection (pneumonia/S aureus), Inflammation (RA,vasculitis,PAN), Cancer (gastric/lung/breast/adrenal), Leukemoid reaction, Myeloproliferative, Lithium

Odd causes of transient neutrophilia: Ovulation, Exercise, Seizures, Vomiting, CO poisoning, Recovery from hypothermia

Odd causes of chronic neutrophilia: Pregnancy, Lactation, Chronic acidosis, Anxiety, Down's syndrome

 

POLYCYTHEMIA VERA

PVSG Criteria, Diagnostic Algorithm, Treatment

PVSG Criteria:

A1. RBC mass > 25% above mean predicted value. (Hct > 60% is diagnostic of increased RBC mass)

A2. Absence of causes of secondary erythrocytosis

A3. Palpable splenomegaly

A4. Abnormal marrow karyotype as clonality marker

 

B1. Platelet count > 400

B2. Neutrophilic leukocytosis > 10K

B3. Radiographic splenomegaly

B4. Reduced erythropoietin level

 

Diagnosis:

A1+A2+A3 or

A1+A4 or

A1+A2 + any 2 B criteria

Note: These criteria are not universally agreed upon. Some advocate a "working definition" which consists of documenting a Hct that is above the upper limit of normal, especially if accompanied by aquagenic pruritis, large-vessel thrombosis, splenomegaly, erythromelalgia, microcytosis, or persistent leukocytosis or thrombocytosis. In the absence of these, one can simply recheck the Hct in 3-6 months. If any of these are present, then check serum erythropoietin level and check a bone marrow with cytogenetics. Immunostain for megakaryocyte c-mpl may help distinguish between P Vera and secondary erythrocytosis (Blood 96:771, 2000) and may make measurement of RBC mass unnecessary.

Course: Proliferative phase -> spent phase -> myelofibrosis.

Prognosis: 20% have thrombotic events, especially when Hct > 50%. The risk increases significantly with age, especially in patients with atherosclerotic conditions. Arterial and venous thromboses occur with equal frequency. Hemorrhage is second most common complication, occurring in 20% of uncontrolled patients. 10% develop post-polycythemic myeloid metaplasia after 15 years, and 50% after 20 years. 2% develop AML.

Treatment:

< 70yo: Phlebotomy. Goal is to get Hct < 45 in males, < 42 in females. Can do twice a week initially, then decrease frequency to once every 1-3 months as needed.

>70yo: Hydrea (In this group risk for thrombosis may be higher if treated only with phlebotomy). Alternate: IFN

Treatment -- Based primarily upon the observations of the PVSG, the mainstay of therapy in PV remains phlebotomy to keep the hematocrit below 45 percent in men and 42 percent in women. For special groups, our approach consists of:

·         Supplement phlebotomy with hydroxyurea (starting dose 15 to 20 mg/kg per day) in patients who are at high-risk for thrombosis (age over 70, prior thrombosis, platelet count >1,500,000/΅L, presence of cardiovascular risk factors).

·         If there is a clinical indication for the use of aspirin, it can be given in a low dose of 75 to 100 mg/day. Treatment with doses higher than this should be avoided.

·         Although the use of 32P (starting dose 2.5 mCi/m2 IV not more often than every 12 weeks) is discouraged in young patients, this agent may have a role in noncompliant patients and in those whose life expectancy is less than 10 years.

·         It is reasonable to use IFNa (starting dose 3 million units subcutaneously three times per week) in patients with refractory pruritis, in high-risk women of childbearing potential, and in the patient refractory to all other medications.

·         Anagrelide (starting dose 0.5 mg four times per day) is used mainly to manage thrombocytosis in patients refractory to other treatments. It must be given with caution in patients with known or suspected heart disease, which may limit its utility in the elderly.

·         Allopurinol (usual daily dose 300 mg) is given to patients with symptomatic hyperuricemia or urinary uric acid excretion above 1100 mg/day, a level which carries a 50 percent risk of uric acid calculi. Allopurinol should not be started in patients with a history of gout until colchicine prophylaxis has been given.

 

PAROXYSMAL COLD HEMOGLOBINURIA

Rare. Accounts for < 1% of all AIHA.

Historically it occurred after cold exposure in patients with congenital syphilis or patients in late stages of syphilis.

Currently, more common in kids.

Most patients have history of recent viral or flu-like illness.

Almost all reports describe an acute, transient, non-recurring hemolytic anemia that is not related to cold exposure.

Usually resolves spontaneously after several wks and doesn't recur

DAT may be only weakly positive for C3d, if positive at all.

Donath-Landsteiner Test: Can get false negative with P- cells, so mix P+ cells with serum at cold temp (to allow Abs to affix). Then warm blood to allow complement activation. Test is + if hemolysis occurs.

 

RBCs: PERIPHERAL SMEAR FINDINGS

Target Cells: Thallassemia, Hb C, Liver disease, Hb E (also MCV: mimics thallassemia), HbSC - suspect if target cells with normal MCV

Acanthocytes (multiple irreg projections. Fr Gk acantha=thorn):

Liver disease --> preferential accumulation of cholesterol in outer hemileaflet of lipid bilayer. Don't do splenectomy.

Abetalipoproteinemia--> cholesterol, retinitis pigmentosa, neuro abnormalities (ataxia, tremors)

   Chorea-acanthocytosis syndrome

McLeod phenotype - RBCs lack Kx antigen--> expression of Kell antigen. X-linked

Stomatocytosis (>5% on smear):

Acquired (alcohol, liver disease)

Rh deficiency (Rh null, Rh mod) --> increased osmotic fragility. Treatment: splenectomy

 

INHERITED PLATELET DYSFUNCTION

(Bernard-Soulier, Glanzman’s, Storage Pool Disease)

--Adhesion----             --------Aggregation----

|

|-vWF-GPIb=Platelet=GPIIb/IIIa-Fibrinogen-GPIIb/IIIa=Platelet

|                                                           |

|-Collagen------------|

 

BERNARD-SOULIER

Decreased GPIb --> decreased adhesion and decreased Factor VIII binding

AutoR. Giant platelets on smear. Clot retraction normal.

Platelet function tests: All NORMAL EXCEPT ristocetin (ristocetin binds to same receptor as vWF). Can be acquired (lymphoma)

 

GLANZMAN'S THROMBASTHENIA

Decreased GPIIb/IIIa --> decreased aggregation and decreased fibrinogen binding

AutoR. Normal platelet morphology. Prolonged bleeding time. Decreased clot retraction. Decreased PF3.

Platelet function tests: All ABNORMAL EXCEPT ristocetin (they aggregate, then de-aggregate). Decreased or absent PlA1 antigen

 

STORAGE POOL DISEASE

Decreased storage pool nucleotides or enzymes (cyclo-oxygenase, thromboxane synthetase)

Platelet morphology varies. Mild-mod bleeding. Bleeding time varies. Normal clot retraction.

Platelet function tests:

Low ADP - aggregate but disperse. High ADP - Normal.
Ristocetin - Normal.
All others - Abnormal (collagen, epi, arachidonate).
Decreased total ADP.
Decreased granules & dense bodies. Pigmented macrophages in marrow

 

(Figure depicting site of defect)

Delta granules - store ADP

Alpha granules - store fibrinogen, Factor V, vWF

-------------------------------------------------------(Figure depicting platelet enzymes)         

           

PNH (Marchiafava-Micheli Syndrome)
(
MKSAP)

Consider if unexplained hemolysis, unusual thrombotic events (especially hepatic veins), or marrow aplasia. Acquired clonal stem cell disorder that does NOT affect LCs.

Affects children and adults with peak incidence in early-mid adulthood. NOT familial, and NO KNOWN RISK FACTORS except that it is more common in the Far East.

Usually has insidious onset, presenting with thrombosis or anemia. Only 25% actually have hemoglobinuria.

Degree of anemia varies, but may be severe. See lots of urinary iron loss (which may still occur without hemoglobinuria). Retic count usually not as high as would expect for degree of anemia present.

BM is variable, ranging from aplastic to hypercellular. May see megaloblastic changes. Megas and WBCs usually appear normal, but can see dysplastic changes. Progressive dysplasia during a patients course is a bad sign that may herald change to leukemia.

Lab: see LAP and RBC acetylcholinesterase

NO specific chromosome abnormality associated with PNH.

See low WBC and platelet count in 2/3 of patients due to decreased production. 10% of deaths attributable to infection.

Patients often thrombocytopenic, but still thrombose bec platelets are not normal. Most common sites of thrombosis:

hepatic veins (Budd-Chiari)
central veins & venous sinuses of brain
intra-abdominal
venules of skin

Think PNH if:

Thrombocytopenia + thrombosis
Thrombocytopenia + abd pain

Associated with small but increased risk of leukemia - usually 5yrs after diagnosis

Clonal abnormality occurs somewhere after the pluripotent stem cell (so LCs not affected). Patients still have some normal cells. Types of cells:

PNH I - normal

PNH II - mildly abnormal (3-5 x as sensitive to complement)

PNH III - markedly abnormal (15-25 x as sensitive)

85% of patients are mix of I & III. Severity of condition depends upon the mix between PNH-I and PNH-III cells:

<20% PNH-III - no hemolysis
20-50 - intermittent hemolysis
>50      - chronic hemolysis

Abnormality seems to be the failure to express certain membrane proteins that carry phosphatidylinositol-glycan (PIG) anchor in cells derived from the affected clone. These include:

DAF (decay accelerating factor) [CD 55] - normally destabilizes C3 and C5 convertases

MIRL (membrane inhibitor of reactive lysis) [CD 59] - also called protectin. Vital membrane inhibitor of complement activation.

Lab diagnosis:

Ham Test (acidified serum test) - acidified serum activates alternate complement pathway--> increased C3 fixed to cells that lack DAF and MIRL --> hemolysis. Test is specific, but not as sensitive as ...

Sugar water test (sucrose hemolysis) - high sucrose concentration --> activates complement PLUS exerts osmotic stress, making it a more sensitive but less specific test.

Flow cytometry: to demonstrate deficiency of PIG proteins, specifically CD55 and CD59. Although RBCs most noticed, the platelets and WBCs are actually more affected. Flow is more sensitive, and less likely to have false negative caused by transfusion.

Treatment:

Corticosteroids 15-45mg qd or qod.

Androgens: Fluoxymesterone 5-40mg /d

Iron - may be needed to replace urinary losses

Folate

Transfuse - when above measures fail. Use washed or deglycerolizes cells to avoid complement activation.

To prevent thrombosis:

If occurs, treatment as usually with heparin, but start coumadin as quickly as possible because heparin can worsen hemolysis in some patients.

Throbolytic therapy IS ok.

For hepatic v thrombosis, continue warfarin for at least 1yr.

Prognosis:

50% live long-term without big problems.

Patients with BM aplasia or thromboses are at high risk of early death. They may benefit from BMT. ATG has been tried, but not adequately tested.

 

POEMS SYNDROME

Myeloma variant

P-polyneuropathy
O-organomegaly
E-endocrinopathy ( low testosterone, impotence, hypercalcemia, hyperglycemia, hyperprolactinemia)
M-monoclonal protein
S-skin lesions (hyperpigment, hypertrichosis, white nails)

 

POLYCYTHEMIA

(See Williams 5th ed. p. 694 for Hct vs Blood volume graph.)

P Vera, Normal volumes, Diagnostic Algorithm

Normal Volumes

            Whole blood: 60-80
            Red cell:  21-31
            Plasma: 39-49

PVera is when RBC mass is > 36 (male) or > 32 (female), OR if normalized to BSA, then > 25% above mean

Diagnostic Algorithm:

1.   PV-related features present? (elevated RBC mass, splenomegaly, O2 sat > 92, thrombocytosis, leukocytosis)  Yes   No

2.   Check serum EPO: High = Secondary erythrocytosis;  Low = P Vera;  Normal

3.   Bone marrow: Diagnostic for PVera: (panmyelosis, especially with megakaryocytosis and absent iron stores with evidence of clonality or cytogenetic abnormality). If non-diagnostic

4.   EEC (endogenous erythroid colony) study: Positive = PVera. Negative = repeat serum EPO and Hb in 3 months.

A.  Check serum EPO: High = Secondary erythrocytosis;  Normal;   Low

B.   Bone marrow:  Diagnostic = PVera.  Non-diagnostic = Repeat serum EPO and Hb in 3 months.

C.  Hb < 18.5 (male) or < 16.5 (female) = Repeat Hb in 3 months
Hb > 18.5 (male) or > 16.5 (female)

D.  Workup for secondary erythrocytosis: Positive = Secondary Erythrocytosis  Negative

 

Secondary Polycythemia

Tissue hypoxia

Smoking (CO)

Increased carboxyhemoglobin

CO intoxication

Some methemoglobinemias

High altitude residence

R->L cardiopulmonary shunts

O2 sat < 90%

Chronic cobalt ingestion

High O2-affinity Hb (These have normal O2 sat, but decreased P50 ie, O2 curve shifted to the left. Order: Oxygen dissociation P50 assay.)

Increased Epo production

Renal cell ca

Renal cysts

Renal artery stenosis

Post renal transplant polycythemia (Occurs in 10-14%. Treatment: ACE inhibitors.)

Hepatocellular ca

Uterine fibroids

Hemangioblastoma of cerebellum

Androgen administration

Androgen-producing tumors (rare)

Increased sensitivity to Erythropoietin (Familial [case reports] - due to mutation of Epo receptor)

 

NEUTROPHIL MORPHOLOGY

Hypersegmentation: Folate or B12 deficiency

Pelger-Huet: MDS

Dohle bodies: Sepsis, May-Hegglin anomaly

Absence of secondary granules: MDS

Giant granules: Chediak-Higashi

 

PURE RBC APLASIA

SELF-LIMITED

Transient erythroblastopenia of childhood
Transient aplastic crisis of hemolysis (parvo B19)

FETAL RBC APLASIA

Nonimmune hydrops fetalis (in utero parvo infx)

HEREDITARY PURE RBC APLASIA
(Blackfan-Diamond)

ACQUIRED PURE RBC APLASIA

Malignancy
Thymoma
Lymphoma, especially CLL (especially T-cell variant)
MDS
Paraneoplastic syndromes
Collagen vascular disease (SLE et al)
Virus (Parvo B19, Hepatitis, HTLV, EBV)
Pregnancy
Drugs

Antileptics (especially Dilantin), Azathioprine, Sulfonamides, Isoniazid, Procainamide)

Idiopathic

 

SCHILLING TEST

1. Give Labeled B12 0.5-2.0 mcg po. Begin 24hr urine. At 2hr give unlabeled B12 1mg IM (to saturate binding proteins).

Normal: excrete > 7% of radioactive dose in 24hrs

2. If low output, wait 5 days. Repeat above, except give 60mg active hog IF. Will miss patients who can't get B12 from food (eg, acid)

Modified: source of labeled B12 is omelet of eggs fed radioactive cobalamin.

False Positive cause by:

- Inadequate urine collection (should excrete > 15mg/kg/d creatinine)

- Renal disease (so do whole body count)

- Inadequate saturation of B12 binding protein by labeled B12

- Inactive IF

- Anti-IF Abs in stomach

- Malabsorption in ileum

False Negatives:

- Isotope from previous test still present

 

SMEAR FINDINGS - PLATELETS

Giant Platelets: Mediterranean macrothrombocytopenia, Epstein's syndrome, Bernard-Soulier, May-Hegglin anomaly

Small Platelets: Wiskott-Aldrich

 

SPLENECTOMY IN ITP: INDICATIONS
(ITP Guidelines: Blood 88:3 7/1/96)

Patients with diagnosis for 6 weeks and platelet count <10 and no bleeding symptoms

Patients with diagnosis for 3 months and platelet count <30 and who had transient or incomplete response to primary treatment. May or may not be bleeding.

Splenectomy not recommended if: Non-bleeding patients with diagnosis > 6 months and platelet count >50K

If do splenectomy, it is appropriate to provide pre-op IVIG to reduce risk in patients with platelet count <20K.

Inappropriate to use IVIG or steroids if platelet count is >50K.

Platelet transfusion inappropriate as preop prophylaxis for platelet count >10.

Patients should be immunized for pneumococcus, H flu, and quadrivalent meningococcal polysaccharide vaccine at least two weeks prior to procedure.

 

T GAMMA or LARGE GRANULAR LYMPHOCYTE (LGL) SYNDROME

Large granular lymphocytes (LGLs) comprise 5 percent of the population of peripheral blood mononuclear cells. These cells are 15 to 18 microns in diameter, have round or indented nuclei, pale blue cytoplasm, and azurophilic granules containing acid hydrolases. LGLs are a heterogeneous population of cells, and include natural killer cells (CD3+ CD8+ CD57+) and cytotoxic T cells. They usually lack surface immunoglobulin, but express receptors for the Fc portion of IgG. Certain surface phenotypes are also frequently expressed such as CD 2, 3, 8, 16, and 57.

Chronic lymphoproliferative disease related to clonal or nonclonal ("reactive") expansion of LGLs is characterized by mild to moderate lymphocytosis, bone marrow infiltration, splenomegaly, granulocytopenia (neutropenia), and anemia. Up to one-third of patients with the LGL syndrome also have polyarthritis resembling rheumatoid arthritis (RA) and may fulfill the clinical criteria for Felty's syndrome (FS). In an academic referral center, a routine search for LGL was performed in all patients with RA and neutropenia over a 10-year period; 33 and 66 percent had the LGL syndrome and FS, respectively.

LGL syndrome and FS have generally been regarded as separate conditions with many clinical similarities. Differentiation is based upon the demonstration in the LGL syndrome of a characteristic, expanded (and usually clonal) lymphocyte population. The superimposition of polyarthritis does not affect the course of the hematologic disorder (described below), but results in a clinical picture which may be difficult to distinguish from FS. The arthritis often fulfills clinical criteria for RA and has similar immunogenetic associations as FS. HLA-DR genotyping, for example, has revealed that patients with the LGL syndrome and arthropathy have an increased frequency of HLA-DR4 compared with normal controls; this frequency approaches that found in FS. In contrast, the frequency of HLA-DR4 in LGL patients without arthropathy is not different from controls. This suggests that FS and LGL syndrome have a similar immunogenetic basis.

Our understanding of the relationship between FS and LGL syndrome continues to evolve. Evidence of a gradation from FS to the LGL syndrome has not been clearly demonstrated.

CLINICAL MANIFESTATIONS — The mean age of onset of the LGL syndrome is 55 years, with a slight female preponderance. Hematologic abnormalities and, less often, arthritis are the main clinical findings.

Hematologic abnormalities — Approximately 85 percent of patients have neutropenia which, in 40 percent, is severe enough to result in recurrent pyogenic infections. Infection is the leading cause of death in this disorder. Patients who do not die from infection generally have a long survival. However, a few patients succumb to progressive lymphoproliferative disease or to leukemia with large numbers of LGLs in the peripheral blood. Autopsy information is limited, but lymphocytic infiltration of various organs can occur.

The bone marrow is infiltrated with lymphocytes in almost 90 percent of patients, three-fourths of whom have mild to moderate absolute lymphocytosis. Anemia (50 percent), thrombocytopenia (20 percent), and ANA positivity (65 to 80 percent) occur with about the same frequency as in FS. However, rheumatoid factor is absent in at least a third of patients, whereas seronegativity is quite rare in FS.

Arthritis — Arthritis occurs in about one-third of patients with the LGL syndrome. There is a broad range of severity, from mild intermittent joint swelling to progressive, deforming arthropathy. In some patients, the arthritis precedes the neutropenia by many years, as is usual in FS, but a simultaneous onset can occur. In one report, the LGL syndrome began after splenectomy. Extraarticular manifestations of RA appear to be less frequent than in FS.

PATHOGENESIS — The LGL syndrome generally appears to be a lymphoproliferative disease of clonal origin. There is, however, considerable controversy about its homogeneity and the existence of "reactive" and neoplastic subsets. It is possible, for example, that clonal disease is present in all patients but is not detectable in some with current immunophenotyping techniques.

One recent report compared patients with FS with and without the LGL syndrome. No significant clinical or hematologic differences were noted, suggesting that a discrete transforming event may be required for LGL expansion.

Several factors appear to contribute to the neutropenia in the LGL syndrome. LGLs in the bone marrow may inhibit myelopoiesis directly by local production of cytokines. In addition, antibody mediated mechanisms may also play a role.

DIFFERENTIAL DIAGNOSIS — The combination of rheumatoid arthritis and neutropenia is seen in both the LGL syndrome and FS. The distinction between these disorders is based mainly upon demonstration of an expanded lymphocyte population in the peripheral blood and/or bone marrow, and the identification of typical LGL cells. As noted above, immunophenotyping will confirm the presence of a population of lymphocytes expressing surface markers (CD 2, 3, 8, 16, and 57) that is infrequent in FS. The demonstration of clonal cytogenetic abnormalities or clonal TCR gene recombination in patients with characteristic lymphocyte phenotypes provides additional diagnostic confirmation of the LGL syndrome.

Clinical differences between FS and the LGL syndrome are interesting, but lack specificity and therefore have limited diagnostic value. The LGL syndrome is more likely when the onset of arthritis and neutropenia are simultaneous, when the arthritis is mild, when extraarticular manifestations are absent, and when rheumatoid factor is absent. However, some reports do not support those distinctions. A poor response to splenectomy has also been noted in the LGL syndrome but considerations of therapy are generally similar. Both arthropathy and hematologic disorder may respond to methotrexate or prednisone.

 

THROMBOCYTOSIS

PHYSIOLOGIC: Exercise, Partuition

HEMATOPOIETIC DISORDERS: Myeloproliferative (ET, PV, CML, idiopathic myelofibrosis), Rapid marrow regeneration (post bleeding, hemolytic anemia), Rebound thrombocytosis after marrow suppression, Miscellaneous: (Fe deficiency anemia, Other chronic anemias, Hemophilia, Myeloma)

ASPLENIC STATES: Splenectomy, agenesis, atrophy, splenic vein thrombosis

INFX & INFLAMMATION: Acute infection, Chronic infection (TB, osteomyelitis), Vasculitis, IBD, sarcoidosis, CTD

CANCER

MISC: trauma, surgery, osteoporosis, nephrotic syndrome, CRF, Cushing's, vincristine

 

ESSENTIAL THROMBOCYTHEMIA

Differential Diagnosis: Must rule out polycythemia vera, iron deficiency, CML, idiopathic myelofibrosis, and reactive thrombocytosis.

Diagnostic Criteria:

  1. Platelet count > 600K. (This criterion has been challenged because complications may occur in patients with only slightly elevated platelet counts. Some use > 400K)
  2. Normal Hb or Normal RBC mass (males < 36 ml/kg, females < 32 mg/kg)
  3. No evidence of iron deficiency (stainable marrow iron or failure of iron trial [< 1 g/dl rise in Hb after 1 month of iron therapy])
  4. No Philadelphia chromosome
  5. No marrow fibrosis (absent or < 1/3 of biopsy area without both splenomegaly and leukoerythroblastic reaction)
  6. No known cause for reactive thrombocytosis. (Check ESR, CRP)

 

IDIOPATHIC MYELOFIBROSIS

Clonal disorder characterized by anemia, mild neutrophilia, thrombocytosis and splenomegaly. 2/3 also have hepatomegaly.

The peripheral smear typically shows immature myeloid and erythroid precursors, teardrop-shaped RBCs, and large platelets.

The marrow has increased reticulin fibers, and this reactive fibroplasia is the result of cytokines released locally by the numerous abnormal megakaryocytes.

The disease may be complicated by portal hypertension, as a result of very large splenic blood flow and the loss of compliance of hepatic vessels, and by fibrohemopoietic tumors that can develop in any tissue and lead to symptoms by compression of vital structures.

Treatment may include hydroxyurea for thrombocytosis and massive splenomegaly, transfusions for anemia, local irradiation of fibrohemopoietic tumors or of the spleen, and splenectomy. Portosystemic shunt surgery may be required for gastroesophageal variceal bleeding.

Prognosis: The disease may remain indolent for years or may progress rapidly by further deterioration in hematopoiesis, by massive splenic enlargement and its sequellae, or by transformation to AML. Overall median survival is about 5 years.

THROMBOTIC THROMBOCYTOPENIC PURPUA (TTP)

Suggestive Clinical Findings:

- Neuro abnormalities
- Purpura
- Abd pain (micro infarcts)
- Jaundice & pallor (hemolytic anemia)
- Fever

Hgb < 8
Platelets < 20K
Schistocytes & NRBCs on smear
DAT negative
BUN high in > 50%
Proteinuria/micro hematuria
Normal coags (may see high FDP or Fdp)

Associations:

Viral illness
BCPs
Pregnancy
Cancer
Chemotherapy
Infections: HUS in kids is associated with gastroenteritis due to E coli or Shigella
Chemo: Mitomycin, cyclosporine, combined CDDP/Bleo/vinca

Differential Diagnosis:

Evans' syndrome
Vasculitis
DIC
Pre-eclampsia/eclampsia
Malignant HBP
HELLP

TTP is thought to be caused by the absence of plasma proteases that normally break down large vWF multimers. This results in the presence of "unusually large" vWF multimers that more efficiently bind GPIb as well as IIb/IIIa. They can also bind RBCs which may contribute to the anemia.

Treatment
(From
Blood, 15 August 2000, Vol. 96, No. 4, pp. 1223-1229, James George, Univ of Okla)

Plasma exchange (3-4 L/d) - continue until platelet count and LDH normalize. If no response or poor response, can increase to a single 1.5 volume exchange, but it is probably better to increase to twice daily exchanges using 1 volume each time. Some recommend using plasma cryosupernatant (cryo-poor FFP) because it contains no vWF.

It may take several days for the platelet count to begin to improve. Renal function may also lag. It is not uncommon for hemolysis to initially worsen requiring transfusion. The platelet count is the most important parameter to follow. A drop in the count may herald worsening of other symptoms.

Relapses are common during first month as begin to taper exchanges.

Some use prednisone 200 mg/d or IV prednisolone 0.75 mg/kg q12h. Others use aspirin and dipyridamole. However, neither steroids nor antiplatelet therapies have been proven to add any benefit to plasma exchange alone.

If no exchange readily available, try FFP 30 cc/kg. Watch volume.

Less proven: splenectomy, VCR, azathioprine, CTX.

Platelet transfusions should be given only if there is life-threatening bleeding or if need to do invasive procedures. In TTP, when the platelet count is < 20K, the risk of bleeding from subclavian catheter insertion is only about 20%.

Relapses occur in up to 20% of survivors.

 

WALDENSTROM'S

Generally discussed under heading of myeloma, but may be more closely related to CLL in terms of lymphoid progenitor development.

CD10+, CD20+, CD5+

Usually affects older patients. Present with:

Anemia
lymphadenopathy
splenomegaly
elevated serum viscosity
neuro defects
cryopathic symptoms

Waldenstrom's per se describes a particular presentation: Massive organomegaly and IgM > 5g/dl

If IgM < 5 g/dl but still abnormal, use term "macroglobulinemic lymphoma."

Patients usually observed until develop:

Symptoms
Cytopenia
Hyperviscosity

Treatment:

For hyperviscosity --> plasmapheresis

For underlying disease --> 2CDA, fludarabine, alkylators, and/or steroids.

CHEMO:

NEW:

Combination Therapy:

2CDA 1.5 mg/m2 SQ TID x 7 days
Oral CTX 40 mg/m2 BID x 7 days
Rituxan 375 mg/m2 IV weekly x 4 wks.

(Alexanian, PROC ASH #551, 1999) Worked well as 2nd line therapy.

STANDARD:

Chlorambucil 8 mg/m2 + Prednisone 40 mg/m2 daily x 10, repeated q 6 wks until maximal reduction in IgM seen.

Response: Reduction by 75% or more of IgM synthesis, lymphadenopathy, and splenomegaly.

Can also use:

Melphalan 6 mg/m2/d + Cytoxan 125 mg/m2/d + prednisone 40 mg/m2/d x 7 days at 4-6 wk intervals.

Can also use:

CHOP

Fludara 25 mg/m2 IV x 5 days q 4 weeks

2-cda (0.1 mg/kg/d CIV x 7 days). May only need 1 or 2 courses of 2-cda.

 

INDICATIONS FOR TRANSPLANT

AML in CR1 and beyond (with exceptions)
ALL in CR2 and beyond or CR1 if Ph+
CML in Chronic Phase
Myeloma in first relapse
NHL in first relapse or CR2 and beyond
HD in first relapse or CR2 and beyond

 

HEPARIN-INDUCED THROMBOCYTOPENIA

Pretest Scoring System

Platelet count fall > 50% and nadir > 20            2
Platelet count fall 30-50% or nadir 10-19          1
Platelet count fall < 30% or nadir < 10  0

Clear onset between days 5-10 or platelet count fall one day or less if prior heparin exposure within last 30 days                        2
Consistent with fall at 5-10 days, but not clear (eg missing platelet counts) or onset after day 10 or fall one day or less with prior heparin exposure 30-100 days ago                       1
Platelet count fall at less than 4 days without recent exposure     0

Confirmed new thrombosis, skin necrosis, or acute systemic reaction post-IV unfractionated heparin bolus                     2
Progressive of recurrent thrombosis, non-necrotizing skin lesions, or suspected thrombosis which has not been proven    1
No thrombosis or other sequellae                      0

No other causes of thrombocytopenia apparent 2
Possible other causes present                1
Definite other causes present                 0

0-3       Low pretest probability
4-5       Intermediate
6-8       High pretest probability of HIT

Discussion

Occurs in 3-5% of patients receiving unfractionated heparin, and 1% of patients receiving LMWH.

Up to 30% of patients receiving bovine heparin may develop mild thrombocytopenia (HIT Type I). This is non-immune and is probably caused by mild, direct platelet activation by heparin. It is not associated with thrombosis. The platelet count does not usually fall below 100K. It typically occurs in patients receiving high-dose heparin, but it does not necessitate discontinuation of the heparin.

HIT with thrombosis (HIT Type II) is caused by an antibody (HIT-Ig) whose target antigen is usually a complex of PF4 and heparin. Some unfractionated heparin molecules are able to wrap around the PF4 molecule thereby eliminating the anticoagulation properties of the heparin and facilitating complex formation. The resulting antigen/antibody complexes activate platelets by binding to the Fc receptors. They then generate procoagulant platelet microparticles, and activate endothelial cells.

Note, however, that only about one-third of patients who develop strong HIT antibodies actually develop thrombocytopenia, and these seem to be the ones most at risk for thrombosis. In all, 0-10% of patients on heparin may develop HIT-Ig antibodies. 30-50% of these may develop thrombocytopenia. 30-80% of those with thrombocytopenia may develop isolated thrombosis which may also be related to other clinical factors such as atherosclerosis, recent vascular trauma, catheters, etc. Finally, 0.01 – 0.1% of patients will develop the full-blown multiple thrombosis (white clot) syndrome. (These facts form the “iceberg model” of HIT.)

Although platelet activation is an important aspect, the most important effect is an increase in thrombin generation and activation (resulting in increased D-dimer levels). This accounts for the thrombotic events as much or more than the platelet-Ab complexes by themselves. This is also why anti-thrombin agents are important in the treatment of HIT.

Suspect HIT in patients who have a fall in the platelet count of 50% or greater within 5-10 days of heparin exposure and in whom there is the absence of other causes of thrombocytopenia. (A more rapid drop in platelets may also occur if the patient received heparin in the previous three months. In these cases, residual circulating antibodies may react upon heparin exposure inducing the rapid fall in the platelet count.) The thrombocytopenia is usually mild-moderate (20-150K). By comparison, thrombocytopenia induced by sulfa drugs and quinine is usually much more severe.

The “HIT Syndrome” may consist of:

·        Mild to moderate thrombocytopenia, often in conjunction with thrombosis.

·        Adrenal hemorrhagic infarction (caused by adrenal vein thrombosis)

·        Coumadin-induced venous limb gangrene

·        Fever, chills, flushing, or transient amnesia beginning 5-30 minutes after an IV heparin bolus

·        Heparin-induced skin lesions associated with HIT antibodies, even in the absence of thrombocytopenia. 75% of patients who develop heparin-induced skin lesions do not develop thrombocytopenia. Thus thrombosis may also occur in patients on heparin without significant thrombocytopenia, so be aware of this.

HIT is a prothrombotic state, so consider prophylactic treatment for patients with suspected HIT, even in the absence of thrombosis. The risk is probably >50%. The thrombotic events tend to be venous rather than arterial (4:1), usually involving large veins. Thromboembolic events are common, and HIT-associated mortality is high (~18%).

Treatment should be with heparin substitutes (lepirudin, danaproid, argatroban). Ancrod is probably not that good because it does not lower thrombin levels.

Warfarin should be avoided because it lowers protein C levels so that there is no longer a balancing anticoagulation between it and the increased thrombin being generated. The clue that this may be occurring is a rising INR to levels greater than 4, yielding worsening thrombosis in the face of a rising INR. 

Adjunctive therapies include plasmapheresis to reduce the titer of HIT antibodies. Replacement of anticoagulants, such as Protein C or antithrombin, may help. Thromboembolectomy can be limb-saving in patients with arterial occlusion.

Aspirin has been used but, unfortunately, has only a modest inhibitory effect on platelet activation by HIT antibodies.

 

 

CAUSES OF THROMBOCYTOPENIA

1. Increased Platelet Destruction

Non-immune: Sepsis, inflammation, DIC, TTP

Immune: Autoimmune (ITP or secondary ITP)
Alloimmune (Post-transfusion purpura)
Drugs-induced: (Heparin, see List)

2.  Decreased Platelet Production: Alcohol, cytotoxic drugs, aplastic anemia, leukemia, MDS, myelophthisis, certain infections

3.  Hypersplenism

4.  Hemodilution: infusion of blood products, colloids, or crystalloids

 

MDS – INTERNATIONAL PROGNOSTIC SCORING SYSTEM
(Blood 89:2079 1997, erratum 91:1100, 1998)

Blasts: < 5% = 0 points, 5-10% = 0.5, 11-20% = 1.5, 21-30 = 2.0

Cytogenetics: Normal, -Y alone, 5q- alone = 0 points; Three or more abnormalities = 1.0 point; Any other = 0.5 points

Cytopenias (ANC < 1.8, Platelet < 100, Hb < 10): 0-1 = 0 points, 2-3 = 0.5

Total: 0 = Low risk, 0.5-1 = Int-1 risk, 1.5-2.0 = Int-2 risk, 2.5+ = High risk

Management: Low/Int-1 --> Observe/support. Int2/High --> BMT or treat as AML.

 

FLOW CYTOMETRY PATTERNS IN LYMPHOCYTOSIS

CD3- CD19- CD38+ = Plasmacytic tumor
CD3- CD16+ and/or CD56+ = NK LGL leukemia

CD3+ CD4+ CD25- = Peripheral T-cell lymphoma or T-cell Prolymphocytic Leukemia (PLL)

CD3+ CD4+ CD25+ = Adult T cell lymphoma/leukemia (ATLL)

CD3+ CD8+ (CD57 or CD16 or CD56+) and HIV- = T Large Granular Leukemia (LGL)

 

CD5+ CD10- CD19+ CD20+/- CD23+ sIg+/- = CLL/SLL

CD5+ CD10- CD19+ CD20++ CD23- sIg++ = Mantle Cell Lymphoma, Lymphomatous polyposis

 

CD19+ CD5- CD10+ = Follicular NHL, Burkitt’s

CD19+ CD5- CD10- CD11c+ CD25++ CD103++ = Hairy Cell Leukemia

CD19+ CD5- CD10- CD11c+ CD25- = Marginal Zone Lymphoma, MALToma, Splenic lymphoma with villous lymphocytes

 

BLOOD GROUP SYSTEMS

Individual blood groups of the red cell membrane are serologically linked antigenic structures, due to their presence on the same biochemically-related lipids or proteins. Some antigens, such as those of the ABO blood group, are surface structures, while others (eg, Rh) are carried on molecules which span the width of the red cell membrane. This topic review will discuss the basic biochemistry of each of the major blood groups, along with unusual phenotypes within each system. An emphasis will be placed on disease association as well as the clinical significance of antibodies produced against each blood group. Finally, the cell lines and tissues on which the blood group is expressed will be listed; the potential deleterious effect of antibodies produced against a given blood group on transplanted organs will also be discussed. Additional reading for subjects not covered here is available to the interested reader.

ABO (ABH) BLOOD GROUP SYSTEM — The four common blood groups in the ABO/ABH system are O, A, B, and AB. These occur In the Caucasian population at frequencies of 44, 40, 12, and 4 percent, respectively. These percentages vary somewhat among different ethnic groups. When an individual lacks the A and/or B antigen on the red cells, the plasma will contain naturally-occurring antibodies to the missing antigen(s). Thus, a group A individual will have anti-B antibodies, a group AB individual will have neither anti-A nor anti-B, and a group O patient will have both anti-A and anti-B antibodies. The function of the ABH antigens is unknown.

Basic biochemistry — The ABO blood groups are defined by the presence of immunodominant sugars: n-acetylgalactosamine for the "A" antigen and D-galactose for the "B" antigen. Both sugars are built upon the "H" antigen, the immunodominant sugar of which is L-fucose. If the "H" antigen is unmodified, the individual types as Group O. All of these sugars are attached to oligosaccharides carried on glycosphingolipid and glycoprotein chains. Thus, ABH antigens are not an integral part of the membrane and actually extend out above the red cell surface.

Unusual phenotypes — The products of the ABH genes are glycosyl transferases, which transfer the immunodominant sugar of each blood group to the backbone chain. In the Bombay phenotype, fucosyl transferase, which conveys H antigen specificity, is lacking. Since the H antigen is the building block for the A and B antigens, neither A nor B can be produced, even in the presence of their respective transferase enzymes. Thus, red cells of the Bombay phenotype lack A, B and H antigens. These individuals have naturally occurring anti-A, anti-B, and broad thermal range anti-H, and can only be transfused with blood from other individuals of the Bombay phenotype (usually a relative), or risk a severe hemolytic transfusion reaction.

Weak subgroups may also occur within the ABO blood group system, in which the A or B antigens are difficult to detect. It is advisable to transfuse group O red cells when the ABO blood group is uncertain because of such weak A or B antigens.

Disease associations

  Acquired B antigen — Acquisition of the B antigen occurs in approximately 5 percent of patients with genetic A1, a strong A antigen found in 80 percent of A individuals, resulting from production of a deacetylase enzyme by bacteria such as E. coli K-12 or Clostridium tertium. When these bacteria gain a foothold in necrotic malignant lesions and/or gastro-intestinal obstructions and elaborate the enzymes into the circulation, a B-like antigen is generated at the expense of the A1 antigen. The resulting ABO discrepancy may be the first indication to the clinician that an ongoing infection and/or malignancy exists. Once the infection is successfully treated, the patient's ABO group will return to group A1.

Patients with the acquired B antigen should never be transfused with AB or B blood, as such blood is likely to be cleared from circulation at a rapid rate, because of the existence of pre-formed anti-B.

A, B, and H antigens often act as tumor markers, and may be weakened or lost in various hematologic malignancies (eg, acute leukemia, myeloproliferative disorders, myelodysplasia), chromosomal aberrations, and thalassemia. Gastric cancers appear to be more prevalent in group A individuals, while gastric and duodenal ulcers occur more often in those who are Group O.

Expression of antigens — Lymphocytes and platelets have ABH antigens which are absorbed from the plasma. There is broad tissue distribution as well, particularly on glandular epithelial and endothelial cells. The strength of the A and B antigens is not fully developed on cord cells.

Clinical significance of antibodies — Most deaths from blood transfusion are the result of transfusing ABO incompatible blood, since the A and B antigens are the most immunogenic and are often strongly hemolytic both in vitro and in vivo. These errors tend to be primarily clerical in nature.

As opposed to the severe nature of transfusion reactions, hemolytic disease of the newborn (HDN) caused by ABO antibodies tends to be relatively mild in nature, since the A and B antigens are not well developed at birth. The exception is in group B African-American newborns of group O mothers. The B antigen appears to be more developed at birth in this ethnic group, and ABO HDN can be of a more severe nature, sometimes requiring exchange transfusion.

Effect of antibodies on transplantation — Donor kidneys should be ABO compatible with the recipient's ABO antibodies. However, ABO compatibility is not required between donor and recipient for bone marrow transplantation. ABO mismatches at the time of bone marrow transplantation may lead to self-limited hemolysis or graft versus host disease, where, for example, group O donor lymphocytes produce anti-A antibodies against red cells and tissues of a group A recipient.

Rh BLOOD GROUP SYSTEM — The Rh antigens are located on non-glycosylated polypeptides. They are transmembrane proteins and thus integral components of the red cell membrane. The antigens commonly recognized on the red cell include D, C, c, E, and e. Each chromosome will be either D positive or D negative (there is no "d" antigen), C or c positive, and E or e positive. Rh antigens (eg, the Rh haplotype) are inherited as a linked group, with the most common phenotype in Caucasians and blacks being DCe and Dce, respectively.

Unusual phenotypes — Rh deletion cells exist in the population, which result in one or more of the Rh antigen sites being absent from the red cell membrane. These deletions can occur at the Ee locus, at both the Cc and Ee loci, or at all three loci. In the latter circumstances, no Rh antigens are produced, a condition referred to as "Rh null." Patients with these deletion types, when transfused, may form one or more antibodies to high frequency Rh antigens, which makes it extremely difficult to procure compatible blood.

Aberrations in the Rh system appear to be more common in the African-American population, particularly at the D and e loci. Such patients may actually type as D+ and demonstrate anti-D or type as e+ and have anti-e-like activity. Interestingly, once one mosaicism is identified (a portion of the antigen appearing to be absent), other mosaicisms will often be detected at a later date. Uniformly weakened expression of the Rh antigens (ie, Rh mod) has also been reported.

Disease associations — Perhaps the most dramatic disease association is the compensated hemolytic anemia seen in most individuals with Rh null or Rh mod red cells. This is not unexpected, since the carrier molecules for the antigens project through the red cell membrane, and the absence of such proteins would be expected to affect the material properties of the red cell, causing loss of red cell membrane, and reducing the ratio of surface area to volume. As a result, Rh null red cells do not appear as biconcave discs, but rather as stomatocytes (cup shaped) and spherocytes, and demonstrate increased osmotic fragility. The anemia is usually mild in nature.

Blood group function and expression — Rh antigens (or more properly, their carrier molecule) are specific to the erythroid cell line and appear to be expressed on cord cells; their function is unknown.

Clinical significance of Rh antibodies — Although a number of examples of anti-E and anti-Cw (a replacement antigen at the Cc loci) can be naturally occurring IgM specificities, the majority of the antibodies are IgG in nature; all are capable of causing significant hemolytic transfusion reactions (HTRs) as well as hemolytic disease of the newborn (HDN).

Anti-D, the originally described anti-Rh, causes the most severe form of HDN, sometimes resulting in hydrops and, on occasion, fetal demise. Fortunately, since the advent of Rh immune globulin, HDN due to anti-D has become much less prevalent. Currently, anti-c and anti-E are the antibodies most often responsible for HDN, and they are frequent causes of delayed HTRs. Since Rh antibodies rarely, if ever, bind complement, red cell destruction is mediated almost exclusively via splenic trapping.

Anti-C can be difficult to detect and yet has been reported on more than one occasion to cause hemolysis and hemoglobinuria. Anti-e, which reacts with 98 percent of random donors, presents a problem with availability of compatible units, particularly in patients needing chronic transfusions.

Patients with sickle cell disease who have aberrations at the e locus present particularly difficult transfusion problems, especially if they have already formed anti-E antibody and then develop an e-like alloantibody. Screening of blood donors (particularly siblings) using the patient's serum may allow for detection of compatible donors.

Anti-Cw and anti-V are antibodies directed against low frequency antigens that often appear in multi-sensitized patients. Anti-Cw is usually seen in conjunction with anti-c. Anti-V is often found in conjunction with anti-D + C. In immunized sickle cell patients (eg, the presence of anti-C, E, Jkb, Fya, K, S), the detection of anti-V will significantly reduce the number of available black donors, since even though the V antigen is infrequent in the random donor population (1 percent), it is present in 30 percent of random black donors.

In autoimmune hemolytic anemia, a high percentage of autoantibodies demonstrate Rh system specificity, with anti-e being most commonly seen. In the absence of hemolysis, e negative blood need not be given and should be reserved for patients who have formed allo anti-e.

Effect of antibody on transplantation — On occasion, production of anti-D by lymphocytes from an Rh negative bone marrow transplant donor may cause self-limited graft-versus-host disease in an Rh positive recipient. Conversely, should the recipient be Rh negative and receive marrow from an Rh positive donor, the recipient may produce anti-D and develop a chronic hemolytic anemia, although production of anti-D may ultimately diminish over time.

LEWIS, P1, AND I BLOOD GROUP SYSTEMS — All of these antigens are structurally related, having identical red cell glycosphingolipid backbones.

  • Lewis antigens are absorbed onto the red cells from the plasma and are located on type 1 glycosphingolipids
  • Paragloboside is the precursor antigen for the P1 antigen, which has a terminal galactose as its immunodominant sugar
  • The I and i determinants are carbohydrate structures on lipids integral to red cells, and are also present on proteins in the plasma. The I antigen, found primarily on adult red cells, is located on branched chains, while the i antigen, found primarily on cord/infant red cells, is found on unbranched chains.

Unusual phenotypes — The I negative adult phenotype (also called i positive) occurs in less than 1 percent of the population. Such individuals will form naturally occurring anti-I, which often has an increased thermal range. Its clinical significance is variable, and RBC survival studies would need to be undertaken before deciding whether I negative red cells would be required for transfusion of an anemic I negative patient.

Disease associations — Lewis antigens may disappear from red cells in infectious mononucleosis-associated hemolysis, severe alcohol-induced cirrhosis, or pancreatitis. The P1 antigen may be weakened in various types of carcinoma. Varied expression of both I and i have been associated with Tk polyagglutination, leukemia and other hemolytic conditions.

Anti-I is often the causative antibody responsible for chronic cold agglutinin disease, as well as the acute, self-limited hemolysis sometimes seen subsequent to mycoplasma pneumonia infection. Anti-i has been associated with hemolysis in conjunction with infectious mononucleosis and lymphoma.

Blood group function — The Lewis antigen is thought to be the receptor for Helicobacter pylori, although this has been brought into doubt. The P antigen is a receptor site for certain strains of E. coli; rare subjects with the P-null phenotype are resistant to infection with these strains. The function of the I antigen is not known.

Expression of antigens — P1 antigens can be found on lymphocytes, granulocytes, monocytes, and platelets. Interestingly, the P1 antigen is also a component of pigeon egg white as well as hydatid and echinococcus cyst fluid. Lewis antigens are found in all body fluids (except cerebrospinal fluid) and on lymphocytes, monocytes, and platelets. They are also widely distributed in body tissues, including the renal cortex, gastric mucosa and the small and large intestine. The I/i system antigens can be found in most body fluids, all blood cell lines, and virtually all body tissues. P1, Lewis and I antigens are weak or absent on cord blood cells.

Clinical significance of antibodies — Anti-P1 is generally considered to be of little clinical significance. It is often naturally occurring and does not cause hemolytic disease of the newborn (HDN). On rare occasions, the antibody may show a broad thermal range, particularly in pigeon breeders who are P1 negative, or those with hydatid or echinococcus cysts. Transfusion of P1 negative blood may be advisable in such instances, or when the antibody is IgG.

Lewis antibodies, like anti-P1, are common during gestation and the immediate postpartum period, and are formed primarily by individuals who type as Le(a-b-). The antibodies do not cause HDN, and rarely if ever are responsible for transfusion reactions. Anti-Le(a), when hemolytic in vitro, may have clinical significance. In such cases, "crossmatch compatible" donor units would be acceptable for transfusion.

The rare instance of hemolytic or IgG reactive anti-Le(a) and anti-Le(b) in a group O Le(a-b-) individual scheduled for a surgical procedure requiring a large number of red cell transfusions presents a special problem. Rather than attempting to obtain Le(a-b-) units, which are found in only six percent of the random donor population, in vivo neutralization of the anti-Le(b) may be a viable option. One to two units of fresh or fresh frozen plasma from a Le(b+) donor may be used to neutralize the anti-Le(b) antibody activity. Transfusion can then be accomplished using Le(a-b+) donor units, since almost four out of five random ABO compatible donor units will type as Le(a-b+). These units will shed their Le(b) antigens and take on the Le(a-b-) phenotype of the recipient within 24 hours post-transfusion. If and when the patient's anti-Le(b) antibody activity returns, which normally occurs within 48 to 72 hours, the transfused red cells will no longer be subject to red cell destruction.

Most examples of anti-I (anti-i is rare) are IgM in nature, reactive at room temperature or below, and rarely cause problems from a transfusion standpoint. Anti-I does not cause HDN. In the rare I negative individual with the adult i phenotype, increased red cell destruction may ensue if random donor units are transfused. In rare instances of patients with auto anti-I with an increased thermal range (> 31ΊC), the use of a blood warmer may be required. The patient's core body temperature should be maintained as close to 37ΊC as possible, in order to avoid cold-induced exacerbation of the hemolysis.

Effect of antibodies or transplantation — Renal graft survival appears to be reduced in recipients whose red cells type as Le(a-b-), suggesting that Le(a+) or Le(b+) donor kidneys may be rejected at an accelerated rate in the presence of Lewis antibodies.

MNSs BLOOD GROUP SYSTEM

Basic biochemistry — The M and N antigens are carried on glycophorin A and the S and s antigens are carried on glycophorin B, both of which are sialoglycoproteins. MN antigens are closely linked with the Ss antigens and generally will be inherited together.

Unusual phenotypes — The extremely rare En(a-) phenotype results from the absence of glycophorin A. It is seen more frequently in Finland. Glycophorin B is lacking from the red cells of U-negative individuals. The S-s- phenotype (also U negative in the majority of cases) is found in approximately 1 percent of Afro-Americans. The exceedingly rare Mk/Mk phenotype results in the absence of both glycophorin A and B. Such red cells are severely deficient in sialic acid residues, but do not undergo hemolysis.

Disease associations — There is a sialic acid dependent site for Plasmodium falciparum invasion located primarily on glycophorin A, as well as a receptor for some E. coli species and viruses.

Blood group function — Glycophorin can act as a complement receptor. The net negative charge of the red cell membrane is primarily due to sialic acid residues carried on Glycophorin A and B. Glycophorin A also associates with band 3 of the RBC membrane.

Expression of antigens — MNSs antigens have not been described on other blood cells, but have been found in the epithelium and endothelium of the kidney. All MNSs antigens are well developed on cord cells.

Clinical significance of antibodies — Anti-M is usually an IgM or cold reactive IgG antibody. It can be naturally occurring and will sometimes react better if the patient's serum is acidified. The antibody rarely is the cause of transfusion reactions unless it is reactive at body temperature. HDN has only rarely been reported, primarily in U-M- individuals.

Anti-N is generally considered to have little if any clinical significance. It is almost always IgM in nature, and may be found as an autoantibody in dialysis patients whose equipment was sterilized with formaldehyde. Anti-En(a) is capable of causing severe hemolytic transfusion reactions (HTRs) and HDN. Fortunately, the En(a-) phenotype is extremely rare.

Both anti-S and anti-s are capable of causing hemolytic transfusion reactions, although the majority are mild to moderate in nature. Anti-S and anti-s are mainly IgG, and thus can cross the placental barrier and cause HDN. Severe cases are rare. Anti-S will frequently be found as an underlying alloantibody in patients with warm autoantibodies. Anti-U can cause both severe HTRs and severe HDN. Since 1 percent of blacks lack the U antigen, anti-U can present as a relatively common serologic problem. If no anti-U is available to screen for compatible donors and a relatively large black donor population exists, soybean lectin may be used for screening, since it will react with red cells containing reduced amounts of sialic acid (eg, U(-) RBCs).

Effect of antibodies on transplantation — Although no transplant rejection reports have been reported in association with MNSs system antibodies, theoretically they could have an effect on kidney transplants because of antigen expression on renal epithelium and endothelium.

THE KELL BLOOD GROUP SYSTEM

Basic biochemistry — The 22 Kell system antigens are located on a highly folded membrane glycoprotein. The Kell proteins require intact disulfide bonds to maintain antigenic integrity.

Unusual phenotypes — The antigens which are most readily recognized are K and its alternate allele k (cellano), Kpa and Kpb, and Jsa and Jsb. In a phenotype called Kmod, there is marked reduction of all Kell system antigens (only detectable via adsorption/elution techniques). The Ko phenotype results in no production of K system antigens, and an increase in what is thought to be precursor substance, Kx.

The McLeod phenotype (see below), also results in marked reduction of all Kell system antigens, and the absence of Kx from the red cell. The sensitized Ko individual will produce an antibody called anti-Ku as will Kmod patients. Both of these types of individuals must receive only Ko blood. Ko units, being exceedingly rare, should be procured through the American Rare Donor File located in Philadelphia, PA.

Disease associations — The Kx gene appears to be closely linked with x-linked chronic granulomatous disease (CGD) in males. There is no evidence to suggest, however, that the absence of the Kx antigen is responsible for the white blood cell defect seen in CGD.

The McLeod phenotype is associated with uniformly weak K system antigens and red cell morphology which demonstrates profound acanthocytosis, and a compensated hemolytic anemia. The McLeod phenotype can occur with or without CGD. In CGD-/McLeod+ patients, the antibody found after transfusion is anti-Km; these patients can receive McLeod or Ko blood. In patients who are CGD+/McLeod+, sensitized patients form an antibody called anti-KL (anti-Km and anti-Kx), and can only be transfused with blood from ABO compatible donors who also have the McLeod phenotype. Non-CGD McLeod males also suffer muscular and neurological abnormalities, as well as cardiomyopathy.

Approximately 1/250 patients with warm-reacting autoimmune hemolytic anemia will have autoantibody directed against Kell blood group system antigens. Microbial infection will sometimes cause transient depression of Kell system antigens (particularly Kpb).

Blood group function — The system as a whole may have some enzymatic properties, possibly activating and inactivating bioactive peptides.

Antigen expression — Kell system antigens appear to be red cell specific. All Kell system antigens are expressed on cord cells. There is no evidence that Kx antigen is found on granulocytes, either in normal individuals or patients with x-linked CGD.

Clinical significance of Kell system antibodies — Anti-K is capable of causing severe HTRs and HDN. In fact, anti-K, along with anti-c and anti-E, currently make up the great majority of cases of clinically significant HDN.

Bacteria are capable of eliciting production of anti-K, probably due to the presence of cross-reactive antigens. Anti-k is one of the most common antibodies directed against high frequency antigens (approximately 1/500 random individuals lack k) and like anti-Jsb and anti-Kpb, can cause mild to moderate HDN and HTRs. Sometimes, in autoimmune hemolytic anemia, the Kpb antigen becomes transiently depressed and the autoantibody "appears" to be an alloantibody. This can create a dilemma as to whether the patient should receive Kp(b-) blood. Radiochromium red cell survival studies may be useful in determining the antibody's clinical significance at a particular point in time.

Anti-Jsb is produced primarily by African-American patients. Anti-Jsa and anti-Kpa are directed against low frequency antigens in the random donor population. Both can cause mild to moderate HTRs and HDN. Formation of anti-Jsa in multisensitized sickle cell patients, who are receiving blood primarily from black donors, may reduce the available donor pool by approximately 20 percent (the frequency of the Jsa antigen in blacks). Because of the scarcity of reagent grade anti-Jsa, the patient's serum may need to be used for finding Js(a-) donor units, providing the antibody is reactive stronger than 1+ in the antiglobulin phase of testing.

Effect of antibody on transplantation — The presence of Kell system antibodies does not appear to affect kidney transplantation. If a bone marrow recipient were to have produced anti-K from previous transfusions, it would be advisable to select a K-negative marrow donor.

DUFFY BLOOD GROUP SYSTEM

Basic biochemistry — The carrier molecule of the Duffy blood group antigens is a multi-pass membrane glycoprotein. The antigens themselves have not yet been fully characterized.

Unusual phenotypes — The Fy(a-b-) phenotype, while extremely rare in the random donor population, occurs in 68 percent of the black population. Fy(a-b-) red cells lack the Duffy protein and Fy(a-b-) subjects may occasionally form anti-Fy5 in response to transfusion, which will react with any red cell positive for Fya and/or Fyb, but not with Fy(a-b-) red cells. Although Fy(a-b-) blacks also lack Fy3 in their red cells, anti-Fy3 is rarely found in blacks. Anti-Fy3 has been produced in three Caucasians and one Crete Indian who had the Fy(a-b-) genotype; anti-Fy3 is usually proceeded by the formation of anti-Fya. The production of anti-Fyb has only rarely been seen in Fy(a-b-) blacks.

Disease associations — Perhaps the most interesting association is between the Duffy blood group and malaria. Approximately 70 percent of West African blacks type as Fy(a-b-) and the same percentage is resistant to infection with Plasmodium vivax. In some fascinating experiments, it was confirmed that merozoites from P. vivax and P. knowlesi would not be internalized into the red cells of Fy(a-b-) individuals, but the membranes of Fya+ and/or Fyb+ red cells would invaginate and allow the merozoites to enter the red cells. Thus the Duffy antigen is the receptor site which allows invasion of both vivax and knowlesi. Although the merozoite attaches to Fy(a-b-) red cells, it will not enter the cell and eventually detaches, leaving widespread deformation of the red cell in its wake.

Function of Duffy blood groups — The Duffy antigens are thought to be receptors for some pro-inflammatory cytokines, such as IL-8.

Antigen expression — Duffy antigens are not present on other blood cells, but can be found in brain, colon, endothelium, lung, spleen, thyroid, thymus and kidney tissue. Duffy antigens are exposed on cord blood cells.

Clinical significance of antibodies — Anti-Fya can cause significant HTRs as well as HDN. Anti-Fyb, on the other hand, is not commonly found, since the Fyb antigen is a poor immunogen. The antibody is usually produced in patients who have already been sensitized to multiple blood group antigens. HDN caused by anti-Fyb is rare and, when present, is mild in nature; anti-Fyb can occasionally cause HTRs.

Anti-Fy5 is distinguished from anti-Fy3 by the fact that, unlike anti-Fy3, anti-Fy5 will not react with Rh null cells of common Duffy type. HDN rarely occurs with either of these two antibodies. Significant transfusion reactions have occurred with anti-Fy3. Anti-Fy5 caused a mild, delayed HTR in one reported case. In the presence of either anti-Fy3 or anti-Fy5, Fy(a-b-) donor units should be transfused. Such units can be readily located by screening units from black donors.

Effect of antibody on transplantation — Although there is no report in the literature, anti-Fya could theoretically interfere with kidney transplant survival if the recipient had formed anti-Fy(a) and the kidney was from an Fy(a+) donor.

KIDD BLOOD GROUP SYSTEM

Basic biochemistry — The biochemistry of the Kidd blood group system has yet to be elucidated; the carrier molecule is known to be a multipass membrane protein.

Unusual phenotypes — The Jk(a-b-) phenotype, also known as Jk(3-), is seen in approximately 1 percent of Polynesians (peoples of the South Pacific). All of the Jk(3-) individuals we have encountered have been of Filipino origin. Jk(a-b-) red cells are resistant to urea lysis, a procedure used in some automated hematology analyzers to lyse red cells prior to performing a white blood cell (WBC) or platelet count. This can result in an abnormally high machine count; however, a blood smear prepared for the differential count will not demonstrate large numbers of WBCs or platelets, confirming this as a technical artifact.

Disease associations — Jk(a-b-) individuals are unable to maximally concentrate their urine.

Blood group function — The Kidd blood group system is thought to be responsible for transporting urea across the red cell membrane.

Expression of antigens — The Jk3 antigen is present in kidney tissue. Kidd antigens are expressed on cord blood cells.

Clinical significance of Kidd system blood group antibodies — Anti-Jka, and to a lesser extent anti-Jkb, are responsible for a great percentage of serious hemolytic transfusion reactions. The reasons are threefold:

  • Kidd antibodies, which are usually IgG, will bind complement and can cause in vivo hemolysis.
  • The antibodies will often react much better with cells containing a double dose of the antigen (eg, Jk(a+b-) than a single dose (Jk(a+b+), and in fact a Jk(a+b+) donor unit might be compatible in vitro, but incompatible in vivo.
  • Kidd antibodies tend to disappear rapidly from the circulation and may not be detectable using very sensitive serologic techniques the next time the patient requires transfusion.

Transfusion of antigen positive "compatible blood" will therefore often result in a dramatic anamnestic response, and rapid red cell destruction may ensue 3 to 14 days post transfusion. If multiple units are involved, renal shutdown and/or coagulopathies may occur. For these reasons, we recommend that anyone who has had anti-Jka or anti-Jkb identified in their serum obtain a Medic Alert bracelet stating that they have formed anti-Jka or anti-Jkb in the past.

Anti-Jka and anti-Jkb usually only cause mild hemolytic disease in the newborn. Anti-Jk3 can cause a significant HTR and in vivo hemolysis, but HDN has only rarely been reported.

Effect of antibodies on transplantation — We have seen a Jk(a-b-) Filipino male who was transplanted with a kidney from a non-Jk(a-b-) donor, who went on to form anti-Jk3. The antibody was not present prior to transplant. The presence of anti-Jk3 did not appear to have a deleterious effect on the transplanted kidney.

MISCELLANEOUS BLOOD GROUPS

Lutheran blood group — There are 18 antigens in the Lutheran system, with Lu(a) and Lu(b) being the most commonly recognized. Lu(a) occurs in approximately 7.5 percent of the population while Lu(b) is absent from the red cells of approximately 1/500 random individuals. Lutheran antigens are resistant to enzyme treatment (ficin or papain), but are inactivated by the sulfhydryl reagent 0.2 Molar dithiothreitol (DTT). The antigens are only weakly expressed on cord cells. Lutheran antigens may have some adhesive properties and mediate intracellular signaling.

Anti-Lu(a) characteristically shows a mixed field appearance versus antigen positive red cells (ie, the presence of agglutinated cells against a background of unagglutinated cells). The great majority of Lutheran antibodies do not cause HDN. Anti-Lu(b) can cause mild to moderate transfusion reactions, but there is no data or only limited case reports of mild HTRs for other Lutheran system antibodies. Anti-Lu(a), however does not cause HTRs. Sera in which anti-Lu(a) is detected often contain HLA antibodies.

The null type Lu(a-b-) can arise from either a dominant or recessive background. Anti-Lu3 is formed only by those individuals of the recessive Le(a-b-) genotype. No concrete data is available on the clinical significance of anti-Lu3.

Vel blood group — The Vel antigen is of high frequency, is enhanced by enzyme treatment, and is resistant to treatment with dithiothreitol. The antigen is only weakly expressed on cord cells and consequently, anti-Vel does not cause HDN. The antibody, which is often a mix of IgM and IgG components and capable of binding complement, can cause severe in vivo hemolysis when Vel+ blood is transfused into a patient with anti-Vel. The antigen is variably expressed on adult red cells, such that donors with weak Vel antigens may be mistyped as Vel-.

When present solely as an IgM antibody, anti-Vel in a group O patient may at first glance appear to be a harmless cold agglutinin (ie, anti-I), since it will be non-reactive with cord red cells, which do not express the Vel or I antigens (). Group O Vel-positive donor units, however, will be incompatible at room temperature, and the patient's autocontrol (the patient's own red cells in the patient's own serum) will be non-reactive at room termperature. These findings would suggest that further serologic work is required before calling the antibody a harmless cold agglutinin, which in this case would be incorrect and would have potentially disastrous consequences were the patient to be transfused with Vel positive blood.

Globoside blood group (P and Pk) — Globoside is universally found in body tissues as well as all blood cells except platelets, basophils, and eosinophils. Globoside is the receptor site for Parvovirus and pyelonephritogenic E. coli. Both the P and Pk antigens are produced from lactosyl ceramide. The null phenotype pp lacks P and PK and the antibody, anti-Tja (anti-P, P1, PK) can cause severe intravascular transfusion reactions. Cytotoxic IgM or IgG3 antibody directed against the P and PK antigens has been associated with an increased rate of spontaneous abortion in women of the pp, P1K and P2K phenotypes, the latter two lacking P, but not PK.

Anti-P when seen as an alloantibody is often naturally occurring and may be IgM or IgG in nature. It can cause serious HTRs and may be hemolytic in vivo. The P system antibodies react better with enzyme treated cells and also react with 0.2 Molar DTT- treated red cells. Although the P and Pk antigen are well expressed on cord cells, HDN in the presence of anti-P is not severe. It is important to keep in mind that neither anti-P nor anti-Tja (anti-P1, PK,P) will react with pp red cells, such that anti-P may be mistaken for anti-Tja. Anti-Tja, however, will react with P1K and P1K red cells.

Auto anti-P is often the causative antibody in paroxysmal cold hemoglobinuria (PCH), where it acts as a biphasic hemolysin, the classical Donath-Landsteiner antibody. This cold reactive IgG antibody binds to red cells at reduced temperatures and causes complement mediated hemolysis as the serum/cell mixture is warmed to body temperature.

Chido-Rodgers blood group — The Chido and Rodgers antigens are located on the C4d complement components of C4a and C4b, and are adsorbed onto the red cell from the surrounding plasma. The antigens are absent from cord cells and are inactivated by proteolytic enzymes, but not DTT. Both antigens are present in greater than 90 percent of the population.

The antibodies demonstrate fragile agglutination at the indirect antiglobulin stage that dissipates relatively easily. Anti-Chido and anti-Rodgers are neutralizable by pooled plasma, and both react strongly with in vitro prepared C4d coated RBCs. The antibodies are of little clinical significance, except that an anaphylactic reaction has occurred in one patient with anti-Chido 3. The etiology of the reaction remains uncertain.

The null phenotype (CH/RG –1, -2, -3) results in C4-deficient RBCs. Inherited low levels of C4 may predispose individuals towards such diseases as autoimmune chronic active hepatitis and insulin dependent diabetes. Lack of C4B (Ch-) results in increased susceptibility to bacterial meningitis in children, while lack of C4A (Rg-) results in a predisposition to develop systemic lupus erythematosus. There is an association between the Rg- phenotype and the HLA haplotype 1-8.

Gerbich blood group system — The Gerbich antigens are located on membrane sialoglycoproteins glycophorin A and glycophorin B. The Ge 2 and 4 antigens are sensitive to proteolytic enzyme treatment, while Ge3 is resistant. All components are unaffected by DTT treatment. Gerbich is expressed on cord cells. HDN has not been reported, but anti-Ge2 and anti-Ge3 have caused HTRs, some of which have resulted in intravascular hemolysis.

Gerbich-negative phenotypes have been seen more commonly in Melanesians and in the Hispanic population in the United States; the only examples of anti-Ge which we have encountered have been in Mexican-Americans. Anti-Gerbich is the most commonly encountered antibody to a high frequency antigen in the Hispanic population of Southern California.

Not all Gerbich negative donors are compatible with individual examples of anti-Gerbich antibody, since the specificity can be directed against various portions of the Ge 2,3,4 complex. It is thus always important to check sample segments from frozen Ge-negative red cell units for compatibility with a given patient's antibody, prior to deglycerolyzing the entire donor blood unit.

The Gerbich-null cell (which most Mexican-American donors appear to be) is called the Leach phenotype (Ge-2-3-4). Plasmodium falciparum parasites have a reduced ability to invade the red cells of the Leach phenotype. Hereditary elliptocytosis without hemolysis has been associated with marked reduction of glycophorin C and the presence of the Leach phenotype.

Auto anti-Gerbich has been reported to cause severe hemolytic anemia. However, in other patients with anti-Gerbich, red cell survival was essentially normal when Ge-positive red cells were transfused. Allo-anti-Ge2 may be naturally occurring.

 

BLOOD TRANSFUSION REACTIONS

Febrile, Acute Hemolytic, Delayed Hemolytic, Anaphylactic, Urticaria, Lung Injury, Post-transfusion Purpura

Unwanted, untoward effects of blood transfusion are not unexpected and in many cases are benign. However, some reactions cause serious morbidity and are potentially fatal. The problem is that the initial symptoms of reactions with serious or relatively benign consequences are often similar.

Adverse reactions occur in 1 to 6 percent of all blood transfusions and are more frequent (10 percent) in patients with hematologic and oncologic diseases. They can be categorized as immunologic, infectious, chemical, and physical; some also subdivide these reactions into acute and delayed types. The "classic" transfusion reactions are immunologic in nature and result from the interaction(s) of inherited or acquired antibodies with foreign antigens associated with cellular or humoral components of transfused blood products.

The major immunologic reactions to blood transfusion will be discussed here:

  • Febrile nonhemolytic transfusion reactions
  • Acute hemolytic transfusion reactions
  • Delayed hemolytic transfusion reactions
  • Anaphylactic transfusion reactions
  • Urticarial transfusion reactions
  • Transfusion-related acute lung injury
  • Posttransfusion purpura

Another immunologic reaction, graft-versus-host disease, is a rare complication that primarily occurs in immunocompromised patients and in immunocompetent subjects when the recipient is heterozygous for an HLA haplotype for which the donor is homozygous. The latter observation has led to the requirement for irradiation of blood products (to prevent lymphocyte proliferation and therefore GvHD) from a family donor or with HLA-matched platelets.

In addition to immunologic reactions, blood transfusions also are associated with infectious risks and with a number of chemical and physical complications (eg, citrate toxicity, hyperkalemia, hypokalemia, hypothermia, and iron overload). These problems are discussed separately.

FEBRILE NONHEMOLYTIC TRANSFUSION REACTIONS — The most common transfusion reaction is a febrile, nonhemolytic transfusion reaction (FNHTR). The clinical manifestations of this reaction include fever, often a chill, and sometimes mild dyspnea within one to six hours after transfusion of red cells or platelets. FNHTRs are benign, causing no lasting sequelae, but are uncomfortable and sometimes frightening to the patient. Furthermore, since fever, with or without a chill, also may be the sign of a severe, acute hemolytic transfusion reaction (see below), FNHTRs cannot be ignored. Only about 15 percent of patients who have an FNHTR will have a second reaction with further transfusions.

FNHTRs have been thought to be immune in nature since they have been associated with class I HLA antibodies (or sometimes granulocyte specific antibodies) directed against contaminating leukocytes in red cell concentrates However, such antibodies are not always found. FNHTRs also occur after platelet transfusions.

It is now clear that FNHTRs are commonly caused by cytokines, such as interleukin (IL)-1, IL-8, and tumor necrosis factor-alpha (TNFa). These cytokines are generated and accumulate during the storage of blood components. In one series, for example, the dominant factor determining the risk of a reaction was the age of the component. In another report, the mean IL-8 concentration increased 100-fold between days two and five of storage and rose further with continued storage.

It has been proposed that an interaction between donor leukocytes and recipient antibody leads to interleukin-1 (IL-1) release from donor leukocytes or recipient monocytes. IL-1 can then cause fever by stimulating prostaglandin E2 production in the hypothalamus. Two observations are compatible with the primary role for donor leukocytes: cytokine accumulation is reduced by prestorage leukocyte reduction, and leukoreduction is an effective preventive therapy.

An alternative proposed mechanism is release of platelet-derived CD154 (CD40 ligand), which is capable of inducing production of proinflammatory cytokines from fibroblasts, epithelial cells, and endothelial cells.

Prevention and treatment — FNHTRs can be avoided or minimized by using leukoreduction to reduce the number of leukocytes transfused.. Components that are leukoreduced prior to storage are preferred over bedside filtered products. In one study, the overall incidence of acute reactions and the incidence of severe reactions to WBC-reduced platelets were 11 to 13 percent and one to two percent, respectively.

The management of FNHTRs consists of the following steps:

  • Stopping the transfusion and determining that a hemolytic reaction is not taking place (see below)
  • Administration of antipyretics (aspirin should be avoided in thrombocytopenic patients) and moderate doses of meperidine in patients with severe chills and rigors

ACUTE HEMOLYTIC TRANSFUSION REACTIONS — Acute hemolytic transfusion reactions (AHTRs) result from the destruction of donor erythrocytes by preformed recipient antibodies. They are usually due to ABO incompatibility which is most often the result of clerical error. Some acquired alloantibodies, such as anti-Rh or anti-Jka, are occasionally implicated, but AHTRs more typically occur when a group O recipient is given non-group O red cells. These reactions involve naturally occurring IgM anti-A and anti-B, which fix complement and cause rapid intravascular hemolysis leading to disseminated intravascular coagulation (DIC), shock, and acute renal failure due to acute tubular necrosis.

The classic presenting triad of fever, flank pain, and red or brown urine is rarely seen. Fever and chills may be the only manifestations and, in patients under anesthesia or in coma, DIC may be the presenting mode, with oozing from puncture sites and hemoglobinuria. As noted above, fever is also the presenting sign of a "benign" FNHTR. Thus, whenever fever occurs after a red cell transfusion, one must first rule out a hemolytic reaction and take the following steps:

  • Stop the transfusion, but leave intravenous line attached
  • Begin an infusion of normal saline (not Ringer's or dextrose)
  • From the other arm, obtain a sample for a direct antiglobulin test and for plasma free hemoglobin
  • Save a urine sample for hemoglobin testing, alert the blood bank, and check for clerical error

Every hospital has a protocol for evaluating transfusion reactions. The plasma of a patient with an AHTR will generally be pink (plasma free hemoglobin >25 to 40 mg/dL) and the direct antiglobulin (Coombs) test will be positive, unless all the red cells have been destroyed and/or removed.

Treatment — If there is any suggestion — clerical mistake, hypotension, and pink plasma or urine — that an AHTR is possible, generous fluid replacement with saline (100 to 200 mL/h) to support a urine output above 100 mL/h should be initiated while lab tests are awaited in an attempt to prevent the development of acute renal failure. The beneficial effect of urinary alkalinization in patients with marked hemoglobinuria is uncertain.

Vigorous supportive care is also important for the treatment of AHTRs. Cautious heparinization (10 U/kg per hour) for the next 12 to 24 hours may prevent DIC if it has not occurred and a vasopressor such as dopamine may be required. If massive hemolysis has already occurred, hyperkalemia is likely and cardiac monitoring and acute hemodialysis may be required.

DELAYED HEMOLYTIC TRANSFUSION REACTIONS — Delayed hemolytic transfusion reactions (DHTRs) are due to an anamnestic antibody response occurring after reexposure to a foreign red cell antigen previously encountered by transfusion, transplantation, or pregnancy. The antibody, often of the Kidd or Rh system, is undetectable on pretransfusion testing but increases rapidly in titer following the transfusion.

DHTRs reactions are seen generally within 2 to 10 days after transfusion. Hemolysis is usually extravascular, gradual, and less severe than with acute reactions, but rapid hemolysis can occur. A falling hematocrit, slight fever, mild increase in serum unconjugated bilirubin, and spherocytosis on the blood smear may be noted. The diagnosis is often made by the blood bank when a positive direct antiglobulin test and antibody screen are found when more blood is ordered.

Treatment — No treatment is required in the absence of brisk hemolysis. However, future transfusions containing the same antigenic red cells could be harmful. It is therefore important to make the diagnosis, inform the patient, and maintain appropriate records.

ANAPHYLACTIC TRANSFUSION REACTIONS — Rapid onset of anaphylaxis, manifested by shock, hypotension, angioedema, and respiratory distress, in a transfusion recipient requires rapid recognition and concomitant action since it is a life-threatening occurrence. An anaphylactic transfusion reaction (ATR) may occur within a few seconds to a few minutes following the initiation of a transfusion that contains plasma, such as frozen or liquid plasma, red cells, platelets, granulocytes, cryoprecipitate, or gamma globulin; they are not generally seen following the administration of normal serum albumin, plasma protein fraction, or coagulation factors. It is the rapid onset that is characteristic of an ATR.

Severe anaphylactic reactions have a reported incidence of 1:20,000 to 50,000, but are believed to be more common. They are almost always due to the presence of class-specific IgG, anti-IgA antibodies in patients who are IgA deficient. Selective IgA deficiency is not uncommon, occurring in about 1 in 300 to 500 people. Fortunately, not all IgA deficient patients have developed antibodies. Anaphylaxis via a similar mechanism has been described after blood product transfusion in patients with anhaptoglobinemia with antihaptoglobin antibodies; this disorder primarily occurs in East Asians.

Severe laryngeal edema or bronchospasm appears to be more common in recipients of plasma exchange, occurring in 1 in every 500 to 1000 plasma exchanges in some studies. Some of these reactions may be due to ethylene oxide (or other) allergy, as noted in some hemapheresis donors.

Treatment — Treatment of an ATR consists of the following steps:

  • Immediate cessation of the transfusion
  • Epinephrine, 0.3 mL of a 1:1000 solution intramuscularly
  • Preparation, for possible administration, of an intravenous epinephrine drip
  • Airway maintenance, oxygenation
  • Volume maintenance with saline
  • Vasopressors (eg, dopamine), if necessary

Prevention consists of establishing the diagnosis after the fact and using either IgA-deficient blood products, which can be obtained through large, regional blood centers, or "ultra-washed" red cell or platelet products.

URTICARIAL TRANSFUSION REACTIONS — Urticarial transfusion reactions (UTRs) occur when soluble allergenic substances in the plasma of the donated blood product react with preexisting IgE antibodies in the recipient. This causes mast cells and basophils to release histamine, leading to hives or urticaria.

A UTR is the only transfusion reaction in which the blood product can be continued. However, the transfusion should first be stopped and 50 mg of diphenhydramine given intravenously since a UTR may be the first sign of a more serious reaction. If the urticaria wanes and neither dyspnea nor anaphylaxis is apparent, the transfusion may be resumed.

TRANSFUSION-RELATED ACUTE LUNG INJURY — Transfusion-related acute lung injury (TRALI) is a not uncommon reaction, occurring in 1:2000 transfusions at a university hospital. This complication is characterized by acute respiratory distress, hypoxemia, hypotension, fever, and pulmonary edema, initially without signs of left ventricular failure. The central venous pressure is normal, separating TRALI from transfusion-associated circulatory overload. Symptoms usually begin within two to four hours of beginning the transfusion. TRALI is life-threatening and is clinically indistinguishable from acute respiratory distress syndrome. However, it has a much more favorable prognosis: death occurs in no more than 10 percent of cases and recovery is generally complete within 96 hours of onset.

Pathogenesis — TRALI is a pulmonary agglutinin reaction. In initial reports, donor lymphocytotoxic, HLA-specific, or antigranulocyte antibodies were found in the plasma of the blood product being infused in about 85 percent of cases. However, other studies found a lower incidence of antibodies in patients with TRALI and only a few patients with these antibodies develop TRALI. It is now thought that TRALI may require two sequential insults:

  • A stimulus that primes neutrophils and activates endothelial cells, leading to increased expression of adhesion molecules (eg, ί2-integrins, selectins, and ICAM-1) and nonspecific adherence of neutrophils to the pulmonary endothelium. Potential stimuli for this process include recent surgery, cytokine administration, massive blood transfusion, or active infection.
  • A subsequent, second stimulus that activates adherent neutrophils causing release of toxic mediators, endothelial damage, and increased capillary permeability. This second process may be initiated by a lipid-soluble species that is formed during the storage of banked blood.

In an animal model, for example, TRALI was induced by endotoxin pretreatment (which causes increased neutrophil adherence) followed by the infusion of plasma from stored packed red blood cells; infusion of fresh plasma did not produce this effect.

Treatment — Treatment is supportive. High-dose steroid therapy has been used but appears to be ineffective.

POSTTRANSFUSION PURPURA — Post-transfusion purpura (PTP) is an uncommon transfusion reaction that occurs primarily in women. Severe thrombocytopenia, lasting days to weeks, develops about 5 to 10 days following transfusion of a platelet-containing product such as red cells, platelets, or granulocyte concentrates. Posttransfusion purpura is an immune thrombocytopenia and may be confused with drug-induced or idiopathic thrombocytopenic purpura, since the blood and bone marrow smears are essentially the same.

Patients with PTP have been sensitized to a foreign antigen by pregnancy or by prior transfusion. The antigen most commonly implicated in this disorder is the platelet antigen PlA1, now known as human platelet antigen 1a, (HPA-1a). It is also the antigen system most commonly implicated in neonatal alloimmune purpura.

The female-to-male ratio of the approximately 250 reported cases is 26:1. Approximately 98 to 99 percent of Caucasian women are PlA1 positive as are their spouses. A woman lacking the antigen can be sensitized by pregnancy, while a man or woman can be sensitized by prior transfusion.

Posttransfusion purpura might be thought of as a DHTR involving the platelets. The difference is that the antigen-negative platelets of the recipient are also involved. The mechanism by which this occurs is not well understood. One possibility is that the patient's platelets adsorb immune complexes and are destroyed, or that an autoantibody, in response to the foreign protein, is elaborated, or that the antigen is passively acquired from donor plasma.

Treatment — The infusion of PlA1-negative platelets is generally not effective because even antigen-negative platelets are destroyed. High dose corticosteroids have been useful in the past, as has exchange transfusion; however, both take two or more weeks to act and are not without negative effects.

The preferred therapy is intravenous immune globulin (IVIG) in high doses (400 to 500 mg/kg per day, usually for five days); alternatively, 1.0 g/kg per day for two days can be given for severe thrombocytopenia. It usually takes about four days for the platelet count to exceed 100,000/mm3. Patients with PTP should receive only washed cells or HPA-1a negative cells in the future.

MYELODYSPLASTIC SYNDROMES

Classification, IPSS, Treatment

FAB/WHO Classification

RA - < 5% marrow blasts, <1% periph blasts, no Auer rods, no monos
RARS – same as RA, but RS in > 15% of nucleated erythroid cells
RAEB – 5-20% marrow blasts, <=5% periph blasts, no Auer rods, may have some RS
CMML – 5-20% marrow blasts, <=5% periph blasts, no Auer rods, abs mono > 1000/uL
RAEB-T – 21-30% blasts OR >5% periph blasts, may have Auer rods, may have inc monos. (WHO considers this to be AML).

International Prognostic Scoring System

A. Marrow Blasts: < 5% = 0, 5-10% = 0.5, 11-20% = 1.5, 21-20% = 2.0
B. Karyotype: Good (normal, -Y, 5q-, 20q-) = 0, Poor (> 2 abnormalities, abnl 7) = 1.0, Intermediate (all others) = 0.5
C. Cytopenias (Hb < 10, ANC < 1.8, Plt < 100): 0/1 = 0, 2-3 = 0.5

Score/Risk Group

0          Low                 MS 6 yrs, 20% risk of AML

0.5-1.0 Intermediate-1            MS 4 yrs, 30% risk of AML

1.5-2.0 Intermediate-2            MS 2 yrs, 50% risk of AML

2.5-3.5 High                MS 1 yr, 80% risk of AML

 

Treatment Algorithm

 

Anemia – use epo, ATG, or lenalidomide (Revlimid)

Neutropenia, Thrombocytopenia – SCT, azacitidine, decitabine, ATG, or AML-like therapy

Increased blasts – SCT, azacitidine, decitabine

 

Azacitidine, a demethylating agent, has 25% RR (7-9% CR, 16-18% PR [small study]), but some improvement in QOL. Given as 75 mg/m2 days 1-7 on a 28 day cycle. Decitabine, is an analogue that has similar results. Postulated that methylation turns off certain genes, such as tumor suppressor genes. By inhibiting the action of DNA methyltransferase, the DNA strands begin to function again. This is unproven. Decitabine was given as 15 mg/m2 IV/3 hrs Q8h x 9 doses every 6 weeks, up to 10 cycles. (Assessed after every 2 cycles, and given CR+2 up to 10.) Higher risk patients seem to benefit the most.